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Title:
FOOD COMPOSITIONS COMPRISING TAILORED OILS
Document Type and Number:
WIPO Patent Application WO/2011/150411
Kind Code:
A1
Abstract:
Methods and compositions for the production of food compositions, oils, fuels, oleochemicals, and other compounds in recombinant microorganisms are provided, including oil-bearing microorganisms and methods of low cost cultivation of such microorganisms. Microalgal cells containing exogenous genes encoding, for example, a lipase, a sucrose transporter, a sucrose invertase, a fructokinase, a polysaccharide-degrading enzyme, a keto acyl-ACP synthase enzyme, a fatty acyl-ACP thioesterase, a fatty acyl-CoA/aldehyde reductase, a fatty acyl-CoA reductase, a fatty aldehyde reductase, a fatty aldehyde decarbonylase, and/or an acyl carrier protein are useful in manufacturing food compositions, and transportation fuels such as renewable diesel, biodiesel, and renewable jet fuel, as well as oleochemicals such as functional fluids, surfactants, soaps and lubricants.

Inventors:
FRANKLIN, Scott (225 Gateway Boulevard, South San Francisco, California, 94080, US)
SOMANCHI, Aravind (225 Gateway Boulevard, South San Francisco, California, 94080, US)
WEE, Janice (225 Gateway Boulevard, South San Francisco, California, 94080, US)
RUDENKO, George (225 Gateway Boulevard, South San Francisco, California, 94080, US)
MOSELEY, Jeffrey L (225 Gateway Boulevard, South San Francisco, California, 94080, US)
RAKITSKY, Walt (225 Gateway Boulevard, South San Francisco, California, 94080, US)
Application Number:
US2011/038464
Publication Date:
December 01, 2011
Filing Date:
May 27, 2011
Export Citation:
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Assignee:
SOLAZYME, INC. (225 Gateway Boulevard, South San Francisco, California, 94080, US)
FRANKLIN, Scott (225 Gateway Boulevard, South San Francisco, California, 94080, US)
SOMANCHI, Aravind (225 Gateway Boulevard, South San Francisco, California, 94080, US)
WEE, Janice (225 Gateway Boulevard, South San Francisco, California, 94080, US)
RUDENKO, George (225 Gateway Boulevard, South San Francisco, California, 94080, US)
MOSELEY, Jeffrey L (225 Gateway Boulevard, South San Francisco, California, 94080, US)
RAKITSKY, Walt (225 Gateway Boulevard, South San Francisco, California, 94080, US)
International Classes:
A23D9/00; C11B5/00
Domestic Patent References:
2010-04-22
Foreign References:
US20090061493A12009-03-05
Attorney, Agent or Firm:
TERMES, Lance A. et al. (Alston & Bird LLP, Bank of America Plaza Suite 4000 101 South Tryon StreetCharlotte, North Carolina, 28280-4000, US)
Download PDF:
Claims:
WHAT IS CLAIMED IS:

1. A food composition comprising at least 0.1% w/w recombinant algal biomass and one or more other ingredient, wherein the recombinant algal biomass comprises at least about 10% triglyceride oil by weight and is cultured under heterotrophic conditions.

2. The food composition of claim 1, wherein the triglyceride oil comprises a fatty acid profile selected from the group consisting of at least about 1% C8:0, at least about 1% C10:0, at least about 1% C12:0, at least about 2% C14:0, at least about 30% C16:0, at least about 5% C18:0, at least about 60% C18: l, less than about 7% C18:2, and at least about 35% saturated fatty acids.

3. The food composition of claim 1, wherein the fatty acid profile of algal triglyceride oil is similar to the fatty acid profile of a naturally occurring oil.

4. The food composition of claim 3, wherein the naturally occurring oil is selected from the group consisting of cocoa butter, coconut oil, palm oil, beef tallow, and lard.

5. The food composition of claim 1, wherein the fatty acid profile of the algal triglyceride oil is at least 60% C18:l.

6. The food composition of claim 5, wherein the fatty acid profile of the algal triglyceride oil is at least 80% C18:l.

7. The food composition of claim 1, wherein the recombinant algal biomass is predominantly intact cells.

8. The food composition of claim 1, wherein the recombinant algal biomass is predominantly lysed cells.

9. The food composition of claim 8, wherein the recombinant algal biomass is a homogenate.

10. The food composition of claim 8, wherein the recombinant algal biomass is a powder.

11. The food composition of claim 8, wherein the recombinant algal biomass is in the form of a powder, and wherein the recombinant algal biomass comprises at least about 40% triglyceride oil by dry weight.

12. The food composition of claim 1, that is a salad dressing, egg product, baked good, bread, bar, snack chip, pasta, sauce, soup, beverage, frozen dessert, butter or spread.

13. The food composition of claim 10, wherein average particle size of the powder is from about 0.2 to about 20 microns.

14. The food composition of claim 1, wherein the recombinant algal biomass is a species of the genus Chlorella or Prototheca.

15. The food composition of claim 14, wherein the recombinant algal biomass is selected from the group consisting of Chlorella protothecoides and Prototheca moriformis.

16. A method of making a food composition comprising combining heterotrophically cultivated recombinant algal biomass comprising at least 10% triglyceride oil by weight with at least one other ingredient.

17. The method of claim 16, wherein said triglyceride oil comprises a fatty acid profile selected from the group consisting of at least about 1% C8:0, at least about 1% C10:0, at least about 1% C12:0, at least about 2% C14:0, at least about 30% C16:0, at least about 5% C18:0, at least about 60% C18:l, less than about 7% C18:2, and at least about 35% saturated fatty acids.

18. The method of claim 16 comprising the steps of:

a. determining the amount non-algal oil, non-algal fat or egg in a conventional food product; and

b. replacing a portion of the non-algal oil, non-algal fat or egg or supplementing the non-algal oil, non-algal fat or egg with a specified amount of recombinant algal biomass.

19. The method of claim 18, wherein non-algal oil, non-algal fat or egg is not added to the food composition.

20. The method of claim 18, wherein the amount of recombinant algal biomass is from about 0.25 times to about 4 times the mass or volume of the non-algal oil, non-algal fat or egg in the conventional food product.

21. The method of claim 18, wherein the recombinant algal biomass is predominantly lysed and is in the form of a powder or a homogenate.

22. The method of claim 16, wherein the recombinant algal biomass is a species of the genus Chlorella or Prototheca.

23. The method of claim 22, wherein the recombinant algal biomass is selected from the group consisting of Chlorella protothecoides and Prototheca moriformis.

24. A food composition comprising at least about 0.1% w/w recombinant algal biomass and one or more other ingredient, wherein the recombinant algal biomass is a color mutant and comprises at least about 10% triglyceride oil by dry weight.

25. The food composition of claim 24, wherein the triglyceride oil comprises a fatty acid profile selected from the group consisting of at least about 1% C8:0, at least about 1% C10:0, at least about 1% C12:0, at least about 2% C14:0, at least about 30% C16:0, at least about 5% C18:0, at least about 60% C18: l, less than about 7% C18:2, and at least about 35% saturated fatty acids..

26. The food composition of claim 24, wherein the recombinant algal biomass is a species of the genus Chlorella or Prototheca.

27. The food composition of claim 26, wherein the recombinant algal biomass is selected from the group consisting of Chlorella protothecoides and Prototheca moriformis.

28. The food composition of claim 24, wherein the recombinant algal biomass is cultured under GMP conditions.

29. A food composition comprising at least 0.1% w/w algal triglyceride oil and one or more other ingredient, wherein the algal triglyceride oil is isolated from recombinant algal biomass that is cultured under heterotrophic conditions.

30. The food composition of claim 29, wherein said triglyceride oil comprises a fatty acid profile selected from the group consisting of at least about 1% C8:0, at least about 1% C10:0, at least about 1% C12:0, at least about 2% C14:0, at least about 30% C16:0, at least about 5% C18:0, at least about 60% C18: l, less than about 7% C18:2, and at least about 35% saturated fatty acids.

31. The food composition of claim 29, wherein the profile of the recombinant algal triglyceride oil is similar to the triglyceride profile of a naturally occurring oil.

32. The food composition of claim 31, wherein the naturally occurring oil is selected from the group consisting of cocoa butter, coconut oil, palm oil, beef tallow, and lard.

33. The food composition of claim 29, wherein the profile of the algal triglyceride oil is at least 60% C18:l.

34. The food composition of claim 33, wherein the profile of the algal triglyceride oil is at least 80% C18:l.

35. The food composition of claim 29, wherein the recombinant algal biomass is a species of the genus Chlorella or Prototheca.

36. The food composition of claim 35, wherein the recombinant algal biomass is selected from the group consisting of Chlorella protothecoicies and Prototheca moriformis.

37. The food composition of claim 29 that is a salad dressing, egg product, baked good, bread, bar, snack chip, pasta, sauce, soup, beverage, frozen dessert, butter or spread.

Description:
FOOD COMPOSITIONS COMPRISING TAILORED OILS

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit under 35 U.S.C. 119(e) of US Provisional Patent Application No. 61/349,774, filed May 28, 2010, US Provisional Patent Application No. 61/374,992, filed August 18, 2010, US Provisional Patent Application No. 61/414,393, filed November 16, 2010, and US Provisional Patent Application No. 61/428,192, filed December 29, 2010. Each of these applications is incorporated herein by reference in its entirety for all purposes.

REFERENCE TO A SEQUENCE LISTING

[0002] This application includes a sequence listing as shown in pages 1-195, appended hereto.

FIELD OF THE INVENTION

[0003] The present invention relates to the production of food compositions, oils, fuels, and oleochemicals made from microorganisms. In particular, the disclosure relates to oil-bearing microalgae, methods of cultivating them for the production of biomass and useful compounds, including lipids, fatty acid esters, fatty acids, aldehydes, alcohols, and alkanes, and methods and reagents for genetically altering them to improve production efficiency and alter the type and composition of the oils produced by them.

BACKGROUND OF THE INVENTION

[0004] As the human population continues to increase, there's a growing need for additional food sources, particularly food sources that are inexpensive to produce but nutritious. Moreover, the current reliance on meat as the staple of many diets, at least in the most developed countries, contributes significantly to the release of greenhouse gases, and there' s a need for new foodstuffs that are equally tasty and nutritious yet less harmful to the environment to produce. There remains a need for methods to produce foodstuffs from microorganism, including algae, cheaply and efficiently, at large scale, particularly foodstuffs that are tasty and nutritious. The present invention meets these and other needs

SUMMARY OF THE INVENTION

[0005] The present invention provides oleaginous microbial cells, preferably microalgal cells, having distinct lipid profiles, and includes recombinant cells expressing exogenous genes encoding proteins such as fatty acyl-ACP thioesterases. The present invention also provides methods of making lipids and oil-based products, including fuels such as biodiesel, renewable diesel and jet fuel, from such cells. [0006] In a first aspect, the present invention provides oleaginous microbial cells, preferably microalgal cells, having a lipid profile that is at least 1% or at least 5%, preferably at least 3%, C8:0. In some cases, the lipid profile is at least 10% or at least 15%, preferably at least 12%, C8:0. In some embodiments, the cell is a recombinant cell. In some cases, the recombinant cell comprises an exogenous gene encoding an acyl-ACP thioesterase protein that has hydrolysis activity towards fatty acyl-ACP substrates of chain length C8. In some embodiments, the exogenous gene encodes a Cuphea palustris acyl-ACP thioesterase. In some cases, the cell is a Prototheca cell. In some cases, the cell is of a microalgal genus or species selected from microalgae identified in Table 1.

[0007] In a second aspect, the present invention provides oleaginous microbial cells, preferably microalgal cells, having a lipid profile that is at least 4% C10:0. In some cases, the lipid profile is at least 20%, at least 25% or at least 30%, preferably at least 24%, C10:0. In some cases, the ratio of C10:0 to C12:0 is at least 6: 1. In some embodiments, the cell is a recombinant cell. In some cases, the recombinant cell comprises an exogenous gene encoding an acyl-ACP thioesterase protein that has hydrolysis activity towards fatty acyl- ACP substrates of chain length CIO. In some embodiments, the exogenous gene encodes an acyl-ACP thioesterase protein from a species selected from the group consisting of Cuphea hookeriana and Ulmus americana. In some cases, the cell is a Prototheca cell. In some embodiments, the cell is of a microalgal genus or species selected from microalgae identified in Table 1.

[0008] In a third aspect, the present invention provides oleaginous microbial cells, preferably microalgal cells, having a lipid profile that is at least 10% or at least 15%, prefereably at least 13%, C12:0. In some cases, the lipid profile is at least 30%, at least 35% or at least 40%, preferably at least 34%, C12:0. In some cases, the ratio of C12 to C14 is at least 5: 1. In some cases, the cell is a recombinant cell. In some embodiments, the recombinant cell comprises an exogenous gene encoding an acyl-ACP thioesterase protein that has hydrolysis activity towards fatty acyl-ACP substrates of chain length CI 2. In some cases, the recombinant cell comprises at least two exogenous genes encoding acyl-ACP thioesterase proteins from Umbellularia californica and Cinnamomum camphora that have hydrolysis activity towards fatty acyl-ACP substrates of chain length C12. In some embodiments, the cell is a Prototheca cell.

[0009] In a fourth aspect, the present invention provides oleaginous microbial cells, preferably microalgal cells, having a lipid profile that is at least 5% or at least 15%, preferably at least 10%, C14:0. In some cases, the lipid profile is at least 40%, at least 45%, or at least 50%, preferably at least 43%, C14:0. In some cases, the ratio of C14:0 to C12:0 is at least 7: 1. In some cases, the cell is a recombinant cell. In some embodiments, the recombinant cell comprises an exogenous gene encoding an acyl-ACP thioesterase protein that has hydrolysis activity towards fatty acyl-ACP substrates of chain length CI 4. In some embodiments, the acyl-ACP thioesterase protein is from a species selected from the group consisting of Cinnamomum camphora and Ulmus americana. In some cases, the cell is a Prototheca cell. In some embodiments, the cell is of a microalgal genus or species selected from microalgae identified in Table 1.

[0010] In a fifth aspect, the present invention provides oleaginous microbial cells, preferably microalgal cells, having a lipid profile that is at least 10% or at least 20%, preferably at least 15%, C16:0. In some cases, the lipid profile is at least 30%, at least 35% or at least 40%, preferably at least 37%, C16:0. In some cases, the cell is a recombinant cell. In some embodiments, the recombinant cell comprises an exogenous gene encoding an acyl- ACP thioesterase protein that has hydrolysis activity towards fatty acyl-ACP substrates of chain length C16. In some embodiments, the recombinant cell comprises at least two exogenous genes encoding acyl-ACP thioesterase proteins from Umbellularia californica and Cinnamomum camphora that have hydrolysis activity towards fatty acyl-ACP substrates of chain length C16. In some cases, the cell is a Prototheca cell.

[0011] In a sixth aspect, the present invention provides oleaginous microbial cells, preferably microalgal cells, having a lipid profile that is at least 55% or at least 65%, preferably at least 60%, saturated fatty acids. In some cases the cells,have a lipid profile that is at least 80%, at least 85%, or at least 90%, preferably at least 86%, saturated fatty acids. In some cases, the cell is a recombinant cell. In some embodiments, the recombinant cell comprises an exogenous gene encoding an acyl-ACP thioesterase protein that has hydrolysis activity towards fatty acyl-ACP substrates of chain lengths C10-C16. In some embodiments, the cell comprises an exogenous gene encoding a ketoacyl synthase protein. In some cases, the cell is a Prototheca cell.

[0012] In a seventh aspect, the present invention provides oleaginous microbial cells, preferably microalgal cells, comprising a mutated endogenous desaturase gene, wherein the mutation renders the gene or desaturase inactive. In some cases, the cell has a lipid profile that is at least 40% or at least 50%, preferably at least 45%, saturated fatty acids. In some cases, the cell has a lipid profile that is at least 15%, at least 20% or at least 25%, preferably at least 19%, CI 8:0. In some embodiments, the cell comprises a mutated endogenous desaturase gene that results in at least a 2-fold increase in C18:0 fatty acid, as compared to a wild-type cell. In some cases, the microalgal cell has a lipid profile that is no more than 1% or no more than 5%, preferably no more than 2%, C18:2. In some embodiments, the microalgal cell has a lipid profile that is no more than 5% or no more than 10%, preferably no more than 7%, 18: 1.

[0013] In some embodiments of the recombinant cells discussed herein, the cell comprises a mutated endogenous desaturase gene, wherein the mutation renders the gene or desaturase inactive.

[0014] In a eighth aspect, the present invention provides a method of making lipid. In one embodiment, the method comprises (a) cultivating a cell as discussed above until the cell is at least 15% or at least 25%, preferably at least 20%, lipid by dry weight, and (b) separating the lipid from water-soluble biomass components.

[0015] In a ninth aspect, the present invention provides another method of making lipid. In one embodiment, the method comprises (a) cultivating an oleaginous microbial, preferably a microalgae cell, containing exogenous genes encoding two distinct acyl-ACP thioesterases, wherein the lipid profile of the cell is distinct from (i) the profile of the cell without the exogenous genes and (ii) the profile of the cell with only one of the exogenous genes, and (b) separating the lipid from water-soluble biomass components. In some cases, at least one of the exogenous genes encodes a fatty acyl-ACP thioesterase selected from the group consisting of the thioesterases identified in Table 4.

[0016] In a tenth aspect, the present invention provides a method of making an oil-based product. In one embodiment, the method comprises (a) cultivating a cell as discussed above until the cell is at least 5% or at least 15%, preferably at least 10%, lipid by dry weight, (b) separating the lipid from water-soluble biomass components, and (c) subjecting the lipid to at least one chemical reaction selected from the group consisting of: saponification; metathesis; acid hydrolysis; alkaline hydrolysis; enzymatic hydrolysis; catalytic hydrolysis; hot- compressed water hydrolysis; a catalytic hydrolysis reaction wherein the lipid is split into glycerol and fatty acids; an amination reaction to produce fatty nitrogen compounds; an ozonolysis reaction to produce mono- and dibasic- acids; a triglyceride splitting reaction selected from the group consisting of enzymatic splitting and pressure splitting; a

condensation reaction that follows a hydrolysis reaction; a hydroprocessing reaction; a hydroprocessing reaction and a deoxygenation reaction or a condensation reaction prior to or simultaneous with the hydroprocessing reaction; a gas removal reaction; a deoxygenation reaction selected from the group consisting of a hydrogenolysis reaction, hydrogenation, a consecutive hydrogenation-hydrogenolysis reaction, a consecutive hydrogenolysis - hydrogenation reaction, and a combined hydrogenation-hydrogenolysis reaction; a condensation reaction following a deoxygenation reaction; an esterification reaction; an interestification reaction; a transesterification reaction; a hydroxylation reaction; and a condensation reaction following a hydroxylation reaction, whereby an oil-based product is produced.

[0017] In some cases, the oil-based product is selected from soap or a fuel product. In some embodiments, the oil-based product is a fuel product selected from the group consisting biodiesel, renewable diesel, and jet fuel. In some cases, the fuel product is biodiesel with one or more of the following attributes: (i) 0.01- 0.5 mcg/g, 0.025-0.3 mcg/g, preferably 0.05- 0.244 mcg/g, total carotenoids; (ii) less than 0.01 mcg/g, less than 0.005 mcg/g, preferably less than 0.003 mcg/g, lycopene; (iii) less than 0.01 mcg/g, less than 0.005 mcg/g, preferably less than 0.003 mcg/g, beta carotene; (iv) 0.01-0.5 mcg/g, 0.025-0.3 mcg/g, preferably 0.045- 0.268 mcg/g, chlorophyll A; (v) 1-500 mcg/g, 35-175 mcg/g, preferably 38.3-164 mcg/g, gamma tocopherol; (vi) less than 1%, less than 0.5%, preferably less than 0.25%, brassicasterol, campesterol, stignasterol, or beta-sitosterol; (vii) 100-500 mcg/g, 225-350 mcg/g, preferably 249.6-325.3 mcg/g, total tocotrienols; (viii) 0.001-0.1 mcg/g, 0.0025-0.05 mcg/g, preferably 0.003-0.039 mcg/g, lutein; or (ix) 10-500 mcg/g, 50-300 mcg/g, preferably 60.8-261.7 mcg/g, tocopherols. In some cases, the fuel product is renewable diesel that has a T10-T90 of at least 20°C, 40°C or 60°C. In some cases, the fuel product is jet fuel that meets HRJ-5 and/or ASTM specification D1655.

[0018] In an eleventh aspect, the present invention provides a triglyceride oil comprising (a) a lipid profile of at least 3% C8:0, at least 4% C10:0, at least 13% C12:0, at least 10% C14:0, and/or at least 60% saturated fatty acids, and (b) one or more of the following attributes: (i) 0.01-0.5 mcg/g, 0.025-0.3 mcg/g, preferably 0.05-0.244 mcg/g, total carotenoids; (ii) less than 0.01 mcg/g, less than 0.005 mcg/g, preferably less than 0.003 mcg/g, lycopene; (iii) less than 0.01 mcg/g, less than 0.005 mcg/g, prefereably less than 0.003 mcg/g, beta carotene; (iv) 0.01-0.5 mcg/g, 0.025-0.3 mcg/g, preferably 0.045-0.268 mcg/g, chlorophyll A; (v) 1-300 mcg/g, 35-175 mcg/g, preferably 38.3-164 mcg/g, gamma tocopherol; (vi) less than 1%, less than 0.5%, preferably less than 0.25%, brassicasterol, campesterol, stignasterol, or beta-sitosterol; (vii) 100-500 mcg/g, 225-350 mcg/g, preferably 249.6-325.3 mcg/g, total tocotrienols; (viii) 0.001-0.1 mcg/g, 0.0025-0.05 mcg/g, preferably 0.003-0.039 mcg/g, lutein; or (ix) 10-500 mcg/g, 50-300 mcg/g, preferably 60.8-261.7 mcg/g, tocopherols. [0019] In a twelvth aspect, the present invention provides an isolated oil from microalgae that has a C8:C10 fatty acid ratio of at least 5: 1. In a related aspect, the present invention provides an isolated oil from microalgae with at least 50% to 75%, preferably at least 60%, saturated fatty acids. In another related aspect, the present invention provides an isolated oil from microalgae that has a C16: 14 fatty acid ratio of about 2: 1. In still another related aspect, the present invention provides an isolated oil from microalgae that has a C12:C14 fatty acid ratio of at least 5: 1. In some embodiments, the microalgae contains at least one exogenous gene. In some cases, the microalgae is of the genus Prototheca.

[0020] In a thirteenth aspect, the present invention provides a triglyceride oil comprising (a) a lipid profile of less than 5% or less than 2%, preferably less than 1%, <C12; between 1%-10%, preferably 2%-7%, C14:0; between 20%-35%, preferably 23%-30%, C16:0;

between 5%-20%, preferably 7%-15%, C18:0; between 35-60%, preferably 40-55%, C18: l ; and between l%-20%, preferably 2-15%, C18:2 fatty acids; and (b) one or more of the following attributes: (i) 0.01-0.5 mcg/g, 0.025-0.3 mcg/g, preferably 0.05-0.244 mcg/g, total carotenoids; (ii) less than 0.01 mcg/g, less than 0.005 mcg/g, preferably less than 0.003 mcg/g, lycopene; (iii) less than 0.01 mcg/g, less than 0.005 mcg/g, preferably less than 0.003 mcg/g, beta carotene; (iv) 0.01-0.5 mcg/g, 0.025-0.3 mcg/g, preferably 0.045-0.268 mcg/g, chlorophyll A; (v) 1-300 mcg/g, 35-175 mcg/g, preferably 38.3-164 mcg/g, gamma tocopherol; (vi) less than 1%, less than 0.5%, preferably less than 0.25%, brassicasterol, campesterol, stignasterol, or beta- sitosterol; (vii) 100-500 mcg/g, 225-350 mcg/g, preferably 249.6-325.3 mcg/g, total tocotrienols; (viii) 0.001-0.1 mcg/g, 0.0025-0.05 mcg/g, preferably 0.003-0.039 mcg/g, lutein; or (ix) 10-500 mcg/g, 50-300, preferably 60.8-261.7 mcg/g, tocopherols.

[0021] In some cases, the triglyceride oil is isolated from a microbe comprising one or more exogenous gene. In some embodiments, the one or more exogenous gene encodes a fatty acyl-ACP thioesterase. In some cases, the fatty acyl-ACP thioesterase has hydrolysis activity towards fatty acyl-ACP substrates of chain length C14. In some embodiments, the microbe further comprises a mutated endogenous desaturase gene, wherein the mutation renders the gene or desaturase inactive.

[0022] In a fourteenth aspect, the present invention provides a method of producing a triglyceride oil comprising a lipid profile of less than 5%, or less than 2%, preferably less than 1%, <C12; between 1%-10%, preferably 2%-7%, C14:0; between 20%-35%, preferably 23%-30%, C16:0; between 5%-20%, preferably 7%-15%, C18:0; between 35%-60%, preferably 40-55%, C18: l ; and between l%-20%, preferably 2-15%, C18:2 fatty acids, wherein the triglyceride oil is isolated from a microbe comprising one or more exogenous gene. In some cases, the triglyceride oil comprises a lipid profile of 1 - 10 , preferably 3- 5%, C14:0; 20 -30 , preferably 25-27%, C16:0; 5%-20%, preferably 10- 15%, C18:0; and 35%-50%, preferably 40-45%, C18: l . In some embodiments, the one or more exogenous gene encodes a fatty acyl-ACP thioesterase. In some cases, the fatty acyl-ACP thioesterase has hydrolysis activity towards fatty acyl-ACP substrates of chain length C14. In some cases, the microbe further comprises a mutated endogenous desaturase gene, wherein the mutation renders the gene or desaturase inactive. In some cases, the one or more exogenous gene is a sucrose invertase. In some embodiments, the mutated endogenous desaturase gene is a stearoyl-acyl carrier protein desaturase (SAD) (e.g., SEQ ID NOs: 199-200). In some embodiments, the mutated endogenous desaturase gene is a fatty acid desaturase (FAD).

[0023] In a fifteenth aspect, the present invention provides a oleaginous microbial cell, preferably a microalgal cell, comprising a triglyceride oil, wherein the fatty acid profile of the triglyceride oil is selected from the group consisting of at least about 1% C8:0, at least about 1% C10:0, at least about 1 % C12:0, at least about 2% C14:0, at least about 30% C16:0, at least about 5% C18:0, at least about 60% C18: l , less than about 7% C18:2, and at least about 35% saturated fatty acids. In some cases, the oleaginous microbial cell comprises an exogenous gene, and optionally, an endogenous desaturase of the oleaginous microbial cell has been inactivated or mutated to have less enzymatic activity.

[0024] In some cases, the fatty acid profile of the triglyceride oil is similar to the fatty acid profile of a naturally occurring oil. In some cases, the naturally occurring oil is selected from the group consisting of cocoa butter, coconut oil, palm oil, palm kernel oil, shea butter, beef tallow and lard. In some cases, the fatty acid profile of the triglyceride oil comprises a profile selected from the group consisting of, the total combined amounts of C8:0 and C10:0 is at least about 10%, the total combined amount of C10:0, C12:0, and C14:0 is at least about 50%, the total combined amount of C16:0, C18:0 and C18: l is at least about 60%, the total combined amount of C18:0, C18: l and C18:2 is at least about 60%, the total combined amount of C14:0, C16:0, C18:0 and C18: l is at least about 60%, and the total combined amount of C18: l and C18:2 is less than about 30%. In some cases, the fatty acid profile of the triglyceride oil comprises a ratio of fatty acids selected from the group consisting of C8:0 to C10:0 ratio of at least about 5 to 1 , C10:0 to C12:0 ratio of at least about 6 to 1 , C12:0 to C14:0 ratio of at least about 5 to 1, C14:0 to C12:0 ratio of at least about 7: 1, and C14:0 to C16:0 ratio of at least about 1 to 2. [0025] In some cases, the endogenous desaturase is selected from the group consisting of stearoyl ACP desaturase and delta 12 fatty acid desaturase. In some cases, the exogenous gene is selected from the group consisting of a gene encoding an acyl-ACP thioesterase. In some cases, the exogenous gene encodes an acyl-ACP thioesterase selected from the group consisting of those identified in Table 4. In some cases, the oleaginous microbial cell further comprises a gene encoding a sucrose invertase.

[0026] In various embodiments, the oleaginous microbial cell is a cell of a microalgal genus or species selected from Achnanthes orientalis, Agmenellum, Amphiprora hyaline, Amphora coffeiformis, Amphora coffeiformis linea, Amphora coffeiformis punctata, Amphora coffeiformis taylori, Amphora coffeiformis tenuis, Amphora delicatissima, Amphora delicatissima capitata, Amphora sp., Anabaena, Ankistrodesmus, Ankistrodesmus falcatus, Boekelovia hooglandii, Borodinella sp., Botryococcus braunii, Botryococcus sudeticus, Carteria, Chaetoceros gracilis, Chaetoceros muelleri, Chaetoceros muelleri subsalsum, Chaetoceros sp., Chlorella anitrata, Chlorella Antarctica, Chlorella aureoviridis, Chlorella Candida, Chlorella capsulate, Chlorella desiccate, Chlorella ellipsoidea, Chlorella emersonii, Chlorella fusca, Chlorella fusca var. vacuolata, Chlorella glucotropha, Chlorella infusionum, Chlorella infusionum var. actophila, Chlorella infusionum var. auxenophila, Chlorella kessleri, Chlorella lobophora (strain SAG 37.88), Chlorella luteoviridis, Chlorella luteoviridis var. aureoviridis, Chlorella luteoviridis var. lutescens, Chlorella miniata, Chlorella minutissima, Chlorella mutabilis, Chlorella nocturna, Chlorella parva, Chlorella photophila, Chlorella pringsheimii, Chlorella protothecoides (including any of UTEX strains 1806, 411, 264, 256, 255, 250, 249, 31, 29, 25, and C CAP strains 211/17 and 21 l/8d), Chlorella protothecoides var. acidicola, Chlorella regularis, Chlorella regularis var.

minima, Chlorella regularis var. umbricata, Chlorella reisiglii, Chlorella saccharophila, Chlorella saccharophila var. ellipsoidea, Chlorella salina, Chlorella simplex, Chlorella sorokiniana, Chlorella sp., Chlorella sphaerica, Chlorella stigmatophora, Chlorella vanniellii, Chlorella vulgaris, Chlorella vulgaris, Chlorella vulgaris f. tertia, Chlorella vulgaris var. autotrophica, Chlorella vulgaris var. viridis, Chlorella vulgaris var. vulgaris, Chlorella vulgaris var. vulgaris f. tertia, Chlorella vulgaris var. vulgaris f. viridis, Chlorella xanthella, Chlorella zofingiensis, Chlorella trebouxioides, Chlorella vulgaris, Chlorococcum infusionum, Chlorococcum sp., Chlorogonium, Chroomonas sp., Chrysosphaera sp., Cricosphaera sp., Cryptomonas sp., Cyclotella cryptica, Cyclotella meneghiniana, Cyclotella sp., Dunaliella sp., Dunaliella bardawil, Dunaliella bioculata, Dunaliella granulate, Dunaliella maritime, Dunaliella minuta, Dunaliella parva, Dunaliella peircei, Dunaliella primolecta, Dunaliella salina, Dunaliella terricola, Dunaliella tertiolecta, Dunaliella viridis, Dunaliella tertiolecta, Eremosphaera viridis, Eremosphaera sp., Ellipsoidon sp., Euglena, Franceia sp., Fragilaria crotonensis, Fragilaria sp., Gleocapsa sp., Gloeothamnion sp., Hymenomonas sp., Isochrysis off. galbana, Isochrysis galbana, Lepocinclis, Micractinium, Micractinium (UTEX LB 2614), Monoraphidium minutum, Monoraphidium sp., Nannochloris sp., Nannochloropsis salina, Nannochloropsis sp., Navicula acceptata, Navicula biskanterae, Navicula pseudotenelloides, Navicula pelliculosa, Navicula saprophila, Navicula sp., Nephrochloris sp., Nephroselmis sp., Nitschia communis, Nitzschia alexandrina, Nitzschia communis, Nitzschia dissipata, Nitzschia frustulum, Nitzschia hantzschiana, Nitzschia inconspicua, Nitzschia intermedia, Nitzschia microcephala, Nitzschia pusilla, Nitzschia pusilla elliptica, Nitzschia pusilla monoensis, Nitzschia quadrangular, Nitzschia sp., Ochromonas sp., Oocystis parva, Oocystis pusilla, Oocystis sp., Oscillatoria limnetica, Oscillatoria sp., Oscillatoria subbrevis, Pascheria acidophila, Pavlova sp., Phagus,

Phormidium, Platymonas sp., Pleurochrysis carterae, Pleurochrysis dentate, Pleurochrysis sp., Prototheca wickerhamii, Prototheca stagnora, Prototheca portoricensis, Prototheca moriformis, Prototheca zopfii, Pyramimonas sp., Pyrobotrys, Sarcinoid chrysophyte, Scenedesmus armatus, Spirogyra, Spirulina platensis, Stichococcus sp., Synechococcus sp., Tetraedron, Tetraselmis sp., Tetraselmis suecica, Thalassiosira weissflogii, and Viridiella fridericiana.

[0027] In some cases, the oleaginous microbial cell is a cell of the genus Prototheca. In some cases, the oleaginous microbial cell is a cell of the genus Prototheca moriformis.

[0028] In some cases, the oleaginous microbial cell is an oleaginous yeast cell. In some cases, the oleaginous microbial cell is an oleaginous bacterial cell.

[0029] In some cases, the naturally occurring oil is cocoa butter and the exogenous gene comprises a Carthamus tinctorus thioesterase gene.. In some cases, the naturally occurring oil is coconut oil. In some cases, the naturally occurring oil is palm oil and the exogenous gene comprises a Elaeis guiniensis thioesterase gene, a Cuphea hookeriana thioesterase gene, a combination of a Cuphea hookeriana KAS IV gene and a Cuphea wrightii FATB2 gene, or a construct designed to disrupt an endogenous KAS II gene.. In some cases, the naturally occurring oil is palm kernel oil and the exogenous gene comprises a combination of a Cuphea wrightii FATB2 gene and a construct designed to disrupt an endogenous SAD2B gene.. In some cases, the naturally occurring oil is shea butter. In some cases, the naturally occurring oil is beef tallow. In some cases, the naturally occurring oil is lard and the exogenous gene comprises a combination of U. californica thioesterase gene and a construct designed to disrupt an endogenous SAD2B gene, a combination of a Garcinia mangostana thioesterase gene and a construct designed to disrupt an endogenous SAD2B gene, a Brassica napus thioesterase gene, or a Cuphea hookeriana thioesterase gene..

[0030] In a sixteenth aspect, the present invention provides an oleaginous microbial triglyceride oil composition, wherein the fatty acid profile of the triglyceride oil is selected from the group consisting of at least about 1% C8:0, at least about 1% C10:0, at least about 1% C12:0, at least about 2% C14:0, at least about 30% C16:0, at least about 5% C18:0, at least about 60% C18: l, less than about 7% C18:2, and at least about 35% saturated fatty acids. In various embodiments, the triglyceride oil composition is produced by cultivating a population of oleaginous microbial cells or recombinant oleaginous microbial cells in a culture medium, wherein the oleaginous microbial cells are as described above, in particular those described above in connection with the fifteeth aspect of the invention.

[0031] In some cases, the oleaginous microbial triglyceride oil composition further comprises an attribute selected from the group consisting of: (i) less than 0.3 mcg/g total carotenoids; (ii) less than 0.005 mcg/g lycopene; (iii) less than 0.005 mcg/g beta carotene; (iv) less than 0.3 mcg/g chlorophyll A; (v) less than 175 mcg/g gamma tocopherol; (vi) less than 0.25% brassicasterol, campesterol, stignasterol, or beta-sitosterol; (vii) less than 350 mcg/g total tocotrienols; (viii) less than 0.05 mcg/g lutein; or (ix) less than 275 mcg/g tocopherols.

[0032] In a seventeenth aspect, the present invention provides a method of producing an oleaginous microbial triglyceride oil composition having a fatty acid profile selected from the group consisting of at least about 1% C8:0, at least about 1% C10:0, at least about 1% C12:0, at least about 2% C14:0, at least about 30% C16:0, at least about 5% C18:0, at least about 60% C18: l, less than about 7% C18:2, and at least about 35% saturated fatty acids, wherein the method comprises the steps of: (a) cultivating a population of oleaginous microbial cells in a culture medium until at least 10% of the dry cell weight of the oleaginous microbial cells is triglyceride oil; and (b) isolating the triglyceride oil composition from the oleaginous microbial cells. In various embodiments, the triglyceride oil composition is produced via cultivation of a population of oleaginous microbial cells or recombinant oleaginous microbial cells as described above, in particular those described above in connection with the fifteenth aspect of the invention.

[0033] In an eighteenth aspect, the present invention provides a method of making an oil- based product, wherein the method comprises the steps of: (a) subjecting the oleaginous microbial triglyceride oil composition, as described above in connection with the sixteenth aspect of the invention, to at least one chemical reaction selected from the group consisting of: saponification; metathesis; acid hydrolysis; alkaline hydrolysis; enzymatic hydrolysis; catalytic hydrolysis; hot-compressed water hydrolysis; a catalytic hydrolysis reaction wherein the lipid is split into glycerol and fatty acids; an amination reaction to produce fatty nitrogen compounds; an ozonolysis reaction to produce mono- and dibasic-acids; a triglyceride splitting reaction selected from the group consisting of enzymatic splitting and pressure splitting; a condensation reaction that follows a hydrolysis reaction; a hydroprocessing reaction; a hydroprocessing reaction and a deoxygenation reaction or a condensation reaction prior to or simultaneous with the hydroprocessing reaction; a gas removal reaction; a deoxygenation reaction selected from the group consisting of a hydrogenolysis reaction, hydrogenation, a consecutive hydrogenation-hydrogenolysis reaction, a consecutive hydrogenolysis-hydrogenation reaction, and a combined hydrogenation-hydrogenolysis reaction; a condensation reaction following a deoxygenation reaction; an esterification reaction; an interestification reaction; a transesterification reaction; a hydroxylation reaction; and a condensation reaction following a hydroxylation reaction; and (b) isolating the product of the reaction from the other components.

[0034] In some cases, the oil-based product is selected from the group consisting of a soap, a fuel, a dielectric fluid, a hydraulic fluid, a plasticizer, a lubricant, a heat transfer fluid, and a metal working fluid. In some cases, the oil-based product is a fuel product selected from the group consisting of: (a) biodiesel; (b) renewable diesel; and (c) jet fuel.

[0035] In some cases, the fuel product is biodiesel with one or more of the following attributes: (i) less than 0.3 mcg/g total carotenoids; (ii) less than 0.005 mcg/g lycopene; (iii) less than 0.005 mcg/g beta carotene; (iv) less than 0.3 mcg/g chlorophyll A; (v) less than 175 mcg/g gamma tocopherol; (vi) less than 0.25% brassicasterol, campesterol, stignasterol, or beta-sitosterol; (vii) less than 350 mcg/g total tocotrienols; (viii) less than 0.05 mcg/g lutein; or (ix) less than 275 mcg/g tocopherols.

[0036] In some cases, the fuel product is renewable diesel that has a T10-T90 of at least 20°C, 40°C or 60°C.

[0037] In some cases, the fuel product is jet fuel that meets HRJ-5 and/or ASTM specification D1655.

[0038] In another aspect, the oleaginous microbial cell of the invention is edible. The triglyceride oils of the invention are also edible. In some cases, the microbial strain is cultivated and processed under good manufacturing process (GMP) conditions. As a food or a food ingredient, the oleaginous microbial cell can be consumed whole. Alternatively, the microbial biomass is processed into a microbial flakes, powder or flour. Microbial flour is prepared by completely or partially lysing the cells in the form of a powder. When processed into microbial flour, the average particle size of lysed microbial biomass is between about 1 to 30 μιη. The lysed microbial cells can agglomerate to form bigger particles of up to 1,000 μιη. In one embodiment, the flour further comprises a flow agent, antioxidants and the like. The microbial cells and the microbial oils of the present invention can be consumed by itself. Alternatively, the microbial cells and the microbial oils of the present invention can be combined with at least one other ingredient. By way of example, the microbial cells and the microbial oils can be combined with edible ingredients, e.g., egg, egg products, milk, dairy products, meats, grains, other edible fats, natural sweetners, artificial sweetners, etc. The microbial cells and the microbial oils can also be combined with preservatives and other ingredients added to processed foods.

[0039] All of the processes described herein can be performed in accordance with GMP or equivalent regulations. In the United States, GMP regulations for manufacturing, packing, or holding human food are codified at 21 C.F.R. 110. These provisions, as well as ancillary provisions referenced therein, are hereby incorporated by reference in their entirety for all purposes. GMP conditions in the Unites States, and equivalent conditions in other jurisdictions, apply in determining whether a food is adulterated (the food has been manufactured under such conditions that it is unfit for food) or has been prepared, packed, or held under unsanitary conditions such that it may have become contaminated or otherwise may have been rendered injurious to health. GMP conditions can include adhering to regulations governing: disease control; cleanliness and training of personnel; maintenance and sanitary operation of buildings and facilities; provision of adequate sanitary facilities and accommodations; design, construction, maintenance, and cleanliness of equipment and utensils; provision of appropriate quality control procedures to ensure all reasonable precautions are taken in receiving, inspecting, transporting, segregating, preparing, manufacturing, packaging, and storing food products according to adequate sanitation principles to prevent contamination from any source; and storage and transportation of finished food under conditions that will protect food against physical, chemical, or undesirable microbial contamination, as well as against deterioration of the food and the container.

[0040] In some embodiments, the oleaginous microbial cells are algal cells of a species of the genus Chlorella or Prototheca. In one embodiment, the algae is Chlorella

protothecoides. In another embodiment, the algae is Prototheca moriformis. In some cases, the biomass is derived from an algae that is a color mutant with reduced color pigmentation compared to the strain from which it was derived. Mutants with reduced color pigmentation are typically prepared using standard mutagenesis techniques. There are many fee-for- service laboratories that will generate mutants with reduced color pigmentation. In some embodiments, the microbial biomass is prepared by cultivating the microorganism

heterotrophically and optionally in the absence of light.

[0041] In one embodiment, the microalgal strain is Chlorella protothecoides 33-55, deposited on October 13, 2009 at the American Type Culture Collection under deposit designation PTA-10397. In one embodiment, the microalgal strain is Chlorella

protothecoides 25-32, deposited on October 13, 2009 at the American Type Culture

Collection under deposit designation PTA- 10396. In some cases, the microalgal strain providing the biomass has been grown and processed under good manufacturing process (GMP) conditions.

[0042] In another aspect, the present invention provides methods for preparing recombinant oleaginous microbial biomass suitable for use as a foodstuff. In these methods, the recombinant oleaginous microbes are fermented under heterotrophic conditions and so lack or have a significantly reduced amount of green pigment that characterizes other recombinant microalgal derived foodstuffs. In one embodiment, the recombinant oleaginous microbes lack or have significantly reduced amounts of any pigment. One aspect of the invention provides recombinant microalgae of the genus Chlorella and Protothecoides. In some embodiments, the invention is a microalgae of the genus recombinant Protothecoides.

Another emboiment provides recombinant Protothecoides moriformis for use in foods and food ingredients. In one embodiment, the fermentation conditions are manipulated to provide a biomass rich in lipid. In another embodiment, the fermentation conditions are manipulated to provide a biomass rich in protein. In all embodiments, the methods can be carried out cheaply and efficiently at large scale (biomass produced in 4500 L or larger fermentors).

[0043] In another aspect, the present invention provides recombinant oleaginous microbial biomass, preferably microalgal biomass, suitable for incorporation into human foodstuffs. In one embodiment, this recombinant microbial biomass is the concentrated biomass resulting directly from the biomass preparation methods of the present invention. In another embodiment, this biomass is in the form of dried flakes resulting from drying, e.g. , drum drying, such biomass preparations. In this latter embodiment, an antioxidant can be added to the biomass prior to the drying step to extend the shelf-life of the biomass and any food product containing such biomass. [0044] Yet another aspect of the invention provides methods for further processing the biomass into flakes or a homogenate. In one embodiment, the dried flakes are rehydrated in deionized water to create a suspension. This suspension is then micronized with a high pressure homogenizer so that the average particle size is less than 20 μιη, preferably 10 μιη in size, creating a homogenate.

[0045] A further aspect of the present invention provides methods for processing the recombinant oleaginous microbial biomass, preferably microalgal biomass, into a food ingredient that is multifunctional in that it provides healthy oils to foods and provides structural benefits to foods such as baked goods. In one embodiment, the processing involves pneumatic drying (e.g. , spray drying or flash drying) the biomass preparation to form a powder that contains a large percentage of intact recombinant cells. In another emobodiment, the biomass is first micronized to disrupt the cells before pneumatic drying to form a flour that contains only a small percentage (or no) intact cells; in some embodiments a flow or dispersal agent is added prior to the drying step.

[0046] In yet another aspect, the present invention is directed to a method of producing an oil or oil-containing recombinant oleaginous microbial biomass suitable for human consumption. In some embodiments, the process involves extracting the lipid (triglyceride) from the biomass to form an oil. In one embodiment, the method comprises providing a microorganism, and culturing the microorganism in the presence of a feedstock that is not derived from a food composition suitable for human consumption, in which the

microorganism converts at least some portion of the feedstock into triglyceride oil. In some cases, the triglyceride oil comprises at least 50%, 60%, 70%, 80% or 90% C18: l lipid.

[0047] The present invention further provides foods that incorporate a recombinant microbial powder, recombinant microbial flour, and/or recombinant microbial oil. In one embodiment, the food is a baked good, dressing, sauce, or mayonnaise in which, relative to the same food produced using conventional recipes, all or a portion of the egg or butter has been replaced by a recombinant microbial flour rich in oil. In another embodiment, the food is a powdered egg product containing a recombinant microbial flour rich in oil. In another embodiment, the food is a liquid egg product containing a microbial flour rich in oil. In another embodiment, the food is a liquid milk product containing microbial protein, fiber, and oil. In another embodiment, the food is a meat product in which, relative to previously available meat products, a portion or all (a meat substitute) of the meat has been replaced by a recombinant microbial flour, recombinant microbial powder, or recombinant microbial flake rich in protein. [0048] The invention also provides methods of inducing satiety by providing recombinant microbial foods or microbial food ingredients containing microbial fiber and optionally microbial protein and/or microbial oil.

[0049] These and other aspects and embodiments of the invention are described in the accompanying drawing, a brief description of which immediately follows, the detailed description of the invention below, and are exemplified in the examples below. Any or all of the features discussed above and throughout the application can be combined in various embodiments of the present invention.

BRIEF DESCRIPTION OF THE DRAWINGS

[0050] Figure 1 shows a chromatogram of renewable diesel produced from Prototheca triglyceride oil.

DETAILED DESCRIPTION OF THE INVENTION

[0051] The present invention arises from the discovery that Prototheca and certain related microorganisms have unexpectedly advantageous properties for the production of oils, fuels, and other hydrocarbon or lipid compositions economically and in large quantities, as well as from the discovery of methods and reagents for genetically altering these microorganisms to improve these properties. The oils produced by these microorganisms can be used in the transportation fuel, oleochemical, and/or food and cosmetic industries, among other applications. Transesterification of lipids yields long-chain fatty acid esters useful as biodiesel. Other enzymatic and chemical processes can be tailored to yield fatty acids, aldehydes, alcohols, alkanes, and alkenes. In some applications, renewable diesel, jet fuel, or other hydrocarbon compounds are produced. The present invention also provides methods of cultivating microalgae for increased productivity and increased lipid yield, and/or for more cost-effective production of the compositions described herein.

[0052] This detailed description of the invention is divided into sections for the convenience of the reader. Section I provides definitions of terms used herein. Section II provides a description of culture conditions useful in the methods of the invention. Section III provides a description of genetic engineering methods and materials. Section IV provides a description of genetic engineering of microorganisms (e.g. , Prototheca) to enable sucrose utilization. Section V provides a description of genetic engineering of microorganisms (e.g. , Prototheca) to modify lipid biosynthesis. Section VI describes methods for making fuels and chemicals. Section VII describes methods for preparing recombinant microbial biomass. Section VIII describes methods for processing recombinant microbial biomass into finished food products. Section IX describes methods for combining recombinant microbial biomass or materials derived therefrom withn other food ingredients. Section X discloses examples and embodiments of the invention. The detailed description of the invention is followed by examples that illustrate the various aspects and embodiments of the invention.

I. DEFINITIONS

[0053] Unless defined otherwise, all technical and scientific terms used herein have the meaning commonly understood by a person skilled in the art to which this invention belongs. The following references provide one of skill with a general definition of many of the terms used in this invention: Singleton et al. , Dictionary of Microbiology and Molecular Biology (2nd ed. 1994); The Cambridge Dictionary of Science and Technology (Walker ed., 1988); The Glossary of Genetics, 5th Ed., R. Rieger et al. (eds.), Springer Verlag (1991); and Hale & Marham, The Harper Collins Dictionary of Biology (1991). As used herein, the following terms have the meanings ascribed to them unless specified otherwise.

[0054] "Active in microalgae" refers to a nucleic acid that is functional in microalgae. For example, a promoter that has been used to drive an antibiotic resistance gene to impart antibiotic resistance to a transgenic microalgae is active in microalgae.

[0055] "Acyl carrier protein" or "ACP" is a protein that binds a growing acyl chain during fatty acid synthesis as a thiol ester at the distal thiol of the 4'-phosphopantetheine moiety and comprises a component of the fatty acid synthase complex.

[0056] "Acyl-CoA molecule" or "acyl-CoA" is a molecule comprising an acyl moiety covalently attached to coenzyme A through a thiol ester linkage at the distal thiol of the 4'- phosphopantetheine moiety of coenzyme A.

[0057] "Area Percent" refers to the area of peaks observed using FAME GC/FID detection methods in which every fatty acid in the sample is converted into a fatty acid methyl ester (FAME) prior to detection. For example, a separate peak is observed for a fatty acid of 14 carbon atoms with no unsaturation (C14:0) compared to any other fatty acid such as C14: l. The peak area for each class of FAME is directly proportional to its percent composition in the mixture and is calculated based on the sum of all peaks present in the sample (i.e. [area under specific peak/ total area of all measured peaks] X 100). When referring to lipid profiles of oils and cells of the invention, "at least 4% C8-C14" means that at least 4% of the total fatty acids in the cell or in the extracted glycerolipid composition have a chain length that includes 8, 10, 12 or 14 carbon atoms.

[0058] "Axenic" is a culture of an organism free from contamination by other living organisms. [0059] "Baked good" means a food item, typically found in a bakery, that is prepared by using an oven. Baked goods include, but are not limited to brownies, cookies, pies, cakes and pastries.

[0060] "Bread" means a food item that contains wheat flour, liquid, and a leavening agent. Breads are usually prepared by baking in an oven, although other methods of cooking are also acceptable. The leavening agent can be chemical or organic in nature. Typically, the organic leavening agent is yeast. In the case where the leavening agent is chemical in nature (such as baking powder and/or baking soda), these food products are referred to as "quick breads".

[0061] "Biodiesel" is a biologically produced fatty acid alkyl ester suitable for use as a fuel in a diesel engine.

[0062] "Biomass" is material produced by growth and/or propagation of cells. Biomass may contain cells and/or intracellular contents as well as extracellular material, includes, but is not limited to, compounds secreted by a cell.

[0063] "Bioreactor" is an enclosure or partial enclosure in which cells are cultured, optionally in suspension.

[0064] "Catalyst" is an agent, such as a molecule or macromolecular complex, capable of facilitating or promoting a chemical reaction of a reactant to a product without becoming a part of the product. A catalyst increases the rate of a reaction, after which, the catalyst may act on another reactant to form the product. A catalyst generally lowers the overall activation energy required for the reaction such that it proceeds more quickly or at a lower temperature. Thus, a reaction equilibrium may be more quickly attained. Examples of catalysts include enzymes, which are biological catalysts; heat, which is a non-biological catalyst; and metals used in fossil oil refining processes.

[0065] "Cellulosic material" is the product of digestion of cellulose, including glucose and xylose, and optionally additional compounds such as disaccharides, oligosaccharides, lignin, furfurals and other compounds. Nonlimiting examples of sources of cellulosic material include sugar cane bagasses, sugar beet pulp, corn stover, wood chips, sawdust and switchgrass.

[0066] "Co-culture", and variants thereof such as "co-cultivate" and "co-ferment", refer to the presence of two or more types of cells in the same bioreactor. The two or more types of cells may both be microorganisms, such as microalgae, or may be a microalgal cell cultured with a different cell type. The culture conditions may be those that foster growth and/or propagation of the two or more cell types or those that facilitate growth and/or proliferation of one, or a subset, of the two or more cells while maintaining cellular growth for the remainder.

[0067] "Cofactor" is any molecule, other than the substrate, required for an enzyme to carry out its enzymatic activity.

[0068] "Complementary DNA" or "cDNA" is a DNA copy of mRNA, usually obtained by reverse transcription of messenger RNA (mRNA) or amplification (e.g., via polymerase chain reaction ("PCR")).

[0069] "Conventional food product" means a composition intended for consumption, e.g. , by a human, that lacks algal biomass or other algal components and includes ingredients ordinarily associated with the food product, particularly a vegetable oil, animal fat, and/or egg(s), together with other edible ingredients. Conventional food products include food products sold in shops and restaurants and those made in the home. Conventional food products are often made by following conventional recipes that specify inclusion of an oil or fat from a non-algal source and/or egg(s) together with other edible ingredient(s).

[0070] "Cooked product" means a food that has been heated, e.g. , in an oven, for a period of time.

[0071] "Creamy salad dressing" means a salad dressing that is a stable dispersion with high viscosity and a slow pour-rate. Generally, creamy salad dressings are opaque.

[0072] "Cultivated", and variants thereof such as "cultured" and "fermented", refer to the intentional fostering of growth (increases in cell size, cellular contents, and/or cellular activity) and/or propagation (increases in cell numbers via mitosis) of one or more cells by use of selected and/or controlled conditions. The combination of both growth and propagation may be termed proliferation. Examples of selected and/or controlled conditions include the use of a defined medium (with known characteristics such as pH, ionic strength, and carbon source), specified temperature, oxygen tension, carbon dioxide levels, and growth in a bioreactor. Cultivate does not refer to the growth or propagation of microorganisms in nature or otherwise without human intervention; for example, natural growth of an organism that ultimately becomes fossilized to produce geological crude oil is not cultivation.

[0073] "Cytolysis" is the lysis of cells in a hypotonic environment. Cytolysis is caused by excessive osmosis, or movement of water, towards the inside of a cell (hyperhydration). The cell cannot withstand the osmotic pressure of the water inside, and so it explodes.

[0074] "Delipidated meal" and "delipidated microbial biomass" is microbial biomass after oil (including lipids) has been extracted or isolated from it, either through the use of mechanical (i.e., exerted by an expeller press) or solvent extraction or both. Delipidated meal has a reduced amount of oil/lipids as compared to before the extraction or isolation of oil/lipids from the microbial biomass but does contain some residual oil/lipid.

[0075] "Dietary fiber" means non-starch carbohydrates found in plants and other organisms containing cell walls, including microalgae. Dietary fiber can be soluble (dissolved in water) or insoluble (not able to be dissolved in water). Soluble and insoluble fiber makes up total dietary fiber.

[0076] "Digestible crude protein" is the portion of protein that is available or can be converted into free nitrogen (amino acids) after digesting with gastric enzymes. In vitro measurement of digestible crude protein is accomplished by using gastric enzymes such as pepsin and digesting a sample and measuring the free amino acid after digestion. In vivo measurement of digestible crude protein is accomplished by measuring the protein levels in a feed/food sample and feeding the sample to an animal and measuring the amount of nitrogen collected in the animal's feces.

[0077] "Dry weight" and "dry cell weight" mean weight determined in the relative absence of water. For example, reference to recombinant microalgal biomass as comprising a specified percentage of a particular component by dry weight means that the percentage is calculated based on the weight of the biomass after substantially all water has been removed.

[0078] "Edible ingredient" means any substance or composition which is fit to be eaten. "Edible ingredients" include, without limitation, grains, fruits, vegetables, proteins, herbs, spices, carbohydrates, and fats.

[0079] "Expression vector" or "expression construct" or "plasmid" or "recombinant DNA construct" refer to a nucleic acid that has been generated via human intervention, including by recombinant means or direct chemical synthesis, with a series of specified nucleic acid elements that permit transcription and/or translation of a particular nucleic acid in a host cell. The expression vector can be part of a plasmid, virus, or nucleic acid fragment. Typically, the expression vector includes a nucleic acid to be transcribed operably linked to a promoter.

[0080] "Exogenous gene" is a nucleic acid that codes for the expression of an RNA and/or protein that has been introduced ("transformed") into a cell. A transformed cell may be referred to as a recombinant cell, into which additional exogenous gene(s) may be introduced. The exogenous gene may be from a different species (and so heterologous), or from the same species (and so homologous), relative to the cell being transformed. Thus, an exogenous gene can include a homologous gene that occupies a different location in the genome of the cell or is under different control, relative to the endogenous copy of the gene. An exogenous gene may be present in more than one copy in the cell. An exogenous gene may be maintained in a cell as an insertion into the genome or as an episomal molecule.

[0081] "Exogenously provided" refers to a molecule provided to the culture media of a cell culture.

[0082] "Expeller pressing" is a mechanical method for extracting oil from raw materials such as soybeans and rapeseed. An expeller press is a screw type machine, which presses material through a caged barrel-like cavity. Raw materials enter one side of the press and spent cake exits the other side while oil seeps out between the bars in the cage and is collected. The machine uses friction and continuous pressure from the screw drives to move and compress the raw material. The oil seeps through small openings that do not allow solids to pass through. As the raw material is pressed, friction typically causes it to heat up.

[0083] "Fat" means a lipid or mixture of lipids that is generally solid at ordinary room temperatures and pressures. "Fat" includes, without limitation, lard and butter.

[0084] "Fatty acyl-ACP thioesterase" is an enzyme that catalyzes the cleavage of a fatty acid from an acyl carrier protein (ACP) during lipid synthesis.

[0085] "Fatty acyl- Co A/aldehyde reductase" is an enzyme that catalyzes the reduction of an acyl-CoA molecule to a primary alcohol.

[0086] "Fatty acyl-CoA reductase" is an enzyme that catalyzes the reduction of an acyl- CoA molecule to an aldehyde.

[0087] "Fatty aldehyde decarbonylase" is an enzyme that catalyzes the conversion of a fatty aldehyde to an alkane.

[0088] "Fatty aldehyde reductase" is an enzyme that catalyzes the reduction of an aldehyde to a primary alcohol.

[0089] "Fiber" means non-starch carbohydrates in the form of polysaccharide. Fiber can be soluble in water or insoluble in water. Many microalgae produce both soluble and insoluble fiber, typically residing in the cell wall.

[0090] "Finished food product" and "finished food ingredient" mean a food composition that is ready for packaging, use, or consumption. For example, a "finished food product" may have been cooked or the ingredients comprising the "finished food product" may have been mixed or otherwise integrated with one another. A "finished food ingredient" is typically used in combination with other ingredients to form a food product.

[0091] "Fixed carbon source" is a molecule(s) containing carbon, typically an organic molecule, that is present at ambient temperature and pressure in solid or liquid form in a culture media that can be utilized by a microorganism cultured therein. [0092] "Food", "food composition", "food product" and "foodstuff mean any composition intended to be or expected to be ingested by humans as a source of nutrition and/or calories. Food compositions are composed primarily of carbohydrates, fats, water and/or proteins and make up substantially all of a person' s daily caloric intake. A "food composition" can have a weight minimum that is at least ten times the weight of a typical tablet or capsule (typical tablet/capsule weight ranges are from less than or equal to 100 mg up to 1500 mg). A "food composition" is not encapsulated or in tablet form.

[0093] "Good manufacturing practice" and "GMP" mean those conditions established by regulations set forth at 21 C.F.R. 110 (for human food) and 111 (for dietary supplements), or comparable regulatory schemes established in locales outside the United States. The U.S. regulations are promulgated by the U.S. Food and Drug Administration under the authority of the Federal Food, Drug, and Cosmetic Act to regulate manufacturers, processors, and packagers of food products and dietary supplements for human consumption.

[0094] "Homogenate" means biomass that has been physically disrupted. Homogenization is a fluid mechanical process that involves the subdivision of particles into smaller and more uniform sizes, forming a dispersion that may be subjected to further processing.

Homogenization is used in treatment of several foods and dairy products to improve stability, shelf-life, digestion, and taste.

[0095] "Hydrocarbon" is (a) a molecule containing only hydrogen and carbon atoms wherein the carbon atoms are covalently linked to form a linear, branched, cyclic, or partially cyclic backbone to which the hydrogen atoms are attached. The molecular structure of hydrocarbon compounds varies from the simplest, in the form of methane (CH 4 ), which is a constituent of natural gas, to the very heavy and very complex, such as some molecules such as asphaltenes found in crude oil, petroleum, and bitumens. Hydrocarbons may be in gaseous, liquid, or solid form, or any combination of these forms, and may have one or more double or triple bonds between adjacent carbon atoms in the backbone. Accordingly, the term includes linear, branched, cyclic, or partially cyclic alkanes, alkenes, lipids, and paraffin. Examples include propane, butane, pentane, hexane, octane, and squalene.

[0096] "Hydrogen: carbon ratio" is the ratio of hydrogen atoms to carbon atoms in a molecule on an atom-to-atom basis. The ratio may be used to refer to the number of carbon and hydrogen atoms in a hydrocarbon molecule. For example, the hydrocarbon with the highest ratio is methane CH 4 (4: 1). [0097] "Hydrophobic fraction" is the portion, or fraction, of a material that is more soluble in a hydrophobic phase in comparison to an aqueous phase. A hydrophobic fraction is substantially insoluble in water and usually non-polar.

[0098] "Increase lipid yield" refers to an increase in the productivity of a microbial culture by, for example, increasing dry weight of cells per liter of culture, increasing the percentage of cells that constitute lipid, or increasing the overall amount of lipid per liter of culture volume per unit time.

[0099] "Inducible promoter" is a promoter that mediates transcription of an operably linked gene in response to a particular stimulus. Examples of such promoters may be promoter sequences that are induced in conditions of changing pH or nitrogen levels.

[0100] "In operable linkage" is a functional linkage between two nucleic acid sequences, such a control sequence (typically a promoter) and the linked sequence (typically a sequence that encodes a protein, also called a coding sequence). A promoter is in operable linkage with an exogenous gene if it can mediate transcription of the gene.

[0101] "In situ" means "in place" or "in its original position".

[0102] "Limiting concentration of a nutrient" is a concentration of a compound in a culture that limits the propagation of a cultured organism. A "non-limiting concentration of a nutrient" is a concentration that supports maximal propagation during a given culture period. Thus, the number of cells produced during a given culture period is lower in the presence of a limiting concentration of a nutrient than when the nutrient is non-limiting. A nutrient is said to be "in excess" in a culture, when the nutrient is present at a concentration greater than that which supports maximal propagation.

[0103] "Lipase" is a water-soluble enzyme that catalyzes the hydrolysis of ester bonds in water-insoluble, lipid substrates. Lipases catalyze the hydrolysis of lipids into glycerols and fatty acids.

[0104] "Lipid modification enzyme" refers to an enayme that alters the covalent structure of a lipid. Examples of lipid modification enzymes include a lipase, a fatty acyl-ACP thioesterase, a fatty acyl-Co A/aldehyde reductase, a fatty acyl-CoA reductase, a fatty aldehyde reductase, a desaturase, including a stearoyl acyl carrier protein desaturase (SAD) and a fatty acyl destaurase (FAD), and a fatty aldehyde decarbonylase.

[0105] "Lipid pathway enzyme" is any enzyme that plays a role in lipid metabolism, i.e., either lipid synthesis, modification, or degradation, and any proteins that chemically modify lipids, as well as carrier proteins. [0106] "Lipids" are a class of molecules that are soluble in nonpolar solvents (such as ether and chloroform) and are relatively or completely insoluble in water. Lipid molecules have these properties, because they consist largely of long hydrocarbon tails which are hydrophobic in nature. Examples of lipids include fatty acids (saturated and unsaturated); glycerides or glycerolipids (such as monoglycerides, diglycerides, triglycerides or neutral fats, and phosphoglycerides or glycerophospholipids); nonglycerides (sphingolipids, sterol lipids including cholesterol and steroid hormones, prenol lipids including terpenoids, fatty alcohols, waxes, and polyketides); and complex lipid derivatives (sugar-linked lipids, or glycolipids, and protein-linked lipids). "Fats" are a subgroup of lipids called

"triacylglycerides."

[0107] "Lysate" is a solution containing the contents of lysed cells.

[0108] "Lysis" is the breakage of the plasma membrane and optionally the cell wall of a biological organism sufficient to release at least some intracellular content, often by mechanical, viral or osmotic mechanisms that compromise its integrity.

[0109] "Lysing" is disrupting the cellular membrane and optionally the cell wall of a biological organism or cell sufficient to release at least some intracellular content.

[0110] "Microalgae" is a eukarytotic microbial organism that contains a chloroplast or plastid, and optionally that is capable of performing photosynthesis, or a prokaryotic microbial organism capable of performing photosynthesis. Microalgae include obligate photoautotrophs, which cannot metabolize a fixed carbon source as energy, as well as heterotrophs, which can live solely off of a fixed carbon source. Microalgae include unicellular organisms that separate from sister cells shortly after cell division, such as Chlamydomonas, as well as microbes such as, for example, Volvox, which is a simple multicellular photosynthetic microbe of two distinct cell types. Microalgae include cells such as Chlorella, Dunaliella, and Prototheca. Microalgae also include other microbial photosynthetic organisms that exhibit cell-cell adhesion, such as Agmenellum, Anabaena, and Pyrobotrys. Microalgae also include obligate heterotrophic microorganisms that have lost the ability to perform photosynthesis, such as certain dinoflagellate algae species and species of the genus Prototheca.

[0111] "Microbial biomass," "Microalgal biomass," "algal biomass," and "biomass" mean a material produced by growth and/or propagation of microbial or microalgal cells. Biomass may contain cells and/or intracellular contents as well as extracellular material. Extracellular material includes, but is not limited to, compounds secreted by a cell. [0112] "Micronized" means biomass that has been homogenized under high pressure (or an equivalent process) so that at least 50% of the particle size is no more 10 μιη in their longest dimension. Typically, at least 50% to 90% or more of such particles are less than 5 μιη in their longest dimension. In any case, the average particle size of micronized biomass is smaller than the intact recombinant microalgal cell.

[0113] "Microorganism" and "microbe" are microscopic unicellular organisms.

[0114] "Naturally co-expressed" with reference to two proteins or genes means that the proteins or their genes are co-expressed naturally in a tissue or organism from which they are derived, e.g., because the genes encoding the two proteins are under the control of a common regulatory sequence or because they are expressed in response to the same stimulus.

[0115] "Oil" means any triacylglyceride, produced by organisms, including microalgae, other plants, and/or animals. "Oil," as distinguished from "fat", refers, unless otherwise indicated, to lipids that are generally liquid at ordinary room temperatures and pressures. For example, "oil" includes vegetable or seed oils derived from plants, including without limitation, an oil derived from soy, rapeseed, canola, palm, palm kernel, coconut, corn, olive, sunflower, cotton seed, cuphea, peanut, camelina sativa, mustard seed, cashew nut, oats, lupine, kenaf, calendula, hemp, coffee, linseed, hazelnut, euphorbia, pumpkin seed, coriander, camellia, sesame, safflower, rice, tung oil tree, cocoa, copra, pium poppy, castor beans, pecan, jojoba, jatropha, macadamia, Brazil nuts, and avocado, as well as combinations thereof.

[0116] "Osmotic shock" is the rupture of cells in a solution following a sudden reduction in osmotic pressure. Osmotic shock is sometimes induced to release cellular components of such cells into a solution.

[0117] "Pasteurization" means a process of heating which is intended to slow microbial growth in food products. Typically pasteurization is performed at a high temperature (but below boiling) for a short amount of time. As described herein, pasteurization can not only reduce the number of undesired microbes in food products, but can also inactivate certain enzymes present in the food product.

[0118] "Polys accharide-degrading enzyme" is any enzyme capable of catalyzing the hydrolysis, or saccharification, of any polysaccharide. For example, cellulases catalyze the hydrolysis of cellulose.

[0119] "Polysaccharides" or "glycans" are carbohydrates made up of monosaccharides joined together by glycosidic linkages. Cellulose is a polysaccharide that makes up certain plant cell walls. Cellulose can be depolymerized by enzymes to yield monosaccharides such as xylose and glucose, as well as larger disaccharides and oligosaccharides.

[0120] "Port" means an opening in a bioreactor that allows influx or efflux of materials such as gases, liquids, and cells; a port is usually connected to tubing.

[0121] "Predominantly encapsulated" means that more than 50% and typically more than

75% to 90% of a referenced component, e.g. , algal oil, is sequestered in a referenced container, which can include, e.g. , a recombinant microalgal cell.

[0122] "Predominantly intact cells" and "predominantly intact biomass" mean a population of cells that comprise more than 50, and oftern more than 75, 90, and 98% intact cells.

"Intact", in this context, means that the physical continuity of the cellular membrane and/or cell wall enclosing the intracellular components of the cell has not been disrupted in any manner that would release the intracellular components of the cell to an extent that exceeds the permeability of the cellular membrane in culture.

[0123] "Predominantly lysed" means a population of cells in which more than 50%, and typically more than 75 to 90%, of the cells have been disrupted such that the intracellular components of the cell are no longer completely enclosed within the cell membrane.

[0124] "Proliferation" means a combination of both growth and propagation.

[0125] "Promoter" is a nucleic acid control sequence that directs transcription of a nucleic acid. As used herein, a promoter includes necessary nucleic acid sequences near the start site of transcription, such as, in the case of a polymerase II type promoter, a TATA element. A promoter also optionally includes distal enhancer or repressor elements, which can be located as much as several thousand base pairs from the start site of transcription.

[0126] "Propagation" means an increase in cell number via mitosis or other cell division.

[0127] "Proximate analysis" means analysis of foodstuffs for fat, nitrogen/protein, crude fiber (cellulose and lignin as main components), moisture and ash. Soluble carbohydrate (total dietary fiber and free sugars) can be calculated by subtracting the total of the known values of the proximate analysis from 100 (carbohydrate by difference).

[0128] "Recombinant" is a cell, nucleic acid, protein or vector, that has been modified due to the introduction of an exogenous nucleic acid or the alteration of a native nucleic acid. Thus, e.g., recombinant cells express genes that are not found within the native (non- recombinant) form of the cell or express native genes differently than those genes are expressed by a non-recombinant cell. A "recombinant nucleic acid" is a nucleic acid originally formed in vitro, in general, by the manipulation of nucleic acid, e.g., using polymerases and endonucleases, or otherwise is in a form not normally found in nature. Recombinant nucleic acids may be produced, for example, to place two or more nucleic acids in operable linkage. Thus, an isolated nucleic acid or an expression vector formed in vitro by ligating DNA molecules that are not normally joined in nature, are both considered recombinant for the purposes of this invention. Once a recombinant nucleic acid is made and introduced into a host cell or organism, it may replicate using the in vivo cellular machinery of the host cell; however, such nucleic acids, once produced recombinantly, although subsequently replicated intracellularly, are still considered recombinant for purposes of this invention. Similarly, a "recombinant protein" is a protein made using recombinant techniques, i.e., through the expression of a recombinant nucleic acid.

[0129] "Recombinant microbial oil," "Recombinant microalgal oil" and "algal oil" mean any of the lipid components produced by recombinant microbial or microalgal cells or algal cells, respectively, including triacylglycerols.

[0130] "Renewable diesel" is a mixture of alkanes (such as C10:0, C12:0, C14:0, C16:0 and CI 8:0) produced through hydrogenation and deoxygenation of lipids.

[0131] "Saccharification" is a process of converting biomass, usually cellulosic or lignocellulosic biomass, into monomeric sugars, such as glucose and xylose. "Saccharified" or "depolymerized" cellulosic material or biomass refers to cellulosic material or biomass that has been converted into monomeric sugars through saccharification.

[0132] The term "similar," when used in the context of a comparison to a naturally occurring oil, without further qualification, means that the oil being compared to the naturally occurring oil contains about +/- 15%, or +/- 10% of the top two triglycerides of the naturally occurring oil. For example, Shea butter (the oil of B. Parkii) contains 41.2-56.8% C18:0 and 34.0-46.9% C18: l as the two most common triglyceride components (see Table 5). A "similar" oil that is within +/- 10% would contain from about 37% to about 62% C18:0 and from 31% to about 52% C18: l as the two most common triglyceride components. When used in this context, the term "similar" includes +/- 9%, +/- 8%, +/- 7%, +/- 6%, +/- 5%, +/- 4%, +/- 3%, +/- 2%, or +/- 1%, and can further represent a comparison to the top three or top four triglycerides of the naturally occurring oil, or two out of the top three triglycerides, or three out of the top four triglycerides.

[0133] "Sonication" is a process of disrupting biological materials, such as a cell, by use of sound wave energy.

[0134] "Species of furfural" is 2-furancarboxaldehyde or a derivative that retains the same basic structural characteristics. [0135] "Stover" is the dried stalks and leaves of a crop remaining after a grain has been harvested.

[0136] "Sucrose utilization gene" is a gene that, when expressed, aids the ability of a cell to utilize sucrose as an energy source. Proteins encoded by a sucrose utilization gene are referred to herein as "sucrose utilization enzymes" and include sucrose transporters, sucrose invertases, and hexokinases such as glucokinases and fructokinases.

[0137] "Suitable for human consumption" means a composition can be consumed by humans as dietary intake without ill health effects and can provide significant caloric intake due to uptake of digested material in the gastrointestinal tract.

[0138] "Uncooked product" means a composition that has not been subjected to heating but may include one or more components previously subjected to heating.

[0139] "V/V" or "v/v", in reference to proportions by volume, means the ratio of the volume of one substance in a composition to the volume of the composition. For example, reference to a composition that comprises 5% v/v recombinant microalgal oil means that 5% of the composition's volume is composed of recombinant microalgal oil (e.g. , such a composition having a volume of 100 mm 3 would contain 5 mm 3 of recombinant microalgal oil), and the remainder of the volume of the composition (e.g. , 95 mm 3 in the example) is composed of other ingredients.

[0140] "W/W" or "w/w", in reference to proportions by weight, means the ratio of the weight of one substance in a composition to the weight of the composition. For example, reference to a composition that comprises 5% w/w recombinant microalgal biomass means that 5% of the composition's weight is composed of recombinant microalgal biomass (e.g. , such a composition having a weight of 100 mg would contain 5 mg of recombinant microalgal biomass) and the remainder of the weight of the composition (e.g. , 95 mg in the example) is composed of other ingredients.

II. CULTIVATION

[0141] The present invention generally relates to cultivation of microorganisms

(e.g. , microalgae, oleaginous yeast, fungi, and bacteria), particularly recombinant microalgal strains, including Prototheca strains, for the production of lipid. For the convenience of the reader, this section is subdivided into subsections. Subsection 1 describes Prototheca species and strains and how to identify new Prototheca species and strains and related microalgae by genomic DNA comparison, as well as other microorganisms. Subsection 2 describes bioreactors useful for cultivation. Subsection 3 describes media for cultivation. Subsection 4 describes oil production in accordance with illustrative cultivation methods of the invention. These descriptions are also more generally applicable to other microorganisms.

1. Prototheca species and strains and other microorganisms

[0142] Prototheca is a remarkable microorganism for use in the production of lipid, because it can produce high levels of lipid, particularly lipid suitable for fuel production. The lipid produced by Prototheca has hydrocarbon chains of shorter chain length and a higher degree of saturation than that produced by other microalgae. Moreover, Prototheca lipid is generally free of pigment (low to undetectable levels of chlorophyll and certain carotenoids) and in any event contains much less pigment than lipid from other microalgae. Moreover, recombinant Prototheca cells provided by the invention can be used to produce lipid in greater yield and efficiency, and with reduced cost, relative to the production of lipid from other microorganisms. Illustrative Prototheca strains for use in the methods of the invention include In addition, this microalgae grows heterotrophically and can be genetically engineered as Prototheca wickerhamii, Prototheca stagnora (including UTEX 327),

Prototheca portoricensis, Prototheca moriformis (including UTEX strains 1441, 1435), and Prototheca zopfii. Species of the genus Prototheca are obligate heterotrophs.

[0143] Species of Prototheca for use in the invention can be identified by amplification of certain target regions of the genome. For example, identification of a specific Prototheca species or strain can be achieved through amplification and sequencing of nuclear and/or chloroplast DNA using primers and methodology using any region of the genome, for example using the methods described in Wu et ah, Bot. Bull. Acad. Sin. (2001) 42: 115-121 Identification of Chlorella spp. isolates using ribosomal DNA sequences. Well established methods of phylogenetic analysis, such as amplification and sequencing of ribosomal internal transcribed spacer (ITS1 and ITS2 rDNA), 23S rRNA, 18S rRNA, and other conserved genomic regions can be used by those skilled in the art to identify species of not only Prototheca, but other hydrocarbon and lipid producing organisms with similar lipid profiles and production capability. For examples of methods of identification and classification of algae also see for example Genetics, 2005 Aug;170(4): 1601-10 and RNA, 2005

Apr;l l(4):361-4.

[0144] Thus, genomic DNA comparison can be used to identify suitable species of microalgae to be used in the present invention. Regions of conserved genomic DNA, such as but not limited to DNA encoding for 23S rRNA, can be amplified from microalgal species and compared to consensus sequences in order to screen for microalgal species that are taxonomically related to the preferred microalgae used in the present invention. Examples of such DNA sequence comparison for species within the Prototheca genus are shown below. Genomic DNA comparison can also be useful to identify microalgal species that have been misidentified in a strain collection. Often a strain collection will identify species of microalgae based on phenotypic and morphological characteristics. The use of these characteristics may lead to miscategorization of the species or the genus of a microalgae. The use of genomic DNA comparison can be a better method of categorizing microalgae species based on their phylogenetic relationship.

[0145] Microalgae for use in the present invention typically have genomic DNA sequences encoding for 23S rRNA that have at least 99%, least 95%, at least 90%, or at least 85% nucleotide identity to at least one of the sequences listed in SEQ ID NOs: 11-19.

[0146] For sequence comparison to determine percent nucleotide or amino acid identity, typically one sequence acts as a reference sequence, to which test sequences are compared. When using a sequence comparison algorithm, test and reference sequences are input into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. The sequence comparison algorithm then calculates the percent sequence identity for the test sequence(s) relative to the reference sequence, based on the designated program parameters.

[0147] Optimal alignment of sequences for comparison can be conducted, e.g., by the local homology algorithm of Smith & Waterman, Adv. Appl. Math. 2:482 (1981), by the homology alignment algorithm of Needleman & Wunsch, /. Mol. Biol. 48:443 (1970), by the search for similarity method of Pearson & Lipman, Proc. Nat'l. Acad. Sci. USA 85:2444 (1988), by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, WI), or by visual inspection (see generally Ausubel et al., supra).

[0148] Another example algorithm that is suitable for determining percent sequence identity and sequence similarity is the BLAST algorithm, which is described in Altschul et al, J. Mol. Biol. 215:403-410 (1990). Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information (at the web address www.ncbi.nlm.nih.gov). This algorithm involves first identifying high scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence, which either match or satisfy some positive-valued threshold score T when aligned with a word of the same length in a database sequence. T is referred to as the neighborhood word score threshold (Altschul et al , supra.). These initial neighborhood word hits act as seeds for initiating searches to find longer HSPs containing them. The word hits are then extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Cumulative scores are calculated using, for nucleotide sequences, the parameters M (reward score for a pair of matching residues; always > 0) and N (penalty score for mismatching residues; always < 0). For amino acid sequences, a scoring matrix is used to calculate the cumulative score.

Extension of the word hits in each direction are halted when: the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below due to the accumulation of one or more negative-scoring residue alignments; or the end of either sequence is reached. For identifying whether a nucleic acid or polypeptide is within the scope of the invention, the default parameters of the BLAST programs are suitable. The BLASTN program (for nucleotide sequences) uses as defaults a word length (W) of 11, an expectation (E) of 10, M=5, N=-4, and a comparison of both strands. For amino acid sequences, the BLASTP program uses as defaults a word length (W) of 3, an expectation (E) of 10, and the BLOSUM62 scoring matrix. The TBLATN program (using protein sequence for nucleotide sequence) uses as defaults a word length (W) of 3, an expectation (E) of 10, and a BLOSUM 62 scoring matrix, (see Henikoff & Henikoff, Proc. Natl. Acad. Sci. USA 89: 10915 (1989)).

[0149] In addition to calculating percent sequence identity, the BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, e.g. , Karlin & Altschul, Proc. Nat'l. Acad. Sci. USA 90:5873-5787 (1993)). One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability by which a match between two nucleotide or amino acid sequences would occur by chance. For example, a nucleic acid is considered similar to a reference sequence if the smallest sum probability in a comparison of the test nucleic acid to the reference nucleic acid is less than about 0.1, more preferably less than about 0.01, and most preferably less than about 0.001.

[0150] Other considerations affecting the selection of microorganisms for use in the invention include, in addition to production of suitable lipids or hydrocarbons for production of oils, fuels, and oleochemicals: (1) high lipid content as a percentage of cell weight; (2) ease of growth; (3) ease of genetic engineering; and (4) ease of biomass processing. In particular embodiments, the wild-type or genetically engineered microorganism yields cells that are at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, or at least 70% or more lipid. Preferred organisms grow heterotrophically (on sugars in the absence of light). [0151] Examples of algae that can be used to practice the present invention include, but are not limited to the following algae listed in Table 1.

[0152] Table 1. Examples of algae.

Achnanthes orientalis, Agmenellum, Amphiprora hyaline, Amphora coffeiformis, Amphora coffeiformis linea, Amphora coffeiformis punctata, Amphora coffeiformis taylori, Amphora coffeiformis tenuis, Amphora delicatissima, Amphora delicatissima capitata, Amphora sp., Anabaena, Ankistrodesmus, Ankistrodesmus falcatus, Boekelovia hooglandii, Borodinella sp., Botryococcus braunii, Botryococcus sudeticus, Carteria, Chaetoceros gracilis, Chaetoceros muelleri, Chaetoceros muelleri subsalsum, Chaetoceros sp., Chlorella anitrata, Chlorella Antarctica, Chlorella aureoviridis, Chlorella Candida, Chlorella capsulate, Chlorella desiccate, Chlorella ellipsoidea, Chlorella emersonii, Chlorella fusca, Chlorella fusca var. vacuolata, Chlorella glucotropha, Chlorella infusionum, Chlorella infusionum var. actophila, Chlorella infusionum var. auxenophila, Chlorella kessleri, Chlorella lobophora (strain SAG 37.88), Chlorella luteoviridis, Chlorella luteoviridis var. aureoviridis, Chlorella luteoviridis var. lutescens, Chlorella miniata, Chlorella minutissima, Chlorella mutabilis, Chlorella nocturna, Chlorella parva, Chlorella photophila, Chlorella pringsheimii, Chlorella protothecoides (including any of UTEX strains 1806, 411, 264, 256, 255, 250, 249, 31, 29, 25, and CCAP strains 211/17 and 21 l/8d), Chlorella protothecoides var. acidicola, Chlorella regularis, Chlorella regularis var. minima, Chlorella regularis var. umbricata, Chlorella reisiglii, Chlorella saccharophila, Chlorella saccharophila var. ellipsoidea, Chlorella salina, Chlorella simplex, Chlorella sorokiniana, Chlorella sp., Chlorella sphaerica, Chlorella stigmatophora, Chlorella vanniellii, Chlorella vulgaris, Chlorella vulgaris, Chlorella vulgaris f. tertia, Chlorella vulgaris var. autotrophica, Chlorella vulgaris var. viridis, Chlorella vulgaris var. vulgaris, Chlorella vulgaris var. vulgaris f. tertia, Chlorella vulgaris var. vulgaris f. viridis, Chlorella xanthella, Chlorella zofingiensis, Chlorella trebouxioides, Chlorella vulgaris, Chlorococcum infusionum, Chlorococcum sp., Chlorogonium,

Chroomonas sp., Chrysosphaera sp., Cricosphaera sp., Cryptomonas sp., Cyclotella cryptica, Cyclotella meneghiniana, Cyclotella sp., Dunaliella sp., Dunaliella bardawil, Dunaliella bioculata, Dunaliella granulate, Dunaliella maritime, Dunaliella minuta, Dunaliella parva, Dunaliella peircei, Dunaliella primolecta, Dunaliella salina, Dunaliella terricola, Dunaliella tertiolecta, Dunaliella viridis, Dunaliella tertiolecta, Eremosphaera viridis, Eremosphaera sp., Ellipsoidon sp., Euglena, Franceia sp., Fragilaria crotonensis, Fragilaria sp., Gleocapsa sp., Gloeothamnion sp., Hymenomonas sp., Isochrysis off galbana, Isochrysis galbana, Lepocinclis, Micractinium, Micractinium (UTEX LB 2614), Monoraphidium minutum, Monoraphidium sp., Nannochloris sp., Nannochloropsis salina, Nannochloropsis sp., Navicula acceptata, Navicula biskanterae, Navicula pseudotenelloides, Navicula pelliculosa, Navicula saprophila, Navicula sp., Nephrochloris sp., Nephroselmis sp., Nitschia communis, Nitzschia alexandrina, Nitzschia communis, Nitzschia dissipata, Nitzschia frustulum, Nitzschia hantzschiana, Nitzschia inconspicua, Nitzschia intermedia, Nitzschia microcephala, Nitzschia pusilla, Nitzschia pusilla elliptica, Nitzschia pusilla monoensis, Nitzschia quadrangular, Nitzschia sp., Ochromonas sp., Oocystis parva, Oocystis pusilla, Oocystis sp., Oscillatoria limnetica, Oscillatoria sp., Oscillatoria subbrevis, Pascheria acidophila, Pavlova sp., Phagus, Phormidium, Platymonas sp., Pleurochrysis carterae, Pleurochrysis dentate, Pleurochrysis sp., Prototheca wickerhamii, Prototheca stagnora, Prototheca portoricensis, Prototheca moriformis, Prototheca zopfii, Pyramimonas sp., Pyrobotrys, Sarcinoid chrysophyte, Scenedesmus armatus, Spirogyra, Spirulina platensis, Stichococcus sp., Synechococcus sp., Tetraedron, Tetraselmis sp., Tetraselmis suecica, Thalassiosira weissflogii, and Viridiella fridericiana

[0153] Examples of oleaginous yeast that can be used to practice the present invention include, but are not limited to the following oleaginous yeast listed in Table 26.

[0154] Table 26. Examples of oleaginous yeast.

Cryptococcus curvatus, Cryptococcus terricolus, Candida sp., Lipomyces starkeyi, Lipomyces lipofer, Endomycopsis vernalis, Rhodotorula glutinis, Rhodotorula gracilis, and Yarrowia lipolytica

[0155] Examples of other fungi that can be used to practice the present invention include, but are not limited to the following fungi listed in Table 27.

[0156] Table 27. Examples of fungi.

Mortierella, Mortierrla vinacea, Mortierella alpine, Pythium debaryanum, Mucor circinelloides, Aspergillus ochraceus, Aspergillus terreus, Pennicillium iilacinum, Hensenulo, Chaetomium, Cladosporium, Malbranchea, Rhizopus, and Pythium

[0157] In some embodiments of the present invention, the microorganism is a bacterium. Examples of expression of exogenous genes in bacteria, such as E. coli, are well known; see for example Molecular Cloning: A Laboratory Manual, Sambrook et al. (3d edition, 2001, Cold Spring Harbor Press).

2. Bioreactor

[0158] Microrganisms are cultured both for purposes of conducting genetic manipulations and for production of hydrocarbons (e.g., lipids, fatty acids, aldehydes, alcohols, and alkanes). The former type of culture is conducted on a small scale and initially, at least, under conditions in which the starting microorganism can grow. Culture for purposes of hydrocarbon production is usually conducted on a large scale (e.g., 10,000 L, 40,000 L, 100,000 L or larger bioreactors) in a bioreactor. Microalgae, including Prototheca species are typically cultured in the methods of the invention in liquid media within a bioreactor.

Typically, the bioreactor does not allow light to enter.

[0159] The bioreactor or fermentor is used to culture oleaginous microbial cells, preferably microalgal cells through the various phases of their physiological cycle. Bioreactors offer many advantages for use in heterotrophic growth and propagation methods. To produce biomass for use in food, microalgae are preferably fermented in large quantities in liquid, such as in suspension cultures as an example. Bioreactors such as steel fermentors can accommodate very large culture volumes (40,000 liter and greater capacity bioreactors are used in various embodiments of the invention). Bioreactors also typically allow for the control of culture conditions such as temperature, pH, oxygen tension, and carbon dioxide levels. For example, bioreactors are typically configurable, for example, using ports attached to tubing, to allow gaseous components, like oxygen or nitrogen, to be bubbled through a liquid culture. Other culture parameters, such as the pH of the culture media, the identity and concentration of trace elements, and other media constituents can also be more readily manipulated using a bioreactor.

[0160] Bioreactors can be configured to flow culture media though the bioreactor throughout the time period during which the microalgae reproduce and increase in number. In some embodiments, for example, media can be infused into the bioreactor after inoculation but before the cells reach a desired density. In other instances, a bioreactor is filled with culture media at the beginning of a culture, and no more culture media is infused after the culture is inoculated. In other words, the microalgal biomass is cultured in an aqueous medium for a period of time during which the microalgae reproduce and increase in number; however, quantities of aqueous culture medium are not flowed through the bioreactor throughout the time period. Thus in some embodiments, aqueous culture medium is not flowed through the bioreactor after inoculation. [0161] Bioreactors equipped with devices such as spinning blades and impellers, rocking mechanisms, stir bars, means for pressurized gas infusion can be used to subject microalgal cultures to mixing. Mixing may be continuous or intermittent. For example, in some embodiments, a turbulent flow regime of gas entry and media entry is not maintained for reproduction of microalgae until a desired increase in number of said microalgae has been achieved.

[0162] Bioreactor ports can be used to introduce, or extract, gases, solids, semisolids, and liquids, into the bioreactor chamber containing the microalgae. While many bioreactors have more than one port (for example, one for media entry, and another for sampling), it is not necessary that only one substance enter or leave a port. For example, a port can be used to flow culture media into the bioreactor and later used for sampling, gas entry, gas exit, or other purposes. Preferably, a sampling port can be used repeatedly without altering compromising the axenic nature of the culture. A sampling port can be configured with a valve or other device that allows the flow of sample to be stopped and started or to provide a means of continuous sampling. Bioreactors typically have at least one port that allows inoculation of a culture, and such a port can also be used for other purposes such as media or gas entry.

[0163] Bioreactors ports allow the gas content of the culture of microalgae to be manipulated. To illustrate, part of the volume of a bioreactor can be gas rather than liquid, and the gas inlets of the bioreactor to allow pumping of gases into the bioreactor. Gases that can be beneficially pumped into a bioreactor include air, air/C0 2 mixtures, noble gases, such as argon, and other gases. Bioreactors are typically equipped to enable the user to control the rate of entry of a gas into the bioreactor. As noted above, increasing gas flow into a bioreactor can be used to increase mixing of the culture.

[0164] Increased gas flow affects the turbidity of the culture as well. Turbulence can be achieved by placing a gas entry port below the level of the aqueous culture media so that gas entering the bioreactor bubbles to the surface of the culture. One or more gas exit ports allow gas to escape, thereby preventing pressure buildup in the bioreactor. Preferably a gas exit port leads to a "one-way" valve that prevents contaminating microorganisms from entering the bioreactor.

3. Media

[0165] Microalgal culture media typically contains components such as a fixed nitrogen source, a fixed carbon source, trace elements, optionally a buffer for pH maintenance, and phosphate (typically provided as a phosphate salt). Other components can include salts such as sodium chloride, particularly for seawater microalgae. Nitrogen sources include organic and inorganic nitrogen sources, including, for example, without limitation, molecular nitrogen, nitrate, nitrate salts, ammonia (pure or in salt form, such as, (NH 4 ) 2 S0 4 and NH4OH), protein, soybean meal, cornsteep liquor, and yeast extract. Examples of trace elements include zinc, boron, cobalt, copper, manganese, and molybdenum in, for example, the respective forms of ZnCl 2 , H 3 BO 3 , CoCl 2 -6H 2 0, CuCl 2 -2H 2 0, MnCl 2 H 2 0 and

(ΝΗ 4 ) 6 Μο 7 0 24 ·4Η 2 0.

[0166] Microorganisms useful in accordance with the methods of the present invention are found in various locations and environments throughout the world. As a consequence of their isolation from other species and their resulting evolutionary divergence, the particular growth medium for optimal growth and generation of lipid and/or hydrocarbon constituents can be difficult to predict. In some cases, certain strains of microorganisms may be unable to grow on a particular growth medium because of the presence of some inhibitory component or the absence of some essential nutritional requirement required by the particular strain of microorganism.

[0167] Solid and liquid growth media are generally available from a wide variety of sources, and instructions for the preparation of particular media that is suitable for a wide variety of strains of microorganisms can be found, for example, online at

http://www.utex.org/, a site maintained by the University of Texas at Austin, 1 University Station A6700, Austin, Texas, 78712-0183, for its culture collection of algae (UTEX). For example, various fresh water and salt water media include those described in PCT Pub. No. 2008/151149, incorporated herein by reference.

[0168] In a particular example, Proteose Medium is suitable for axenic cultures, and a 1L volume of the medium (pH -6.8) can be prepared by addition of lg of proteose peptone to 1 liter of Bristol Medium. Bristol medium comprises 2.94 mM NaN0 3 , 0.17 mM CaCl 2 -2H 2 0, 0.3 mM MgS0 4 -7H 2 0, 0.43 mM, 1.29 mM KH 2 P0 4 , and 1.43 mM NaCl in an aqueous solution. For 1.5% agar medium, 15 g of agar can be added to 1 L of the solution. The solution is covered and autoclaved, and then stored at a refrigerated temperature prior to use. Another example is the Prototheca isolation medium (PIM), which comprises lOg/L postassium hydrogen phthalate (KHP), 0.9g/L sodium hydroxide, O. lg/L magnesium sulfate, 0.2g/L potassium hydrogen phosphate, 0.3g/L ammonium chloride, lOg/L glucose O.OOlg/L thiamine hydrochloride, 20g/L agar, 0.25g/L 5-fluorocytosine, at a pH in the range of 5.0 to 5.2 (see Pore, 1973, App. Microbiology, 26: 648-649). Other suitable media for use with the methods of the invention can be readily identified by consulting the URL identified above, or by consulting other organizations that maintain cultures of microorganisms, such as SAG, CCAP, or CCALA. SAG refers to the Culture Collection of Algae at the University of Gottingen (Gottingen, Germany), CCAP refers to the culture collection of algae and protozoa managed by the Scottish Association for Marine Science (Scotland, United Kingdom), and CCALA refers to the culture collection of algal laboratory at the Institute of Botany (Trebon, Czech Republic). Additionally, US Patent No. 5,900,370 describes media formulations and conditions suitable for heterotrophic fermentation of Prototheca species.

[0169] For oil production, selection of a fixed carbon source is important, as the cost of the fixed carbon source must be sufficiently low to make oil production economical. Thus, while suitable carbon sources include, for example, acetate, floridoside, fructose, galactose, glucuronic acid, glucose, glycerol, lactose, mannose, N-acetylglucosamine, rhamnose, sucrose, and/or xylose, selection of feedstocks containing those compounds is an important aspect of the methods of the invention. Suitable feedstocks useful in accordance with the methods of the invention include, for example, black liquor, corn starch, depolymerized cellulosic material, milk whey, molasses, potato, sorghum, sucrose, sugar beet, sugar cane, rice, and wheat. Carbon sources can also be provided as a mixture, such as a mixture of sucrose and depolymerized sugar beet pulp. The one or more carbon source(s) can be supplied at a concentration of at least about 50 μΜ, at least about 100 μΜ, at least about 500 μΜ, at least about 5 mM, at least about 50 mM, and at least about 500 mM, of one or more exogenously provided fixed carbon source(s). Carbon sources of particular interest for purposes of the present invention include cellulose (in a depolymerized form), glycerol, sucrose, and sorghum, each of which is discussed in more detal below.

[0170] In accordance with the present invention, microorganisms can be cultured using depolymerized cellulosic biomass as a feedstock. Cellulosic biomass (e.g., stover, such as corn stover) is inexpensive and readily available; however, attempts to use this material as a feedstock for yeast have failed. In particular, such feedstocks have been found to be inhibitory to yeast growth, and yeast cannot use the 5-carbon sugars produced from cellulosic materials (e.g., xylose from hemi-cellulose). By contrast, microalgae can grow on processed cellulosic material. Cellulosic materials generally include about 40-60% cellulose; about 20- 40% hemicellulose; and 10-30% lignin.

[0171] Suitable cellulosic materials include residues from herbaceous and woody energy crops, as well as agricultural crops, i.e., the plant parts, primarily stalks and leaves, not removed from the fields with the primary food or fiber product. Examples include agricultural wastes such as sugarcane bagasse, rice hulls, corn fiber (including stalks, leaves, husks, and cobs), wheat straw, rice straw, sugar beet pulp, citrus pulp, citrus peels; forestry wastes such as hardwood and softwood thinnings, and hardwood and softwood residues from timber operations; wood wastes such as saw mill wastes (wood chips, sawdust) and pulp mill waste; urban wastes such as paper fractions of municipal solid waste, urban wood waste and urban green waste such as municipal grass clippings; and wood construction waste.

Additional cellulosics include dedicated cellulosic crops such as switchgrass, hybrid poplar wood, and miscanthus, fiber cane, and fiber sorghum. Five-carbon sugars that are produced from such materials include xylose.

[0172] Cellulosic materials are treated to increase the efficiency with which the microbe can utilize the sugar(s) contained within the materials. The invention provides novel methods for the treatment of cellulosic materials after acid explosion so that the materials are suitable for use in a heterotrophic culture of microbes (e.g., microalgae and oleaginous yeast). As discussed above, lignocellulosic biomass is comprised of various fractions, including cellulose, a crystalline polymer of beta 1,4 linked glucose (a six-carbon sugar), hemicellulose, a more loosely associated polymer predominantly comprised of xylose (a five-carbon sugar) and to a lesser extent mannose, galactose, arabinose, lignin, a complex aromatic polymer comprised of sinapyl alcohol and its derivatives, and pectins, which are linear chains of an alpha 1 ,4 linked polygalacturonic acid. Because of the polymeric structure of cellulose and hemicellulose, the sugars (e.g., monomeric glucose and xylose) in them are not in a form that can be efficiently used (metabolized) by many microbes. For such microbes, further processing of the cellulosic biomass to generate the monomeric sugars that make up the polymers can be very helpful to ensuring that the cellulosic materials are efficiently utilized as a feedstock (carbon source).

[0173] Celluose or cellulosic biomass is subjected to a process, termed "explosion", in which the biomass is treated with dilute sulfuric (or other) acid at elevated temperature and pressure. This process conditions the biomass such that it can be efficiently subjected to enzymatic hydrolysis of the cellulosic and hemicellulosic fractions into glucose and xylose monomers. The resulting monomeric sugars are termed cellulosic sugars. Cellulosic sugars can subsequently be utilized by microorganisms to produce a variety of metabolites (e.g., lipid). The acid explosion step results in a partial hydrolysis of the hemicellulose fraction to constitutent monosaccharides. These sugars can be completely liberated from the biomass with further treatment. In some embodiments, the further treatment is a hydrothermal treatment that includes washing the exploded material with hot water, which removes contaminants such as salts. This step is not necessary for cellulosic ethanol fermentations due to the more dilute sugar concentrations used in such processes. In other embodiments, the further treatment is additional acid treatment. In still other embodiments, the further treatment is enzymatic hydrolysis of the exploded material. These treatments can also be used in any combination. The type of treatment can affect the type of sugars liberated (e.g., five carbon sugars versus six carbon sugars) and the stage at which they are liberated in the process. As a consequence, different streams of sugars, whether they are predominantly five-carbon or six- carbon, can be created. These enriched five-carbon or six-carbon streams can thus be directed to specific microorganisms with different carbon utilization cabilities.

[0174] The methods of the present invention typically involve fermentation to higher cell densities than what is achieved in ethanol fermentation. Because of the higher densities of the cultures for heterotrophic cellulosic oil production, the fixed carbon source (e.g., the cellulosic derived sugar stream(s)) is preferably in a concentrated form. The glucose level of the depolymerized cellulosic material is preferably at least 300 g/liter, at least 400 g/liter, at least 500 g/liter or at least 600 g/liter prior to the cultivation step, which is optionally a fed batch cultivation in which the material is fed to the cells over time as the cells grow and accumulate lipid. Cellulosic sugar streams are not used at or near this concentration range in the production of cellulosic ethanol. Thus, in order to generate and sustain the very high cell densities during the production of lignocellulosic oil, the carbon feedstock(s) must be delivered into the heterotrophic cultures in a highly concentrated form. However, any component in the feedstream that is not a substrate for, and is not metabolized by, the oleaginous microorganism will accumulate in the bioreactor, which can lead to problems if the component is toxic or inhibitory to production of the desired end product. While ligin and lignin-derived by-products, carbohydrate-derived byproducts such as furfurals and hydroxymethyl furfurals and salts derived from the generation of the cellulosic materials (both in the explosion process and the subsequent neutralization process), and even non- metabolized pentose/hexose sugars can present problems in ethanolic fermentations, these effects are amplified significantly in a process in which their concentration in the initial feedstock is high. To achieve sugar concentrations in the 300g/L range (or higher) for six- carbon sugars that may be used in large scale production of lignocellulosic oil described in the present invention, the concentration of these toxic materials can be 20 times higher than the concentrations typically present in ethanolic fermentations of cellulosic biomass.

[0175] The explosion process treatment of the cellulosic material utilizes significant amounts of sulfuric acid, heat and pressure, thereby liberating by-products of carbohydrates, namely furfurals and hydroxymethyl furfurals. Furfurals and hydroxymethyl furfurals are produced during hydrolysis of hemicellulose through dehydration of xylose into furfural and water. In some embodiments of the present invention, these by-products (e.g., furfurals and hydroxymethyl furfurals) are removed from the saccharified lignocellulosic material prior to introduction into the bioreactor. In certain embodiments of the present invention, the process for removal of the by-products of carbohydrates is hydrothermal treatment of the exploded cellulosic materials. In addition, the present invention provides methods in which strains capable of tolerating compounds such as furfurals or hydroxymethyl furfurals are used for lignocellulosic oil production. In another embodiment, the present invention also provides methods and microorganisms that are not only capable of tolerating furfurals in the fermentation media, but are actually able to metabolize these by-products during the production of lignocellulosic oil.

[0176] The explosion process also generates significant levels of salts. For example, typical conditions for explosion can result in conductivites in excess of 5 mS/cm when the exploded cellulosic biomass is resuspended at a ratio of 10: 1 watensolids (dry weight). In certain embodiments of the present invention, the diluted exploded biomass is subjected to enzymatic saccharification, and the resulting supernatant is concentrated up to 25 fold for use in the bioreactor. The salt level (as measured by conductivity) in the concentrated sugar stream(s) can be unacceptably high (up to 1.5 M Na + equivalents). Additional salts are generated upon neutralization of the exploded materials for the subsequent enzymatic saccharification process as well. The present invention provides methods for removing these salts so that the resulting concentrated cellulosic sugar stream(s) can be used in heterotrophic processes for producing lignocellulosic oil. In some embodiments, the method of removing these salts is deionization with resins, such as, but not limited to, DOWEX Marathon MR3. In certain embodiments, the deionization with resin step occurs before sugar concentration or pH adjustment and hydrothermal treatment of biomass prior to saccharification, or any combination of the preceding; in other embodiments, the step is conducted after one or more of these processes. In other embodiments, the explosion process itself is changed so as to avoid the generation of salts at unacceptably high levels. For example, a suitable alternative to sulfuric acid (or other acid) explosion of the cellulosic biomass is mechanical pulping to render the cellulosic biomass receptive to enzymatic hydrolysis (saccharification). In still other embodiments, native strains of microorganisms resistant to high levels of salts or genetically engineered strains with resistance to high levels of salts are used. [0177] A preferred embodiment for the process of preparing of exploded cellulosic biomass for use in heterotrophic lignocellulosic oil production using oleaginous microbes. A first step comprises adjusting the pH of the resuspended exploded cellulosic biomass to the range of 5.0-5.3 followed by washing the cellulosic biomass three times. This washing step can be accomplished by a variety of means including the use of desalting and ion exchange resins, reverse omosis, hydrothermal treatment (as described above), or just repeated re-suspension and centrifugation in deionized water. This wash step results in a cellulosic stream whose conductivity is between 100-300 μ8 :ιη and the removal of significant amounts of furfurals and hydroxymethyl furfurals. Decants from this wash step can be saved to concentrate five- carbon sugars liberated from the hemicellulose fraction. A second step comprises enzymatic saccharification of the washed cellulosic biomass. In a preferred embodiment, Accellerase (Genencor) is used. A third step comprises the recovery of sugars via centrifugation or decanting and rinsing of the saccharified biomass. The resulting biomass (solids) is an energy dense, lignin rich component that can be used as fuel or sent to waste. The recovered sugar stream in the centrifugation/decanting and rinse process is collected. A fourth step comprises microfiltration to remove contaminating solids with recovery of the permeate. A fifth step comprises a concentration step which can be accomplished using a vacuum evaporator. This step can optionally include the addition of antifoam agents such as P'2000 (Sigma/Fluka), which is sometimes necessary due to the protein content of the resulting sugar feedstock.

[0178] In another embodiment of the methods of the invention, the carbon source is glycerol, including acidulated and non- acidulated glycerol byproduct from biodiesel transesterification. In one embodiment, the carbon source includes glycerol and at least one other carbon source. In some cases, all of the glycerol and the at least one other fixed carbon source are provided to the microorganism at the beginning of the fermentation. In some cases, the glycerol and the at least one other fixed carbon source are provided to the microorganism simultaneously at a predetermined ratio. In some cases, the glycerol and the at least one other fixed carbon source are fed to the microbes at a predetermined rate over the course of fermentation.

[0179] Some microalgae undergo cell division faster in the presence of glycerol than in the presence of glucose (see PCT Pub. No. 2008/151149). In these instances, two-stage growth processes in which cells are first fed glycerol to rapidly increase cell density, and are then fed glucose to accumulate lipids can improve the efficiency with which lipids are produced. The use of the glycerol byproduct of the transesterification process provides significant economic advantages when put back into the production process. Other feeding methods are provided as well, such as mixtures of glycerol and glucose. Feeding such mixtures also captures the same economic benefits. In addition, the invention provides methods of feeding alternative sugars to microalgae such as sucrose in various combinations with glycerol.

[0180] In another embodiment of the methods of the invention, the carbon source is invert sugar. Invert sugar is produced by splitting the sucrose into its monosaccharide components, fructose and glucose. Production of invert sugar can be achieved through several methods that are known in the art. One such method is heating an aqueous solution of sucrose. Often, catalysts are employed in order to accelerate the conversion of sucrose into invert sugar. These catalysts can be biological, for example enzymes such as invertases and sucrases can be added to the sucrose to accelerate the hydrolysis reaction to produce invert sugar. Acid is an example of non-biological catalyst, when paired with heat, can accelerate the hydrolysis reaction. Once the invert sugar is made, it is less prone to crystallization compared to sucrose and thus, provides advantages for storage and in fed batch fermentation, which in the case of heterotrophic cultivation of microbes, including microalgae, there is a need for concentrated carbon source. In one embodiment, the carbon source is invert sugar, preferably in a concentrated form, preferably at least 800g/liter, at least 900 g/liter, at least 1000 g/liter or at least 1100 g/liter prior to the cultivation step, which is optionally a fed batch cultivation. The invert sugar, preferably in a concentrated form, is fed to the cells over time as the cells grow and accumulate lipid.

[0181] In another embodiment of the methods of the invention, the carbon source is sucrose, including a complex feedstock containing sucrose, such as thick cane juice from sugar cane processing. Because of the higher densities of the cultures for heterotrophic oil production, the fixed carbon source (e.g., sucrose, glucose, etc.) is preferably in a

concentrated form, preferably at least 500 g/liter, at least 600 g/liter, at least 700 g/liter or at least 800 g/liter of the fixed carbon source prior to the cultivation step, which is optionally a fed batch cultivation in which the material is fed to the cells over time as the cells grow and accumulate lipid. In the some cases, the carbon source is sucrose in the form of thick cane juice, preferably in a concentrated form, preferably at least 60% solids or about 770 g/liter sugar, at least 70% solids or about 925 g/liter sugar, or at least 80% solids or about 1125 g/liter sugar prior to the cultivation step, which is optionally a fed batch cultivation. The concentrated thick cane juice is fed to the cells over time as the cells grow and accumulate lipid.

[0182] In one embodiment, the culture medium further includes at least one sucrose utilization enzyme. In some cases, the culture medium includes a sucrose invertase. In one embodiment, the sucrose invertase enzyme is a secrectable sucrose invertase enzyme encoded by an exogenous sucrose invertase gene expressed by the population of microorganisms. Thus, in some cases, as described in more detail in Section IV, below, the microalgae has been genetically engineered to express a sucrose utilization enzyme, such as a sucrose transporter, a sucrose invertase, a hexokinase, a glucokinase, or a fructokinase.

[0183] Complex feedstocks containing sucrose include waste molasses from sugar cane processing; the use of this low- value waste product of sugar cane processing can provide significant cost savings in the production of hydrocarbons and other oils. Another complex feedstock containing sucrose that is useful in the methods of the invention is sorghum, including sorghum syrup and pure sorghum. Sorghum syrup is produced from the juice of sweet sorghum cane. Its sugar profile consists of mainly glucose (dextrose), fructose and sucrose.

4. Oil production

[0184] For the production of oil in accordance with the methods of the invention, it is preferable to culture cells in the dark, as is the case, for example, when using extremely large (40,000 liter and higher) fermentors that do not allow light to strike the culture. Prototheca species are grown and propagated for the production of oil in a medium containing a fixed carbon source and in the absence of light; such growth is known as heterotrophic growth.

[0185] As an example, an inoculum of lipid-producing oleaginous microbial cells, preferably microalgal cells are introduced into the medium; there is a lag period (lag phase) before the cells begin to propagate. Following the lag period, the propagation rate increases steadily and enters the log, or exponential, phase. The exponential phase is in turn followed by a slowing of propagation due to decreases in nutrients such as nitrogen, increases in toxic substances, and quorum sensing mechanisms. After this slowing, propagation stops, and the cells enter a stationary phase or steady growth state, depending on the particular environment provided to the cells. For obtaining lipid rich biomass, the culture is typically harvested well after then end of the exponential phase, which may be terminated early by allowing nitrogen or another key nutrient (other than carbon) to become depleted, forcing the cells to convert the carbon sources, present in excess, to lipid. Culture condition parameters can be manipulated to optimize total oil production, the combination of lipid species produced, and/or production of a specific oil.

[0186] As discussed above, a bioreactor or fermentor is used to allow cells to undergo the various phases of their growth cycle. As an example, an inoculum of lipid-producing cells can be introduced into a medium followed by a lag period (lag phase) before the cells begin growth. Following the lag period, the growth rate increases steadily and enters the log, or exponential, phase. The exponential phase is in turn followed by a slowing of growth due to decreases in nutrients and/or increases in toxic substances. After this slowing, growth stops, and the cells enter a stationary phase or steady state, depending on the particular environment provided to the cells. Lipid production by cells disclosed herein can occur during the log phase or thereafter, including the stationary phase wherein nutrients are supplied, or still available, to allow the continuation of lipid production in the absence of cell division.

[0187] Preferably, microorganisms grown using conditions described herein and known in the art comprise at least about 20% by weight of lipid, preferably at least about 40% by weight, more preferably at least about 50% by weight, and most preferably at least about 60% by weight. Process conditions can be adjusted to increase the yield of lipids suitable for a particular use and/or to reduce production cost. For example, in certain embodiments, a microalgae is cultured in the presence of a limiting concentration of one or more nutrients, such as, for example, nitrogen, phosphorous, or sulfur, while providing an excess of fixed carbon energy such as glucose. Nitrogen limitation tends to increase microbial lipid yield over microbial lipid yield in a culture in which nitrogen is provided in excess. In particular embodiments, the increase in lipid yield is at least about: 10%, 50%, 100%, 200%, or 500%. The microbe can be cultured in the presence of a limiting amount of a nutrient for a portion of the total culture period or for the entire period. In particular embodiments, the nutrient concentration is cycled between a limiting concentration and a non- limiting concentration at least twice during the total culture period. Lipid content of cells can be increased by continuing the culture for increased periods of time while providing an excess of carbon, but limiting or no nitrogen.

[0188] In another embodiment, lipid yield is increased by culturing a lipid-producing microbe (e.g., microalgae) in the presence of one or more cofactor(s) for a lipid pathway enzyme (e.g., a fatty acid synthetic enzyme). Generally, the concentration of the cofactor(s) is sufficient to increase microbial lipid (e.g., fatty acid) yield over microbial lipid yield in the absence of the cofactor(s). In a particular embodiment, the cofactor(s) are provided to the culture by including in the culture a microbe (e.g., microalgae) containing an exogenous gene encoding the cofactor(s). Alternatively, cofactor(s) may be provided to a culture by including a microbe (e.g., microalgae) containing an exogenous gene that encodes a protein that participates in the synthesis of the cofactor. In certain embodiments, suitable cofactors include any vitamin required by a lipid pathway enzyme, such as, for example: biotin, pantothenate. Genes encoding cofactors suitable for use in the invention or that participate in the synthesis of such cofactors are well known and can be introduced into microbes (e.g., microalgae), using contracts and techniques such as those described above.

[0189] The specific examples of bioreactors, culture conditions, and heterotrophic growth and propagation methods described herein can be combined in any suitable manner to improve efficiencies of microbial growth and lipid and/or protein production.

[0190] Microalgal biomass with a high percentage of oil/lipid accumulation by dry weight has been generated using different methods of culture, which are known in the art (see PCT Pub. No. 2008/151149). Microalgal biomass generated by the culture methods described herein and useful in accordance with the present invention comprises at least 10% microalgal oil by dry weight. In some embodiments, the microalgal biomass comprises at least 25%, at least 50%, at least 55%, or at least 60% microalgal oil by dry weight. In some embodiments, the microalgal biomass contains from 10-90% microalgal oil, from 25-75% microalgal oil, from 40-75% microalgal oil, or from 50-70% microalgal oil by dry weight.

[0191] The microalgal oil of the biomass described herein, or extracted from the biomass for use in the methods and compositions of the present invention can comprise glycerolipids with one or more distinct fatty acid ester side chains. Glycerolipids are comprised of a glycerol molecule esterified to one, two or three fatty acid molecules, which can be of varying lengths and have varying degrees of saturation. The length and saturation

characteristics of the fatty acid molecules (and the microalgal oils) can be manipulated to modify the properties or proportions of the fatty acid molecules in the microalgal oils of the present invention via culture conditions or via lipid pathway engineering, as described in more detail in Section IV, below. Thus, specific blends of algal oil can be prepared either within a single species of algae by mixing together the biomass or algal oil from two or more species of microalgae, or by blending algal oil of the invention with oils from other sources such as soy, rapeseed, canola, palm, palm kernel, coconut, corn, waste vegetable, Chinese tallow, olive, sunflower, cottonseed, chicken fat, beef tallow, porcine tallow, microalgae, macroalgae, microbes, Cuphea, flax, peanut, choice white grease, lard, Camelina sativa, mustard seed, cashew nut, oats, lupine, kenaf, calendula, help, coffee, linseed (flax), hazelnut, euphorbia, pumpkin seed, coriander, camellia, sesame, safflower, rice, tung tree, cocoa, copra, pium poppy, castor beans, pecan, jojoba, macadamia, Brazil nuts, avocado, petroleum, or a distillate fraction of any of the preceding oils.

[0192] The oil composition, i.e. , the properties and proportions of the fatty acid

consitutents of the glycerolipids, can also be manipulated by combining biomass or oil from at least two distinct species of microalgae. In some embodiments, at least two of the distinct species of microalgae have different glycerolipid profiles. The distinct species of microalgae can be cultured together or separately as described herein, preferably under heterotrophic conditions, to generate the respective oils. Different species of microalgae can contain different percentages of distinct fatty acid consituents in the cell's glycerolipids.

[0193] Generally, Prototheca strains have very little or no fatty acids with the chain length C8-C14. For example, Prototheca moriformis (UTEX 1435), Prototheca krugani (UTEX 329), Prototheca stagnora (UTEX 1442) and Prototheca zopfii (UTEX 1438) contains no (or undectable amounts) C8 fatty acids, between 0-0.01% CIO fatty acids, between 0.03-2.1% C12 fatty acids and between 1.0-1.7% C14 fatty acids.

[0194] In some cases, the Prototheca strains containing a transgene encoding a fatty acyl- ACP thioesterase that has activity towards fatty acyl-ACP substrate of chain lengths C8 or C8-10 has at least 1%, at least 1.5%, at least 2%, at least 3%, at least 4%, at least 5%, at least 10%, at least 12%, or at least 15% or more, fatty acids of chain length C8 . In other instances, the Prototheca strains containing a transgene encoding a fatty acyl ACP thioesterase that has activity towards fatty acyl-ACP substrate of chain lengths CIO has at least at least 1%, at least 5%, at least 10%, at least 15%, at least 20%, at least 24%, or at least 25% or more, fatty acids of chain length CIO. In other instances, the Prototheca strains containing a transgene encoding a fatty acyl-ACP thioesterase that has activity towards fatty acyl-ACP substrate of chain length C12 has at least 1%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 34%, at least 35% or at least 40% or more, fatty acids of the chain length C12. In other cases, the Prototheca strains containing a transgene encoding a fatty acyl-ACP thioesterase that has activity towards fatty acyl-ACP substrate of chain length C14 has at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 6%, at least 7%, at least 10%, at least 15%, at least 30%, at least 43%, or at least 45% or more, fatty acids of the chain length C14.

[0195] In non-limiting examples, the Prototheca strains containing a transgene encoding a fatty acyl-ACP thioesterase that has activity towards fatty acyl-ACP substrate of chain length C8 has between l%-25%, or between 1%-15%, preferably 1.8-12.29%, fatty acids of chain length C8. In other non-limiting examples, Prototheca strains containing a transgene encoding a fatty acyl-ACP thioesterase that has activity towards fatty acyl-ACP substrate of chain length CIO has between l%-50%, or between l%-25%, preferably 1.91-23.97% fatty acids of chain length CIO. In other non-limiting examples, Prototheca strains containing a transgene encoding a fatty acyl-ACP thioesterase that has activity towards fatty acyl-ACP substrate of chain length C12 has between 5%-50%, or between 10%-40, preferably 13.55- 34.01%, fatty acids of the chain length C12. In other non-limiting examples, Prototheca strains containing a transgene encoding a fatty acyl-ACP thioesterase that has activity towards fatty acyl-ACP substrate of chain length C14 has between l%-60%, or between 2%- 45%, preferably 2.59-43.27 %, fatty acids of the chain length C14. In other non-limiting examples, Prototheca strains containing a transgene encoding a fatty acyl-ACP thioesterase that has broad specificity towards fatty acyl-ACP substrates of varying carbon chain length has up to 30%, up to 35%, or preferably up to 39.45% fatty acids of the chain length C16. In some cases, the Prototheca strains containing a transgene encoding a fatty acyl-ACP thioesterase that has activity towards fatty acyl-ACP substrate of chain lengths between C8 and C14 have between l%-75%, or between 2%-60%, preferably 2.69- 57.98%, medium chain (C8-C14) fatty acids. In some cases, the Prototheca strains containing a transgene encoding a fatty acyl-ACP thioesterase that has activity towards fatty acyl-ACP substrates of chain lengths between C12 and C14 have at least 30%, at least 40%, or at least 49% C12-C14 fatty acids. In some instances, keeping the transgenic Prototheca strains under constant and high selective pressure to retain exogenous genes is advantageous due to the increase in the desired fatty acid of a specific chain length. High levels of exogenous gene retention can also be achieved by inserting exogenous genes into the nuclear chromosomes of the cells using homologous recombination vectors and methods disclosed herein. Recombinant cells containing exogenous genes integrated into nuclear chromosomes are an object of the invention.

[0196] Microalgal oil can also include other constituents produced by the microalgae, or incorporated into the microalgal oil from the culture medium. These other constituents can be present in varying amount depending on the culture conditions used to culture the microalgae, the species of microalgae, the extraction method used to recover microalgal oil from the biomass and other factors that may affect microalgal oil composition. Non-limiting examples of such constituents include carotenoids, present from 0.01-0.5 mcg/g, 0.025-0.3 mcg/g, preferably 0.05 to 0.244 micrograms/gram, of oil; chlorophyll A present from 0.01-0.5 mcg/g, 0.025-0.3 mcg/g, preferably 0.045 to 0.268 micrograms/gram, of oil; total chlorophyll of less than 0.1 mcg/g, less than 0.05 mcg/g, preferably less than 0.025 micrograms/gram, of oil; gamma tocopherol present from 1-300 mcg/g, 35-175 mcg/g, preferably 38.3-164 micrograms/gram, of oil; total tocopherols present from 10-500 mcg/g, 50-300 mcg/g, preferably 60.8 to 261.7 microgram/gram, of oil; less than 1%, less than 0.5%, preferably less than 0.25% brassicasterol, campesterol, stigmasterol, or betasitosterol; total tocotrienols less than 400 mcg/g, preferably less than 300 micrograms/gram, of oil; or total tocotrienols present from 100-500 mcg/g, 225-350 mcg/g, preferably 249.6 to 325.3 micrograms/gram, of oil.

[0197] The other constituents can include, without limitation, phospholipids, tocopherols, tocotrienols, carotenoids (e.g., alpha-carotene, beta-carotene, lycopene, etc.), xanthophylls (e.g., lutein, zeaxanthin, alpha-cryptoxanthin and beta-cry toxanthin), and various organic or inorganic compounds. In some cases, the oil extracted from Prototheca species comprises betweenO.001-0.01 mcg/g, 0.0025-0.05 mcg/g, preferably 0.003 to 0.039 microgram lutein/gram, of oil, less than 0.01 mcg/g, less than 0.005 mcg/g, preferably less than 0.003 micrograms lycopene/gram, of oil; and less than 0.01 mcg/g, less than 0.005 mcg/g, preferably less than 0.003 microgram beta carotene/gram, of oil.

[0198] In some embodiments, the present invention provides an oleaginous microbial cell comprising a triglyceride oil, wherein the fatty acid profile of the triglyceride oil is selected from the group consisting of: at least about 1%, at least about 2%, at least about 5%, at least about 7%, at least about 10%, or at least about 15%, C8:0; at least about 1%, at least about 5%, at least about 15%, at least about 20%, at least about 25%, or at least about 30%, C10:0; at least about 1%, at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, or at least about 80%, C12:0; at least about 2%, at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, or at least about 50%, C14:0; at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, or at least about 90%, C16:0; at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, or at least about 50%, C18:0; at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, or at least about 90%, C18: l; less than about 7%, less than about 5%, less than about 3%, less than about 1%, or about 0%, C18:2; and at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, or at least about 90%, saturated fatty acids.

[0199] In some embodiments, the oleaginous microbial cell comprises triglyceride oil comprising a fatty acid profile selected from the group consisting of: total combined amounts of C8:0 and C10:0 of at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or about 100%; total combined amounts of C10:0, C12:0, and C14:0 of at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or about 100%; total combined amounts of C16:0, C18:0 and C18: l of at least about 60%, at least about 70%, at least about 80%, at least about 90%, or about 100%; total combined amounts of C18:0, C18: l and C18:2 of at least about 60%, at least about 70%, at least about 80%, at least about 90%, or about 100%; total combined amounts of C14:0, C16:0, C18:0 and C18: 1 of at least about 60%, at least about 70s%, at least about 80%, at least about 90%, or about 100%; and total combined amounts of C18: l and C18:2 of less than about 30%, less than about 25%, less than about 20%, less than about 15%, less than about 10%, less than about 5%, or about 0%,

[0200] In some embodiments, the oleaginous microbial cell comprises triglyceride oil having a fatty acid profile comprising a ratio of fatty acids selected from the group consisting of: a C8:0 to C10:0 ratio of at least about 5 to 1, at least 6 to 1, at least 7 to 1, at least 8 to 1, at least 9 to 1, or at least 10 to 1; a C10:0 to C12:0 ratio of at least about 6 to 1, at least 7 to 1, at least 8 to 1, at least 9 to 1, or at least 10 to 1; a C12:0 to C14:0 ratio of at least about 5 to 1, at least 6 to 1, at least 7 to 1, at least 8 to 1, at least 9 to 1, or at least 10 to 1 ; a C14:0 to C12:0 ratio of at least 7 to 1, at least 8 to 1, at least 9 to 1, or at least 10 to 1; and a C14:0 to C16:0 ratio of at least 1 to 2, at least 1 to 3, at least 1 to 4, at least 1 to 5, at leasdt 1 to 6, at least 1 to 7, at least 1 to 8, at least 1 to 9, or at least 1 to 10.

[0201] In some embodiments, the present invention provides an oleaginous microbial triglyceride oil composition, wherein the fatty acid profile of the triglyceride oil is selected from the group consisting of: at least about 1%, at least about 2%, at least about 5%, at least about 7%, at least about 10%, or at least about 15%, C8:0; at least about 1%, at least about 5%, at least about 15%, at least about 20%, at least about 25%, or at least about 30% C10:0; at least about 1%, at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, or at least about 80%, C12:0; at least about 2%, at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, or at least about 50%, C14:0; at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, or at least about 90%, C16:0; at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, or at least about 50%, C18:0; at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, or at least about 90%, C18: l; less than about 7%, less than about 5%, less than about 3%, less than about 1%, or about 0%, C18:2; and at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, or at least about 90%, saturated fatty acids.

[0202] In some embodiments, the oleaginous microbial triglyceride oil composition comprises triglyceride oil comprising a fatty acid profile in which: the total combined amount of C10:0, C12:0 and C14:0 is at least about 50%, at least bout 60%, at least about 70%, at least about 80%, at least about 90%, or about 100%; the total combined amount of C16:0, C18:0 and C18: l is at least about 60%, at least about 70%, at least about 80%, at least about 90%, or about 100%; the total combined amount of C18:0, C18: l and C18:2 is at least about 60%, at least about 70%, at least about 80%, at least about 90%, or about 100%; the total combined amount of C14:0, C16:0, C18:0 and C18: l is at least about 60%, at least about 70%, at least about 80%, at least about 90%, or about 100%; the total combined amounts of C8:0 and C10:0 is less than about 50%, less than about 45%, less than about 40%, less than about 35%, less than about 30%, less than about 25%, less than about 20%, less than about 15%, less than about 10%, less than about 5%, or about 0%.

[0203] In some embodiments, the oleaginous microbial triglyceride oil composition comprises triglyceride oil having a fatty acid profile comprising a ratio of fatty acids selected from the group consisting of: a C8:0 to C10:0 ratio of at least about 5 to 1, at least about 6 to 1, at least about 7 to 1, at least about 8 to 1, at least about 9 to 1, or at least about 10 to 1 ; a C10:0 to C12:0 ratio of at least about 6 to 1, at least about 7 to 1, at least about 8 to 1, at least about 9 to 1, or at least about 10 to 1; a C12:0 to C14:0 ratio of at least about 5 to 1, at least about 6 to 1, at least about 7 to 1, at least about 8 to 1, at least about 9 to 1, or at least about 10 to 1 ; a C14:0 to C12:0 ratio of at least about 7 to 1, at least about 8 to 1, at least about 9 to 1, or at least about 10 to 1 ; a C14:0 to C16:0 ratio of at least about 1 to 2, at least about 1 to 3, at least about 1 to 4, at least about 1 to 5, at least about 1 to 6, at least about 1 to 7, at least about 1 to 8, at least about 1 to 9, or at least about 1 to 10.

[0204] In some embodiments, the present invention provides a method of producing an oleaginous microbial triglyceride oil composition having a fatty acid profile selected from the group consisting of: at least about 1%, at least about 2%, at least about 5%, at least about 7%, at least about 10%, or at least about 15%, C8:0; at least about 1%, at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, or at least about 30%, C 10:0; at least about 1%, at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, or at least about 80%, C12:0; at least about 2%, at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, or at least about 50%, C14:0; at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, or at least about 90%, C16:0; at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, or at least about 50% C18:0; at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, or at least about 90%, C18: l; less than about 7%, less than about 5%, less than about 3%, less than about 1%, or about 0%, C18:2; and at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, or at least about 90%, saturated fatty acids, wherein the method comprises the steps of: (a) cultivating a population of oleaginous microbial cells in a culture medium until at least 10% of the dry cell weight of the oleaginous microbial cells is triglyceride oil; and (b) isolating the triglyceride oil composition from the oleaginous microbial cells.

[0205] In some embodiments, the method of producing oleaginous microbial triglyceride oil compositions yields triglyceride oils comprising a fatty acid profile in which: the total combined amount of C10:0, C12:0 and C14:0 is at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or about 100%; the total combined amount of C16:0, C18:0 and C18: l is at least about 60%, at least about 70%, at least about 80%, at least about 90%, or about 100%; the total combined amount of C18:0, C18: l and C18:2 is at least about 60%, at least about 70%, at least about 80%, at least about 90%, or about 100%; the total combined amount of C14:0, C16:0, C18:0 and C18: l is at least about 60%, at least about 70%, at least about 80%, at least about 90%, or about 100%; the total combined amount of C8:0 and C10:0 is less than about 50%, less than about 45%, less than about 40%, less than about 35%, less than about 30%, less than about 25%, less than about 20%, less than about 15%, less than about 10%, less than about 5%, or about 0%.

[0206] In some embodiments, the method of producing oleaginous microbial triglyceride oil compositions yields triglyceride oils having a fatty acid profile comprising a ratio of triglyceride oils selected from the group consisting of: a C8:0 to C10:0 ratio of at least about 5 to 1, at least about 6 to 1, at least about 7 to 1, at least about 8 to 1, at least about 9 to 1, or at least about 10 to 1 ; a C10:0 to C12:0 ratio of at least about 6 to 1, at least about 7 to 1, at least about 8 to 1, at least about 9 to 1, or at least about 10 to 1; a C12:0 to C14:0 ratio of at least about 5 to 1, at least about 6 to 1, at least about 7 to 1, at least about 8 to 1, at least about 9 to 1, or at least about 10 to 1 ; a C14:0 to C12:0 ratio of at least about 7 to 1, at least about 8 to 1, at least about 9 to 1, or at least about 10 to 1 ; and a C14:0 to C16:0 ratio of at least about 1 to 2, at least about 1 to 3, at least about 1 to 4, at least about 1 to 5, at least about 1 to 6, at least about 1 to 7, at least about 1 to 8, at least about 1 to 9, or at least about 1 to 10.

III. GENETIC ENGINEERING METHODS AND MATERIALS

[0207] The present invention provides methods and materials for genentically modifying microorganisms, including Prototheca cells and recombinant host cells, useful in the methods of the present invention, including but not limited to recombinant Prototheca moriformis, Prototheca zopfii, Prototheca krugani, and Prototheca stagnora host cells. The description of these methods and materials is divided into subsections for the convenience of the reader. In subsection 1, transformation methods are described. In subsection 2, genetic engineering methods using homologous recombination are described. In subsection 3, expression vectors and components are described.

[0208] In certain embodiments of the present invention it is desirable to genentically modify a microorganism to enhance lipid production, modify the properties or proportions of components generated by the microorganism, or to improve or provide de novo growth characteristics on a variety of feedstock materials. Chlorella, particularly Chlorella protothecoides, Chlorella minutissima, Chlorella sorokiniana, Chlorella ellipsoidea, Chlorella sp., and Chlorella emersonii are preferred microorganisms for use in the genetic engineering methods described herein, although other Chlorella species as well as other varieties of microorganisms can be used.

[0209] Promoters, cDNAs, and 3'UTRs, as well as other elements of the vectors, can be generated through cloning techniques using fragments isolated from native sources (see for example Molecular Cloning: A Laboratory Manual, Sambrook et al. (3d edition, 2001, Cold Spring Harbor Press; and U.S. Patent 4,683,202). Alternatively, elements can be generated synthetically using known methods (see for example Gene. 1995 Oct 16;164(l):49-53).

1. En2ineerin2 Methods - Transformation

[0210] Cells can be transformed by any suitable technique including, e.g., biolistics, electroporation (see Maruyama et al. (2004), Biotechnology Techniques 8:821-826), glass bead transformation and silicon carbide whisker transformation. Another method that can be used involves forming protoplasts and using CaCl 2 and polyethylene glycol (PEG) to introduce recombinant DNA into microalgal cells (see Kim et al. (2002), Mar. Biotechnol. 4:63-73, which reports the use of this method for the transformation of Chorella ellipsoidea). Co-transformation of microalgae can be used to introduce two distinct vector molecules into a cell simultaneously (see for example Protist 2004 Dec;155(4):381-93).

[0211] Biolistic methods (see, for example, Sanford, Trends In Biotech. (1988) 6:299 302, U.S. Patent No. 4,945,050; electroporation (Fromm et al., Proc. Nat'l. Acad. Sci. (USA) (1985) 82:5824 5828); use of a laser beam, microinjection or any other method capable of introducing DNA into a microalgae can also be used for transformation of a Prototheca cell.

[0212] Any convenient technique for introducing a transgene into a microorganism, such as Chorella, can be employed in the present invention. Dawson et al. (1997) (supra) described the use of micro-projectile bombardment to introduce the nitrate reductase (NR) gene from Chlorella vulgaris into NR-deficient Chlorella sorokiniana mutants, resulting in stable transformants. Briefly, 0.4 micron tungsten beads were coated with plasmid; 3 X 10 7 C. sorokiniana cells were spread in the center third of a non-selective agar plate and bombarded with the PDS-1000/He Biolistic Particle Delivery® system (Bio-Rad).

[0213] A preferred method for introducing a transgene into a microorganism, such as Chlorella, is the method described by Kim et al. (2002), Mar. Biotechnol. 4:63-73. Kim reports the transformation of Chorella ellipsoidea protoplasts using CaCl 2 and polyethylene glycol (PEG). In particular, protoplasts were prepared by growing C. ellipsoidea cells to a density of 1-2 X 10 8 /M1. Cells were recovered and washed by centrifugation for 5 minutes at 1600 g and resuspended in 5 Ml of phosphate buffer (Ph 6.0) containing 0.6 M sorbitol, 0.6 M mannitol, 4% (weight/volume) cellulose (Calbiochem), 2% (weight/volume) macerase (Calbiochem), and 50 units pectinase (Sigma). The cell suspension was incubated at 25°C for 16 hours in the dark with gentle shaking. The resultant protoplasts were recovered by centrifugation at 400 g for 5 minutes. The pellet was gently resuspended in 5 Ml of f/2 medium containing 0.6 M sorbitol and 0.6 M mannitol and centrifuged at 400 g for 5 minutes. This pellet was resuspended in 1 Ml of 0.6 M sorbitol/mannitol solution containing 50 mMCaCl 2 . Then, 5 mg of transgene DNA was added, along with 25 μg calf thymus DNA (Sigma), to 10 7 -10 8 protoplasts in 0.4 Ml. After 15 minutes at room temperature, 200 of PNC (40% polyethylene glycol 4000, 0.8 M NaCl, 50 Mm CaCl 2 ) was added and mixed gently for 30 minutes at room temperature. After this, 0.6 Ml of f/2 medium supplemented with 0.6 M sorbitol/mannitol solution, 1% yeast extract and 1% glucose was added, and the transformed cells were incubated at 25 °C for 12 hours in the dark for cell wall regeneration. A similar method was used by Huang et al. (2007) (supra) to introduce a transgene encoding mercuric reductase into Chlorella sp. DT.

[0214] Electorporation has also been employed to transform microorganisms, such as Chorella. As reported by Maruyama et al. (2004), Biotechnology Techniques 8:821-826 (incorporated by reference herein in its entirety), this technique was used to introduce a transgene into protoplasts of Chlorella saccharophila c-211-la prepared from the cells in the stationary phase. Transient expression of the introduced plasmid was observed under a field strength of between 600 and 900 V/cm, and a pulse duration of around 400 ms, where high membrane permeability to 70-kDa FITC-dextran was ascertained.

[0215] Examples of expression of transgenes in microorganisms, such as Chlorella, can be found in the literature (see for example Current Microbiology Vol. 35 (1997), pp. 356-362; Sheng Wu Gong Cheng Xue Bao. 2000 Jul;16(4):443-6; Current Microbiology Vol. 38 (1999), pp. 335-341 ; Appl Microbiol Biotechnol (2006) 72: 197-205; Marine Biotechnology 4, 63-73, 2002; Current Genetics 39:5, 365-370 (2001); Plant Cell Reports 18:9, 778-780, (1999); Biologia Plantarium 42(2): 209-216, (1999); Plant Pathol. J 21(1): 13-20, (2005)). Also see Examples herein.

[0216] Examples of expression of transgenes in oleaginous yeast (e.g., Yarrowia lipolytica) can be found in the literature (see, for example, Bordes et al., J Microbiol Methods, Jun 27 (2007)). Examples of expression of transgenes in fungi (e.g., Mortierella alpine, Mucor circinelloides, and Aspergillus ochraceus) can also be found in the literature (see, for example, Microbiology, Jul; 153(Pt. 7):2013-25 (2007); Mol Genet Genomics, Jun;

271(5):595-602 (2004); Curr Genet, Mar;21(3):215-23 (1992); Current Microbiology, 30(2):83-86 (1995); Sakuradani, NISR Research Grant, "Studies of Metabolic Engineering of Useful Lipid-producing Microorganisms" (2004); and PCT/JP2004/012021). Examples of expression of exogenous genes in bacteria such as E. coli are well known; see for example Molecular Cloning: A Laboratory Manual, Sambrook et al. (3d edition, 2001, Cold Spring Harbor Press.

[0217] Vectors for transformation of microorganisms in accordance with the present invention can be prepared by known techniques familiar to those skilled in the art. The nucleotide sequence of the construct used for transformation of multiple Chlorella species corresponds to SEQ ID NO: 8. In one embodiment, an exemplary vector design for expression of a lipase gene in a microorganism such as a microalgae contains a gene encoding a lipase in operable linkage with a promoter active in microalgae. Alternatively, if the vector does not contain a promoter in operable linkage with the gene of interest, the gene can be transformed into the cells such that it becomes operably linked to an endogenous promoter at the point of vector integration. The promoterless method of transformation has been proven to work in microalgae (see for example Plant Journal 14:4, (1998), pp.441-447). The vector can also contain a second gene that encodes a protein that, e.g., imparts resistance to an antibiotic or herbicide, i.e., a selectable marker. Optionally, one or both gene(s) is/are followed by a 3' untranslated sequence containing a polyadenylation signal. Expression cassettes encoding the two genes can be physically linked in the vector or on separate vectors. Co-transformation of microalgae can also be used, in which distinct vector molecules are simultaneously used to transform cells (see for example Protist 2004 Dec; 155(4):381-93). The transformed cells can be optionally selected based upon the ability to grow in the presence of the antibiotic or other selectable marker under conditions in which cells lacking the resistance cassette would not grow.

2. En2ineerin2 Methods - Homolo2Qus Recombination

[0218] Homologous recombination is the ability of complementary DNA sequences to align and exchange regions of homology. Transgenic DNA ("donor") containing sequences homologous to the genomic sequences being targeted ("template") is introduced into the organism and then undergoes recombination into the genome at the site of the corresponding genomic homologous sequences. The mechanistic steps of this process, in most casees, include: (1) pairing of homologous DNA segments; (2) introduction of double- stranded breaks into the donor DNA molecule; (3) invasion of the template DNA molecule by the free donor DNA ends followed by DNA synthesis; and (4) resolution of double-strand break repair events that result in final recombination products.

[0219] The ability to carry out homologous recombination in a host organism has many practical implications for what can be carried out at the molecular genetic level and is useful in the generation of an oleaginous microbe that can produced tailored oils. By its very nature homologous recombination is a precise gene targeting event, hence, most transgenic lines generated with the same targeting sequence will be essentially identical in terms of phenotype, necessitating the screening of far fewer transformation events. Homologous recombination also targets gene insertion events into the host chromosome, resulting in excellent genetic stability, even in the absence of genetic selection. Because different chromosomal loci will likey impact gene expression, even from heterologous

promoters/UTRs, homologous recombination can be a method of querying loci in an unfamiliar genome environment and to assess the impact of these environments on gene expression.

[0220] Particularly useful genetic engineering applications using homologous

recombination is to co-opt specific host regulatory elements such as promoters/UTRs to drive heterologous gene expression in a highly specific fashion. For example, ablation or knockout of desaturase genes/gene families with a heterologous gene encoding a selective marker might be expected to increase overall percentage of saturated fatty acids produced in the host cell. Example 11 describes the homologous recombination targeting constructs and a working example of such desaturase gene ablations or knockouts generated in Prototheca moriformis.

[0221] Because homologous recombination is a precise gene targeting event, it can be used to precisely modify any nucleotide(s) within a gene or region of interest, so long as sufficient flanking regions have been identified. Therefore, homologous recombination can be used as a means to modify regulatory sequences impacting gene expression of RNA and/or proteins. It can also be used to modify protein coding regions in an effort to modify enzyme activites such as substrate specificity, affinities and Km, and thus affecting the desired change in metabolism of the host cell. Homologous recombination provides a powerful means to manipulate the gost genome resulting in gene targeting, gene conversion, gene deletion, gene duplication, gene inversion and exchanging gene expression regulatory elements such as promoters, enhancers and 3'UTRs.

[0222] Homologous recombination can be achieve by using targeting constructs containing pieces of endogenous sequences to "target" the gene or region of interest within the endogenous host cell genome. Such targeting sequences can either be located 5' of the gene or region of interest, 3 ' of the gene/region of interest or even flank the gene/region of interest. Such targeting constructs can be transformed into the host cell either as a supercoiled plasmid DNA with additional vector backbone, a PCR product with no vector backbone, or as a linearized molecule. In some cases, it may be advantageous to first expose the homologous sequences within the transgenic DNA (donor DNA) with a restriction enzyme. This step can increase the recombination efficiency and decrease the occurance of undesired events. Other methods of increasing recombination efficiency include using PCR to generate transforming transgenic DNA containing linear ends homologous to the genomic sequences being targeted.

[0223] For purposes of non- limiting illustration, regions of donor DNA sequences that are useful for homologous recombination include the KE858 region of DNA in Prototheca moriformis. KE858 is a 1.3 kb, genomic fragment that encompasses part of the coding region for a protein that shares homology with the transfer RNA (tRNA) family of proteins.

Southern blots have shown that the KE858 sequence is present in a single copy in the Prototheca moriformis (UTEX 1435) genome. This region and Examples of using this region for homologous recombination targeting has been described in PCT Application No.

PCT/US2009/66142. Another region of donor DNA that is useful is portions of the 6S rRNA genomic sequence. The use of this sequence in homologous recombination in Prototheca morifomis are described below in the Examples.

3. Vectors and Vector Components

[0224] Vectors for transformation of microorganisms in accordance with the present invention can be prepared by known techniques familiar to those skilled in the art in view of the disclosure herein. A vector typically contains one or more genes, in which each gene codes for the expression of a desired product (the gene product) and is operably linked to one or more control sequences that regulate gene expression or target the gene product to a particular location in the recombinant cell. To aid the reader, this subsection is divided into subsections. Subsection A describes control sequences typically contained on vectors as well as novel control sequences provided by the present invention. Subsection B describes genes typically contained in vectors as well as novel codon optimization methods and genes prepared using them provided by the invention.

A. Control Sequences

[0225] Control sequences are nucleic acids that regulate the expression of a coding sequence or direct a gene product to a particular location in or outside a cell. Control sequences that regulate expression include, for example, promoters that regulate transcription of a coding sequence and terminators that terminate transcription of a coding sequence.

Another control sequence is a 3 ' untranslated sequence located at the end of a coding sequence that encodes a polyadenylation signal. Control sequences that direct gene products to particular locations include those that encode signal peptides, which direct the protein to which they are attached to a particular location in or outside the cell. [0226] Thus, an exemplary vector design for expression of an exogenous gene in a microalgae contains a coding sequence for a desired gene product (for example, a selectable marker, a lipid pathway modification enzyme, or a sucrose utilization enzyme) in operable linkage with a promoter active in microalgae. Alternatively, if the vector does not contain a promoter in operable linkage with the coding sequence of interest, the coding sequence can be transformed into the cells such that it becomes operably linked to an endogenous promoter at the point of vector integration. The promoterless method of transformation has been proven to work in microalgae (see for example Plant Journal 14:4, (1998), pp.441-447).

[0227] Many promoters are active in microalgae, including promoters that are endogenous to the algae being transformed, as well as promoters that are not endogenous to the algae being transformed (i.e., promoters from other algae, promoters from higher plants, and promoters from plant viruses or algae viruses). Illustrative exogenous and/or endogenous promoters that are active in microalgae (as well as antibiotic resistance genes functional in microalgae) are described in PCT Pub. No. 2008/151149 and references cited therein).

[0228] The promoter used to express an exogenous gene can be the promoter naturally linked to that gene or can be a heterologous gene. Some promoters are active in more than one species of microalgae. Other promoters are species-specific. Illustrative promoters include promoters such as β-tubulin from Chlamydomonas reinhardtii, used in the Examples below,and viral promoters, such as cauliflower mosaic virus (CMV) and chlorella virus, which have been shown to be active in multiple species of microalgae (see for example Plant Cell Rep. 2005 Mar;23(10-l l):727-35; J Microbiol. 2005 Aug;43(4):361-5; Mar Biotechnol (NY). 2002 Jan;4(l):63-73). Another promoter that is suitable for use for expression of exogenous genes in Prototheca is the Chlorella sorokiniana glutamate dehydrogenase promoter/5 'UTR. Optionally, at least 10, 20, 30, 40, 50, or 60 nucleotides or more of these sequences containing a promoter are used. Illustrative promoters useful for expression of exogenous genes in Prototheca are listed in the sequence listing of this application, such as the promoter of the Chlorella HUP1 gene (SEQ ID NO: l) and the Chlorella ellipsoidea nitrate reductase promoter (SEQ ID NO:2). Chlorella virus promoters can also be used to express genes in Prototheca, such as SEQ ID NOs: 1-7 of U.S. Patent 6,395,965. Additional promoters active in Prototheca can be found, for example, in Biochem Biophys Res

Commun. 1994 Oct 14;204(l): 187-94; Plant Mol Biol. 1994 Oct;26(l):85-93; Virology. 2004 Aug 15;326(l): 150-9; and Virology. 2004 Jan 5;318(l):214-23. Other useful promoters are described in detail in the Examples below. [0229] A promoter can generally be characterized as either constitutive or inducible.

Constitutive promoters are generally active or function to drive expression at all times (or at certain times in the cell life cycle) at the same level. Inducible promoters, conversely, are active (or rendered inactive) or are significantly up- or down-regulated only in response to a stimulus. Both types of promoters find application in the methods of the invention. Inducible promoters useful in the invention include those that mediate transcription of an operably linked gene in response to a stimulus, such as an exogenously provided small molecule (e.g, glucose, as in SEQ ID NO: 1), temperature (heat or cold), lack of nitrogen in culture media, etc. Suitable promoters can activate transcription of an essentially silent gene or upregulate, preferably substantially, transcription of an operably linked gene that is transcribed at a low level. Examples below describe additional inducible promoters that are useful in Prototheca cells.

[0230] Inclusion of termination region control sequence is optional, and if employed, then the choice is be primarily one of convenience, as the termination region is relatively interchangeable. The termination region may be native to the transcriptional initiation region (the promoter), may be native to the DNA sequence of interest, or may be obtainable from another source. See, for example, Chen and Orozco, Nucleic Acids Res. (1988) 16:8411.

[0231] The present invention also provides control sequences and recombinant genes and vectors containing them that provide for the compartmentalized expression of a gene of interest. Organelles for targeting are chloroplasts, plastids, mitochondria, and endoplasmic reticulum. In addition, the present invention provides control sequences and recombinant genes and vectors containing them that provide for the secretion of a protein outside the cell.

[0232] Proteins expressed in the nuclear genome of Prototheca can be targeted to the plastid using plastid targeting signals. Plastid targeting sequences endogenous to Chlorella are known, such as genes in the Chlorella nuclear genome that encode proteins that are targeted to the plastid; see for example GenBank Accession numbers AY646197 and AF499684, and in one embodiment, such control sequences are used in the vectors of the present invention to target expression of a protein to a Prototheca plastid.

[0233] The Examples below describe the use of algal plastid targeting sequences to target heterologous proteins to the correct compartment in the host cell. cDNA libraries were made using Prototheca moriformis and Chlorella protothecodies cells and are described in PCT Application No. PCT/US2009/066142.

[0234] In another embodiment of the present invention, the expression of a polypeptide in Prototheca is targeted to the endoplasmic reticulum. The inclusion of an appropriate retention or sorting signal in an expression vector ensure that proteins are retained in the endoplasmic reticulum (ER) and do not go downstream into Golgi. For example, the

IMPACTVECTOR1.3 vector, from Wageningen UR- Plant Research International, includes the well known KDEL retention or sorting signal. With this vector, ER retention has a practical advantage in that it has been reported to improve expression levels 5-fold or more. The main reason for this appears to be that the ER contains lower concentrations and/or different proteases responsible for post-translational degradation of expressed proteins than are present in the cytoplasm. ER retention signals functional in green microalgae are known. For example, see Proc Natl Acad Sci U S A. 2005 Apr 26;102(17):6225-30.

[0235] In another embodiment of the present invention, a polypeptide is targeted for secretion outside the cell into the culture media. See Hawkins et al., Current Microbiology Vol. 38 (1999), pp. 335-341 for examples of secretion signals active in Chlorella that can be used, in accordance with the methods of the invention, in Prototheca.

[0236] Many promoters are active in microalgae, including promoters that are endogenous to the algae being transformed, as well as promoters that are not endogenous to the algae being transformed (i.e., promoters from other algae, promoters from higher plants, and promoters from plant viruses or algae viruses). Exogenous and/or endogenous promoters that are active in microalgae, and antibiotic resistance genes functional in microalgae are described by e.g., Curr Microbiol. 1997 Dec;35(6):356-62 (Chlorella vulgaris); Mar

Biotechnol (NY). 2002 Jan;4(l):63-73 (Chlorella ellipsoidea); Mol Gen Genet. 1996 Oct 16;252(5):572-9 (Phaeodactylum tricornutum); Plant Mol Biol. 1996 Apr;31(l): l-12 (Volvox carteri); Proc Natl Acad Sci U S A. 1994 Nov 22;91(24): 11562-6 (Volvox carteri); Falciatore A, Casotti R, Leblanc C, Abrescia C, Bowler C, PMID: 10383998, 1999

May;l(3):239-251 (Laboratory of Molecular Plant Biology, Stazione Zoologica, Villa Comunale, 1-80121 Naples, Italy) (Phaeodactylum tricornutum and Thalassiosira weissflogii); Plant Physiol. 2002 May; 129(l):7-12. (Porphyridium sp.); Proc Natl Acad Sci U S A. 2003 Jan 21 ;100(2):438-42. (Chlamydomonas reinhardtii); Proc Natl Acad Sci U S A. 1990 Feb;87(3): 1228-32. (Chlamydomonas reinhardtii); Nucleic Acids Res. 1992 Jun 25;20(12):2959-65; Mar Biotechnol (NY). 2002 Jan;4(l):63-73 (Chlorella); Biochem Mol Biol Int. 1995 Aug;36(5): 1025-35 (Chlamydomonas reinhardtii); J Microbiol. 2005

Aug;43(4):361-5 (Dunaliella); Yi Chuan Xue Bao. 2005 Apr;32(4):424-33 (Dunaliella); Mar Biotechnol (NY). 1999 May;l(3):239-251. (Thalassiosira and Phaedactylum); Koksharova, Appl Microbiol Biotechnol 2002 Feb;58(2): 123-37 (various species); Mol Genet Genomics. 2004 Feb;271(l):50-9 (Thermosynechococcus elongates); J. Bacteriol. (2000), 182, 211-215; FEMS Microbiol Lett. 2003 Apr 25;221(2): 155-9; Plant Physiol. 1994 Jun;105(2):635-41 ; Plant Mol Biol. 1995 Dec;29(5):897-907 (Synechococcus PCC 7942); Mar Pollut Bull.

2002;45(1-12): 163-7 (Anabaena PCC 7120); Proc Natl Acad Sci U S A. 1984

Mar;81(5): 1561-5 (Anabaena (various strains)); Proc Natl Acad Sci U S A. 2001 Mar 27;98(7):4243-8 (Synechocystis); Wirth, Mol Gen Genet 1989 Mar;216(l): 175-7 (various species); Mol Microbiol, 2002 Jun;44(6): 1517-31 and Plasmid, 1993 Sep;30(2):90-105 (Fremyella diplosiphon); Hall et al. (1993) Gene 124: 75-81 (Chlamydomonas reinhardtii); Gruber et al. (1991). Current Micro. 22: 15-20; Jarvis et al. (1991) Current Genet. 19: 317- 322 (Chlorella); for additional promoters see also table 1 from US Patent 6,027,900).

[0237] The promoter used to express an exogenous gene can be the promoter naturally linked to that gene or can be a heterologous gene. Some promoters are active in more than one species of microalgae. Other promoters are species-specific. Preferred promoters include promoters such as RBCS2 from Chlamydomonas reinhardtii and viral promoters, such as cauliflower mosaic virus (CMV) and chlorella virus, which have been shown to be active in multiple species of microalgae (see for example Plant Cell Rep. 2005 Mar;23(10- l l):727-35; J Microbiol. 2005 Aug;43(4):361-5; Mar Biotechnol (NY). 2002 Jan;4(l):63-73). In other embodiments, the Botryococcus malate dehydrogenase promoter, such a nucleic acid comprising any part of SEQ ID NO: 150, or the Chlamydomonas reinhardtii RBCS2 promoter (SEQ ID NO: 151) can be used. Optionally, at least 10, 20, 30, 40, 50, or 60 nucleotides or more of these sequences containing a promoter are used. Preferred promoters endogenous to species of the genus Chlorella are SEQ ID NO: l and SEQ ID NO:2.

[0238] Preferred promoters useful for expression of exogenous genes in Chlorella are listed in the sequence listing of this application, such as the promoter of the Chlorella HUP1 gene (SEQ ID NO: l) and the Chlorella ellipsoidea nitrate reductase promoter (SEQ ID NO:2). Chlorella virus promoters can also be used to express genes in Chlorella, such as SEQ ID NOs: 1-7 of U.S. Patent 6,395,965. Additional promoters active in Chlorella can be found, for example, in Biochem Biophys Res Commun. 1994 Oct 14;204(1): 187-94; Plant Mol Biol. 1994 Oct;26(l):85-93; Virology. 2004 Aug 15;326(1): 150-9; and Virology. 2004 Jan 5;318(l):214-23.

B. Genes and Codon Optimization

[0239] Typically, a gene includes a promoter, coding sequence, and termination control sequences. When assembled by recombinant DNA technology, a gene may be termed an expression cassette and may be flanked by restriction sites for convenient insertion into a vector that is used to introduce the recombinant gene into a host cell. The expression cassette can be flanked by DNA sequences from the genome or other nucleic acid target to facilitate stable integration of the expression cassette into the genome by homologous recombination. Alternatively, the vector and its expression cassette may remain unintegrated, in which case, the vector typically includes an origin of replication, which is capable of providing for replication of the heterologous vector DNA.

[0240] A common gene present on a vector is a gene that codes for a protein, the expression of which allows the recombinant cell containing the protein to be differentiated from cells that do not express the protein. Such a gene, and its corresponding gene product, is called a selectable marker. Any of a wide variety of selectable markers can be employed in a transgene construct useful for transforming Prototheca. Examples of suitable selectable markers include the G418 resistance gene, the nitrate reductase gene (see Dawson et al. (1997), Current Microbiology 35:356-362), the hygromycin phosphotransferase gene (HPT; see Kim et al. (2002), Mar. Biotechnol. 4:63-73), the neomycin phosphotransferase gene, the ble gene, which confers resistance to phleomycin (Huang et al. (2007), Appl. Microbiol. Biotechnol. 72: 197-205), and the aminoglycoside-3'-0-phosphotransferase (SEQ ID NO: 194), which confers resistance to kanamycin. Methods of determining sensitivity of microalgae to antibiotics are well known. For example, Mol Gen Genet. 1996 Oct

16;252(5):572-9.

[0241] Other selectable markers that are not antibiotic-based can alsobe employed in a transgene construct useful for transforming microalgae in general, including Prototheca species. Genes that confers the ability to utilize certain carbon sources that were previously unable to be utilized by the microalgae can also be used as a selectable marker. By way of illustration, Prototheca moriformis strains typically grow poorly, if at all, on sucrose. Using a construct containing a sucrose invertase gene can confer the ability of positive

transformants to grow on sucrose as a carbon substrate. Additional details on using sucrose utilization as a selectable marker along with other selectable markers are discussed in Section IV below.

[0242] For purposes of the present invention, the expression vector used to prepare a recombinant host cell of the invention will include at least two, and often three, genes, if one of the genes is a selectable marker. For example, a genetically engineered Prototheca of the invention can be made by transformation with vectors of the invention that comprise, in addition to a selectable marker, one or more exogenous genes, such as, for example, sucrose invertase gene or acyl ACP-thioesterase gene. One or both genes can be expressed using an inducible promoter, which allows the relative timing of expression of these genes to be controlled to enhance the lipid yield and conversion to fatty acid esters. Expression of the two or more exogenous genes may be under control of the same inducible promoter or under control of different inducible (or constitutive) promoters. In the latter situation, expression of a first exogenous gene can be induced for a first period of time (during which expression of a second exogenous gene may or may not be induced) and expression of a second exogenous gene can be induced for a second period of time (during which expression of a first exogenous gene may or may not be induced).

[0243] In other embodiments, the two or more exogenous genes (in addition to any selectable marker) are: a fatty acyl-ACP thioesterase and a fatty acyl- Co A/aldehyde reductase, the combined action of which yields an alcohol product. Further provided are other combinations of exogenous genes, including without limitation, a fatty acyl-ACP thioesterase and a fatty acyl-CoA reductase to generate aldehydes. In one embodiment, the vector provides for the combination of a fatty acyl-ACP thioesterase, a fatty acyl-CoA reductase, and a fatty aldehyde decarbonylase to generate alkanes. In each of these embodiments, one or more of the exogenous genes can be expressed using an inducible promoter.

[0244] Other illustrative vectors of the invention that express two or more exogenous genes include those encoding both a sucrose transporter and a sucrose invertase enzyme and those encoding both a selectable marker and a secreted sucrose invertase. The recombinant Prototheca transformed with either type of vector produce lipids at lower manufacturing cost due to the engineered ability to use sugar cane (and sugar cane-derived sugars) as a carbon source. Insertion of the two exogenous genes described above can be combined with the disruption of polysaccharide biosynthesis through directed and/or random mutagenesis, which steers ever greater carbon flux into lipid production. Individually and in combination, trophic conversion, engineering to alter lipid production and treatment with exogenous enzymes alter the lipid composition produced by a microorganism. The alteration can be a change in the amount of lipids produced, the amount of one or more hydrocarbon species produced relative to other lipids, and/or the types of lipid species produced in the

microorganism. For example, microalgae can be engineered to produce a higher amount and/or percentage of TAGs.

[0245] For optimal expression of a recombinant protein, it is beneficial to employ coding sequences that produce mRNA with codons preferentially used by the host cell to be transformed. Thus, proper expression of transgenes can require that the codon usage of the transgene matches the specific codon bias of the organism in which the transgene is being expressed. The precise mechanisms underlying this effect are many, but include the proper balancing of available aminoacylated tRNA pools with proteins being synthesized in the cell, coupled with more efficient translation of the transgenic messenger RNA (mRNA) when this need is met. When codon usage in the transgene is not optimized, available tRNA pools are not sufficient to allow for efficient translation of the heterologous mRNA resulting in ribosomal stalling and termination and possible instability of the transgenic mRNA.

[0246] The present invention provides codon-optimized nucleic acids useful for the successful expression of recombinant proteins in Prototheca. Codon usage in Prototheca species was analyzed by studying cDNA sequences isolated from Prototheca moriformis. This analysis represents the interrogation over 24, 000 codons and resulted in Table 2 below.

[0247] Table 2. Preferred codon usage in Prototheca strains.

Ala GCG 345 (0.36) Asn AAT 8 (0.04)

GCA 66 (0.07) AAC 201 (0.96)

GCT 101 (0.11)

GCC 442 (0.46) Pro CCG 161 (0.29)

CCA 49 (0.09)

Cys TGT 12 (0.10) CCT 71 (0.13)

TGC 105 (0.90) CCC 267 (0.49)

Asp GAT 43 (0.12) Gin CAG 226 (0.82)

GAC 316 (0.88) CAA 48 (0.18)

Glu GAG 377 (0.96) Arg AGG 33 (0.06)

GAA 14 (0.04) AGA 14 (0.02)

CGG 102 (0.18)

Phe TTT 89 (0.29) CGA 49 (0.08)

TTC 216 (0.71) CGT 51 (0.09)

CGC 331 (0.57)

Gly GGG 92 (0.12)

GGA 56 (0.07) Ser AGT 16 (0.03)

GGT 76 (0.10) AGC 123 (0.22)

GGC 559 (0.71) TCG 152 (0.28)

TCA 31 (0.06)

His CAT 42 (0.21) TCT 55 (0.10)

CAC 154 (0.79) TCC 173 (0.31)

He ATA 4 (0.01) Thr ACG 184 (0.38)

ATT 30 (0.08) ACA 24 (0.05)

ATC 338 (0.91) ACT 21 (0.05)

ACC 249 (0.52)

Lys AAG 284 (0.98)

AAA 7 (0.02) Val GTG 308 (0.50)

GTA 9 (0.01)

Leu TTG 26 (0.04) GTT 35 (0.06)

TTA 3 (0.00) GTC 262 (0.43)

CTG 447 (0.61) CTA 20 (0.03) Trp TGG 107 (1.00)

CTT 45 (0.06)

CTC 190 (0.26) Tyr TAT 10 (0.05)

TAC 180 (0.95)

Met ATG 191 (1.00)

Stop TGA/TAG/TAA

[0248] In other embodiments, the gene in the recombinant vector has been codon- optimized with reference to a microalgal strain other than a Prototheca strain. For example, methods of recoding genes for expression in microalgae are described in U.S. Patent 7,135,290. Additional information for codon optimization is available, e.g., at the codon usage database of GenBank.

[0249] Other non-limiting examples of codon usage in Chlorella pyrenoidosa, Dunaliella salina, and Chlorella protothecoides are shown in Tables 28, 29, and 30, respectively.

[0250] Table 28. Codon usage in Chlorella pyrenoidosa.

Phe uuu 39 (0.82) Ser ucu 50 (1.04)

uuc 56 (1.18) ucc 60 (1.25)

Leu UUA 10 (0.20) UCA 6 (0.96)

UUG 46 (0.91) UCG 43 (0.89)

Tyr UAU 15 (0.59) Cys UGU 46 (0.77)

UAC 36 (1.41) UGC 73 (1.23)

ter UAA 9 (0.00) ter UGA 43 (0.00)

ter UAG 15 (0.00) Trp UGG 69 (1.00)

Leu CUU 49 (0.97) Pro ecu 80 (0.98)

cue 73 (1.45) CCC 88 (1.08)

CUA 22 (0.44) CCA 93 (1.14)

CUG 103 (2.04) CCG 65 (0.80)

His CAU 50 (0.88) Arg CGU 39 (0.76)

CAC 3 (1.12) CGC 63 (1.23)

Gin CAA 59 (0.84) CGA 46 (0.90)

CAG 2 (1.16) CGG 47 (0.92)

He AUU 24 (0.69) Thr ACU 32 (0.67)

AUC 61 (1.76) ACC 76 (1.60)

AUA 19 (0.55) ACA 41 (0.86)

Met AUG 42 (1.00) ACG 41 (0.86)

Asn AAU 26 (0.75) Ser AGU 23 (0.48)

AAC 3 (1.25) AGC 67 (1.39)

Lys AAA 32 (0.54) Arg AGA 51 (1.00)

AAG 86 (1.46) AGG 61 (1.19)

Val GUU 36 (0.75) Ala GCU 57 (0.79)

GUC 54 (1.13) GCC 97 (1.34)

GUA 30 (0.63) GCA 89 (1.23)

GUG 71 (1.49) GCG 47 (0.65)

Asp GAU 60 (0.95) Gly GGU 35 (0.60)

GAC 66 (1.05) GGC 78 (1.33) Glu GAA 41 (0.68) GGA 54 (0.92)

GAG 80 (1.32) GGG 67 (1.15)

[0251] Table 29. Preferred codon usage in Dunaliella salina.

TTC (Phe) TAC (Tyr) TGC (Cys) TAA (Stop)

TGG (Tip) CCC (Pro) CAC (His) CGC (Arg)

CTG (Leu) CAG (Gin) ATC (lie) ACC (Thr)

AAC (Asn) AGC (Ser) ATG (Met) AAG (Lys)

GCC (Ala) GAC (Asp) GGC (Gly) GTG (Val)

GAG (Glu)

[0252] Table 30. Preferred codon usage in Chlorella protothecoides.

TTC (Phe) TAC (Tyr) TGC (Cys) TGA (Stop)

TGG (Trp) CCC (Pro) CAC (His) CGC (Arg)

CTG (Leu) CAG (Gin) ATC (lie) ACC (Thr)

GAC (Asp) TCC (Ser) ATG (Met) AAG (Lys)

GCC (Ala) AAC (Asn) GGC (Gly) GTG (Val)

GAG (Glu)

C. Inducible Expression

[0253] The present invention also provides for the use of an inducible promoter to express a gene of interest. In particular, the use of an inducible promoter to express a lipase gene permits production of the lipase after growth of the microorganism when conditions have been adjusted, if necessary, to enhance transesterification, for example, after disruption of the cells, reduction of the water content of the reaction mixture, and/or addition sufficient alcohol to drive conversion of TAGs to fatty acid esters.

[0254] Inducible promoters useful in the invention include those that mediate transcription of an operably linked gene in response to a stimulus, such as an exogenously provided small molecule (e.g, glucose, as in SEQ ID NO: l), temperature (heat or cold), light, etc. Suitable promoters can activate transcription of an essentially silent gene or upregulate, preferably substantially, transcription of an operably linked gene that is transcribed at a low level. In the latter case, the level of transcription of the lipase preferably does not significantly interfere with the growth of the microorganism in which it is expressed.

[0255] Expression of transgenes in Chlorella can be performed inducibly through promoters such as the promoter that drives the Chlorella hexose transporter gene (SEQ ID NO: l). This promoter is strongly activated by the presence of glucose in the culture media.

D. Expression of Two or More Exogenous Genes [0256] Further, a genetically engineered microorganism, such as a microalgae, may comprise and express two or more exogenous genes, such as, for example, a lipase and a lytic gene, e.g., one encoding a polysaccharide-degrading enzyme. One or both genes can be expressed using an inducible promoter, which allows the relative timing of expression of these genes to be controlled to enhance the lipid yield and conversion to fatty acid esters. Expression of the two or more exogenous genes may be under control of the same inducible promoter or under control of a different inducible promoters. In the latter situation, expression of a first exogenous gene can be induced for a first period of time (during which expression of a second exogenous gene may or may not be induced) and expression of a second exogenous gene can be induced for a second period of time (during which expression of a first exogenous gene may or may not be induced). Provided herein are vectors and methods for engineering lipid-producing microbes to metabolize sucrose, which is an advantageous trait because it allows the engineered cells to convert sugar cane feedstocks into lipids.

[0257] Also provided herein are genetically engineered strains of microbes (e.g., microalgae, oleaginous yeast, bacteria, or fungi) that express two or more exogenous genes, such as, for example, a fatty acyl-ACP thioesterase and a fatty acyl-CoA/aldehyde reductase, the combined action of which yields an alcohol product. Further provided are other combinations of exogenous genes, including without limitation, a fatty acyl-ACP thioesterase and a fatty acyl-CoA reductase to generate aldehydes. In addition, this application provides for the combination of a fatty acyl-ACP thioesterase, a fatty acyl-CoA reductase, and a fatty aldehyde decarbonylase to generate alkanes. One or more of the exogenous genes can be expressed using an inducible promoter.

[0258] Examples of further modifications suitable for use in the present invention are include genetically engineering strains of microalgae to express two or more exogenous genes, one encoding a transporter of a fixed carbon source (such as sucrose) and a second encoding a sucrose invertase enzyme. The resulting fermentable organisms produce hydrocarbons at lower manufacturing cost than what has been obtainable by previously known methods of biological hydrocarbon production. Insertion of the two exogenous genes described above can be combined with the disruption of polysaccharide biosynthesis through directed and/or random mutagenesis, which steers ever greater carbon flux into hydrocarbon production. Individually and in combination, trophic conversion, engineering to alter hydrocarbon production and treatment with exogenous enzymes alter the hydrocarbon composition produced by a microorganism. The alteration can be a change in the amount of hydrocarbons produced, the amount of one or more hydrocarbon species produced relative to other hydrocarbons, and/or the types of hydrocarbon species produced in the microorganism. For example, microalgae can be engineered to produce a higher amount and/or percentage of TAGs.

E. Compartmentalized Expression

[0259] The present invention also provides for compartmentalized expression of a gene of interest. In particular, it can be advantageous, in particular embodiments, to target expression of the lipase to one or more cellular compartments, where it is sequestered from the majority of cellular lipids until initiation of the transesterification reaction. Preferred organelles for targeting are chloroplasts, mitochondria, and endoplasmic reticulum.

(1) Expression in Chloroplasts

[0260] In one embodiment of the present invention, the expression of a polypeptide in a microorganism is targeted to chloroplasts. Methods for targeting expression of a

heterologous gene to the chloroplast are known and can be employed in the present invention. Methods for targeting foreign gene products into chloroplasts are described in Shrier et al., EMBO J. (1985) 4:25 32. See also Tomai et al. Gen. Biol. Chem. (1988) 263: 15104 15109 and U.S. Pat. No. 4,940,835 for the use of transit peptides for translocating nuclear gene products into the chloroplast. Methods for directing the transport of proteins to the chloroplast are also reviewed in Kenauf TIBTECH (1987) 5:40 47. Chloroplast targeting sequences endogenous to Chlorella are known, such as genes in the Chlorella nuclear genome that encode proteins that are targeted to the chloroplast; see for example GenBank Accession numbers AY646197 and AF499684.

[0261] Wageningen UR- Plant Research International sells an IMPACTVECTORl .4 vector, which uses the secretion signal of the Chrysanthemum morifolium small subunit protein to deliver a heterologous protein into the chloroplast stroma (cytoplasmic) environment, shuttling across a double membrane system. The protein is fused to the first 11 amino acids of the mature rubisco protein in order to allow proper processing of the signal peptide (Wong et al., Plant Molecular Biology 20: 81-93 (1992)). The signal peptide contains a natural intron from the RbcS gene.

[0262] In another approach, the chloroplast genome is genetically engineered to express the heterologous protein. Stable transformation of chloroplasts of Chlamydomonas reinhardtii (a green alga) using bombardment of recipient cells with high- velocity tungsten microprojectiles coated with foreign DNA has been described. See, for example, Boynton et al., Science (1988) 240: 1534 1538; Blowers et al. Plant Cell (1989) 1 : 123 132 and Debuchy et al., EMBO J. (1989) 8: 2803 2809. The transformation technique, using tungsten microprojectiles, is described by Klein et al., Nature (London) (1987) 7:70 73. Other methods of chloroplast transformation for both plants and microalgae are known. See for example U.S. Patents 5,693,507; 6,680,426; and Plant Physiol. 2002 May;129(l):7-12; and Plant Biotechnol J. 2007 May;5(3):402-12.

[0263] As described in U.S. Patent No. 6,320,101 (issued November 20, 2001 to Kaplan et al.; which is incorporated herein by reference), cells can be chemically treated so as to reduce the number of chloroplasts per cell to about one. Then, the heterologous nucleic acid can be introduced into the cells via particle bombardment with the aim of introducing at least one heterologous nucleic acid molecule into the chloroplasts. The heterologous nucleic acid is selected such that it is integratable into the chloroplast's genome via homologous

recombination which is readily effected by enzymes inherent to the chloroplast. To this end, the heterologous nucleic acid includes, in addition to a gene of interest, at least one nucleic acid sequence that is derived from the chloroplast' s genome. In addition, the heterologous nucleic acid typically includes a selectable marker. Further details relating to this technique are found in U.S. Patent. Nos. 4,945,050 and 5,693,507 which are incorporated herein by reference. A polypeptide can thus be produced by the protein expression system of the chloroplast.

[0264] U.S. Patent No. 7,135,620 (issued November 14, 2006 to Daniell et al.; incorporated herein by reference) describes chloroplast expression vectors and related methods.

Expression cassettes are DNA constructs including a coding sequence and appropriate control sequences to provide for proper expression of the coding sequence in the chloroplast.

Typical expression cassettes include the following components: the 5' untranslated region from a microorganism gene or chloroplast gene such as psbA which will provide for transcription and translation of a DNA sequence encoding a polypeptide of interest in the chloroplast; a DNA sequence encoding a polypeptide of interest; and a translational and transcriptional termination region, such as a 3' inverted repeat region of a chloroplast gene that can stabilize RNA of introduced genes, thereby enhancing foreign gene expression. The cassette can optionally include an antibiotic resistance gene.

[0265] Typically, the expression cassette is flanked by convenient restriction sites for insertion into an appropriate genome. The expression cassette can be flanked by DNA sequences from chloroplast DNA to facilitate stable integration of the expression cassette into the chloroplast genome, particularly by homologous recombination. Alternatively, the expression cassette may remain unintegrated, in which case, the expression cassette typically includes a chloroplast origin of replication, which is capable of providing for replication of the heterologous DNA in the chloroplast.

[0266] The expression cassette generally includes a promoter region from a gene capable of expression in the chloroplast. The promoter region may include promoters obtainable from chloroplast genes, such as the psbA gene from spinach or pea, or the rbcL and atpB promoter region from maize and Rrna promoters. Examples of promoters are described in Hanley- Bowdoin and Chua, TIBS (1987) 12:67 70; Mullet et al., Plant Molec Biol. (1985) 4: 39 54; Hanley-Bowdoin (1986) PhD. Dissertation, the Rockefeller University; Krebbers et al., Nucleic Acids Res. (1982) 10: 4985 5002; Zurawaki et al., Nucleic Acids Res. (1981) 9:3251 3270; and Zurawski et al., Proc. Nat'l Acad Sci. U.S.A. (1982) 79: 7699 7703. Other promoters can be identified and the relative strength of promoters so identified evaluated, by placing a promoter of interest 5 ' to a promoterless marker gene and observing its

effectiveness relative to transcription obtained from, for example, the promoter from the psbA gene, a relatively strong chloroplast promoter. The efficiency of heterologus gene expression additionally can be enhanced by any of a variety of techniques. These include the use of multiple promoters inserted in tandem 5' to the heterologous gente, for example a double psbA promoter, the addition of enhancer sequences and the like.

[0267] Numerous promoters active in the Chlorella chloroplast can be used for expression of exogenous genes in the Chlorella chloroplast, such as those found in GenBank accession number NC_001865 (Chlorella vulgaris chloroplast, complete genome),

[0268] Where it is desired to provide for inducible expression of the heterologous gene, an inducible promoter and/or a 5 ' untranslated region containing sequences which provide for regulation at the level of transcription and/or translation (at the 3' end) may be included in the expression cassette. For example, the 5' untranslated region can be from a gene wherein expression is regulatable by light. Similarly, 3 ' inverted repeat regions could be used to stabilize RNA of heterologous genes. Inducible genes may be identified by enhanced expression in response to a particular stimulus of interest and low or absent expression in the absence of the stimulus. For example, a light-inducible gene can be identified where enhanced expression occurs during irradiation with light, while substantially reduced expression or no expression occurs in low or no light. Light regulated promoters from green microalgae are known (see for example Mol Genet Genomics. 2005 Dec;274(6):625-36).

[0269] The termination region which is employed will be primarily one of convenience, since the termination region appears to be relatively interchangeable among chloroplasts and bacteria. The termination region may be native to the transcriptional initiation region, may be native to the DNA sequence of interest, or may be obtainable from another source. See, for example, Chen and Orozco, Nucleic Acids Res. (1988) 16:8411.

[0270] The expression cassettes may be transformed into a plant cell of interest by any of a number of methods. These methods include, for example, biolistic methods (See, for example, Sanford, Trends In Biotech. (1988) 6:299 302, U.S. Patent No. 4,945,050;

electroporation (Fromm et al., Proc. Nat'l. Acad. Sci. (USA) (1985) 82:5824 5828); use of a laser beam, microinjection or any other method capable of introducing DNA into a chloroplast.

[0271] Additional descriptions of chloroplast expression vectors suitable for use in microorganisms such as microalgae are found in U.S. Patent Nos. 7,081,567 (issued July 25, 2006 to Xue et al.); 6,680,426 (issued January 20, 2004 to Daniell et al.); and 5,693,507 (issued December 2, 1997 to Daniell et al.).

[0272] Proteins expressed in the nuclear genome of Chlorella can be targeted to the chloroplast using chloroplast targeting signals. Chloroplast targeting sequences endogenous to Chlorella are known, such as genes in the Chlorella nuclear genome that encode proteins that are targeted to the chloroplast; see for example GenBank Accession numbers AY646197 and AF499684. Proteins can also be expressed in the Chlorella chloroplast by insertion of genes directly into the chloroplast genome. Chloroplast transformation typically occurs through homologous recombination, and can be performed if chloroplast genome sequences are known for creation of targeting vectors (see for example the complete genome sequence of a Chlorella chloroplast; Genbank accession number NC_001865). See previous sections herein for details of chloroplast transformation.

(2) Expression in Mitochondria

[0273] In another embodiment of the present invention, the expression of a polypeptide in a microorganism is targeted to mitochondria. Methods for targeting foreign gene products into mitochnodria (Boutry et al. Nature (London) (1987) 328:340 342) have been described, including in green microalgae (see for example Mol Gen Genet. 1993 Jan;236(2-3):235-44).

[0274] For example, an expression vector encoding a suitable secretion signal can target a heterologus protein to the mitochondrion. The IMP ACT VECTOR 1.5 vector, from

Wageningen UR- Plant Research International, uses the yeast CoxIV secretion signal, which was shown to deliver proteins in the mitochondrial matrix. The protein is fused to the first 4 amino acids of the yeast CoxIV protein in order to allow proper processing of the signal peptide (Kohler et al. Plant J 11 : 613-621 (1997)). Other mitochondrial targeting sequences are known, including those functional in green microalgae. For example, see FEBS Lett. 1990 Jan 29;260(2): 165-8; and J Biol Chem. 2002 Feb 22;277(8):6051-8.

[0275] Proteins expressed in the nuclear genome of Chlorella can be targeted to the mitochondria using mitochondrial targeting signals. See previous sections herein for details of mitochondrial protein targeting and transformation.

(3) Expression in Endoplasmic Reticulum

[0276] In another embodiment of the present invention, the expression of a polypeptide in a microorganism is targeted to the endoplasmic reticulum. The inclusion of an appropriate retention or sorting signal in an expression vector ensure that proteins are retained in the endoplasmic reticulum (ER) and do not go downstream into Golgi. For example, the IMPACTVECTOR1.3 vector, from Wageningen UR- Plant Research International, includes the well known KDEL retention or sorting signal. With this vector, ER retention has a practical advantage in that it has been reported to improve expression levels 5-fold or more. The main reason for this appears to be that the ER contains lower concentrations and/or different proteases responsible for post-translational degradation of expressed proteins than are present in the cytoplasm. ER retention signals functional in green microalgae are known. For example, see Proc Natl Acad Sci U S A. 2005 Apr 26;102(17):6225-30.

[0277] While the methods and materials of the invention allow for the introduction of any exogenous gene into a microorganism, for example Prototheca, genes relating to sucrose utilization and lipid pathway modification are of particular interest, as discussed in the following sections.

IV. SELECTABLE MARKERS

1. Sucrose Utilization

[0278] In embodiment, the recombinant Prototheca cell of the invention further contains one or more exogenous sucrose utilization genes. In various embodiments, the one or more genes encode one or more proteins selected from the group consisting of a fructokinase, a glucokinase, a hexokinase, a sucrose invertase, a sucrose transporter. For example, expression of a sucrose transporter and a sucrose invertase allows Prototheca to transport sucrose into the cell from the culture media and hydrolyze sucrose to yield glucose and fructose. Optionally, a fructokinase can be expressed as well in instances where endogenous hexokinase activity is insufficient for maximum phosphorylation of fructose. Examples of suitable sucrose transporters are Genbank accession numbers CAD91334, CAB92307, and CAA53390. Examples of suitable fructokinases are Genbank accession numbers P26984, P26420 and CAA43322. [0279] In one embodiment, the present invention provides a Prototheca host cell that secretes a sucrose invertase. Secretion of a sucrose invertase obviates the need for expression of a transporter that can transport sucrose into the cell. This is because a secreted invertase catalyzes the conversion of a molecule of sucrose into a molecule of glucose and a molecule of fructose, both of which can be transported and utilized by microbes provided by the invention. For example, expression of a sucrose invertase (such as SEQ ID NO:3) with a secretion signal (such as that of SEQ ID NO: 4 (from yeast), SEQ ID NO: 5 (from higher plants), SEQ ID NO: 6 (eukaryotic consensus secretion signal), and SEQ ID NO: 7

(combination of signal sequence from higher plants and eukaryotic consensus) generates invertase activity outside the cell. Expression of such a protein, as enabled by the genetic engineering methodology disclosed herein, allows cells already capable of utilizing extracellular glucose as an energy source to utilize sucrose as an extracellular energy source.

[0280] Prototheca species expressing an invertase in media containing sucrose are a preferred microalgal species for the production of oil. The expression and extracellular targeting of this fully active protein allows the resulting host cells to grow on sucrose, whereas their non-transformed counterparts cannot. Thus, the present invention provides Prototheca recombinant cells with a codon-optimized invertase gene, including but not limited to the yeast invertase gene, integrated into their genome such that the invertase gene is expressed as assessed by invertase activity and sucrose hydrolysis. The present invention also provides invertase genes useful as selectable markers in Prototheca recombinant cells, as such cells are able to grow on sucrose, while their non-transformed counterparts cannot; and methods for selecting recombinant host cells using an invertase as a powerful, selectable marker for algal molecular genetics.

[0281] The successful expression of a sucrose invertase in Prototheca also illustrates another aspect of the present invention in that it demonstrates that heterologous

(recombinant) proteins can be expressed in the algal cell and successfully transit outside of the cell and into the culture medium in a fully active and functional form. Thus, the present invention provides methods and reagents for expressing a wide and diverse array of heterologous proteins in microalgae and secreting them outside of the host cell. Such proteins include, for example, industrial enzymes such as, for example, lipases, proteases, cellulases, pectinases, amylases (e.g. , SEQ ID NO: 190-191), esterases, oxidoreductases, transferases, lactases, isomerases, and invertases, as well as therapeutic proteins such as, for example, growth factors, cytokines, full length antibodies comprising two light and two heavy chains, Fabs, scFvs (single chain variable fragment), camellid-type antibodies, antibody fragments, antibody fragment-fusions, antibody-receptor fusions, insulin, interferons, and insulin-like growth factors.

[0282] The successful expression of a sucrose invertase in Prototheca also illustrates another aspect of the present invention in that it provides methods and reagents for the use of fungal transit peptides in algae to direct secretion of proteins in Prototheca; and methods and reagents for determining if a peptide can function, and the ability of it to function, as a transit peptide in Prototheca cells. The methods and reagents of the invention can be used as a tool and platform to identify other transit peptides that can successfully traffic proteins outside of a cell, and that the yeast invertase has great utility in these methods. As demonstrated in this example, removal of the endogenous yeast invertase transit peptide and its replacement by other transit peptides, either endogenous to the host algae or from other sources (eukaryotic, prokaryotic and viral), can identify whether any peptide of interest can function as a transit peptide in guiding protein egress from the cell.

[0283] Examples of suitable sucrose invertases include those identified by Genbank accession numbers CAB95010, NP_012104 and CAA06839. Non-limiting examples of suitable invertases are listed below in Table 3. Amino acid sequences for each listed invertase are included in the Sequence Listing below. In some cases, the exogenous sucrose utilization gene suitable for use in the methods and vectors of the invention encodes a sucrose invertase that has at least 40, 50, 60, 75, or 90% or higher amino acid identity with a sucrose invertase selected from Table 3.

[0284] Table 3. Sucrose invertases.

Invertase Beta vulgaris subsp. AJ278531 SEQ ID NO:26

Vulgaris beta-fructofuranosidase Bifidobacterium breve AAT28190 SEQ ID NO:27

(invertase) UCC2003

Invertase Saccharomyces cerevisiae NP_012104 SEQ ID NO:8 (nucleotide)

SEQ ID NO:28 (amino acid)

Invertase A Zymomonas mobilis AA038865 SEQ ID NO:29

Invertase Arabadopsis thaliana NP_566464 SEQ ID NO:188

[0285] The secretion of an invertase to the culture medium by Prototheca enable the cells to grow as well on waste molasses from sugar cane processing as they do on pure reagent- grade glucose; the use of this low- value waste product of sugar cane processing can provide significant cost savings in the production of lipids and other oils. Thus, the present invention provides a microbial culture containing a population of Prototheca microorganisms, and a culture medium comprising (i) sucrose and (ii) a sucrose invertase enzyme. In various embodiments the sucrose in the culture comes from sorghum, sugar beet, sugar cane, molasses, or depolymerized cellulosic material (which may optionally contain lignin). In another aspect, the methods and reagents of the invention significantly increase the number and type of feedstocks that can be utilized by recombinant Prototheca. While the microbes exemplified here are altered such that they can utilize sucrose, the methods and reagents of the invention can be applied so that feedstocks such as cellulosics are utilizable by an engineered host microbe of the invention with the ability to secrete cellulases, pectinases, isomerases, or the like, such that the breakdown products of the enzymatic reactions are no longer just simply tolerated but rather utilized as a carbon source by the host. An example of this is described below and in the Examples of microbes engineered to express a secretable a- galactosidase, conferring the ability to hydrolyze a-galactosyl bonds in oligosaccharides such as those contained in raffinose and stachyose which are two oligosaccharides found in agricultural waste streams.

2. Alpha-galaetosiclase Expression

[0286] While the expression of a sucrose invertase, as described above, confers the ability for Prototheca cells to more efficiently utilize sucrose as a carbon source (via the enzyme hydrolyzing the a-linkage between fructose and glucose molecules in the disaccharide sucrose), the expression of other enzymes that hydrolyze other types of a-linkages in oligosaccharides can confer the ability for Prototheca cells to utilize other carbon sources. The expression of these enzymes (and the resulting ability to utilize carbon sources that Prototheca and other microalgal cells ordinarily would not be able to) can be used as a selectable marker for these transgenic Prototheca cells by allowing for the selection of positive clones that are able to grow on these carbon sources.

[0287] In an embodiment, the recombinant Prototheca cell of the invention further contains one or more exogenous genes encoding polysaccharide-degrading enzymes. In various embodiments, the one or more genes encoding a polysaccharide-degrading enzyme is a gene encoding a secreted a-galactosidase. The expression of an exogenous secreted a- galactosidase in a Prototheca cell confers the ability of such transformed strains to grow on sugars (carbon sources) containing D-galactosyl linkages, such as a-linkages between galactose and glucose monosaccharide units. Prototheca strains expressing an exogenous, secreted a-galactosidase will be able to utilize disaccharides such as melibiose (disaccharide composed of a-D-galactose-glucose).

[0288] Sugars such as raffinose (a trisaccharide comprised of a-linked galactose-glucose- fructose) and stachyose (a tetrasaccharide composed to two a-linked D-galactose units, followed by a-linked glucose and fructose) are present in significant proportions in agricultural waste streams such as beet pulp (raffinose) and soybean meal (stachyose). Such agricultural residues represent a significant untapped carbon source for the conversion into oil by microbes (including Prototheca) capable of utilizing them.

[0289] Prototheca strains are unable to utilize oligosaccharides such as raffinose and stachyose in any significant quantity or at all. In the case of raffinose and stachyose, although transgenic strains expressing a sucrose invertase (as described above) have the ability to hydrolyze the a-linkage between fructose and glucose in a-galactosyl derivatives of sucrose, but the remainder of the oligosaccharide remains unutilized, as sucrose invertase will not cleave the remaining a-linkages in such sugars and the resulting disaccharides are not utilizable. In another embodiment, the recombinant Prototheca cell of the invention comprises both an exogenous gene encoding a sucrose invertase and an exogenous gene encoding an α-galactosidase. Thus, strains expressing both a sucrose invertase and an a- galactosidase will be capable of fully hydrolyzing oligosaccharides such as raffinose and stachyose, enabling the consumption of the component monomers. In addition, a- galactosidase encoding genes may be used as a selectable marker for transformation. Clones containing the the exogenous α-galactosidase gene will have the ability to grow on melibiose. Examples of suitable α-galactosidase genes for use in Prototheca strains include the MEL1 gene from Saccharomyces carlbergensis, the AglC gene from Aspergilus niger. Interestingly, not all a-galactosidase genes are functional in Prototheca species, even if the genes are optimized according to the preferred codon usage in Prototheca strains. The Examples below demonstrates the ability of transgenic Prototheca cells to grow on melibiose when transformed with codon-optimized MELl gene from S. carlbergensis and the AglC gene from A. niger, but not an a-galactosidase encoding gene from the higher plant, Cyamopsis tetragonobola (Guar bean).

3. Thiamine Auxotrophv Complementation

[0290] Prototheca strains including Prototheca moriformis are known to be thiamine auxotrophic (See, for example, Ciferri, O. (1956) Nature, v.178, pp. 1475-1476), meaning that these strains require thiamine in the nutrient media for growth. Thiamine auxotrophy can be the result of mutations or lack of expression of enzymes in the thiamine biosynthetic pathway. Complemented transgenic strains expressing the missing enzyme(s) in the thiamine biosynthetic pathway can then be grown without added thiamine, thus reducing the cost of the nutrient media as well as rendering the resulting microalgal biomass more desireable from an animal nutrition perspective. Complementation with a thiamine biosynthetic pathway enzyme can also be used as a selectable marker as the transgenic gene confers the ability to grow on plates/media that does not contain thiamine.

[0291] In an embodiment, the recombinant Prototheca cell of the invention further contains one or more exogenous genes encoding thiamine biosynthetic pathway enzyme. In another embodiment, the recombinant Prototheca cell of the invention comprises an exogenous gene encoding hydroxymethylpyrimidine phosphate synthases (e.g., SEQ ID NO: 192) from algal, plant or cyanobacterial sources. In still other embodiments, the hydroxymethylpyrimidine phosphate synthase is encoded by a THIC gene. In still other embodiments, the THIC gene is the Coccomyxa C-169 THIC, Arabidopsis thaliana THIC, the Synechocystis sp. PCC 6803 THIC, or the Salmonella enterica subsp. enterica serovar Typhimurium str. THIC (SEQ ID NO: 193). The Examples below details the engineering of Prototheca moriformis UTEX 1435 with restored thiamine prototrophy.

4. Other Selectable Markers

[0292] Any of a wide variety of selectable markers can be employed in a transgene construct useful for transforming microorganisms, such as Chlorella. Examples of suitable selectable markers include the nitrate reductase gene, the hygromycin phosphotransferase gene (HPT), the neomycin phosphotransferase gene, and the ble gene, which confers resistance to phleomycin. Methods of determining sensitivity of microalgae to antibiotics are well known. For example, Mol Gen Genet. 1996 Oct 16;252(5):572-9.

[0293] More specifically, Dawson et al. (1997), Current Microbiology 35:356-362

(incorporated by reference herein in its entirety), described the use of the nitrate reductase

(NR) gene from Chlorella vulgaris as a selectable marker for NR-deficient Chlorella sorokiniana mutants. Kim et al. (2002), Mar. Biotechnol. 4:63-73 (incorporated by reference herein in its entirety), disclosed the use of the HPT gene as a selectable marker for transforming Chorella ellipsoidea. Huang et al. (2007), Appl. Microbiol. Biotechnol.

72: 197-205 (incorporated by reference herein in its entirety), reported on the use of Sh ble as a selectable marker for Chlorella sp. DT.

V. LIPID PATHWAY ENGINEERING

[0294] In addition to altering the ability of microorganisms (e.g., microalgae, oleaginous yeast, fungi, or bacteria), such as Prototheca to utilize feedstocks such as sucrose-containing feedstocks, the present invention also provides recombinant microorganisms (e.g. ,

Prototheca) that have been modified to alter the properties and/or proportions of lipids produced. The pathway can further, or alternatively, be modified to alter the properties and/or proportions of various lipid molecules produced through enzymatic processing of lipids and intermediates in the fatty acid pathway. In various embodiments, the recombinant microorganisms (e.g. , Prototheca cells) of the invention have, relative to their untransformed counterparts, optimized lipid yield per unit volume and/or per unit time, carbon chain length (e.g., for renewable diesel production or for industrial chemicals applications requiring lipid feedstock), reduced number of double or triple bonds, optionally to zero, and increasing the hydrogen:carbon ratio of a particular species of lipid or of a population of distinct lipid. In addition, microorganisms that produce desirable hydrocarbons can be engineered to produce such components in higher quantities, or with greater specificity.

[0295] In the case of microalgae, some wild-type cells already have good growth characteristics but do not produce the desired types or quantities of lipids. Examples include, without limitation, Pyrobotrys, Phormidium, Agmenellum, Carteria, Lepocinclis, Pyrobotrys, Nitzschia, Lepocinclis, Anabaena, Euglena, Spirogyra, Chlorococcum, Tetraedron,

Oscillatoria, Phagus, and Chlorogonium, which have the desirable growth characteristic of growing in municipal sewage or wastewater. Such cells, as well as species of Chlorella, Prototheca and other microbes, can be engineered to have improved lipid production characteristics. Desired characteristics include optimizing lipid yield per unit volume and/or per unit time, carbon chain length (e.g., for biodiesel production or for industrial applications requiring hydrocarbon feedstock), reducing the number of double or triple bonds, optionally to zero, removing or eliminating rings and cyclic structures, and increasing the

hydrogen:carbon ratio of a particular species of lipid or of a population of distinct lipid. In addition, microalgae that produce appropriate hydrocarbons can also be engineered to have even more desirable hydrocarbon outputs. Examples of such microalgae include species of the genus Chlorella and the genus Prototheca.

[0296] In particular embodiments, one or more key enzymes that control branch points in metabolism to fatty acid synthesis have been up-regulated or down-regulated to improve lipid production. Up-regulation can be achieved, for example, by transforming cells with expression constructs in which a gene encoding the enzyme of interest is expressed, e.g., using a strong promoter and/or enhancer elements that increase transcription. Such constructs can include a selectable marker such that the transformants can be subjected to selection, which can result in amplification of the construct and an increase in the expression level of the encoded enzyme. Examples of enzymes suitable for up-regulation according to the methods of the invention include pyruvate dehydrogenase, which plays a role in converting pyruvate to acetyl-CoA (examples, some from microalgae, include Genbank accession numbers NP_415392; AAA53047; Q1XDM1 ; and CAF05587). Up-regulation of pyruvate dehydrogenase can increase production of acetyl-CoA, and thereby increase fatty acid synthesis. Acetyl-CoA carboxylase catalyzes the initial step in fatty acid synthesis.

Accordingly, this enzyme can be up-regulated to increase production of fatty acids

(examples, some from microalgae, include Genbank accession numbers BAA94752;

AAA75528; AAA81471 ; YP_537052; YP_536879; NP_045833; and BAA57908). Fatty acid production can also be increased by up-regulation of acyl carrier protein (ACP), which carries the growing acyl chains during fatty acid synthesis (examples, some from microalgae, include Genbank accession numbers A0T0F8; P51280; NP_849041 ; YP_874433). Glycerol-3- phosphate acyltransferase catalyzes the rate-limiting step of fatty acid synthesis. Up- regulation of this enzyme can increase fatty acid production (examples, some from microalgae, include Genbank accession numbers AAA74319; AAA33122; AAA37647; P44857; and AB094442).

[0297] Up- and/or down-regulation of genes can be applied to global regulators controlling the expression of the genes of the fatty acid biosynthetic pathways. Accordingly, one or more global regulators of fatty acid synthesis can be up- or down-regulated, as appropriate, to inhibit or enhance, respectively, the expression of a plurality of fatty acid synthetic genes and, ultimately, to increase lipid production. Examples include sterol regulatory element binding proteins (SREBPs), such as SREBP-la and SREBP-lc (for examples see Genbank accession numbers NP_035610 and Q9WTN3).

[0298] The present invention also provides recombinant microorganisms (e.g., Prototheca cells) that have been modified to contain one or more exogenous genes encoding lipid modification enzymes such as, for example, fatty acyl-ACP thioesterases (e.g. , C. callophylla (SEQ ID NO: 145 and SEQ ID NO: 146; see also Table 4), fatty acyl- Co A/aldehyde reductases (see Table 6), fatty acyl-CoA reductases (see Table 7), fatty aldehyde

decarbonylase (see Table 8), fatty aldehyde reductases, desaturases (such as stearoyl- ACP desaturases (e.g. , a codon optimized R. communis SAD, SEQ ID NO: 147 and SEQ ID NO: 148) and fatty acyl desaturases and squalene synthases (see GenBank Accession number AF205791). In some embodiments, genes encoding a fatty acyl-ACP thioesterase and a naturally co-expressed acyl carrier protein are transformed into a Prototheca cell, optionally with one or more genes encoding other lipid modification enzymes. In other embodiments, the ACP and the fatty acyl-ACP thioesterase may have an affinity for one another that imparts an advantage when the two are used together in the microbes and methods of the present invention, irrespective of whether they are or are not naturally co-expressed in a particular tissue or organism. Thus, the present invention contemplates both naturally co- expressed pairs of these enzymes as well as those that share an affinity for interacting with one another to facilitate cleavage of a length- specific carbon chain from the ACP.

[0299] In still other embodiments, an exogenous gene encoding a desaturase is transformed into the microorganism (e.g. , a Prototheca cell) in conjunction with one or more genes encoding other lipid modification enzymes to provide modifications with respect to lipid saturation. In other embodiments, an endogenous desaturase gene is overexpressed (e.g., through the introduction of additonal copies off the gene) in the microorganism (e.g. , a Prototheca cell). Stearoyl- ACP desaturase (see, e.g., GenBank Accession numbers

AAF15308; ABM45911; and AAY86086), for example, catalyzes the conversion of stearoyl- ACP to oleoyl-ACP. Up-regulation of this gene can increase the proportion of

monounsaturated fatty acids produced by a cell; whereas down-regulation can reduce the proportion of monounsaturates. For illustrative purposes, stearoyl-ACP desaturases (SAD) are responsible for for the synthesis of C18: 1 fatty acids from C18:0 precursors. Another family of desaturases are the fatty acyl desaturases (FAD), including delta 12 fatty acid desaturases (Δ12 FAD). These desaturases also provide modifications with respect to lipid saturation. For illustrative purposes, delta 12 fatty acid desaturases are responsible for the synthesis of C18:2 fatty acids from C18: l precursors. Similarly, the expression of one or more glycerolipid desaturases can be controlled to alter the ratio of unsaturated to saturated fatty acids such as ω-6 fatty acid desaturase, ω-3 fatty acid desaturase, or ω-6-oleate desaturase. In some embodiments, the desaturase can be selected with reference to a desired carbon chain length, such that the desaturase is capable of making location specific modifications within a specified carbon-length substrate, or substrates having a carbon-length within a specified range. In another embodiment, if the desired fatty acid profile is an increase in monounsaturates (such as C16: l and/or C18: l) overexpression of a SAD or expression of a heterologous SAD can be coupled with the silencing or inactivation (e.g., through mutation, RNAi, knockout of an endogenous desaturase gene, etc.) of a fatty acyl desaturase (FAD).

[0300] In other embodiments, the microorganism (e.g. , Prototheca cell) has been modified to have a mutated endogenous desaturase gene, wherein the mutation renders the gene or desaturase enzyme inactive. In some cases, the mutated endogenous desaturase gene is a fatty acid desaturase (FAD). In other cases, the mutated endogenous desaturase gene is a stearoyl Acyl carrier protein desaturase (SAD). Example 11 below describes the targeted ablation or knockout of stearoyl- ACP desaturases and delta 12 fatty acid desaturases.

[0301] In some cases, it may be advantageous to pair one or more of the genetic engineering techniques in order to achieve a trangenic cell that produces the desired lipid profile. In one embodiment, a microorganism (e.g. , a Prototheca cell) comprises a mutated endogenous desaturase gene and one or more exogenous gene. In non-limiting examples, a Prototheca cell with a mutated endogenous desaturase gene can also express an exogenous fatty acyl- ACP thioesterase gene and/or a sucrose invertase gene. Example 11 below describes a transgenic Prototheca cell containing a targeted ablation or knockout of an endogenous SAD and also expresses a Cinnamomum camphora C14-pref erring thioesterase and a sucrose invertase. In this case, the transgenic Prototheca cell produces a lipid profile that closely approximates the lipid profile found in tallow. Tallow is typically derived from rendered beef or mutton fat, is solid at room temperature and is utilized in a variety of applications in the food, cosmetics, and chemicals industries. The fatty acid profile of tallow is: 4% C14:0; 26% C16:0; 3% C16: l; 14% C18:0; 41% C18: l; 3% C18:2; and 1% C18:3. As is shown in Example 11 below, clones of transgenic Prototheca cells with a targeted ablation or knockout of an endogenous SAD and expressing a C. camphora C14-preferring thioesterase have lipid profiles of: less than 1% C12 and shorter carbon chain length fatty acids; 2.74% to 6.13% C14:0; 23.07% to 25.69% C16:0; 7.02% to 11.08% C18:0; 42.03% to 51.21% C18: l ; and 9.37% to 13.45% C18:2 (expressed in area percent). In some cases, the transgenic Prototheca cells have lipid profiles of: 3-5% C14:0; 25-27% C16:0; 10-15% C18:0; and 40-45% C18: l.

[0302] Thus, in particular embodiments, microbes of the present invention are genetically engineered to express one or more exogenous genes selected from an acyl-ACP thioesterase, an acyl- Co A/aldehyde reductase, a fatty acyl-CoA reductase, a fatty aldehyde reductase, a fatty aldehyde decarbonylase, or a naturally co-expressed acyl carrier protein. Suitable expression methods are described above with respect to the expression of a lipase gene, including, among other methods, inducible expression and compartmentalized expression. A fatty acyl-ACP thioesterase cleaves a fatty acid from an acyl carrier protein (ACP) during lipid synthesis. Through further enzymatic processing, the cleaved fatty acid is then combined with a coenzyme to yield an acyl-CoA molecule. This acyl-CoA is the substrate for the enzymatic activity of a fatty acyl-CoA reductase to yield an aldehyde, as well as for a fatty acyl- Co A/aldehyde reductase to yield an alcohol. The aldehyde produced by the action of the fatty acyl-CoA reductase identified above is the substrate for further enzymatic activity by either a fatty aldehyde reductase to yield an alcohol, or a fatty aldehyde decarbonylase to yield an alkane or alkene.

[0303] In some embodiments, fatty acids, glycerolipids, or the corresponding primary alcohols, aldehydes, alkanes or alkenes, generated by the methods described herein, contain 8, 10, 12,or 14 carbon atoms. Preferred fatty acids for the production of diesel, biodiesel, renewable diesel, or jet fuel, or the corresponding primary alcohols, aldehydes, alkanes and alkenes, for industrial applications contain 8 to 14 carbon atoms. In certain embodiments, the above fatty acids, as well as the other corresponding hydrocarbon molecules, are saturated (with no carbon-carbon double or triple bonds); mono unsaturated (single double bond); poly unsturated (two or more double bonds); are linear (not cyclic) or branched. For fuel production, greater saturation is preferred.

[0304] The enzymes described directly above have a preferential specificity for hydrolysis of a substrate containing a specific number of carbon atoms. For example, a fatty acyl-ACP thioesterase may have a preference for cleaving a fatty acid having 12 carbon atoms from the ACP. In some embodiments, the ACP and the length-specific thioesterase may have an affinity for one another that makes them particularly useful as a combination (e.g., the exogenous ACP and thioesterase genes may be naturally co-expressed in a particular tissue or organism from which they are derived). Therefore, in various embodiments, the recombinant Prototheca cell of the invention can contain an exogenous gene that encodes a protein with specificity for catalyzing an enzymatic activity (e.g., cleavage of a fatty acid from an ACP, reduction of an acyl-CoA to an aldehyde or an alcohol, or conversion of an aldehyde to an alkane) with regard to the number of carbon atoms contained in the substrate. The enzymatic specificity can, in various embodiments, be for a substrate having from 8 to 34 carbon atoms, preferably from 8 to 18 carbon atoms, and more preferably from 8 to 14 carbon atoms. A preferred specificity is for a substrate having fewer, i.e., 12, rather than more, i.e., 18, carbon atoms.

[0305] Other fatty acyl-ACP thioesterases suitable for use with the microbes and methods of the invention include, without limitation, those listed in Table 4.

[0306] Table 4. Fatty acyl-ACP thioesterases and GenBank accession numbers.

Umbellularia californica fatty acyl-ACP thioesterase (GenBank #AAC49001) (SEQ ID NO: 203)

Cinnamomum camphora fatty acyl-ACP thioesterase (GenBank #Q39473)

Umbellularia californica fatty acyl-ACP thioesterase (GenBank #Q41635)

Myristicafragrans fatty acyl-ACP thioesterase (GenBank #AAB71729) (SEQ ID NO: 224) Myristicafragrans fatty acyl-ACP thioesterase (GenBank #AAB71730) (SEQ ID NO: 222) Elaeis guineensis fatty acyl-ACP thioesterase (GenBank #ABD83939) (SEQ ID NO: 204) Elaeis guineensis fatty acyl-ACP thioesterase (GenBank #AAD42220)

Populus tomentosa fatty acyl-ACP thioesterase (GenBank #ABC47311) (SEQ ID NO: 207) Arabidopsis thaliana fatty acyl-ACP thioesterase (GenBank #NP_172327) (SEQ ID NO: 208) Arabidopsis thaliana fatty acyl-ACP thioesterase (GenBank #CAA85387) (SEQ ID NO: 209) Arabidopsis thaliana fatty acyl-ACP thioesterase (GenBank #CAA85388) (SEQ ID NO: 210) Gossypium hirsutum fatty acyl-ACP thioesterase (GenBank #Q9SQI3) (SEQ ID NO: 211) Cuphea lanceolata fatty acyl-ACP thioesterase (GenBank #CAA54060) (SEQ ID NO: 212) Cuphea hookeriana fatty acyl-ACP thioesterase (GenBank #AAC72882) (SEQ ID NO: 202) Cuphea calophylla subsp. mesostemon fatty acyl-ACP thioesterase (GenBank #ABB71581) (SEQ ID NO: 213)

Cuphea lanceolata fatty acyl-ACP thioesterase (GenBank #CAC19933)

Elaeis guineensis fatty acyl-ACP thioesterase (GenBank #AAL15645) (SEQ ID NO: 206)

Cuphea hookeriana fatty acyl-ACP thioesterase (GenBank #Q39513)

Gossypium hirsutum fatty acyl-ACP thioesterase (GenBank #AAD01982) (SEQ ID NO: 214) Vitis vinifera fatty acyl-ACP thioesterase (GenBank #CAN81819) (SEQ ID NO: 215) Garcinia mangostana fatty acyl-ACP thioesterase (GenBank #AAB51525)

Brassicajuncea fatty acyl-ACP thioesterase (GenBank #ABI18986) (SEQ ID NO: 216) Madhuca longifolia fatty acyl-ACP thioesterase (GenBank #AAX51637) (SEQ ID NO: 217) Brassica napus fatty acyl-ACP thioesterase (GenBank #ABH11710)

Oryza sativa (indica cultivar-group) fatty acyl-ACP thioesterase (GenBank #EAY86877) (SEQ ID NO: 218)

Oryza sativa (japonica cultivar-group) fatty acyl-ACP thioesterase (GenBank

#NP_001068400) (SEQ ID NO: 219)

Oryza sativa (indica cultivar-group) fatty acyl-ACP thioesterase (GenBank #EAY99617) (SEQ ID NO: 220)

Cuphea hookeriana fatty acyl-ACP thioesterase (GenBank #AAC49269)

Ulmus Americana fatty acyl-ACP thioesterase (GenBank #AAB71731)

Cuphea lanceolata fatty acyl-ACP thioesterase (GenBank #CAB60830) (SEQ ID NO: 221)

Cuphea palustris fatty acyl-ACP thioesterase (GenBank #AAC49180) Iris germanica fatty acyl-ACP thioesterase (GenBank #AAG43858)

Iris germanica fatty acyl-ACP thioesterase (GenBank #AAG43858.1)

Cuphea palustris fatty acyl-ACP thioesterase (GenBank #AAC49179)

Myristica fragrans fatty acyl-ACP thioesterase (GenBank# AAB71729)

Myristica fragrans fatty acyl-ACP thioesterase (GenBank# AAB717291.1)

Cuphea hookeriana fatty acyl-ACP thioesterase (GenBank #U39834) (SEQ ID NO: 197)

Umbelluaria californica fatty acyl-ACP thioesterase (GenBank # M94159) (SEQ ID NO:

285)

Cinnamomum camphora fatty acyl-ACP thioesterase (GenBank #U31813) (SEQ ID NO: 223) Cuphea wrightii fatty acyl-ACOP thioesterase (GenBank #U56103) (SEQ ID NO: 183) Ricinus communis fatty acyl-ACP thioesterase (GenBank #ABS30422) (SEQ ID NO: 198)

[0307] The Examples below describe the successful targeting and expression of

heterologous fatty acyl-ACP thioesterases from Cuphea hookeriana, Umbellularia

californica, Cinnamomun camphora, Cuphea palustris, Cuphea lanceolata, Iris germanica, Myristica fragrans and Ulmus americana in Prototheca species. Additionally, alterations in fatty acid profiles were confirmed in the host cells expression these heterologous fatty acyl- ACP thioesterases. These results were quite unexpected given the lack of sequence identity between algal and higher plant thioesterases in general, and between Prototheca moriformis fatty acyl-ACP thioesterase and the above listed heterologous fatty acyl-ACP thioesterases. As shown in the Examples, the expression of these heterologous thioesterases in Prototheca generates a transgenic microalgae that is able to produce oil/lipids with truly unique fatty acid profiles that are currently not available from commercial seed crops, even through the blending of several seed crop oils. Table 5 shows the fatty acid profiles of common commercial seed oils. All commercial seed oil data below were compiled from the US Pharmacopeias Food and Chemicals Codes, 7 th Ed. 2010-2011. Tallow data is from the National Research Council: Fat Content and Composition of Animal Products (1976).

[0308] Table 5. Lipid profiles of commercial seed oils (in percentages).

C8:0 C10:0 C12:0 C14:0 C16:0 C18:0 C18:l C18:0- C18:l- C18:2 C18:3 a diOH OH

R. communis 0 0 0 0 0.9- 1.0- 3.7- 0.4- 83.6- 0 0.2-0.6

(Castor oil) 1.6 1.8 6.7 1.3 89.0

C. nucifera 5.0- 4.0- 44-52 15-21 8.0- 1.0- 5.0- 0 0 0-2.5 0

(Coconut oil) 9.0 8.0 11.0 4.0 8.0

Z. mays 0 0 0 < 1.0 8.0- 0.5- 19-50 0 0 38-65 < 2.0

(Corn oil) 19.0 4.0

G. 0 0 < 0.1 0.5- 17-29 1.0- 13-44 0 0 40-63 0.1-2.1 barbadense 2.0 4.0

(Cottonseed

oil)

B. rapa, B 0 0 < 0.1 < 0.2 < 6.0 < 2.5 > 50 0 0 < 40 < 14 napus, B. juncea

(Canola)

0. europea 0 0 0 < 0.1 6.5- 0.5- 56-85 0 0 3.5- < 1.2

(Olive) 20.0 5.0 20.0

A. hypogaea 0 0 < 0.1 < 0.2 7.0- 1.3- 35-72 0 0 13.0- < 0.6

(Peanut) 16.0 6.5 43

E. guineensis 3.0- 2.5- 40-52 14.0- 7.0- 1.0- 11.0- 0 0 0.5- 0

(Palm kernel) 5.0 6.0 18.0 10.0 3.0 19.0 4.0

E. guineensis 0 0 0 0.5- 32.0- 2.0- 34-44 0 0 7.2- 0

(Palm) 5.9 47.0 8.0 12.0

C. tinctorus 0 0 < 0.1 < 0.1 2.0- 1.0- 7.0- 0 0 72-81 < 1.5

(Safflower) 10.0 10.0 16.0

H. annus 0 0 < 0.1 < 0.5 3.0- 1.0- 14-65 0 0 20-75 < 0.5

(Sunflower) 10.0 10.0

G. max 0 0 < 0.1 < 0.5 7.0- 2.0- 19-30 0 0 48-65 5.0-10.0

(Soybean) 12.0 5.5

L 0 0 < 0.1 < 0.5 2.0- 2.0- 8.0-60 0 0 40-80 < 5.0 usitatissimum 9.0 5.0

(Solin-Flax)

B. parkii 0 0 0 0 3.8- 41.2- 34.0- 0 0 3.7- 0

(Sheanut) 4.1 56.8 46.9 6.5

Cocoa Butter 0-1 0-1 0-4 22-30 24-37 29-38 0-3

Tallow 3-4 23-28 14-23 36-43 1-4 < 1

Lard 1-2 22-26 13-18 39-45 8-15 0.5-1.5

[0309] As an example, none of these common seed oils contain high amounts of C8 or CIO fatty acids, with coconut oil and palm kernel oil being the largest sources, but both a ratio of 1 : 1 (C8:C10 fatty acids). As shown in the Examples, Prototheca transformed with Cuphea palustris C:8 preferring thioesterase was able to achieve not only a C8 fatty acid levels of over 12%, but also, the ratio of C8:C10 fatty acids were about a 5: 1. Changes in fatty acid levels are useful for producing oils containing a tailored fatty acid profile for a variety of commercial applications. Additionally, changes of ratios between different fatty acid chain lengths is something has not been available commercially in oils that have not been through further costly chemical processes (such as esterification, distillation, fractionation, and re- esterification). As another example, palm oil is the highest C16:0 fatty acid (32-47%) containing oils, but palm oil has very little C14:0 fatty acids. Prototheca containing the U. americana thioesterase achieved about 33-38% C16:0 fatty acids and about a 10-16% C14:0 fatty acids (about a 2: 1 C16:0 to C14:0 ratio). This fatty acid profile is unachievable through blending of existing oils at a commercial level because the seed oils that are high in 16:0 fatty acids usually do not contain much 14:0 fatty acids.

[0310] The Examples below also describe, for the first time, the successful targeting and expression of at least two fatty acyl-ACP thioesterases in one clone. The alterations in the fatty acid profiles were confirmed in these clones and depending on which two thioesterases were co-expressed in one clone, the fatty acid profiles were impacted in different ways. As an example, from Table 5 above, both coconut oil and palm kernel oil have C12:C14 ratios of roughly 3: 1. As described in the Examples below, a Prototheca transformant containing two heterologous thioesterase genes was able to produce C12:C14 fatty acid levels at a ratio of roughly 5: 1. This kind of ratio of C12:C14 fatty acids has been, up to now, unachievable at commercial levels (i.e., through blending of seed oils).

[0311] Another novel aspect of the oils produced by transgenic microalgae is the degree of saturation of the fatty acids. Palm oil is currently the largest source of saturated oil, with a total saturates to unsaturates of 52% to 48%. As shown in the Examples below, Prototheca with heterologous thioesterases from U. americana and C. camphora achieved total saturates levels of over 60% in the oil that it produced. Also shown in the Examples below, Prototheca with heterologous thioesterase from U. americana achieved total saturates level of over 86% in the oil that it produced.

[0312] Fatty acyl- Co A/aldehyde reductases suitable for use with the microbes and methods of the invention include, without limitation, those listed in Table 6.

[0313] Table 6. Fatty acyl- Co A/aldehyde reductases listed by GenBank accession numbers.

AAC45217, YP_047869, BAB85476, YP_001086217, YP_580344, YP_001280274, YP_264583, YP_436109, YP_959769, ZP_01736962, ZP_01900335, ZP_01892096, ZP_01103974, ZP_01915077, YP_924106, YP_130411, ZP_01222731, YP_550815, YP_983712, YP_001019688, YP_524762, YP_856798, ZP_01115500, YP_001141848, NP_336047, NP_216059, YP_882409, YP_706156, YP_001136150, YP_952365,

ZP_01221833, YP_130076, NP_567936, AAR88762, ABK28586, NP_197634,

CAD30694, NP_001063962, BAD46254, NP_001030809, EAZ10132, EAZ43639, EAZ07989, NP_001062488, CAB88537, NP_001052541, CAH66597, CAE02214, CAH66590, CAB88538, EAZ39844, AAZ06658, CAA68190, CAA52019, and

BAC84377

[0314] Fatty acyl-CoA reductases suitable for use with the microbes and methods of the invention include, without limitation, those listed in Table 7.

[0315] Table 7. Fatty acyl-CoA reductases listed by GenBank accession numbers.

NP_187805, AB014927, NP_001049083, CAN83375, NP_191229, EAZ42242,

EAZ06453, CAD30696, BAD31814, NP_190040, AAD38039, CAD30692, CAN81280, NP_197642, NP_190041, AAL15288, and NP_190042

[0316] Fatty aldehyde decarbonylases suitable for use with the microbes and methods of the invention include, without limitation, those listed in Table 8.

[0317] Table 8. Fatty aldehyde decarbonylases listed by GenBank accession numbers. NP_850932, ABN07985, CAN60676, AAC23640, CAA65199, AAC24373, CAE03390, ABD28319, NP_181306, EAZ31322, CAN63491, EAY94825, EAY86731, CAL55686, XP_001420263, EAZ23849, NP_200588, NP_001063227, CAN83072, AAR90847, and AAR97643

[0318] Combinations of naturally co-expressed fatty acyl-ACP thioesterases and acyl carrier proteins are suitable for use with the microbes and methods of the invention.

[0319] Additional examples of hydrocarbon or lipid modification enzymes include amino acid sequences contained in, referenced in, or encoded by nucleic acid sequences contained or referenced in, any of the following US patents: 6,610,527; 6,451,576; 6,429,014;

6,342,380; 6,265,639; 6,194,185; 6,114,160; 6,083,731; 6,043,072 ; 5,994,114; 5,891,697; 5,871,988; 6,265,639, and further described in GenBank Accession numbers: AA018435; ZP 00513891; Q38710; AAK60613; AAK60610; AAK60611 ; NP_113747; CAB75874; AAK60612; AAF20201 ; BAA11024; AF205791 ; and CAA03710.

[0320] Other enzymes in the lipid biosynthetic pathways are also suitable for use with microbes and methods of the invention. For example, keto acyl-ACP synthase (Kas) enzymes work in conjunction with some of the above listed enzymes in the lipid biosynthetic pathway. There different classes of Kas enzymes: Kas I participates in successive condensation steps between the ever-growing acyl ACP chains and malonyl-ACP. Kas II typically participates in the final condensation step leading from C16:0-ACP to C18:0-ACP incorporating malonyl-ACP. As such, in higher plants and some microalgae species/strains that synthesize predominantly C16-C18:0 fatty acids (and their unsaturated derivatives), Kas II enzymes interact with products of FatA genes (acyl-ACP thioesterases).

[0321] Acyl-ACP thioesterases are the terminators of higher plant (and some microalgal species) fatty acid biosynthesis, and in most plant species, this is carried out by members of the FatA gene family, whose role is to terminate elongation at the C16:0 to C18:0 stage. In species that synthesize shorter chain fatty acids (such as Cuphea, Elaeis, Myristica, or Umbellularia), a different group of acyl-ACP thioesterases encoded by FatB genes carry out this termination step (see e.g. , the codon optimized coding region of Cocos nucifera FatB3-B, SEQ ID NO: 189). The interaction between Kas II enzymes and acyl-Acp thioesterases is important for the correct termination of fatty acid chain elongation. As a consequence, in higher plant species (and microalgal species) that have evolved FatB genes capable of shorter chain lipid biosynthesis, there has been a corresponding co-evolution of an additional class of Kas genes, termed Kas IV genes. Kas IV genes are responsible for chain length elongation of a specific size range of fatty acids, 4-14 carbons in length.

[0322] Other suitable enzymes for use with the microbes and the methods of the invention include those that have at least 70% amino acid identity with one of the proteins listed in Tables 4, 6-8, and that exhibit the corresponding desired enzymatic activity (e.g., cleavage of a fatty acid from an acyl carrier protein, reduction of an acyl-CoA to an aldehyde or an alcohol, or conversion of an aldehyde to an alkane). In additional embodiments, the enzymatic activity is present in a sequence that has at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 99% identity with one of the above described sequences, all of which are hereby incorporated by reference as if fully set forth.

[0323] By selecting the desired combination of exogenous genes to be expressed, one can tailor the product generated by the microbe, which may then be extracted from the aqueous biomass. For example, the microbe can contain: (i) an exogenous gene encoding a fatty acyl- ACP thioesterase; and, optionally, (ii) a naturally co-expressed acyl carrier protein or an acyl carrier protein otherwise having affinity for the fatty acyl-ACP thioesterase (or conversely); and, optionally, (iii) an exogenous gene encoding a fatty acyl-Co A/aldehyde reductase or a fatty acyl-CoA reductase; and, optionally, (iv) an exogenous gene encoding a fatty aldehyde reductase or a fatty aldehyde decarbonylase. The microbe, under culture conditions described herein, synthesizes a fatty acid linked to an ACP and the fatty acyl-ACP thioesterase catalyzes the cleavage of the fatty acid from the ACP to yield, through further enzymatic processing, a fatty acyl-CoA molecule. When present, the fatty acyl-CoA/aldehyde reducatase catalyzes the reduction of the acyl-CoA to an alcohol. Similarly, the fatty acyl- CoA reductase, when present, catalyzes the reduction of the acyl-CoA to an aldehyde. In those embodiments in which an exogenous gene encoding a fatty acyl-CoA reductase is present and expressed to yield an aldehyde product, a fatty aldehyde reductase, encoded by the third exogenous gene, catalyzes the reduction of the aldehyde to an alcohol. Similarly, a fatty aldehyde decarbonylase catalyzes the conversion of the aldehyde to an alkane or an alkene, when present. [0324] In another embodiment, the microbe can contain: (i) an exogenous gene encoding a fatty acyl-ACP thioesterase; (ii) optionally, a naturally co-expressed acyl carrier protein or an acyl carrier protein having affinity for the fatty acid acyl-ACP thioesterase; (iii) a mutated endogenous desaturase gene, wherein the mutation renders the desaturase gene or desaturase protein inactive, such as a desaturase knockout; (iv) overexpression of an endogenous stearoyl acyl carrier protein desaturase or the expression of a heterologous SAD; and (v) any combination of the foregoing.

[0325] Genes encoding such enzymes, such as fatty acyl ACP thioesterases, can be obtained from cells already known to exhibit significant lipid production such as Chlorella protothec oides. Genes already known to have a role in lipid production, e.g., a gene encoding an enzyme that saturates double bonds, can be transformed individually into recipient cells. However, to practice the invention it is not necessary to make a priori assumptions as to which genes are required. Methods for identifiying genes that can alter (improve) lipid production in microalgae are described in PCT Pub. No.2008/151149.

[0326] Thus, the present invention provides a microorganism {e.g. , a Prototheca cell) that has been genetically engineered to express a lipid pathway enzyme at an altered level compared to a wild-type cell of the same species. In some cases, the cell produces more lipid compared to the wild-type cell when both cells are grown under the same conditions. In some cases, the cell has been genetically engineered and/or selected to express a lipid pathway enzyme at a higher level than the wild-type cell. In some cases, the lipid pathway enzyme is selected from the group consisting of pyruvate dehydrogenase, acetyl-CoA carboxylase, acyl carrier protein, and glycerol-3 phosphate acyltransf erase. In some cases, the cell has been genetically engineered and/or selected to express a lipid pathway enzyme at a lower level than the wild-type cell. In at least one embodiment in which the cell expresses the lipid pathway enzyme at a lower level, the lipid pathway enzyme comprises citrate synthase.

[0327] In some embodiments, the cell has been genetically engineered and/or selected to express a global regulator of fatty acid synthesis at an altered level compared to the wild-type cell, whereby the expression levels of a plurality of fatty acid synthetic genes are altered compared to the wild-type cell. In some cases, the lipid pathway enzyme comprises an enzyme that modifies a fatty acid. In some cases, the lipid pathway enzyme is selected from a stearoyl- ACP desaturase and a glycerolipid desaturase. In some cases, the cell has been genetically engineered and/or selected to express a lower level of a lipid pathway enzyme, or not to express a specific lipid pathway enzyme at all (i.e., wherein a lipid pathway enzyme has been knockout, or replaced with an exogenous gene).

[0328] Some microalgae produce significant quantities of non-lipid metabolites, such as, for example, polysaccharides. Because polysaccharide biosynthesis can use a significant proportion of the total metabolic energy available to cells, mutagenesis of lipid-producing cells followed by screening for reduced or eliminated polysaccharide production generates novel strains that are capable of producing higher yields of lipids.

[0329] In other embodiments, the present invention is directed to an oil-producing microbe containing one or more exogenous genes, wherein the exogenous genes encode protein(s) selected from the group consisting of a fatty acyl-ACP thioesterase, a fatty acyl-CoA reductase, a fatty aldehyde reductase, a fatty acyl- Co A/aldehyde reductase, a fatty aldehyde decarbonylase, a desaturase, and an acyl carrier protein. In another embodiment, an endogenous desaturase gene is overexpressed in a micro containing one or more of the above exogenous genes. In one embodiment, the exogenous gene is in operable linkage with a promoter, which is inducible or repressible in response to a stimulus. In some cases, the stimulus is selected from the group consisting of an exogenously provided small molecule, heat, cold, and limited or no nitrogen in the culture media. In some cases, the exogenous gene is expressed in a cellular compartment. In some embodiments, the cellular compartment is selected from the group consisting of a chloroplast, a plastid and a mitochondrion. In some embodiments the microbe is Prototheca moriformis, Prototheca krugani, Prototheca stagnora or Prototheca zopfii.

[0330] In one embodiment, the exogenous gene encodes a fatty acid acyl-ACP thioesterase. In some cases, the thioesterase encoded by the exogenous gene catalyzes the cleavage of an 8 to 18-carbon fatty acid from an acyl carrier protein (ACP). In some cases, the thioesterase encoded by the exogenous gene catalyzes the cleavage of a 10 to 14-carbon fatty acid from an ACP. In one embodiment, the thioesterase encoded by the exogenous gene catalyzes the cleavage of a 12-carbon fatty acid from an ACP.

[0331] In one embodiment, the exogenous gene encodes a fatty acyl-CoA/aldehyde reductase. In some cases, the reductase encoded by the exogenous gene catalyzes the reduction of an 8 to 18-carbon fatty acyl-CoA to a corresponding primary alcohol. In some cases, the reductase encoded by the exogenous gene catalyzes the reduction of a 10 to 14- carbon fatty acyl-CoA to a corresponding primary alcohol. In one embodiment, the reductase encoded by the exogenous gene catalyzes the reduction of a 12-carbon fatty acyl-CoA to dodecanol. [0332] The present invention also provides a recombinant Prototheca cell containing two exogenous genes, wherein a first exogenous gene encodes a fatty acyl-ACP thioesterase and a second exogenous gene encodes a protein selected from the group consisting of a fatty acyl- CoA reductase, a fatty acyl-CoA/aldehyde reductase, and an acyl carrier protein. In some cases, the two exogenous genes are each in operable linkage with a promoter, which is inducible in response to a stimulus. In some cases, each promoter is inducible in response to an identical stimulus, such as limited or no nitrogen in the culture media. Limitation or complete lack of nitrogen in the culture media stimulates oil production in some

microorganisms such as Prototheca species, and can be used as a trigger to inducec oil production to high levels. When used in combination with the genetic engineering methods disclosed herein, the lipid as a percentage of dry cell weight can be pushed to high levels such as at least 30%, at least 40%, at least 50%, at least 60%, at least 70% and at least 75%;

methods disclosed herein provide for cells with these levels of lipid, wherein the lipid is at least l%-5%, preferably at least 4%, C8-C14, at least 0.25%-l%, preferably at least 0.3%, C8, at least l%-5%, preferably at least 2%, CIO, at least l%-5%, preferably at least 2%, C12, and at least l%-5%, preferably at least 2%, CI 4. In some embodiments the cells are over 10%, over 15%, over 20%, or over 25% lipid by dry cell weight and contain lipid that is at least 5%, at least 10% or at least 15% C8-C14, at least 10%, at least 15%, at least 20%, at least 25% or at least 30% C8-C14, at least 20%, at least 25%, at least 30%, at least 35% or at least 40%, C8-C14, 5%-40%, preferably 10-30%, C8-C14 and 10%-40%, preferably 20-30%, C8-C14.

[0333] The novel oils disclosed herein are distinct from other naturally occurring oils that are high in mid-chain fatty acids, such as palm oil, palm kernel oil, and coconut oil. For example, levels of contaminants such as carotenoids are far higher in palm oil and palm kernel oil than in the oils of the invention. Palm and palm kernel oils in particular contain alpha and beta carotenes and lycopene in much higher amounts than is in the oils of the invention. In addition, over 20 different carotenoids are found in palm and palm kernel oil, whereas the Examples demonstrate that the oils of the invention contain very few carotenoids species and very low levels. In addition, the levels of vitamin E compounds such as tocotrienols are far higher in palm, palm kernel, and coconut oil than in the oils of the invention.

[0334] In one embodiment, the thioesterase encoded by the first exogenous gene catalyzes the cleavage of an 8 to 18-carbon fatty acid from an ACP. In some embodiments, the second exogenous gene encodes a fatty acyl- Co A/aldehyde reductase which catalyzes the reduction of an 8 to 18-carbon fatty acyl-CoA to a corresponding primary alcohol. In some cases, the thioesterase encoded by the first exogenous gene catalyzes the cleavage of a 10 to 14-carbon fatty acid from an ACP, and the reductase encoded by the second exogenous gene catalyzes the reduction of a 10 to 14-carbon fatty acyl-CoA to the corresponding primary alcohol, wherein the thioesterase and the reductase act on the same carbon chain length. In one embodiment, the thioesterase encoded by the first exogenous gene catalyzes the cleavage of a 12-carbon fatty acid from an ACP, and the reductase encoded by the second exogenous gene catalyzes the reduction of a 12-carbon fatty acyl-CoA to dodecanol. In some embodiments, the second exogenous gene encodes a fatty acyl-CoA reductase which catalyzes the reduction of an 8 to 18-carbon fatty acyl-CoA to a corresponding aldehyde. In some embodiments, the second exogenous gene encodes an acyl carrier protein that is naturally co-expressed with the fatty acyl-ACP thioesterase.

[0335] In some embodiments, the second exogenous gene encodes a fatty acyl-CoA reductase, and the microbe further contains a third exogenous gene encoding a fatty aldehyde decarbonylase. In some cases, the thioesterase encoded by the first exogenous gene catalyzes the cleavage of an 8 to 18-carbon fatty acid from an ACP, the reductase encoded by the second exogenous gene catalyzes the reduction of an 8 to 18-carbon fatty acyl-CoA to a corresponding fatty aldehyde, and the decarbonylase encoded by the third exogenous gene catalyzes the conversion of an 8 to 18-carbon fatty aldehyde to a corresponding alkane, wherein the thioesterase, the reductase, and the decarbonylase act on the same carbon chain length.

[0336] In some embodiments, the second exogenous gene encodes an acyl carrier protein, and the microbe further contains a third exogenous gene encoding a protein selected from the group consisting of a fatty acyl-CoA reductase and a fatty acyl- Co A/aldehyde reductase. In some cases, the third exogenous gene encodes a fatty acyl-CoA reductase, and the microbe further contains a fourth exogenous gene encoding a fatty aldehyde decarbonylase.

[0337] The present invention also provides methods for producing an alcohol comprising culturing a population of recombinant microorganisms (e.g. , Prototheca cells) in a culture medium, wherein the cells contain (i) a first exogenous gene encoding a fatty acyl-ACP thioesterase, and (ii) a second exogenous gene encoding a fatty acyl- Co A/aldehyde reductase, and the cells synthesize a fatty acid linked to an acyl carrier protein (ACP), the fatty acyl- ACP thioesterase catalyzes the cleavage of the fatty acid from the ACP to yield, through further processing, a fatty acyl-CoA, and the fatty acyl-CoA/aldehyde reductase catalyzes the reduction of the acyl-CoA to an alcohol. [0338] The present invention also provides methods of producing a lipid molecule in a microorganism (e.g. , a Prototheca cell). In one embodiment, the method comprises culturing a population of Prototheca cells in a culture medium, wherein the cells contain (i) a first exogenous gene encoding a fatty acyl-ACP thioesterase, and (ii) a second exogenous gene encoding a fatty acyl-CoA reductase, and wherein the microbes synthesize a fatty acid linked to an acyl carrier protein (ACP), the fatty acyl-ACP thioesterase catalyzes the cleavage of the fatty acid from the ACP to yield, through further processing, a fatty acyl-CoA, and the fatty acyl-CoA reductase catalyzes the reduction of the acyl-CoA to an aldehyde.

[0339] The present invention also provides methods of producing a fatty acid molecule having a specified carbon chain length in a microorganism (e.g. , a Prototheca cell). In one embodiment, the method comprises culturing a population of lipid-producing Prototheca cells in a culture medium, wherein the microbes contain an exogenous gene encoding a fatty acyl-ACP thioesterase having an activity specific or preferential to a certain carbon chain length, such as 8, 10, 12 or 14 carbon atoms, and wherein the microbes synthesize a fatty acid linked to an acyl carrier protein (ACP) and the thioesterase catalyzes the cleavage of the fatty acid from the ACP when the fatty acid has been synthesized to the specific carbon chain length.

[0340] In the various embodiments described above, the microorganism (e.g., a Prototheca cell) can contain at least one exogenous gene encoding a lipid pathway enzyme. In some cases, the lipid pathway enzyme is selected from the group consisting of a stearoyl-ACP desaturase, a glycerolipid desaturase, a pyruvate dehydrogenase, an acetyl-CoA carboxylase, an acyl carrier protein, and a glycerol-3 phosphate acyltransferase. In other cases, the microorganism (e.g. , Prototheca cell) contains a lipid modification enzyme selected from the group consisting of a fatty acyl-ACP thioesterase, a fatty acyl- Co A/aldehyde reductase, a fatty acyl-CoA reductase, a fatty aldehyde reductase, a fatty aldehyde decarbonylase, and/or an acyl carrier protein.

[0341] A number of exemplary transformation cassettes or constructs used to express a variety of the lipid pathway enzymes and lipid modification enzymes discussed herein are presented in the Examples. Other useful constructs, without limitation, are listed in Table 37, below.

[0342] Table 37. Exemplary transformation constructs, codon-optimized coding regions, and enzymes. C. hookeriana C10:0 specific thioesterase construct 243 coding region for C. hookeriana C10:0 specific thioesterase (codon- 244 optimized)

C. hookeriana KAS IV enzyme construct 245 coding region for C. hookeriana KAS IV enzyme (codon-optimized) 246

C. hookeriana KAS IV enzyme 247

C. hookeriana C10:0 specific thioesterase plus C. hookeriana KAS IV 248 enzyme construct

coding region for C. lanceolata C10:0 specific thioesterase with UTEX 1435 249 Δ12 fatty acid desaturase

U. californica C12:0 specific thioesterase construct 250 coding region for U. californica C12:0 specific thioesterase (codon- 251 optimized)

G. mangostana C16:0 thioesterase construct 252 coding region for G. mangostana C16:0 thioesterase (codon-optimized) 253

B. napus C18:0 thioesterase construct 254 coding region for B. napus C18:0 thioesterase (codon-optimized) 255

0. europaea stearoyl-ACP desaturase construct 256 coding region for 0. europaea stearoyl-ACP desaturase (codon-optimized) 257

C. hookeriana C16:0 thioesterase construct 258 coding region for C. hookeriana C16:0 thioesterase (codon-optimized) 259

E. guineensis C16:0 thioesterase construct 260 coding region for E. guineensis C16:0 thioesterase (codon-optimized) 261

C. tinctorius ACP-thioesterase at Δ12 fatty acid desaturase locus construct 262 coding region for C. tinctorius ACP-thioesterase (codon-optimized) 263

M. fragrans 04:0-08:0 broad specificity thioesterase construct 264 coding region for M. fragrans 04:0-08:0 broad specificity thioesterase 265 (codon-optimized)

coding region for M. fragrans C: 14:0 specific thioesterase 266

M. fragrans 04:0 specfic thioesterase with Δ12 FAD transit peptide 267

Ricinus communis ACP-thioesterase construct 268 coding region for Ricinus communis ACP-thioesterase (codon-optimized) 269

C. camphor a 04:0 thioesterase construct 270 coding region for C. camphora 04:0 thioesterase (codon-optimized) 271

C. camphora 04:0 specific thioesterase construct 272 C. camphor a C14:0 specific thioesterase construct 273

U. Americana 00:0-06:0 specific thioesterase in a SAD locus 274 coding region for U. Americana 00:0-06:0 specific thioesterase (codon- 275 optimized)

C. wrightii KASA1 + C. wrightii FatB2 thioesterase + suc2 construct 276 coding region for C. wrightii KASA1 (codon-optimized) 277 coding region for C. wrightii FatB2 thioesterase (codon-optimized) 278

VI. FUELS AND CHEMICALS PRODUCTION

[0343] For the production of fuel in accordance with the methods of the invention lipids produced by cells of the invention are harvested, or otherwise collected, by any convenient means. Lipids can be isolated by whole cell extraction. The cells are first disrupted, and then intracellular and cell membrane/cell wall- associated lipids as well as extracellular hydrocarbons can be separated from the cell mass, such as by use of centrifugation as described above. Intracellular lipids produced in microorganisms are, in some embodiments, extracted after lysing the cells of the microorganism. Once extracted, the lipids are further refined to produce oils, fuels, or oleochemicals.

[0344] After completion of culturing, the microorganisms can be separated from the fermentation broth. Optionally, the separation is effected by centrifugation to generate a concentrated paste. Centrifugation does not remove significant amounts of intracellular water from the microorganisms and is not a drying step. The biomass can then optionally be washed with a washing solution (e.g., DI water) to get rid of the fermentation broth and debris.

Optionally, the washed microbial biomass may also be dried (oven dried, lyophilized, etc.) prior to cell disruption. Alternatively, cells can be lysed without separation from some or all of the fermentation broth when the fermentation is complete. For example, the cells can be at a ratio of less than 1 : 1 v:v cells to extracellular liquid when the cells are lysed.

[0345] Microorganisms containing a lipid can be lysed to produce a lysate. As detailed herein, the step of lysing a microorganism (also referred to as cell lysis) can be achieved by any convenient means, including heat-induced lysis, adding a base, adding an acid, using enzymes such as proteases and polysaccharide degradation enzymes such as amylases, using ultrasound, mechanical lysis, using osmotic shock, infection with a lytic virus, and/or expression of one or more lytic genes. Lysis is performed to release intracellular molecules which have been produced by the microorganism. Each of these methods for lysing a microorganism can be used as a single method or in combination simultaneously or sequentially. The extent of cell disruption can be observed by microscopic analysis. Using one or more of the methods described herein, typically more than 70% cell breakage is observed. Preferably, cell breakage is more than 80%, more preferably more than 90% and most preferred about 100%.

[0346] In particular embodiments, the microorganism is lysed after growth, for example to increase the exposure of cellular lipid and/or hydrocarbon for extraction or further processing. The timing of lipase expression (e.g., via an inducible promoter) or cell lysis can be adjusted to optimize the yield of lipids and/or hydrocarbons. Below are described a number of lysis techniques. These techniques can be used individually or in combination.

[0347] In one embodiment of the present invention, the step of lysing a microorganism comprises heating of a cellular suspension containing the microorganism. In this

embodiment, the fermentation broth containing the microorganisms (or a suspension of microorganisms isolated from the fermentation broth) is heated until the microorganisms, i.e., the cell walls and membranes of microorganisms degrade or breakdown. Typically, temperatures applied are at least 50°C. Higher temperatures, such as, at least 30°C at least 60°C, at least 70°C, at least 80°C, at least 90°C, at least 100°C, at least 110°C, at least 120°C, at least 130°C or higher are used for more efficient cell lysis. Lysing cells by heat treatment can be performed by boiling the microorganism. Alternatively, heat treatment (without boiling) can be performed in an autoclave. The heat treated lysate may be cooled for further treatment. Cell disruption can also be performed by steam treatment, i.e., through addition of pressurized steam. Steam treatment of microalgae for cell disruption is described, for example, in U.S. Patent No. 6,750,048. In some embodiments, steam treatment may be achieved by sparging steam into the fermentor and maintaining the broth at a desired temperature for less than about 90 minutes, preferably less than about 60 minutes, and more preferably less than about 30 minutes.

[0348] In another embodiment of the present invention, the step of lysing a microorganism comprises adding a base to a cellular suspension containing the microorganism. The base should be strong enough to hydrolyze at least a portion of the proteinaceous compounds of the microorganisms used. Bases which are useful for solubilizing proteins are known in the art of chemistry. Exemplary bases which are useful in the methods of the present invention include, but are not limited to, hydroxides, carbonates and bicarbonates of lithium, sodium, potassium, calcium, and mixtures thereof. A preferred base is KOH. Base treatment of microalgae for cell disruption is described, for example, in U.S. Patent No. 6,750,048. [0349] In another embodiment of the present invention, the step of lysing a microorganism comprises adding an acid to a cellular suspension containing the microorganism. Acid lysis can be effected using an acid at a concentration of 10-500 mN or preferably 40-160 nM. Acid lysis is preferably performed at above room temperature (e.g., at 40-160°, and preferably a temperature of 50-130°. For moderate temperatures (e.g., room temperature to 100°C and particularly room temperature to 65°, acid treatment can usefully be combined with sonication or other cell disruption methods.

[0350] In another embodiment of the present invention, the step of lysing a microorganism comprises lysing the microorganism by using an enzyme. Preferred enzymes for lysing a microorganism are proteases and polysaccharide-degrading enzymes such as hemicellulase (e.g., hemicellulase from Aspergillus niger; Sigma Aldrich, St. Louis, MO; #H2125), pectinase (e.g., pectinase from Rhizopus sp:, Sigma Aldrich, St. Louis, MO; #P2401), Mannaway 4.0 L (Novozymes), cellulase (e.g., cellulose from Trichoderma viride; Sigma Aldrich, St. Louis, MO; #C9422), and driselase (e.g., driselase from Basidiomycetes sp.; Sigma Aldrich, St. Louis, MO; #D9515.

[0351] In other embodiments of the present invention, lysis is accomplished using an enzyme such as, for example, a cellulase such as a polysaccharide-degrading enzyme, optionally from Chlorella or a Chlorella virus, or a proteases, such as Streptomyces griseus protease, chymotrypsin, proteinase K, proteases listed in Degradation of Polylactide by Commercial Proteases, Oda Yet al., Journal of Polymers and the Environment, Volume 8, Number 1, January 2000 , pp. 29-32(4), Alcalase 2.4 FG (Novozymes), and Flavourzyme 100 L (Novozymes). Any combination of a protease and a polysaccharide-degrading enzyme can also be used, including any combination of the preceding proteases and polysaccharide- degrading enzymes.

[0352] In another embodiment, lysis can be performed using an expeller press. In this process, biomass is forced through a screw-type device at high pressure, lysing the cells and causing the intracellular lipid to be released and separated from the protein and fiber (and other components) in the cell.

[0353] In another embodiment of the present invention, the step of lysing a microorganism is performed by using ultrasound, i.e., sonication. Thus, cells can also by lysed with high frequency sound. The sound can be produced electronically and transported through a metallic tip to an appropriately concentrated cellular suspension. This sonication (or ultrasonication) disrupts cellular integrity based on the creation of cavities in cell suspension. [0354] In another embodiment of the present invention, the step of lysing a microorganism is performed by mechanical lysis. Cells can be lysed mechanically and optionally

homogenized to facilitate hydrocarbon (e.g., lipid) collection. For example, a pressure disrupter can be used to pump a cell containing slurry through a restricted orifice valve. High pressure (up to 1500 bar) is applied, followed by an instant expansion through an exiting nozzle. Cell disruption is accomplished by three different mechanisms: impingement on the valve, high liquid shear in the orifice, and sudden pressure drop upon discharge, causing an explosion of the cell. The method releases intracellular molecules. Alternatively, a ball mill can be used. In a ball mill, cells are agitated in suspension with small abrasive particles, such as beads. Cells break because of shear forces, grinding between beads, and collisions with beads. The beads disrupt the cells to release cellular contents. Cells can also be disrupted by shear forces, such as with the use of blending (such as with a high speed or Waring blender as examples), the french press, or even centrifugation in case of weak cell walls, to disrupt cells.

[0355] In another embodiment of the present invention, the step of lysing a microorganism is performed by applying an osmotic shock.

[0356] In another embodiment of the present invention, the step of lysing a microorganism comprises infection of the microorganism with a lytic virus. A wide variety of viruses are known to lyse microorganisms suitable for use in the present invention, and the selection and use of a particular lytic virus for a particular microorganism is within the level of skill in the art. For example, Paramecium bursaria chlorella virus (PBCV-1) is the prototype of a group (family Phycodnaviridae, genus Chlorovirus) of large, icosahedral, plaque-forming, double- stranded DNA viruses that replicate in, and lyse, certain unicellular, eukaryotic chlorella- like green algae. Accordingly, any susceptible microalgae can be lysed by infecting the culture with a suitable chlorella virus. Methods of infecting species of Chlorella with a chlorella virus are known. See for example Adv. Virus Res. 2006;66:293-336; Virology, 1999 Apr 25;257(l): 15-23; Virology, 2004 Jan 5;318(l):214-23; Nucleic Acids Symp. Ser.

2000;(44):161-2; /. Virol. 2006 Mar;80(5):2437-44; and Annu. Rev. Microbiol. 1999;53:447- 94.

[0357] In another embodiment of the present invention, the step of lysing a microorganism comprises autolysis. In this embodiment, a microorganism according to the invention is genetically engineered to produce a lytic protein that will lyse the microorganism. This lytic gene can be expressed using an inducible promoter so that the cells can first be grown to a desirable density in a fermentor, followed by induction of the promoter to express the lytic gene to lyse the cells. In one embodiment, the lytic gene encodes a polysaccharide-degrading enzyme. In certain other embodiments, the lytic gene is a gene from a lytic virus. Thus, for example, a lytic gene from a Chlorella virus can be expressed in an algal cell; see Virology 260, 308-315 (1999); FEMS Microbiology Letters 180 (1999) 45-53; Virology 263, 376-387 (1999); and Virology 230, 361-368 (1997). Expression of lytic genes is preferably done using an inducible promoter, such as a promoter active in microalgae that is induced by a stimulus such as the presence of a small molecule, light, heat, and other stimuli.

[0358] Various methods are available for separating lipids from cellular lysates produced by the above methods. For example, lipids and lipid derivatives such as fatty aldehydes, fatty alcohols, and hydrocarbons such as alkanes can be extracted with a hydrophobic solvent such as hexane (see Frenz et al. 1989, Enzyme Microb. Technol., 11 :717). Lipids and lipid derivatives can also be extracted using liquefaction (see for example Sawayama et al. 1999, Biomass and Bioenergy 17:33-39 and Inoue et al. 1993, Biomass Bioenergy 6(4):269-274); oil liquefaction (see for example Minowa et al. 1995, Fuel 74(12): 1735-1738); and supercritical C0 2 extraction (see for example Mendes et al. 2003, Inorganica Chimica Acta 356:328-334). Miao and Wu describe a protocol of the recovery of microalgal lipid from a culture of Chlorella prototheocoides in which the cells were harvested by centrifugation, washed with distilled water and dried by freeze drying. The resulting cell powder was pulverized in a mortar and then extracted with n-hexane. Miao and Wu, Biosource

Technology (2006) 97:841-846.

[0359] Thus, lipids, lipid derivatives and hydrocarbons generated by the microorganisms of the present invention can be recovered by extraction with an organic solvent. In some cases, the preferred organic solvent is hexane. Typically, the organic solvent is added directly to the lysate without prior separation of the lysate components. In one embodiment, the lysate generated by one or more of the methods described above is contacted with an organic solvent for a period of time sufficient to allow the lipid and/or hydrocarbon components to form a solution with the organic solvent. In some cases, the solution can then be further refined to recover specific desired lipid or hydrocarbon components. Hexane extraction methods are well known in the art.

[0360] Lipids and lipid derivatives such as fatty aldehydes, fatty alcohols, and

hydrocarbons such as alkanes produced by cells as described herein can be modified by the use of one or more enzymes, including a lipase, as described above. When the hydrocarbons are in the extracellular environment of the cells, the one or more enzymes can be added to that environment under conditions in which the enzyme modifies the hydrocarbon or completes its synthesis from a hydrocarbon precursor. Alternatively, the hydrocarbons can be partially, or completely, isolated from the cellular material before addition of one or more catalysts such as enzymes. Such catalysts are exogenously added, and their activity occurs outside the cell or in vitro.

[0361] Thus, lipids and hydrocarbons produced by cells in vivo, or enzymatically modified in vitro, as described herein can be optionally further processed by conventional means. The processing can include "cracking" to reduce the size, and thus increase the hydrogen:carbon ratio, of hydrocarbon molecules. Catalytic and thermal cracking methods are routinely used in hydrocarbon and triglyceride oil processing. Catalytic methods involve the use of a catalyst, such as a solid acid catalyst. The catalyst can be silica- alumina or a zeolite, which result in the heterolytic, or asymmetric, breakage of a carbon-carbon bond to result in a carbocation and a hydride anion. These reactive intermediates then undergo either rearrangement or hydride transfer with another hydrocarbon. The reactions can thus regenerate the intermediates to result in a self-propagating chain mechanism. Hydrocarbons can also be processed to reduce, optionally to zero, the number of carbon-carbon double, or triple, bonds therein. Hydrocarbons can also be processed to remove or eliminate a ring or cyclic structure therein. Hydrocarbons can also be processed to increase the hydrogen:carbon ratio. This can include the addition of hydrogen ("hydrogenation") and/or the "cracking" of hydrocarbons into smaller hydrocarbons.

[0362] Thermal methods involve the use of elevated temperature and pressure to reduce hydrocarbon size. An elevated temperature of about 800°C and pressure of about 700kPa can be used. These conditions generate "light," a term that is sometimes used to refer to hydrogen-rich hydrocarbon molecules (as distinguished from photon flux), while also generating, by condensation, heavier hydrocarbon molecules which are relatively depleted of hydrogen. The methodology provides homolytic, or symmetrical, breakage and produces alkenes, which may be optionally enzymatically saturated as described above.

[0363] Catalytic and thermal methods are standard in plants for hydrocarbon processing and oil refining. Thus hydrocarbons produced by cells as described herein can be collected and processed or refined via conventional means. See Hillen et al. (Biotechnology and Bioengineering, Vol. XXIV: 193-205 (1982)) for a report on hydrocracking of microalgae- produced hydrocarbons. In alternative embodiments, the fraction is treated with another catalyst, such as an organic compound, heat, and/or an inorganic compound. For processing of lipids into biodiesel, a transesterification process is used as described below in this Section. [0364] Hydrocarbons produced via methods of the present invention are useful in a variety of industrial applications. For example, the production of linear alkylbenzene sulfonate (LAS), an anionic surfactant used in nearly all types of detergents and cleaning preparations, utilizes hydrocarbons generally comprising a chain of 10-14 carbon atoms. See, for example, US Patent Nos.: 6,946,430; 5,506,201; 6,692,730; 6,268,517; 6,020,509; 6,140,302;

5,080,848; and 5,567,359. Surfactants, such as LAS, can be used in the manfacture of personal care compositions and detergents, such as those described in US Patent Nos.:

5,942,479; 6,086,903; 5,833,999; 6,468,955; and 6,407,044.

[0365] Increasing interest is directed to the use of hydrocarbon components of biological origin in fuels, such as biodiesel, renewable diesel, and jet fuel, since renewable biological starting materials that may replace starting materials derived from fossil fuels are available, and the use thereof is desirable. There is an urgent need for methods for producing hydrocarbon components from biological materials. The present invention fulfills this need by providing methods for production of biodiesel, renewable diesel, and jet fuel using the lipids generated by the methods described herein as a biological material to produce biodiesel, renewable diesel, and jet fuel.

[0366] Traditional diesel fuels are petroleum distillates rich in paraffinic hydrocarbons. They have boiling ranges as broad as 370° to 780°F, which are suitable for combustion in a compression ignition engine, such as a diesel engine vehicle. The American Society of Testing and Materials (ASTM) establishes the grade of diesel according to the boiling range, along with allowable ranges of other fuel properties, such as cetane number, cloud point, flash point, viscosity, aniline point, sulfur content, water content, ash content, copper strip corrosion, and carbon residue. Technically, any hydrocarbon distillate material derived from biomass or otherwise that meets the appropriate ASTM specification can be defined as diesel fuel (ASTM D975), jet fuel (ASTM D1655), or as biodiesel if it is a fatty acid methyl ester (ASTM D6751).

[0367] After extraction, lipid and/or hydrocarbon components recovered from the microbial biomass described herein can be subjected to chemical treatment to manufacture a fuel for use in diesel vehicles and jet engines.

[0368] Biodiesel is a liquid which varies in color - between golden and dark brown - depending on the production feedstock. It is practically immiscible with water, has a high boiling point and low vapor pressure. Biodiesel refers to a diesel-equivalent processed fuel for use in diesel-engine vehicles. Biodiesel is biodegradable and non-toxic. An additional benefit of biodiesel over conventional diesel fuel is lower engine wear. Typically, biodiesel comprises C14-C18 alkyl esters. Various processes convert biomass or a lipid produced and isolated as described herein to diesel fuels. A preferred method to produce biodiesel is by transesterification of a lipid as described herein. A preferred alkyl ester for use as biodiesel is a methyl ester or ethyl ester.

[0369] Biodiesel produced by a method described herein can be used alone or blended with conventional diesel fuel at any concentration in most modern diesel-engine vehicles. When blended with conventional diesel fuel (petroleum diesel), biodiesel may be present from about 0.1% to about 99.9%. Much of the world uses a system known as the "B" factor to state the amount of biodiesel in any fuel mix. For example, fuel containing 20% biodiesel is labeled B20. Pure biodiesel is referred to as B 100.

[0370] Biodiesel can also be used as a heating fuel in domestic and commercial boilers. Existing oil boilers may contain rubber parts and may require conversion to run on biodiesel. The conversion process is usually relatively simple, involving the exchange of rubber parts for synthetic parts due to biodiesel being a strong solvent. Due to its strong solvent power, burning biodiesel will increase the efficiency of boilers. Biodiesel can be used as an additive in formulations of diesel to increase the lubricity of pure Ultra- Low Sulfur Diesel (ULSD) fuel, which is advantageous because it has virtually no sulfur content. Biodiesel is a better solvent than petrodiesel and can be used to break down deposits of residues in the fuel lines of vehicles that have previously been run on petrodiesel.

[0371] Biodiesel can be produced by transesterification of triglycerides contained in oil- rich biomass. Thus, in another aspect of the present invention a method for producing biodiesel is provided. In a preferred embodiment, the method for producing biodiesel comprises the steps of (a) cultivating a lipid-containing microorganism using methods disclosed herein (b) lysing a lipid-containing microorganism to produce a lysate, (c) isolating lipid from the lysed microorganism, and (d) transesterifying the lipid composition, whereby biodiesel is produced. Methods for growth of a microorganism, lysing a microorganism to produce a lysate, treating the lysate in a medium comprising an organic solvent to form a heterogeneous mixture and separating the treated lysate into a lipid composition have been described above and can also be used in the method of producing biodiesel.

[0372] The lipid profile of the biodiesel is usually highly similar to the lipid profile of the feedstock oil. Other oils provided by the methods and compositions of the invention can be subjected to transesterification to yield biodiesel with lipid profiles including (a) at least 1%- 5%, preferably at least 4%, C8-C14; (b) at least 0.25%-l%, preferably at least 0.3%, C8; (c) at least l -5 , preferably at least 2%, CIO; (d) at least l -5 , preferably at least 2%, C12; and (3) at least 20 -40 , preferably at least 30%, C8-C14.

[0373] Lipid compositions can be subjected to transesterification to yield long-chain fatty acid esters useful as biodiesel. Preferred transesterification reactions are outlined below and include base catalyzed transesterification and transesterification using recombinant lipases. In a base-catalyzed transesterification process, the triacylglycerides are reacted with an alcohol, such as methanol or ethanol, in the presence of an alkaline catalyst, typically potassium hydroxide. This reaction forms methyl or ethyl esters and glycerin (glycerol) as a byproduct.

[0374] Animal and plant oils are typically made of triglycerides which are esters of free fatty acids with the trihydric alcohol, glycerol. In transesterification, the glycerol in a triacylglyceride (TAG) is replaced with a short-chain alcohol such as methanol or ethanol. A typical reaction scheme is as follows: + R 2 C00Et + R 3 C00Et + C 3 H 5 (OH) 3

Ethyl esters of fatty acids Glycerol

Triglyceride

[0375] In this reaction, the alcohol is deprotonated with a base to make it a stronger nucleophile. Commonly, ethanol or methanol is used in vast excess (up to 50-fold).

Normally, this reaction will proceed either exceedingly slowly or not at all. Heat, as well as an acid or base can be used to help the reaction proceed more quickly. The acid or base are not consumed by the transesterification reaction, thus they are not reactants but catalysts. Almost all biodiesel has been produced using the base-catalyzed technique as it requires only low temperatures and pressures and produces over 98% conversion yield (provided the starting oil is low in moisture and free fatty acids).

[0376] Transesterification has also been carried out, as discussed above, using an enzyme, such as a lipase instead of a base. Lipase-catalyzed transesterification can be carried out, for example, at a temperature between the room temperature and 80° C, and a mole ratio of the TAG to the lower alcohol of greater than 1 : 1, preferably about 3: 1. Lipases suitable for use in transesterification include, but are not limited to, those listed in Table 9. Other examples of lipases useful for transesterification are found in, e.g. U.S. Patent Nos. 4,798,793; 4,940,845 5,156,963; 5,342,768; 5,776,741 and WO89/01032. Such lipases include, but are not limited to, lipases produced by microorganisms of Rhizopus, Aspergillus, Candida, Mucor, Pseudomonas, Rhizomucor, Candida, and Humicola and pancreas lipase.

[0377] Table 9. Lipases suitable for use in transesterification. Aspergillus niger lipase ABG73614, Candida antarctica lipase B (novozym-435)

CAA83122, Candida cylindracea lipase AAR24090, Candida lipolytica lipase (Lipase L; Amano Pharmaceutical Co., Ltd.), Candida rugosa lipase (e.g., Lipase-OF; Meito Sangyo Co., Ltd.), Mucor miehei lipase (Lipozyme IM 20), Pseudomonas fluorescens lipase AAA25882, Rhizopus japonicas lipase (Lilipase A-10FG) Q7M4U7_1, Rhizomucor miehei lipase B34959, Rhizopus oryzae lipase (Lipase F) AAF32408, Serratia marcescens lipase (SM Enzyme) ABI13521, Thermomyces lanuginosa lipase CAB58509, Lipase P (Nagase ChemteX Corporation), and Lipase QLM (Meito Sangyo Co., Ltd., Nagoya, Japan)

[0378] One challenge to using a lipase for the production of fatty acid esters suitable for biodiesel is that the price of lipase is much higher than the price of sodium hydroxide (NaOH) used by the strong base process. This challenge has been addressed by using an immobilized lipase, which can be recycled. However, the activity of the immobilized lipase must be maintained after being recycled for a minimum number of cycles to allow a lipase- based process to compete with the strong base process in terms of the production cost.

Immobilized lipases are subject to poisoning by the lower alcohols typically used in transesterification. U.S. Patent No. 6,398,707 (issued June 4, 2002 to Wu et al.) describes methods for enhancing the activity of immobilized lipases and regenerating immobilized lipases having reduced activity. Some suitable methods include immersing an immobilized lipase in an alcohol having a carbon atom number not less than 3 for a period of time, preferably from 0.5-48 hours, and more preferably from 0.5-1.5 hours. Some suitable methods also include washing a deactivated immobilized lipase with an alcohol having a carbon atom number not less than 3 and then immersing the deactivated immobilized lipase in a vegetable oil for 0.5-48 hours.

[0379] In particular embodiments, a recombinant lipase is expressed in the same microorganisms that produce the lipid on which the lipase acts. Suitable recombinant lipases include those listed above in Table 9 and/or having GenBank Accession numbers listed above in Table 9, or a polypeptide that has at least 70% amino acid identity with one of the lipases listed above in Table 9 and that exhibits lipase activity. In additional embodiments, the enzymatic activity is present in a sequence that has at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 99% identity with one of the above described sequences, all of which are hereby incorporated by reference as if fully set forth. DNA encoding the lipase and selectable marker is preferably codon-optimized cDNA. Methods of recoding genes for expression in microalgae are described in US Patent 7,135,290.

[0380] The common international standard for biodiesel is EN 14214. ASTM D6751 is the most common biodiesel standard referenced in the United States and Canada. Germany uses DIN EN 14214 and the UK requires compliance with BS EN 14214. Basic industrial tests to determine whether the products conform to these standards typically include gas

chromatography, HPLC, and others. Biodiesel meeting the quality standards is very nontoxic, with a toxicity rating (LD 50 ) of greater than 50 mL/kg.

[0381] Although biodiesel that meets the ASTM standards has to be non-toxic, there can be contaminants which tend to crystallize and/or precipitate and fall out of solution as sediment. Sediment formation is particularly a problem when biodiesel is used at lower temperatures. The sediment or precipitates may cause problems such as decreasing fuel flow, clogging fuel lines, clogging filters, etc. Processes are well-known in the art that specifically deal with the removal of these contaminants and sediments in biodiesel in order to produce a higher quality product. Examples for such processes include, but are not limited to, pretreatment of the oil to remove contaiminants such as phospholipids and free fatty acids (e.g., degumming, caustic refining and silica adsorbant filtration) and cold filtration. Cold filtration is a process that was developed specifically to remove any particulates and sediments that are present in the biodiesel after production. This process cools the biodiesel and filters out any sediments or precipitates that might form when the fuel is used at a lower temperature. Such a process is well known in the art and is described in US Patent Application Publication No. 2007- 0175091. Suitable methods may include cooling the biodiesel to a temperature of less than about 38°C so that the impurities and contaminants precipitate out as particulates in the biodiesel liquid. Diatomaceous earth or other filtering material may then added to the cooled biodiesel to form a slurry, which may then filtered through a pressure leaf or other type of filter to remove the particulates. The filtered biodiesel may then be run through a polish filter to remove any remaining sediments and diatomaceous earth, so as to produce the final biodiesel product.

[0382] Example 13 describes the production of biodiesel using triglyceride oil from Prototheca moriformis. The Cold Soak Filterability by the ASTM D6751 Al method of the biodiesel produced in Example 13 was 120 seconds for a volume of 300ml. This test involves filtration of 300 ml of B 100, chilled to 40°F for 16 hours, allowed to warm to room temp, and filtered under vacuum using 0.7 micron glass fiber filter with stainless steel support. Oils of the invention can be transesterified to generate biodiesel with a cold soak time of less than 120 seconds, less than 100 seconds, and less than 90 seconds.

[0383] Subsequent processes may also be used if the biodiesel will be used in particularly cold temperatures. Such processes include winterization and fractionation. Both processes are designed to improve the cold flow and winter performance of the fuel by lowering the cloud point (the temperature at which the biodiesel starts to crystallize). There are several approaches to winterizing biodiesel. One approach is to blend the biodiesel with petroleum diesel. Another approach is to use additives that can lower the cloud point of biodiesel.

Another approach is to remove saturated methyl esters indiscriminately by mixing in additives and allowing for the crystallization of saturates and then filtering out the crystals. Fractionation selectively separates methyl esters into individual components or fractions, allowing for the removal or inclusion of specific methyl esters. Fractionation methods include urea fractionation, solvent fractionation and thermal distillation.

[0384] Another valuable fuel provided by the methods of the present invention is renewable diesel, which comprises alkanes, such as C10:0, C12:0, C14:0, C16:0 and C18:0 and thus, are distinguishable from biodiesel. High quality renewable diesel conforms to the ASTM D975 standard. The lipids produced by the methods of the present invention can serve as feedstock to produce renewable diesel. Thus, in another aspect of the present invention, a method for producing renewable diesel is provided. Renewable diesel can be produced by at least three processes: hydrothermal processing (hydrotreating); hydroprocessing; and indirect liquefaction. These processes yield non-ester distillates. During these processes,

triacylglycerides produced and isolated as described herein, are converted to alkanes.

[0385] In one embodiment, the method for producing renewable diesel comprises (a) cultivating a lipid-containing microorganism using methods disclosed herein (b) lysing the microorganism to produce a lysate, (c) isolating lipid from the lysed microorganism, and (d) deoxygenating and hydrotreating the lipid to produce an alkane, whereby renewable diesel is produced. Lipids suitable for manufacturing renewable diesel can be obtained via extraction from microbial biomass using an organic solvent such as hexane, or via other methods, such as those described in US Patent 5,928,696. Some suitable methods may include mechanical pressing and centrifuging.

[0386] In some methods, the microbial lipid is first cracked in conjunction with

hydrotreating to reduce carbon chain length and saturate double bonds, respectively. The material is then isomerized, also in conjunction with hydrotreating. The naptha fraction can then be removed through distillation, followed by additional distillation to vaporize and distill components desired in the diesel fuel to meet an ASTM D975 standard while leaving components that are heavier than desired for meeting the D975 standard. Hydrotreating, hydrocracking, deoxygenation and isomerization methods of chemically modifying oils, including triglyceride oils, are well known in the art. See for example European patent applications EP1741768 (Al); EP1741767 (Al); EP1682466 (Al); EP1640437 (Al);

EP1681337 (Al); EP1795576 (Al); and U.S. Patents 7,238,277; 6,630,066; 6,596,155; 6,977,322; 7,041,866; 6,217,746; 5,885,440; 6,881,873.

[0387] In one embodiment of the method for producing renewable diesel, treating the lipid to produce an alkane is performed by hydrotreating of the lipid composition. In hydrothermal processing, typically, biomass is reacted in water at an elevated temperature and pressure to form oils and residual solids. Conversion temperatures are typically 300° to 660°F, with pressure sufficient to keep the water primarily as a liquid, 100 to 170 standard atmosphere (atm). Reaction times are on the order of 15 to 30 minutes. After the reaction is completed, the organics are separated from the water. Thereby a distillate suitable for diesel is produced.

[0388] In some methods of making renewable diesel, the first step of treating a triglyceride is hydroprocessing to saturate double bonds, followed by deoxygenation at elevated temperature in the presence of hydrogen and a catalyst. In some methods, hydrogenation and deoxygenation occur in the same reaction. In other methods deoxygenation occurs before hydrogenation. Isomerization is then optionally performed, also in the presence of hydrogen and a catalyst. Naphtha components are preferably removed through distillation. For examples, see U.S. Patents 5,475,160 (hydrogenation of triglycerides); 5,091,116

(deoxygenation, hydrogenation and gas removal); 6,391,815 (hydrogenation); and 5,888,947 (isomerization).

[0389] One suitable method for the hydrogenation of triglycerides includes preparing an aqueous solution of copper, zinc, magnesium and lanthanum salts and another solution of alkali metal or preferably, ammonium carbonate. The two solutions may be heated to a temperature of about 20°C to about 85°C and metered together into a precipitation container at rates such that the pH in the precipitation container is maintained between 5.5 and 7.5 in order to form a catalyst. Additional water may be used either initially in the precipitation container or added concurrently with the salt solution and precipitation solution. The resulting precipitate may then be thoroughly washed, dried, calcined at about 300°C and activated in hydrogen at temperatures ranging from about 100°C to about 400°C. One or more triglycerides may then be contacted and reacted with hydrogen in the presence of the above-described catalyst in a reactor. The reactor may be a trickle bed reactor, fixed bed gas- solid reactor, packed bubble column reactor, continuously stirred tank reactor, a slurry phase reactor, or any other suitable reactor type known in the art. The process may be carried out either batchwise or in continuous fashion. Reaction temperatures are typically in the range of from about 170°C to about 250°C while reaction pressures are typically in the range of from about 300 psig to about 2000 psig. Moreover, the molar ratio of hydrogen to triglyceride in the process of the present invention is typically in the range of from about 20: 1 to about 700: 1. The process is typically carried out at a weight hourly space velocity (WHSV) in the range of from about 0.1 hr 1 to about 5 hr 1 . One skilled in the art willrecognize that the time period required for reaction will vary according to the temperature used, the molar ratio of hydrogen to triglyceride, and the partial pressure of hydrogen. The products produced by the such hydrogenation processes include fatty alcohols, glycerol, traces of paraffins and unreacted triglycerides. These products are typically separated by conventional means such as, for example, distillation, extraction, filtration, crystallization, and the like.

[0390] Petroleum refiners use hydroprocessing to remove impurities by treating feeds with hydrogen. Hydroprocessing conversion temperatures are typically 300° to 700°F. Pressures are typically 40 to 100 atm. The reaction times are typically on the order of 10 to 60 minutes. Solid catalysts are employed to increase certain reaction rates, improve selectivity for certain products, and optimize hydrogen consumption.

[0391] Suitable methods for the deoxygenation of an oil includes heating an oil to a temperature in the range of from about 350°F to about 550°F and continuously contacting the heated oil with nitrogen under at least pressure ranging from about atmospeheric to above for at least about 5 minutes.

[0392] Suitable methods for isomerization include using alkali isomerization and other oil isomerization known in the art.

[0393] Hydrotreating and hydroprocessing ultimately lead to a reduction in the molecular weight of the triglyceride feed. The triglyceride molecule is reduced to four hydrocarbon molecules under hydroprocessing conditions: a propane molecule and three heavier hydrocarbon molecules, typically in the C8 to C18 range.

[0394] Thus, in one embodiment, the product of one or more chemical reaction(s) performed on lipid compositions of the invention is an alkane mixture that comprises ASTM D975 renewable diesel. Production of hydrocarbons by microorganisms is reviewed by Metzger et al. Appl Microbiol Biotechnol (2005) 66: 486-496 and A Look Back at the U.S. Department of Energy's Aquatic Species Program: Biodiesel from Algae, NREL/TP-580- 24190, John Sheehan, Terri Dunahay, John Benemann and Paul Roessler (1998). [0395] The distillation properties of a diesel fuel is described in terms of T10-T90

(temperature at 10% and 90%, respectively, volume distilled). Renewable diesel was produced from Prototheca moriformis triglyceride oil and is described in Example 13. The T10-T90 of the material produced in Example 13 was 57.9°C. Methods of hydrotreating, isomerization, and other covalent modification of oils disclosed herein, as well as methods of distillation and fractionation (such as cold filtration) disclosed herein, can be employed to generate renewable diesel compositions with other T10-T90 ranges, such as 20, 25, 30, 35, 40, 45, 50, 60 and 65°C using triglyceride oils produced according to the methods disclosed herein.

[0396] The T10 of the material produced in Example 13 was 242.1°C. Methods of hydrotreating, isomerization, and other covalent modification of oils disclosed herein, as well as methods of distillation and fractionation (such as cold filtration) disclosed herein, can be employed to generate renewable diesel compositions with other T10 values, such as T10 between 180 and 295, between 190 and 270, between 210 and 250, between 225 and 245, and at least 290.

[0397] The T90 of the material produced in Example 13 was 300°C. Methods of hydrotreating, isomerization, and other covalent modification of oils disclosed herein, as well as methods of distillation and fractionation (such as cold filtration) disclosed herein can be employed to generate renewable diesel compositions with other T90 values, such as T90 between 280 and 380, between 290 and 360, between 300 and 350, between 310 and 340, and at least 290.

[0398] The FBP of the material produced in Example 13 was 300°C. Methods of hydrotreating, isomerization, and other covalent modification of oils disclosed herein, as well as methods of distillation and fractionation (such as cold filtration) disclosed herein, can be employed to generate renewable diesel compositions with other FBP values, such as FBP between 290 and 400, between 300 and 385, between 310 and 370, between 315 and 360, and at least 300.

[0399] Other oils provided by the methods and compositions of the invention can be subjected to combinations of hydrotreating, isomerization, and other covalent modification including oils with lipid profiles including (a) at least l%-5%, preferably at least 4%, C8- C14; (b) at least 0.25%-l%, preferably at least 0.3%, C8; (c) at least l%-5%, preferably at least 2%, CIO; (d) at least l%-5%, preferably at least 2%, C12; and (3) at least 20%-40%, preferably at least 30% C8-C14. [0400] A traditional ultra- low sulfur diesel can be produced from any form of biomass by a two-step process. First, the biomass is converted to a syngas, a gaseous mixture rich in hydrogen and carbon monoxide. Then, the syngas is catalytically converted to liquids.

Typically, the production of liquids is accomplished using Fischer- Tropsch (FT) synthesis. This technology applies to coal, natural gas, and heavy oils. Thus, in yet another preferred embodiment of the method for producing renewable diesel, treating the lipid composition to produce an alkane is performed by indirect liquefaction of the lipid composition.

[0401] The present invention also provides methods to produce jet fuel. Jet fuel is clear to straw colored. The most common fuel is an unleaded/paraffin oil-based fuel classified as Aeroplane A-1, which is produced to an internationally standardized set of specifications. Jet fuel is a mixture of a large number of different hydrocarbons, possibly as many as a thousand or more. The range of their sizes (molecular weights or carbon numbers) is restricted by the requirements for the product, for example, freezing point or smoke point. Kerosone-type Aeroplane fuel (including Jet A and Jet A-1) has a carbon number distribution between about 8 and 16 carbon numbers. Wide-cut or naphta-type Aeroplane fuel (including Jet B) typically has a carbon number distribution between about 5 and 15 carbons.

[0402] Both Aeroplanes (Jet A and Jet B) may contain a number of additives. Useful additives include, but are not limited to, antioxidants, antistatic agents, corrosion inhibitors, and fuel system icing inhibitor (FSII) agents. Antioxidants prevent gumming and usually, are based on alkylated phenols, for example, AO-30, AO-31, or AO-37. Antistatic agents dissipate static electricity and prevent sparking. Stadis 450 with dinonylnaphthylsulfonic acid (DINNSA) as the active ingredient, is an example. Corrosion inhibitors, e.g., DCI-4A is used for civilian and military fuels and DCI-6A is used for military fuels. FSII agents, include, e.g., Di-EGME.

[0403] In one embodiment of the invention, a jet fuel is produced by blending algal fuels with existing jet fuel. The lipids produced by the methods of the present invention can serve as feedstock to produce jet fuel. Thus, in another aspect of the present invention, a method for producing jet fuel is provided. Herewith two methods for producing jet fuel from the lipids produced by the methods of the present invention are provided: fluid catalytic cracking (FCC); and hydrodeoxygenation (HDO).

[0404] Fluid Catalytic Cracking (FCC) is one method which is used to produce olefins, especially propylene from heavy crude fractions. The lipids produced by the method of the present invention can be converted to olefins. The process involves flowing the lipids produced through an FCC zone and collecting a product stream comprised of olefins, which is useful as a jet fuel. The lipids produced are contacted with a cracking catalyst at cracking conditions to provide a product stream comprising olefins and hydrocarbons useful as jet fuel.

[0405] In one embodiment, the method for producing jet fuel comprises (a) cultivating a lipid-containing microorganism using methods disclosed herein, (b) lysing the lipid- containing microorganism to produce a lysate, (c) isolating lipid from the lysate, and (d) treating the lipid composition, whereby jet fuel is produced. In one embodiment of the method for producing a jet fuel, the lipid composition can be flowed through a fluid catalytic cracking zone, which, in one embodiment, may comprise contacting the lipid composition with a cracking catalyst at cracking conditions to provide a product stream comprising C2-C5 olefins.

[0406] In certain embodiments of this method, it may be desirable to remove any contaminants that may be present in the lipid composition. Thus, prior to flowing the lipid composition through a fluid catalytic cracking zone, the lipid composition is pretreated.

Pretreatment may involve contacting the lipid composition with an ion-exchange resin. The ion exchange resin is an acidic ion exchange resin, such as Amberlyst™-15 and can be used as a bed in a reactor through which the lipid composition is flowed, either upflow or downflow. Other pretreatments may include mild acid washes by contacting the lipid composition with an acid, such as sulfuric, acetic, nitric, or hydrochloric acid. Contacting is done with a dilute acid solution usually at ambient temperature and atmospheric pressure.

[0407] The lipid composition, optionally pretreated, is flowed to an FCC zone where the hydrocarbonaceous components are cracked to olefins. Catalytic cracking is accomplished by contacting the lipid composition in a reaction zone with a catalyst composed of finely divided particulate material. The reaction is catalytic cracking, as opposed to hydrocracking, and is carried out in the absence of added hydrogen or the consumption of hydrogen. As the cracking reaction proceeds, substantial amounts of coke are deposited on the catalyst. The catalyst is regenerated at high temperatures by burning coke from the catalyst in a regeneration zone. Coke-containing catalyst, referred to herein as "coked catalyst", is continually transported from the reaction zone to the regeneration zone to be regenerated and replaced by essentially coke-free regenerated catalyst from the regeneration zone.

Fluidization of the catalyst particles by various gaseous streams allows the transport of catalyst between the reaction zone and regeneration zone. Methods for cracking

hydrocarbons, such as those of the lipid composition described herein, in a fluidized stream of catalyst, transporting catalyst between reaction and regeneration zones, and combusting coke in the regenerator are well known by those skilled in the art of FCC processes. Exemplary FCC applications and catalysts useful for cracking the lipid composition to produce C2-C5 olefins are described in U.S. Pat. Nos. 6,538,169, 7,288,685, which are incorporated in their entirety by reference.

[0408] Suitable FCC catalysts generally comprise at least two components that may or may not be on the same matrix. In some embodiments, both two components may be circulated throughout the entire reaction vessel. The first component generally includes any of the well- known catalysts that are used in the art of fluidized catalytic cracking, such as an active amorphous clay-type catalyst and/or a high activity, crystalline molecular sieve. Molecular sieve catalysts may be preferred over amorphous catalysts because of their much-improved selectivity to desired products. IN some preferred embodiments, zeolites may be used as the molecular sieve in the FCC processes. Preferably, the first catalyst component comprises a large pore zeolite, such as an Y-type zeolite, an active alumina material, a binder material, comprising either silica or alumina and an inert filler such as kaolin.

[0409] In one embodiment, cracking the lipid composition of the present invention, takes place in the riser section or, alternatively, the lift section, of the FCC zone. The lipid composition is introduced into the riser by a nozzle resulting in the rapid vaporization of the lipid composition. Before contacting the catalyst, the lipid composition will ordinarily have a temperature of about 149°C to about 316°C (300°F to 600°F). The catalyst is flowed from a blending vessel to the riser where it contacts the lipid composition for a time of abort 2 seconds or less.

[0410] The blended catalyst and reacted lipid composition vapors are then discharged from the top of the riser through an outlet and separated into a cracked product vapor stream including olefins and a collection of catalyst particles covered with substantial quantities of coke and generally referred to as "coked catalyst." In an effort to minimize the contact time of the lipid composition and the catalyst which may promote further conversion of desired products to undesirable other products, any arrangement of separators such as a swirl arm arrangement can be used to remove coked catalyst from the product stream quickly. The separator, e.g. swirl arm separator, is located in an upper portion of a chamber with a stripping zone situated in the lower portion of the chamber. Catalyst separated by the swirl arm arrangement drops down into the stripping zone. The cracked product vapor stream comprising cracked hydrocarbons including light olefins and some catalyst exit the chamber via a conduit which is in communication with cyclones. The cyclones remove remaining catalyst particles from the product vapor stream to reduce particle concentrations to very low levels. The product vapor stream then exits the top of the separating vessel. Catalyst separated by the cyclones is returned to the separating vessel and then to the stripping zone. The stripping zone removes adsorbed hydrocarbons from the surface of the catalyst by counter-current contact with steam.

[0411] Low hydrocarbon partial pressure operates to favor the production of light olefins. Accordingly, the riser pressure is set at about 172 to 241 kPa (25 to 35 psia) with a hydrocarbon partial pressure of about 35 to 172 kPa (5 to 25 psia), with a preferred hydrocarbon partial pressure of about 69 to 138 kPa (10 to 20 psia). This relatively low partial pressure for hydrocarbon is achieved by using steam as a diluent to the extent that the diluent is 10 to 55 wt- of lipid composition and preferably about 15 wt- of lipid composition. Other diluents such as dry gas can be used to reach equivalent hydrocarbon partial pressures.

[0412] The temperature of the cracked stream at the riser outlet will be about 510°C to 621°C (950°F to 1150°F). However, riser outlet temperatures above 566°C (1050°F) make more dry gas and more olefins. Whereas, riser outlet temperatures below 566°C (1050°F) make less ethylene and propylene. Accordingly, it is preferred to run the FCC process at a preferred temperature of about 566°C to about 630°C, preferred pressure of about 138 kPa to about 240 kPa (20 to 35 psia). Another condition for the process is the catalyst to lipid composition ratio which can vary from about 5 to about 20 and preferably from about 10 to about 15.

[0413] In one embodiment of the method for producing a jet fuel, the lipid composition is introduced into the lift section of an FCC reactor. The temperature in the lift section will be very hot and range from about 700°C (1292°F) to about 760°C (1400°F) with a catalyst to lipid composition ratio of about 100 to about 150. It is anticipated that introducing the lipid composition into the lift section will produce considerable amounts of propylene and ethylene.

[0414] In another embodiment of the method for producing a jet fuel using the lipid composition or the lipids produced as described herein, the structure of the lipid composition or the lipids is broken by a process referred to as hydrodeoxygenation (HDO). HDO means removal of oxygen by means of hydrogen, that is, oxygen is removed while breaking the structure of the material. Olefinic double bonds are hydrogenated and any sulphur and nitrogen compounds are removed. Sulphur removal is called hydrodesulphurization (HDS). Pretreatment and purity of the raw materials (lipid composition or the lipids) contribute to the service life of the catalyst. [0415] Generally in the HDO/HDS step, hydrogen is mixed with the feed stock (lipid composition or the lipids) and then the mixture is passed through a catalyst bed as a co- current flow, either as a single phase or a two phase feed stock. After the HDO/MDS step, the product fraction is separated and passed to a separate isomerzation reactor. An isomerization reactor for biological starting material is described in the literature (FI 100 248) as a co- current reactor.

[0416] The process for producing a fuel by hydrogenating a hydrocarbon feed, e.g., the lipid composition or the lipids herein, can also be performed by passing the lipid composition or the lipids as a co-current flow with hydrogen gas through a first hydrogenation zone, and thereafter the hydrocarbon effluent is further hydrogenated in a second hydrogenation zone by passing hydrogen gas to the second hydrogenation zone as a counter-current flow relative to the hydrocarbon effluent. Exemplary HDO applications and catalysts useful for cracking the lipid composition to produce C2-C5 olefins are described in U.S. Pat. No. 7,232,935, which is incorporated in its entirety by reference.

[0417] Typically, in the hydrodeoxygenation step, the structure of the biological component, such as the lipid composition or lipids herein, is decomposed, oxygen, nitrogen, phosphorus and sulphur compounds, and light hydrocarbons as gas are removed, and the olefinic bonds are hydrogenated. In the second step of the process, i.e. in the so-called isomerization step, isomerzation is carried out for branching the hydrocarbon chain and improving the performance of the paraffin at low temperatures.

[0418] In the first step, i.e. HDO step, of the cracking process, hydrogen gas and the lipid composition or lipids herein which are to be hydrogenated are passed to a HDO catalyst bed system either as co-current or counter-current flows, said catalyst bed system comprising one or more catalyst bed(s), preferably 1-3 catalyst beds. The HDO step is typically operated in a co-current manner. In case of a HDO catalyst bed system comprising two or more catalyst beds, one or more of the beds may be operated using the counter-current flow principle. In the HDO step, the pressure varies between 20 and 150 bar, preferably between 50 and 100 bar, and the temperature varies between 200 and 500°C, preferably in the range of 300-400°C. In the HDO step, known hydrogenation catalysts containing metals from Group VII and/or VIB of the Periodic System may be used. Preferably, the hydrogenation catalysts are supported Pd, Pt, Ni, NiMo or a C0M0 catalysts, the support being alumina and/or silica. Typically, NiMo/Al 2 0 3 and CoMo/Al 2 0 3 catalysts are used.

[0419] Prior to the HDO step, the lipid composition or lipids herein may optionally be treated by prehydrogenation under milder conditions thus avoiding side reactions of the double bonds. Such prehydrogenation is carried out in the presence of a prehydrogenation catalyst at temperatures of 50-400°C and at hydrogen pressures of 1-200 bar, preferably at a temperature between 150 and 250°C and at a hydrogen pressure between 10 and 100 bar. The catalyst may contain metals from Group VIII and/or VIB of the Periodic System. Preferably, the prehydrogenation catalyst is a supported Pd, Pt, Ni, NiMo or a CoMo catalyst, the support being alumina and/or silica.

[0420] A gaseous stream from the HDO step containing hydrogen is cooled and then carbon monoxide, carbon dioxide, nitrogen, phosphorus and sulphur compounds, gaseous light hydrocarbons and other impurities are removed therefrom. After compressing, the purified hydrogen or recycled hydrogen is returned back to the first catalyst bed and/or between the catalyst beds to make up for the withdrawn gas stream. Water is removed from the condensed liquid. The liquid is passed to the first catalyst bed or between the catalyst beds.

[0421] After the HDO step, the product is subjected to an isomerization step. It is substantial for the process that the impurities are removed as completely as possible before the hydrocarbons are contacted with the isomerization catalyst. The isomerization step comprises an optional stripping step, wherein the reaction product from the HDO step may be purified by stripping with water vapour or a suitable gas such as light hydrocarbon, nitrogen or hydrogen. The optional stripping step is carried out in counter-current manner in a unit upstream of the isomerization catalyst, wherein the gas and liquid are contacted with each other, or before the actual isomerization reactor in a separate stripping unit utilizing counter- current principle.

[0422] After the stripping step the hydrogen gas and the hydrogenated lipid composition or lipids herein, and optionally an n-paraffin mixture, are passed to a reactive isomerization unit comprising one or several catalyst bed(s). The catalyst beds of the isomerization step may operate either in co-current or counter-current manner.

[0423] It is important for the process that the counter-current flow principle is applied in the isomerization step. In the isomerization step this is done by carrying out either the optional stripping step or the isomerization reaction step or both in counter-current manner. In the isomerzation step, the pressure varies in the range of 20-150 bar, preferably in the range of 20-100 bar, the temperature being between 200 and 500°C, preferably between 300 and 400°C. In the isomerization step, isomerization catalysts known in the art may be used. Suitable isomerization catalysts contain molecular sieve and/or a metal from Group VII and/or a carrier. Preferably, the isomerization catalyst contains SAPO-11 or SAP041 or ZSM-22 or ZSM-23 or ferrierite and Pt, Pd or Ni and AI2O 3 or Si0 2 . Typical isomerization catalysts are, for example, Pt/SAPO-l l/Al 2 0 3 , Pt/ZSM-22/Al 2 0 3 , Pt/ZSM-23/Al 2 0 3 and Pt/SAPO-l l/Si0 2 . The isomerization step and the HDO step may be carried out in the same pressure vessel or in separate pressure vessels. Optional prehydrogenation may be carried out in a separate pressure vessel or in the same pressure vessel as the HDO and isomerization steps.

[0424] Thus, in one embodiment, the product of one or more chemical reactions is an alkane mixture that comprises HRJ-5. In another embodiment, the product of the one or more chemical reactions is an alkane mixture that comprises ASTM D1655 jet fuel. In some embodiments, the composition comforming to the specification of ASTM 1655 jet fuel has a sulfur content that is less than 10 ppm. In other embodiments, the composition conforming to the specification of ASTM 1655 jet fuel has a T10 value of the distillation curve of less than 205° C. In another embodiment, the composition conforming to the specification of ASTM 1655 jet fuel has a final boiling point (FBP) of less than 300° C. In another embodiment, the composition conforming to the specification of ASTM 1655 jet fuel has a flash point of at least 38° C. In another embodiment, the composition conforming to the specification of ASTM 1655 jet fuel has a density between 775 K/M 3 and 840K/M 3 . In yet another embodiment, the composition conforming to the specification of ASTM 1655 jet fuel has a freezing point that is below -47° C. In another embodiment, the composition conforming to the specification of ASTM 1655 jet fuel has a net Heat of Combustion that is at least 42.8 MJ/K. In another embodiment, the composition conforming to the specification of ASTM 1655 jet fuel has a hydrogen content that is at least 13.4 mass . In another embodiment, the composition conforming to the specification of ASTM 1655 jet fuel has a thermal stability, as tested by quantitative gravimetric JFTOT at 260° C, that is below 3mm of Hg. In another embodiment, the composition conforming to the specification of ASTM 1655 jet fuel has an existent gum that is below 7 mg/dl.

[0425] Thus, the present invention discloses a variety of methods in which chemical modification of microalgal lipid is undertaken to yield products useful in a variety of industrial and other applications. Examples of processes for modifying oil produced by the methods disclosed herein include, but are not limited to, hydrolysis of the oil,

hydroprocessing of the oil, and esterification of the oil. Other chemical modification of microalgal lipid include, without limitation, epoxidation, oxidation, hydrolysis, sulfations, sulfonation, ethoxylation, propoxylation, amidation, and saponification. The modification of the microalgal oil produces basic oleochemicals that can be further modified into selected derivative oleochemicals for a desired function. In a manner similar to that described above with reference to fuel producing processes, these chemical modifications can also be performed on oils generated from the microbial cultures described herein. Examples of basic oleochemicals include, but are not limited to, soaps, fatty acids, fatty esters, fatty alcohols, fatty nitrogen compounds, fatty acid methyl esters, and glycerol. Examples of derivative oleochemicals include, but are not limited to, fatty nitriles, esters, dimer acids, quats, surfactants, fatty alkanolamides, fatty alcohol sulfates, resins, emulsifiers, fatty alcohols, olefins, drilling muds, polyols, polyurethanes, polyacrylates, rubber, candles, cosmetics, metallic soaps, soaps, alpha-sulphonated methyl esters, fatty alcohol sulfates, fatty alcohol ethoxylates, fatty alcohol ether sulfates, imidazolines, surfactants, detergents, esters, quats, ozonolysis products, fatty amines, fatty alkanolamides, ethoxy sulfates, monoglycerides, diglycerides, triglycerides (including medium chain triglycerides), lubricants, hydraulic fluids, greases, dielectric fluids, mold release agents, metal working fluids, heat transfer fluids, other functional fluids, industrial chemicals (e.g., cleaners, textile processing aids, plasticizers, stabilizers, additives), surface coatings, paints and lacquers, electrical wiring insulation, and higher alkanes.

[0426] Hydrolysis of the fatty acid constituents from the glycerolipids produced by the methods of the invention yields free fatty acids that can be derivatized to produce other useful chemicals. Hydrolysis occurs in the presence of water and a catalyst which may be either an acid or a base. The liberated free fatty acids can be derivatized to yield a variety of products, as reported in the following: US Patent Nos. 5,304,664 (Highly sulfated fatty acids);

7,262,158 (Cleansing compositions); 7,115,173 (Fabric softener compositions); 6,342,208 (Emulsions for treating skin); 7,264,886 (Water repellant compositions); 6,924,333 (Paint additives); 6,596,768 (Lipid-enriched ruminant feedstock); and 6,380,410 (Surfactants for detergents and cleaners).

[0427] With regard to hydrolysis, in one embodiment of the invention, a triglyceride oil is optionally first hydrolyzed in a liquid medium such as water or sodium hydroxide so as to obtain glycerol and soaps. There are various suitable triglyceride hydrolysis methods, including, but not limited to, saponification, acid hydrolysis, alkaline hydrolysis, enzymatic hydrolysis (referred herein as splitting), and hydrolysis using hot-compressed water. One skilled in the art will recognize that a triglyceride oil need not be hydrolyzed in order to produce an oleochemical; rather, the oil may be converted directly to the desired

oleochemical by other known process. For example, the triglyceride oil may be directly converted to a methyl ester fatty acid through esterification. [0428] In some embodiments, catalytic hydrolysis of the oil produced by methods disclosed herein occurs by splitting the oil into glycerol and fatty acids. As discussed above, the fatty acids may then be further processed through several other modifications to obtained derivative oleochemicals. For example, in one embodiment the fatty acids may undergo an amination reaction to produce fatty nitrogen compounds. In another embodiment, the fatty acids may undergo ozonolysis to produce mono- and dibasic-acids.

[0429] In other embodiments hydrolysis may occur via the, splitting of oils produced herein to create oleochemicals. In some preferred embodiments of the invention, a triglyceride oil may be split before other processes is performed. One skilled in the art will recognize that there are many suitable triglyceride splitting methods, including, but not limited to, enzymatic splitting and pressure splitting.

[0430] Generally, enzymatic oil splitting methods use enzymes, lipases, as biocatalysts acting on a water/oil mixture. Enzymatic splitting then slpits the oil or fat, respectively, is into glycerol and free fatty acids. The glycerol may then migrates into the water phase whereas the organic phase enriches with free fatty acids.

[0431] The enzymatic splitting reactions generally take place at the phase boundary between organic and aqueous phase, where the enzyme is present only at the phase boundary. Triglycerides that meet the phase boundary then contribute to or participate in the splitting reaction. As the reaction proceeds, the occupation density or concentration of fatty acids still chemically bonded as glycerides, in comparison to free fatty acids, decreases at the phase boundary so that the reaction is slowed down. In certain embodiments, enzymatic splitting may occur at room temperature. One of ordinary skill in the art would know the suitable conditions for splitting oil into the desired fatty acids.

[0432] By way of example, the reaction speed can be accelerated by increasing the interface boundary surface. Once the reaction is complete, free fatty acids are then separated from the organic phase freed from enzyme, and the residue which still contains fatty acids chemically bonded as glycerides is fed back or recycled and mixed with fresh oil or fat to be subjected to splitting. In this manner, recycled glycerides are then subjected to a further enzymatic splitting process. In some embodiments, the free fatty acids are extracted from an oil or fat partially split in such a manner. In that way, if the chemically bound fatty acids (triglycerides) are returned or fed back into the splitting process, the enzyme consumption can be drastically reduced.

[0433] The splitting degree is determined as the ratio of the measured acid value divided by the theoretically possible acid value which can be computed for a given oil or fat. Preferably, the acid value is measured by means of titration according to standard common methods. Alternatively, the density of the aqueous glycerol phase can be taken as a measure for the splitting degree.

[0434] In one embodiment, the slitting process as described herein is also suitable for splitting the mono-, di- and triglyceride that are contained in the so-called soap-stock from the alkali refining processes of the produced oils. In this manner, the soap-stock can be quantitatively converted without prior saponification of the neutral oils into the fatty acids. For this purpose, the fatty acids being chemically bonded in the soaps are released, preferably before splitting, through an addition of acid. In certain embodiments, a buffer solution is used in addition to water and enzyme for the splitting process.

[0435] In one embodiment, oils produced in accordance with the methods of the invention can also be subjected to saponification as a method of hydrolysis. Animal and plant oils are typically made of triacylglycerols (TAGs), which are esters of fatty acids with the trihydric alcohol, glycerol. In an alkaline hydrolysis reaction, the glycerol in a TAG is removed, leaving three carboxylic acid anions that can associate with alkali metal cations such as sodium or potassium to produce fatty acid salts. In this scheme, the carboxylic acid constituents are cleaved from the glycerol moiety and replaced with hydroxyl groups. The quantity of base (e.g., KOH) that is used in the reaction is determined by the desired degree of saponification. If the objective is, for example, to produce a soap product that comprises some of the oils originally present in the TAG composition, an amount of base insufficient to convert all of the TAGs to fatty acid salts is introduced into the reaction mixture. Normally, this reaction is performed in an aqueous solution and proceeds slowly, but may be expedited by the addition of heat. Precipitation of the fatty acid salts can be facilitated by addition of salts, such as water-soluble alkali metal halides (e.g., NaCl or KC1), to the reaction mixture. Preferably, the base is an alkali metal hydroxide, such as NaOH or KOH. Alternatively, other bases, such as alkanolamines, including for example triethanolamine and

aminomethylpropanol, can be used in the reaction scheme. In some cases, these alternatives may be preferred to produce a clear soap product. In one embodiment the lipid composition subjected to saponification is a tallow mimetic (i.e., lipid composition similar to that of tallow) produced as described herein, or a blend of a tallow mimetic with another triglyceride oil.

[0436] In some methods, the first step of chemical modification may be hydroprocessing to saturate double bonds, followed by deoxygenation at elevated temperature in the presence of hydrogen and a catalyst. In other methods, hydrogenation and deoxygenation may occur in the same reaction. In still other methods deoxygenation occurs before hydrogenation.

Isomerization may then be optionally performed, also in the presence of hydrogen and a catalyst. Finally, gases and naphtha components can be removed if desired. For example, see U.S. Patents 5,475,160 (hydrogenation of triglycerides); 5,091,116 (deoxygenation, hydrogenation and gas removal); 6,391,815 (hydrogenation); and 5,888,947 (isomerization).

[0437] In some embodiments of the invention, the triglyceride oils are partially or completely deoxygenated. The deoxygenation reactions form desired products, including, but not limited to, fatty acids, fatty alcohols, polyols, ketones, and aldehydes. In general, without being limited by any particular theory, the deoxygenation reactions involve a combination of various different reaction pathways, including without limitation:

hydrogenolysis, hydrogenation, consecutive hydrogenation-hydrogenolysis, consecutive hydrogenolysis-hydrogenation, and combined hydrogenation-hydrogenolysis reactions, resulting in at least the partial removal of oxygen from the fatty acid or fatty acid ester to produce reaction products, such as fatty alcohols, that can be easily converted to the desired chemicals by further processing. For example, in one embodiment, a fatty alcohol may be converted to olefins through FCC reaction or to higher alkanes through a condensation reaction.

[0438] One such chemical modification is hydrogenation, which is the addition of hydrogen to double bonds in the fatty acid constituents of glycerolipids or of free fatty acids. The hydrogenation process permits the transformation of liquid oils into semi-solid or solid fats, which may be more suitable for specific applications.

[0439] Hydrogenation of oil produced by the methods described herein can be performed in conjunction with one or more of the methods and/or materials provided herein, as reported in the following: US Patent Nos. 7,288,278 (Food additives or medicaments); 5,346,724 (Lubrication products); 5,475,160 (Fatty alcohols); 5,091,116 (Edible oils); 6,808,737 (Structural fats for margarine and spreads); 5,298,637 (Reduced-calorie fat substitutes); 6,391,815 (Hydrogenation catalyst and sulfur adsorbent); 5,233,099 and 5,233,100 (Fatty alcohols); 4,584,139 (Hydrogenation catalysts); 6,057,375 (Foam suppressing agents); and 7,118,773 (Edible emulsion spreads).

[0440] One skilled in the art will recognize that various processes may be used to hydrogenate carbohydrates. One suitable method includes contacting the carbohydrate with hydrogen or hydrogen mixed with a suitable gas and a catalyst under conditions sufficient in a hydrogenation reactor to form a hydrogenated product. The hydrogenation catalyst generally can include Cu, Re, Ni, Fe, Co, Ru, Pd, Rh, Pt, Os, Ir, and alloys or any combination thereof, either alone or with promoters such as W, Mo, Au, Ag, Cr, Zn, Mn, Sn, B, P, Bi, and alloys or any combination thereof. Other effective hydrogenation catalyst materials include either supported nickel or ruthenium modified with rhenium. In an embodiment, the hydrogenation catalyst also includes any one of the supports, depending on the desired functionality of the catalyst. The hydrogenation catalysts may be prepared by methods known to those of ordinary skill in the art.

[0441] In some embodiments the hydrogenation catalyst includes a supported Group VIII metal catalyst and a metal sponge material (e.g., a sponge nickel catalyst). Raney nickel provides an example of an activated sponge nickel catalyst suitable for use in this invention. In other embodiment, the hydrogenation reaction in the invention is performed using a catalyst comprising a nickel-rhenium catalyst or a tungsten-modified nickel catalyst. One example of a suitable catalyst for the hydrogenation reaction of the invention is a carbon- supported nickel-rhenium catalyst.

[0442] In an embodiment, a suitable Raney nickel catalyst may be prepared by treating an alloy of approximately equal amounts by weight of nickel and aluminum with an aqueous alkali solution, e.g., containing about 25 weight % of sodium hydroxide. The aluminum is selectively dissolved by the aqueous alkali solution resulting in a sponge shaped material comprising mostly nickel with minor amounts of aluminum. The initial alloy includes promoter metals (i.e., molybdenum or chromium) in the amount such that about 1 to 2 weight % remains in the formed sponge nickel catalyst. In another embodiment, the hydrogenation catalyst is prepared using a solution of ruthenium(III) nitrosylnitrate, ruthenium (III) chloride in water to impregnate a suitable support material. The solution is then dried to form a solid having a water content of less than about 1% by weight. The solid may then be reduced at atmospheric pressure in a hydrogen stream at 300°C (uncalcined) or 400°C (calcined) in a rotary ball furnace for 4 hours. After cooling and rendering the catalyst inert with nitrogen, 5% by volume of oxygen in nitrogen is passed over the catalyst for 2 hours.

[0443] In certain embodiments, the catalyst described includes a catalyst support. The catalyst support stabilizes and supports the catalyst. The type of catalyst support used depends on the chosen catalyst and the reaction conditions. Suitable supports for the invention include, but are not limited to, carbon, silica, silica-alumina, zirconia, titania, ceria, vanadia, nitride, boron nitride, heteropoly acids, hydroxyapatite, zinc oxide, chromia, zeolites, carbon nanotubes, carbon fullerene and any combination thereof.

[0444] The catalysts used in this invention can be prepared using conventional methods known to those in the art. Suitable methods may include, but are not limited to, incipient wetting, evaporative impregnation, chemical vapor deposition, wash-coating, magnetron sputtering techniques, and the like.

[0445] The conditions for which to carry out the hydrogenation reaction will vary based on the type of starting material and the desired products. One of ordinary skill in the art, with the benefit of this disclosure, will recognize the appropriate reaction conditions. In general, the hydrogenation reaction is conducted at temperatures of 80°C to 250°C, and preferably at 90°C to 200°C, and most preferably at 100°C to 150°C. In some embodiments, the hydrogenation reaction is conducted at pressures from 500 KPa to 14000 KPa.

[0446] The hydrogen used in the hydrogenolysis reaction of the current invention may include external hydrogen, recycled hydrogen, in situ generated hydrogen, and any combination thereof. As used herein, the term "external hydrogen" refers to hydrogen that does not originate from the biomass reaction itself, but rather is added to the system from another source.

[0447] In some embodiments of the invention, it is desirable to convert the starting carbohydrate to a smaller molecule that will be more readily converted to desired higher hydrocarbons. One suitable method for this conversion is through a hydrogenolysis reaction. Various processes are known for performing hydrogenolysis of carbohydrates. One suitable method includes contacting a carbohydrate with hydrogen or hydrogen mixed with a suitable gas and a hydrogenolysis catalyst in a hydrogenolysis reactor under conditions sufficient to form a reaction product comprising smaller molecules or polyols. As used herein, the term "smaller molecules or polyols" includes any molecule that has a smaller molecular weight, which can include a smaller number of carbon atoms or oxygen atoms than the starting carbohydrate. In an embodiment, the reaction products include smaller molecules that include polyols and alcohols. Someone of ordinary skill in the art would be able to choose the appropriate method by which to carry out the hydrogenolysis reaction.

[0448] In some embodiments, a 5 and/or 6 carbon sugar or sugar alcohol may be converted to propylene glycol, ethylene glycol, and glycerol using a hydrogenolysis catalyst. The hydrogenolysis catalyst may include Cr, Mo, W, Re, Mn, Cu, Cd, Fe, Co, Ni, Pt, Pd, Rh, Ru, Ir, Os, and alloys or any combination thereof, either alone or with promoters such as Au, Ag, Cr, Zn, Mn, Sn, Bi, B, O, and alloys or any combination thereof. The hydrogenolysis catalyst may also include a carbonaceous pyropolymer catalyst containing transition metals (e.g., chromium, molybdemum, tungsten, rhenium, manganese, copper, cadmium) or Group VIII metals (e.g., iron, cobalt, nickel, platinum, palladium, rhodium, ruthenium, iridium, and osmium). In certain embodiments, the hydrogenolysis catalyst may include any of the above metals combined with an alkaline earth metal oxide or adhered to a catalytically active support. In certain embodiments, the catalyst described in the hydrogenolysis reaction may include a catalyst support as described above for the hydrogenation reaction.

[0449] The conditions for which to carry out the hydrogenolysis reaction will vary based on the type of starting material and the desired products. One of ordinary skill in the art, with the benefit of this disclosure, will recognize the appropriate conditions to use to carry out the reaction. In general, they hydrogenolysis reaction is conducted at temperatures of 110°C to 300°C, and preferably at 170°C to 220°C, and most preferably at 200°C to 225°C. In some embodiments, the hydrogenolysis reaction is conducted under basic conditions, preferably at a pH of 8 to 13, and even more preferably at a pH of 10 to 12. In some embodiments, the hydrogenolysis reaction is conducted at pressures in a range between 60 KPa and 16500 KPa, and preferably in a range between 1700 KPa and 14000 KPa, and even more preferably between 4800 KPa and 11000 KPa.

[0450] The hydrogen used in the hydrogenolysis reaction of the current invention can include external hydrogen, recycled hydrogen, in situ generated hydrogen, and any combination thereof.

[0451] In some embodiments, the reaction products discussed above may be converted into higher hydrocarbons through a condensation reaction in a condensation reactor. In such embodiments, condensation of the reaction products occurs in the presence of a catalyst capable of forming higher hydrocarbons. While not intending to be limited by theory, it is believed that the production of higher hydrocarbons proceeds through a stepwise addition reaction including the formation of carbon-carbon, or carbon-oxygen bond. The resulting reaction products include any number of compounds containing these moieties, as described in more detail below.

[0452] In certain embodiments, suitable condensation catalysts include an acid catalyst, a base catalyst, or an acid/base catalyst. As used herein, the term "acid/base catalyst" refers to a catalyst that has both an acid and a base functionality. In some embodiments the condensation catalyst can include, without limitation, zeolites, carbides, nitrides, zirconia, alumina, silica, aluminosilicates, phosphates, titanium oxides, zinc oxides, vanadium oxides, lanthanum oxides, yttrium oxides, scandium oxides, magnesium oxides, cerium oxides, barium oxides, calcium oxides, hydroxides, heteropolyacids, inorganic acids, acid modified resins, base modified resins, and any combination thereof. In some embodiments, the condensation catalyst can also include a modifier. Suitable modifiers include La, Y, Sc, P, B, Bi, Li, Na, K, Rb, Cs, Mg, Ca, Sr, Ba, and any combination thereof. In some embodiments, the condensation catalyst can also include a metal. Suitable metals include Cu, Ag, Au, Pt, Ni, Fe, Co, Ru, Zn, Cd, Ga, In, Rh, Pd, Ir, Re, Mn, Cr, Mo, W, Sn, Os, alloys, and any combination thereof.

[0453] In certain embodiments, the catalyst described in the condensation reaction may include a catalyst support as described above for the hydrogenation reaction. In certain embodiments, the condensation catalyst is self-supporting. As used herein, the term "self- supporting" means that the catalyst does not need another material to serve as support. In other embodiments, the condensation catalyst in used in conjunction with a separate support suitable for suspending the catalyst. In an embodiment, the condensation catalyst support is silica.

[0454] The conditions under which the condensation reaction occurs will vary based on the type of starting material and the desired products. One of ordinary skill in the art, with the benefit of this disclosure, will recognize the appropriate conditions to use to carry out the reaction. In some embodiments, the condensation reaction is carried out at a temperature at which the thermodynamics for the proposed reaction are favorable. The temperature for the condensation reaction will vary depending on the specific starting polyol or alcohol. In some embodiments, the temperature for the condensation reaction is in a range from 80°C to 500°C, and preferably from 125°C to 450°C, and most preferably from 125°C to 250°C. In some embodiments, the condensation reaction is conducted at pressures in a range between 0 Kpa to 9000 KPa, and preferably in a range between 0 KPa and 7000 KPa, and even more preferably between 0 KPa and 5000 KPa.

[0455] The higher alkanes formed by the invention include, but are not limited to, branched or straight chain alkanes that have from 4 to 30 carbon atoms, branched or straight chain alkenes that have from 4 to 30 carbon atoms, cycloalkanes that have from 5 to 30 carbon atoms, cycloalkenes that have from 5 to 30 carbon atoms, aryls, fused aryls, alcohols, and ketones. Suitable alkanes include, but are not limited to, butane, pentane, pentene, 2- methylbutane, hexane, hexene, 2-methylpentane, 3-methylpentane, 2,2,-dimethylbutane, 2,3- dimethylbutane, heptane, heptene, octane, octene, 2,2,4-trimethylpentane, 2,3-dimethyl hexane, 2,3,4-trimethylpentane, 2,3-dimethylpentane, nonane, nonene, decane, decene, undecane, undecene, dodecane, dodecene, tridecane, tridecene, tetradecane, tetradecene, pentadecane, pentadecene, nonyldecane, nonyldecene, eicosane, eicosene, uneicosane, uneicosene, doeicosane, doeicosene, trieicosane, trieicosene, tetraeicosane, tetraeicosene, and isomers thereof. Some of these products may be suitable for use as fuels. [0456] In some embodiments, the cycloalkanes and the cycloalkenes are unsubstituted. In other embodiments, the cycloalkanes and cycloalkenes are mono-substituted. In still other embodiments, the cycloalkanes and cycloalkenes are multi-substituted. In the embodiments comprising the substituted cycloalkanes and cycloalkenes, the substituted group includes, without limitation, a branched or straight chain alkyl having 1 to 12 carbon atoms, a branched or straight chain alkylene having 1 to 12 carbon atoms, a phenyl, and any combination thereof. Suitable cycloalkanes and cycloalkenes include, but are not limited to, cyclopentane, cyclopentene, cyclohexane, cyclohexene, methyl-cyclopentane, methyl-cyclopentene, ethyl- cyclopentane, ethyl-cyclopentene, ethyl-cyclohexane, ethyl-cyclohexene, isomers and any combination thereof.

[0457] In some embodiments, the aryls formed are unsubstituted. In another embodiment, the aryls formed are mono-substituted. In the embodiments comprising the substituted aryls, the substituted group includes, without limitation, a branched or straight chain alkyl having 1 to 12 carbon atoms, a branched or straight chain alkylene having 1 to 12 carbon atoms, a phenyl, and any combination thereof. Suitable aryls for the invention include, but are not limited to, benzene, toluene, xylene, ethyl benzene, para xylene, meta xylene, and any combination thereof.

[0458] The alcohols produced in the invention have from 4 to 30 carbon atoms. In some embodiments, the alcohols are cyclic. In other embodiments, the alcohols are branched. In another embodiment, the alcohols are straight chained. Suitable alcohols for the invention include, but are not limited to, butanol, pentanol, hexanol, heptanol, octanol, nonanol, decanol, undecanol, dodecanol, tridecanol, tetradecanol, pentadecanol, hexadecanol, heptyldecanol, octyldecanol, nonyldecanol, eicosanol, uneicosanol, doeicosanol, trieicosanol, tetraeicosanol, and isomers thereof.

[0459] The ketones produced in the invention have from 4 to 30 carbon atoms. In an embodiment, the ketones are cyclic. In another embodiment, the ketones are branched. In another embodiment, the ketones are straight chained. Suitable ketones for the invention include, but are not limited to, butanone, pentanone, hexanone, heptanone, octanone, nonanone, decanone, undecanone, dodecanone, tridecanone, tetradecanone, pentadecanone, hexadecanone, heptyldecanone, octyldecanone, nonyldecanone, eicosanone, uneicosanone, doeicosanone, trieicosanone, tetraeicosanone, and isomers thereof.

[0460] Another such chemical modification is interesterification. Naturally produced glycerolipids do not have a uniform distribution of fatty acid constituents. In the context of oils, interesterification refers to the exchange of acyl radicals between two esters of different glycerolipids. The interesterification process provides a mechanism by which the fatty acid constituents of a mixture of glycerolipids can be rearranged to modify the distribution pattern. Interesterification is a well-known chemical process, and generally comprises heating (to about 200°C) a mixture of oils for a period (e.g, 30 minutes) in the presence of a catalyst, such as an alkali metal or alkali metal alkylate (e.g., sodium methoxide). This process can be used to randomize the distribution pattern of the fatty acid constituents of an oil mixture, or can be directed to produce a desired distribution pattern. This method of chemical modification of lipids can be performed on materials provided herein, such as microbial biomass with a percentage of dry cell weight as lipid at least 20%.

[0461] Directed interesterification, in which a specific distribution pattern of fatty acids is sought, can be performed by maintaining the oil mixture at a temperature below the melting point of some TAGs which might occur. This results in selective crystallization of these TAGs, which effectively removes them from the reaction mixture as they crystallize. The process can be continued until most of the fatty acids in the oil have precipitated, for example. A directed interesterification process can be used, for example, to produce a product with a lower calorie content via the substitution of longer-chain fatty acids with shorter-chain counterparts. Directed interesterification can also be used to produce a product with a mixture of fats that can provide desired melting characteristics and structural features sought in food additives or products (e.g., margarine) without resorting to hydrogenation, which can produce unwanted trans isomers.

[0462] Interesterification of oils produced by the methods described herein can be performed in conjuction with one or more of the methods and/or materials, or to produce products, as reported in the following: US Patent Nos. 6,080,853 (Nondigestible fat substitutes); 4,288,378 (Peanut butter stabilizer); 5,391,383 (Edible spray oil); 6,022,577 (Edible fats for food products); 5,434,278 (Edible fats for food products); 5,268,192 (Low calorie nut products); 5,258,197 (Reduce calorie edible compositions); 4,335,156 (Edible fat product); 7,288,278 (Food additives or medicaments); 7,115,760 (Fractionation process); 6,808,737 (Structural fats); 5,888,947 (Engine lubricants); 5,686,131 (Edible oil mixtures); and 4,603,188 (Curable urethane compositions).

[0463] In one embodiment in accordance with the invention, transesterification of the oil, as described above, is followed by reaction of the transesterified product with polyol, as reported in US Patent No. 6,465,642, to produce polyol fatty acid polyesters. Such an esterification and separation process may comprise the steps as follows: reacting a lower alkyl ester with polyol in the presence of soap; removing residual soap from the product mixture; water- washing and drying the product mixture to remove impurities; bleaching the product mixture for refinement; separating at least a portion of the unreacted lower alkyl ester from the polyol fatty acid polyester in the product mixture; and recycling the separated unreacted lower alkyl ester.

[0464] Transesterification can also be performed on microbial biomass with short chain fatty acid esters, as reported in U.S. Patent 6,278,006. In general, transesterification may be performed by adding a short chain fatty acid ester to an oil in the presence of a suitable catalyst and heating the mixture. In some embodiments, the oil comprises about 5% to about 90% of the reaction mixture by weight. In some embodiments, the short chain fatty acid esters can be about 10% to about 50% of the reaction mixture by weight. Non-limiting examples of catalysts include base catalysts, sodium methoxide, acid catalysts including inorganic acids such as sulfuric acid and acidified clays, organic acids such as methane sulfonic acid, benzenesulfonic acid, and toluenesulfonic acid, and acidic resins such as Amberlyst 15. Metals such as sodium and magnesium, and metal hydrides also are useful catalysts.

[0465] Another such chemical modification is hydroxylation, which involves the addition of water to a double bond resulting in saturation and the incorporation of a hydroxyl moiety. The hydroxylation process provides a mechanism for converting one or more fatty acid constituents of a glycerolipid to a hydroxy fatty acid. Hydroxylation can be performed, for example, via the method reported in US Patent No. 5,576,027. Hydroxylated fatty acids, including castor oil and its derivatives, are useful as components in several industrial applications, including food additives, surfactants, pigment wetting agents, defoaming agents, water proofing additives, plasticizing agents, cosmetic emulsifying and/or deodorant agents, as well as in electronics, pharmaceuticals, paints, inks, adhesives, and lubricants. One example of how the hydroxylation of a glyceride may be performed is as follows: fat may be heated, preferably to about 30-50°C combined with heptane and maintained at temperature for thirty minutes or more; acetic acid may then be added to the mixture followed by an aqueous solution of sulfuric acid followed by an aqueous hydrogen peroxide solution which is added in small increments to the mixture over one hour; after the aqueous hydrogen peroxide, the temperature may then be increased to at least about 60°C and stirred for at least six hours; after the stirring, the mixture is allowed to settle and a lower aqueous layer formed by the reaction may be removed while the upper heptane layer formed by the reaction may be washed with hot water having a temperature of about 60 °C; the washed heptane layer may then be neutralized with an aqueous potassium hydroxide solution to a pH of about 5 to 7 and then removed by distillation under vacuum; the reaction product may then be dried under vacuum at 100°C and the dried product steam-deodorized under vacuum conditions and filtered at about 50° to 60°C using diatomaceous earth.

[0466] Hydroxylation of microbial oils produced by the methods described herein can be performed in conjuction with one or more of the methods and/or materials, or to produce products, as reported in the following: US Patent Nos. 6,590,113 (Oil-based coatings and ink); 4,049,724 (Hydroxylation process); 6,113,971 (Olive oil butter); 4,992,189 (Lubricants and lube additives); 5,576,027 (Hydroxylated milk); and 6,869,597 (Cosmetics).

[0467] Hydroxylated glycerolipids can be converted to estolides. Estolides consist of a glycerolipid in which a hydroxylated fatty acid constituent has been esterified to another fatty acid molecule. Conversion of hydroxylated glycerolipids to estolides can be carried out by warming a mixture of glycerolipids and fatty acids and contacting the mixture with a mineral acid, as described by Isbell et al., JAOCS 71(2): 169-174 (1994). Estolides are useful in a variety of applications, including without limitation those reported in the following: US Patent Nos. 7,196,124 (Elastomeric materials and floor coverings); 5,458,795 (Thickened oils for high-temperature applications); 5,451,332 (Fluids for industrial applications); 5,427,704 (Fuel additives); and 5,380,894 (Lubricants, greases, plasticizers, and printing inks).

[0468] Another such chemical modification is olefin metathesis. In olefin metathesis, a catalyst severs the alkylidene carbons in an alkene (olefin) and forms new alkenes by pairing each of them with different alkylidine carbons. The olefin metathesis reaction provides a mechanism for processes such as truncating unsaturated fatty acid alkyl chains at alkenes by ethenolysis, cross-linking fatty acids through alkene linkages by self-metathesis, and incorporating new functional groups on fatty acids by cross-metathesis with derivatized alkenes.

[0469] In conjunction with other reactions, such as transesterification and hydrogenation, olefin metathesis can transform unsaturated glycerolipids into diverse end products. These products include glycerolipid oligomers for waxes; short-chain glycerolipids for lubricants; homo- and hetero-bifunctional alkyl chains for chemicals and polymers; short-chain esters for biofuel; and short-chain hydrocarbons for jet fuel. Olefin metathesis can be performed on triacylglycerols and fatty acid derivatives, for example, using the catalysts and methods reported in U.S. Patent No. 7,119,216, US Patent Pub. No. 2010/0160506, and U.S. Patent Pub. No. 2010/0145086.

[0470] Olefin metathesis of bio-oils generally comprises adding a solution of Ru catalyst at a loading of about 10 to 250 ppm under inert conditions to unsaturated fatty acid esters in the presence (cross-metathesis) or absence (self-metathesis) of other alkenes. The reactions are typically allowed to proceed from hours to days and ultimately yield a distribution of alkene products. One example of how olefin metathesis may be performed on a fatty acid derivative is as follows: A solution of the first generation Grubbs Catalyst (dichloro[2(l-methylethoxy- a-0)phenyl]methylene-a-C] (tricyclohexyl-phosphine) in toluene at a catalyst loading of 222 ppm may be added to a vessel containing degassed and dried methyl oleate. Then the vessel may be pressurized with about 60 psig of ethylene gas and maintained at or below about 30°C for 3 hours, whereby approximately a 50% yield of methyl 9-decenoate may be produced.

[0471] Olefin metathesis of oils produced by the methods described herein can be performed in conjunction with one or more of the methods and/or materials, or to produce products, as reported in the following: Patent App. PCT/US07/081427 (a-olefin fatty acids) and U.S. Patent App. Nos. 12/281,938 (petroleum creams), 12/281,931 (paintball gun capsules), 12/653,742 (plasticizers and lubricants), 12/422,096 (bifunctional organic compounds), and 11/795,052 (candle wax).

[0472] Other chemical reactions that can be performed on microbial oils include reacting triacylglycerols with a cyclopropanating agent to enhance fluidity and/or oxidative stability, as reported in U.S. Patent 6,051,539; manufacturing of waxes from triacylglycerols, as reported in U.S. Patent 6,770,104; and epoxidation of triacylglycerols, as reported in "The effect of fatty acid composition on the acrylation kinetics of epoxidized triacylglycerols", Journal of the American Oil Chemists' Society, 79: 1, 59-63, (2001) and Free Radical Biology and Medicine, 37: 1, 104-114 (2004).

[0473] The generation of oil-bearing microbial biomass for fuel and chemical products as described above results in the production of delipidated biomass meal. Delipidated meal is a byproduct of preparing algal oil and is useful as animal feed for farm animals, e.g., ruminants, poultry, swine and aquaculture. The resulting meal, although of reduced oil content, still contains high quality proteins, carbohydrates, fiber, ash, residual oil and other nutrients appropriate for an animal feed. Because the cells are predominantly lysed by the oil separation process, the delipidated meal is easily digestible by such animals. Delipidated meal can optionally be combined with other ingredients, such as grain, in an animal feed. Because delipidated meal has a powdery consistency, it can be pressed into pellets using an extruder or expander or another type of machine, which are commercially available.

[0474] The invention, having been described in detail above, is exemplified in the following examples, which are offered to illustrate, but not to limit, the claimed invention. VII. METHODS FOR PREPARING RECOMBINANT MICROALGAL BIOMASS

[0475] The present invention provides recombinant microbial, preferably algal, biomass suitable for human consumption that is rich in nutrients, including lipid and/or protein constituents, methods of combining the same with ingredients, including edible ingredients and other ingredients and food compositions containing the same. Although much of the following discussion is directed to algal biomass or algal oil, it is intended to apply equally to microbial biomass or microbial oil generally. The invention arose in part from the discoveries that recombinant algal biomass can be prepared with a high oil content and/or with excellent functionality, and the resulting biomass incorporated into food products in which the oil and/or protein content of the biomass can substitute in whole or in part for oils and/or fats and/or proteins present in conventional food products. Algal oil, which can comprise predominantly monosaturated oil, provides health benefits compared with saturated, hydrogenated (trans fats) and polyunsaturated fats often found in conventional food products. Algal oil also can be used as a healthy stable cooking oil free of trans fats. The remainder of the algal biomass can encapsulate the oil at least until a food product is cooked, thereby increasing shelf-life of the oil. In uncooked products, in which cells remain intact, the biomass, along with natural antioxidants found in the oil, also protects the oil from oxidation, which would otherwise create unpleasant odors, tastes, and textures. The biomass also provides several beneficial micro-nutrients in addition to the oil and/or protein, such as algal- derived dietary fibers (both soluble and insoluble carbohydrates), phospholipids,

glycoprotein, phytosterols, tocopherols, tocotrieneols, and selenium.

1. Microalgae for Use in the Methods of the Invention

[0476] A variety species of microalgae that produce suitable oils and/or lipids and/or protein can be used in accordance with the methods of the present invention, although microalgae that naturally produce high levels of suitable oils and/or lipids and/or protein are preferred. Considerations affecting the selection of microalgae for use in the invention include, in addition to production of suitable oils, lipids, or protein for production of food products: (1) high lipid (or protein) content as a percentage of cell weight; (2) ease of growth; (3) ease of propagation; (4) ease of biomass processing; (5) glycerolipid profile; and (6) absence of algal toxins (Example 5 below demonstrates dried recombinant microalgal biomass and oils or lipids extracted from the biomass lacks algal toxins).

[0477] In some embodiments, the cell wall of the microalgae must be disrupted during food processing (e.g. , cooking) to release the active components or for digestion, and, in these embodiments, strains of microalgae with cell walls susceptible to digestion in the gastrointestinal tract of an animal, e.g. , a human or other monogastrics, are preferred, especially if the algal biomass is to be used in uncooked food products. Digestibility is generally decreased for recombinant microalgal strains which have a high content of cellulose/hemicellulose in the cell walls. Digestibility can be evaluated using a standard pepsin digestibility assay.

2. Methods of Generating a Microalgae Strain Lacking or That has Significantly Reduced Pigmentation

[0478] Microalgae, such as Chlorella, can be capable of either photosynthetic or heterotrophic growth. Prototheca is an obligate heterotroph. When grown in heterotrophic conditions where the carbon source is a fixed carbon source and in the absence of light, the normally green colored microalgae has a yellow color, lacking or is significantly reduced in green pigmentation. Microalgae of reduced (or lacking in) green pigmentation can be advantageous as a food ingredient. One advantage of microalgae of reduced (or is lacking) in green pigmentation is that the microalgae has a reduced chlorophyll flavor. Another advantage of microalgae of reduced (or is lacking in) green pigmentation is that as a food ingredient, the addition of the microalgae to foodstuffs will not impart a green color that can be unappealing to the consumer. The reduced green pigmentation of microalgae grown under heterotrophic conditions is transient. When switched back to phototrophic growth, microalgae capable of both phototrophic and heterotrophic growth will regain the green pigmentation. Additionally, even with reduced green pigments, heterotrophically grown microalgae is a yellow color and this may be unsuitable for some food applications where the consumer expects the color of the foodstuff to be white or light in color. Thus, it is advantageous to generate a microalgae strain that is capable of heterotrophic growth (so it is reduced or lacking in green pigmentation) and is also reduced in yellow pigmentation (so that it is a neutral color for food applications).

[0479] One method for generating such microalgae strain lacking in or has significantly reduced pigmentation is through mutagenesis and then screening for the desired phenotype. Several methods of mutagenesis are known and practiced in the art. For example, Urano et al., (Urano et al., J Bioscience Bioengineering (2000) v. 90(5): pp. 567-569) describes yellow and white color mutants of Chlorella ellipsoidea generated using UV irradiation. Kamiya (Kamiya, Plant Cell Physiol. (1989) v. 30(4): 513-521) describes a colorless strain of Chlorella vulgaris, l lh (M125).

[0480] In addition to mutagenesis by UV irradiation, chemical mutagenesis can also be employed in order to generate microalgae with reduced (or lacking in) pigmentation. Chemical mutagens such as ethyl methanesulfonate (EMS) or N-methyl-N'nitro-N- nitroguanidine (NTG) have been shown to be effective chemical mutagens on a variety of microbes including yeast, fungi, mycobacterium and microalgae. Mutagenesis can also be carried out in several rounds, where the microalgae is exposed to the mutagen (either UV or chemical or both) and then screened for the desired reduced pigmentation phenotype.

Colonies with the desired phenotype are then streaked out on plates and reisolated to ensure that the mutation is stable from one generation to the next and that the colony is pure and not of a mixed population.

[0481] In a particular example, Chlorella protothecoides was used to generate strains lacking in or with reduced pigmentation using a combination of UV and chemical mutagenesis. Chlorella protothecoides was exposed to a round of chemical mutagenesis with NTG and colonies were screened for color mutants. Colonies not exhibiting color mutations were then subjected to a round of UV irradiation and were again screened for color mutants. In one embodiment, a Chlorella protothecoides strain lacking in pigmentation was isolated and is Chlorella protothecoides 33-55, deposited on October 13, 2009 at the American Type Culture Collection at 10801 University Boulevard, Manassas, VA 20110-2209, in accordance with the Budapest Treaty, with a Patent Deposit Designation of PTA-XXXX. In another embodiment, a Chlorella protothecoides strain with reduced pigmentation was isolated and is Chlorella protothecoides 25-32, deposited on October 13, 2009 at the American Type Culture Collection at 10801 University Boulevard, Manassas, VA 20110-2209, in accordance with the Budapest Treaty, with a Patent Deposit Designation of PTA-XXXX.

[0482] High lipid biomass from microalgae is an advantageous material for inclusion in food products compared to low lipid biomass, because it allows for the addition of less recombinant microalgal biomass to incorporate the same amount of lipid into a food composition. This is advantageous, because healthy oils from high lipid microalgae can be added to food products without altering other attributes such as texture and taste compared with low lipid biomass. The lipid-rich biomass provided by the methods of the invention typically has at least 25% lipid by dry cell weight. Process conditions can be adjusted to increase the percentage weight of cells that is lipid. For example, in certain embodiments, a microalgae is cultured in the presence of a limiting concentration of one or more nutrients, such as, for example, nitrogen, phosphorous, or sulfur, while providing an excess of a fixed carbon source, such as glucose. Nitrogen limitation tends to increase microbial lipid yield over microbial lipid yield in a culture in which nitrogen is provided in excess. In particular embodiments, the increase in lipid yield is at least about 10%, 50%, 100%, 200%, or 500%. The microbe can be cultured in the presence of a limiting amount of a nutrient for a portion of the total culture period or for the entire period. In some embodiments, the nutrient concentration is cycled between a limiting concentration and a non- limiting concentration at least twice during the total culture period.

[0483] High protein biomass from algae is another advantageous material for inclusion in food products. The methods of the invention can also provide biomass that has at least 20%, 30%, 40% or 50% of its dry cell weight as protein. Growth conditions can be adjusted to increase the percentage weight of cells that is protein. In a preferred embodiment, a microalgae is cultured in a nitrogen rich environment and an excess of fixed carbon energy such as glucose or any of the other carbon sources discussed above. Conditions in which nitrogen is in excess tends to increase microbial protein yield over microbial protein yield in a culture in which nitrogen is not provided in excess. For maximal protein production, the microbe is preferably cultured in the presence of excess nitrogen for the total culture period. Suitable nitrogen sources for microalgae may come from organic nitrogen sources and/or inorganic nitrogen sources.

[0484] Recombinant microalgal cultures generated according to the methods described herein yield recombinant microalgal biomass in fermentation media. To prepare the biomass for use as a food composition, the biomass is concentrated, or harvested, from the fermentation medium. At the point of harvesting the recombinant microalgal biomass from the fermentation medium, the biomass comprises predominantly intact cells suspended in an aqueous culture medium. To concentrate the biomass, a dewatering step is performed.

Dewatering or concentrating refers to the separation of the biomass from fermentation broth or other liquid medium and so is solid-liquid separation. Thus, during dewatering, the culture medium is removed from the biomass (for example, by draining the fermentation broth through a filter that retains the biomass), or the biomass is otherwise removed from the culture medium. Common processes for dewatering include centrifugation, filtration, and the use of mechanical pressure. These processes can be used individually or in any combination.

[0485] After concentration, recombinant microalgal biomass can be processed, as described herein, to produce vacuum-packed cake, algal flakes, algal homogenate, algal powder, algal flour, or algal oil.

3. Chemical Composition of Recombinant Microalgal Biomass

[0486] The recombinant microalgal biomass generated by the culture methods described herein comprises recombinant microalgal oil and/or protein as well as other constituents generated by the microorganisms or incorporated by the microorganisms from the culture medium during fermentation.

[0487] Heterotrophic growth results in relatively low chlorophyll content (as compared to phototrophic systems such as open ponds or closed photobioreactor systems). Reduced chlorophyll content generally improves organoleptic properties of microalgae and therefore allows more algal biomass (or oil prepared therefrom) to be incorporated into a food product. The reduced chlorophyll content found in heterotrophically grown microalgae (e.g.,

Chlorella) also reduces the green color in the biomass as compared to phototrophically grown microalgae. Thus, the reduced chlorophyll content avoids an often undesired green coloring associated with food products containing phototrophically grown microalgae and allows for the incorporation or an increased incorporation of algal biomass into a food product. In at least one embodiment, the food product contains heterotrophically grown microalgae of reduced chlorophyll content compared to phototrophically grown microalgae.

[0488] The recombinant microalgal oil of the biomass described herein (or extracted from the biomass) can comprise glycerolipids with one or more distinct fatty acid ester side chains. Glycerolipids are comprised of a glycerol molecule esterified to one, two, or three fatty acid molecules, which can be of varying lengths and have varying degrees of saturation. Specific blends of algal oil can be prepared either within a single species of algae, or by mixing together the biomass (or algal oil) from two or more species of microalgae.

[0489] Thus, the oil composition, i.e. , the properties and proportions of the fatty acid constituents of the glycerolipids, can also be manipulated by combining biomass (or oil) from at least two distinct species of microalgae. In some embodiments, at least two of the distinct species of microalgae have different glycerolipid profiles. The distinct species of microalgae can be cultured together or separately as described herein, preferably under heterotrophic conditions, to generate the respective oils. Different species of microalgae can contain different percentages of distinct fatty acid constituents in the cell's glycerolipids.

[0490] In some embodiments, the recombinant microalgal oil is primarily comprised of monounsaturated oil. In some cases, the algal oil is at least 20% monounsaturated oil by weight. In various embodiments, the algal oil is at least 25%, 50%, 75% or more

monounsaturated oil by weight or by volume. In some embodiments, the monounsaturated oil is 18: 1, 16: 1, 14: 1 or 12: 1. In some embodiments, the recombinant microalgal oil comprises at least 10%, 20%, 25%, or 50% or more esterified oleic acid or esterified alpha- linolenic acid by weight of by volume. In at least one embodiment, the algal oil comprises less than 10%, less than 5%, less than 3%, less than 2%, or less than 1% by weight or by volume, or is substantially free of, esterified docosahexanoic acid (DHA (22:6)). For examples of production of high DHA-containing microalgae, such as in Crypthecodinium cohnii, see US Patent Nos. 7,252,979, 6,812,009 and 6,372,460.

[0491] High protein recombinant microalgal biomass has been generated using different methods of culture. Recombinant microalgal biomass with a higher percentage of protein content is useful in accordance with the present invention. For example, the protein content of various species of microalgae has been reported (see Table 1 of Becker, Biotechnology Advances (2007) 25:207-210). Controlling the renewal rate in a semi-continous

photoautotrophic culture of Tetraselmis suecica has been reported to affect the protein content per cell, the highest being approximately 22.8% protein (Fabregas, et al., Marine Biotechnology (2001) 3:256-263).

[0492] Recombinant microalgal biomass generated by culture methods described herein and useful in accordance to those embodiments of the present invention relating to high protein typically comprises at least 30% protein by dry cell weight. In some embodiments, the recombinant microalgal biomass comprises at least 40%, 50%, 75% or more protein by dry cell weight. In some embodiments, the recombinant microalgal biomass comprises from 30-75% protein by dry cell weight or from 40-60% protein by dry cell weight. In some embodiments, the protein in the recombinant microalgal biomass comprises at least 40% digestible crude protein. In other embodiments, the protein in the recombinant microalgal biomass comprises at least 50%, 60%, 70%, 80%, or at least 90% digestible crude protein. In some embodiments, the protein in the recombinant microalgal biomass comprises from 40- 90% digestible crude protein, from 50-80% digestible crude protein, or from 60-75% digestible crude protein.

[0493] Recombinant microalgal biomass (and oil extracted therefrom), can also include other constituents produced by the microalgae, or incorporated into the biomass from the culture medium. These other constituents can be present in varying amounts depending on the culture conditions used and the species of microalgae (and, if applicable, the extraction method used to recover recombinant microalgal oil from the biomass). The other constituents can include, without limitation, phospholipids {e.g. , algal lecithin), carbohydrates, soluble and insoluble fiber, glycoproteins, phytosterols {e.g. , β-sitosterol, campesterol, stigmasterol, ergosterol, and brassicasterol), tocopherols, tocotrienols, carotenoids {e.g. , a-carotene, β- carotene, and lycopene), xanthophylls {e.g. , lutein, zeaxanthin, a-cryptoxanthin, and β- cryptoxanthin), proteins, polysaccharides {e.g., arabinose, mannose, galactose, 6-methyl galactose and glucose) and various organic or inorganic compounds {e.g. , selenium). [0494] In some cases, the recombinant microalgal biomass comprises at least 10% soluble fiber. In other embodiments, the recombinant microalgal biomass comprises at least 20% to 25% soluble fiber. In some embodiments, the recombinant microalgal biomass comprises at least 30% insoluble fiber. In other embodiments, the recombinant microalgal biomass comprises at least 50% to at least 70% insoluble fiber. Total dietary fiber is the sum of soluble fiber and insoluble fiber. In some embodiments, the recombinant microalgal biomass comprises at least 40% total dietary fiber. In other embodiments, the recombinant microalgal biomass comprises at least 50%, 55%, 60%, 75%, 80%, 90%, to 95% total dietary fiber. VIII. PROCESSING RECOMBINANT MICROALGAL BIOMASS INTO

FINISHED FOOD INGREDIENTS

[0495] The concentrated recombinant microalgal biomass produced in accordance with the methods of the invention is itself a finished food ingredient and may be used in foodstuffs without further, or with only minimal, modification. For example, the cake can be vacuum- packed or frozen. Alternatively, the biomass may be dried via lyophilization, a "freeze- drying" process, in which the biomass is frozen in a freeze-drying chamber to which a vacuum is applied. The application of a vacuum to the freeze-drying chamber results in sublimation (primary drying) and desorption (secondary drying) of the water from the biomass. However, the present invention provides a variety of recombinant microalgal derived finished food ingredients with enhanced properties resulting from processing methods of the invention that can be applied to the concentrated recombinant microalgal biomass.

[0496] Drying the recombinant microalgal biomass, either predominantly intact or in homogenate form, is advantageous to facilitate further processing or for use of the biomass in the methods and compositions described herein. Drying refers to the removal of free or surface moisture/water from predominantly intact biomass or the removal of surface water from a slurry of homogenized (e.g. , by micronization)biomass. Different textures and flavors can be conferred on food products depending on whether the algal biomass is dried, and if so, the drying method. Drying the biomass generated from the cultured microalgae described herein removes water that may be an undesirable component of finished food products or food ingredients. In some cases, drying the biomass may facilitate a more efficient recombinant microalgal oil extraction process.

[0497] In one embodiment, the concentrated recombinant microalgal biomass is drum dried to a flake form to produce algal flake, as described in part A of this section. In another embodiment, the concentrated micralgal biomass is spray or flash dried (i.e., subjected to a pneumatic drying process) to form a powder containing predominantly intact cells to produce algal powder, as described in part B of this section. In another embodiment, the

concentratedrecombinant microalgal biomass is micronized (homogenized) to form a homogenate of predominantly lysed cells that is then spray or flash dried to produce algal flour, as described in part C of this section. In another embodiment, oil is extracted from the concentrated recombinant microalgal biomass to form algal oil, as described in part D of this section.

1. Algal Flake

[0498] Algal flake of the invention is prepared from concentrated recombinant microalgal biomass that is applied as a film to the surface of a rolling, heated drum. The dried solids are then scraped off with a knife or blade, resulting in a small flakes. U.S. Patent No. 6,607,900 describes drying recombinant microalgal biomass using a drum dryer without a prior centrifugation (concentration) step, and such a process may be used in accordance with the methods of the invention.

[0499] Because the biomass may be exposed to high heat during the drying process, it may be advantageous to add an antioxidant to the biomass prior to drying. The addition of an antioxidant will not only protect the biomass during drying, but also extend the shelf-life of the dried recombinant microalgal biomass when stored. In a preferred embodiment, an antioxidant is added to the recombinant microalgal biomass prior to subsequent processing such as drying or homogenization. Antioxidants that are suitable for use are discussed in detail below.

[0500] Additionally, if there is significant time between the production of the dewatered recombinant microalgal biomass and subsequent processing steps, it may be advantageous to pasteurize the biomass prior to drying. Free fatty acids from lipases may form if there is significant time between producing and drying the biomass. Pasteurization of the biomass inactivates these lipases and prevents the formation of a "soapy" flavor in the resulting dried biomass product. Thus, in one embodiment, the invention provides pasteurized recombinant microalgal biomass. In another embodiment, the pasteurized recombinant microalgal biomass is an algal flake.

2. Algal Powder

[0501] Algal powder of the invention is prepared from concentrated recombinant microalgal biomass using a pneumatic or spray dryer (see for example U.S. Patent No.

6,372,460). In a spray dryer, material in a liquid suspension is sprayed in a fine droplet dispersion into a current of heated air. The entrained material is rapidly dried and forms a dry powder. In some cases, a pulse combustion dryer can also be used to achieve a powdery texture in the final dried material. In other cases, a combination of spray drying followed by the use of a fluid bed dryer is used to achieve the optimal conditions for dried microbial biomass (see, for example, U.S. Patent No. 6,255,505). As an alternative, pneumatic dryers can also be used in the production of algal powder. Pneumatic dryers draw or entrain the material that is to be dried in a stream of hot air. While the material is entrained in the hot air, the moisture is rapidly removed. The dried material is then separated from the moist air and the moist air is then recirculated for further drying.

3. Algal Flour

[0502] Algal flour of the invention is prepared from concentrated recombinant microalgal biomass that has been mechanically lysed and homogenized and the homogenate spray or flash dried (or dried using another pneumatic drying system). The production of algal flour requires that cells be lysed to release their oil and that cell wall and intracellular components be micronized or reduced in particle size to an average size of no more than 20 μιη, preferably 10 μιη. The lysed microbial cells can agglomerate to form bigger particles of up to 1,000 μιη. The resulting oil, water, and micronized particles are emulsified such that the oil does not separate from the dispersion prior to drying. For example, a pressure disrupter can be used to pump a cell containing slurry through a restricted orifice valve to lyse the cells. High pressure (up to 1500 bar) is applied, followed by an instant expansion through an exiting nozzle. Cell disruption is accomplished by three different mechanisms: impingement on the valve, high liquid shear in the orifice, and sudden pressure drop upon discharge, causing an explosion of the cell. The method releases intracellular molecules. A Niro (Niro Soavi GEA) homogenizer (or any other high pressure homogenizer) can be used to process cells to particles predominantly 0.2 to 5 microns in length. Processing of algal biomass under high pressure (approximately 1000 bar) typically lyses over 90% of the cells and reduces particle size to less than 5 microns.

[0503] Alternatively, a ball mill can be used. In a ball mill, cells are agitated in suspension with small abrasive particles, such as beads. Cells break because of shear forces, grinding between beads, and collisions with beads. The beads disrupt the cells to release cellular contents. In one embodiment, algal biomass is disrupted and formed into a stable emulsion using a Dyno-mill ECM Ultra (CB Mills) ball mill. Cells can also be disrupted by shear forces, such as with the use of blending (such as with a high speed or Waring blender as examples), the french press, or even centrifugation in case of weak cell walls, to disrupt cells. A suitable ball mill including specifics of ball size and blade is described in US Patent No. 5,330,913.

[0504] The immediate product of homogenization is a slurry of particles smaller in size than the original cells that is suspended in in oil and water. The particles represent cellular debris. The oil and water are released by the cells. Additional water may be contributed by aqueous media containing the cells before homogenization. The particles are preferably in the form of a micronized homogenate. If left to stand, some of the smaller particles may coalesce. However, an even dispersion of small particles can be preserved by seeding with a microcrystalline stabilizer, such as microcrystalline cellulose.

[0505] To form the algal flour, the slurry is spray or flash dried, removing water and leaving a dry power containing cellular debris and oil. Although the oil content of the powder can be at least 10, 25 or 50% by weight of the dry powder, the powder can have a dry rather than greasy feel and appearance (e.g. , lacking visible oil) and can also flow freely when shaken. Various flow agents (including silica-derived products) can also be added. After drying, the water or moisture content of the powder is typically less than 10%, 5%, 3% or 1% by weight. Other dryers such as pneumatic dryers or pulse combustion dryers can also be used to produce algal flour.

[0506] The oil content of algal flour can vary depending on the percent oil of the algal biomass. Algal flour can be produced from algal biomass of varying oil content. In certain embodiments, the algal flour is produced from algal biomass of the same oil content. In other embodiments, the algal flour is produced from alglal biomass of different oil content. In the latter case, algal biomass of varying oil content can be combined and then the

homogenization step performed. In other embodiments, algal flour of varying oil content is produced first and then blended together in various proportions in order to achieve an algal flour product that contains the final desired oil content. In a further embodiment, algal biomass of different lipid profiles can be combined together and then homogenized to produce algal flour. In another embodiment, algal flour of different lipid profiles is produced first and then blended together in various proportions in order to achieve an algal flour product that contains the final desired lipid profile.

[0507] The algal flour of the invention is useful for a wide range of food preparations. Because of the oil content, fiber content and the micronized particles, algal flour is a multifunctional food ingredient. Algal flour can be used in baked goods, quick breads, yeast dough products, egg products, dressing, sauces, nutritional beverages, algal milk, pasta and gluten free products. Additional details of formulating these food products and more with algal flour is described in the Examples below.

[0508] Algal flour can be used in baked goods in place of convention fat sources (e.g. , oil, butter or margarine) and eggs. Baked goods and gluten free products have superior moisture content and a cumb structure that is indistinguishable from conventional baked goods made with butter and eggs. Because of the superior moisture content, these baked goods have a longer shelf life and retain their original texture longer than conventional baked goods that are produced without algal flour.

[0509] Algal flour can also act as a fat extender with used in smoothies, sauces, or dressings. The composition of algal flour is unique in its ability to convey organoleptic qualities and mouth-feel comparable to a food product with a higher fat content. Dressings, sauces and beverages made with algal flour have a rheology and opacity that is close to conventional higher fat recipes although these food products contains about half the fat/oil levels. Algal flour is also a superior emulsifier and is suitable in use in food preparation that requires thickness, opacity and viscosity, such as, sauces, dressings and soups. Additionally the lipid profile found in algal flour of the inventions described herein does not contain trans- fat and have a higher level of healthy, unsaturated fats as compared to butter or margarine (or other animal fats). Thus, products made with algal flour can have a lower fat content (with healthier fats) without sacrificing the mouthfeel and organoleptic qualities of the same food product that is made using a conventional recipe using a conventional fat source.

[0510] Algal flour can also be added to powdered or liquid eggs, which are typically served in a food service setting. The addition of algal flour improves the appearance, texture and mouthfeel of powdered and liquid eggs and also extends improved appearance, texture and mouthfeel over time, even when the prepared eggs are held on a steam table. Specific formulations and sensory panel results are described below in the Examples.

4. Algal Oil

[0511] In one aspect, the present invention is directed to a method of preparing algal oil by harvesting algal oil from an algal biomass comprising at least 15% oil by dry weight under GMP conditions, in which the algal oil is greater than 50% 18: 1 lipid. In some cases, the algal biomass comprises a mixture of at least two distinct species of microalgae. In some cases, at least two of the distinct species of microalgae have been separately cultured. In at least one embodiment, at least two of the distinct species of microalgae have different glycerolipid profiles. In some cases, the algal biomass is derived from algae grown heterotrophically. In some cases, all of the at least two distinct species of microalgae contain at least 15% oil by dry weight.

[0512] In one aspect, the present invention is directed to a method of making a food composition comprising combining algal oil obtained from algal cells containing at least 10%, or at least 15% oil by dry weight with one or more other edible ingredients to form the food composition. In some cases, the method further comprises preparing the algal oil under GMP conditions.

[0513] Algal oil can be separated from lysed biomass for use in food product (among other applications). The algal biomass remaining after oil extraction is referred to as delipidated meal. Delipidated meal contains less oil by dry weight or volume than the microalgae contained before extraction. Typically 50-90% of oil is extracted so that delipidated meal contains, for example, 10-50% of the oil content of biomass before extraction. However, the biomass still has a high nutrient value in content of protein and other constituents discussed above. Thus, the delipidated meal can be used in animal feed or in human food applications.

[0514] In some embodiments of the method, the algal oil is at least 50% w/w oleic acid and contains less than 5% DHA. In some embodiments of the method, the algal oil is at least 50% w/w oleic acid and contains less than 0.5% DHA. In some embodiments of the method, the algal oil is at least 50% w/w oleic acid and contains less than 5% glycerolipid containing carbon chain length greater than 18. In some cases, the algal cells from which the algal oil is obtained comprise a mixture of cells from at least two distinct species of microalgae. In some cases, at least two of the distinct species of microalgae have been separately cultured. In at least one embodiment, at least two of the distinct species of microalgae have different glycerolipid profiles. In some cases, the algal cells are cultured under heterotrophic conditions. In some cases, all of the at least two distinct species of microalgae contain at least 10%, or at least 15% oil by dry weight.

[0515] In one aspect, the present invention is directed to algal oil containing at least 50% monounsaturated oil and containing less than 1% DHA prepared under GMP conditions. In some cases, the monounsaturated oil is 18: 1 lipid. In some cases, the algal oil is packaged in a capsule for delivery of a unit dose of oil. In some cases, the algal oil is derived from a mixture of at least two distinct species of microalgae. In some cases, at least two of the distinct species of microalgae have been separately cultured. In at least one embodiment, at least two of the distinct species of microalgae have different glycerolipid profiles. In some cases, the algal oil is derived from algal cells cultured under heterotrophic conditions. [0516] In one aspect, the present invention is directed to oil comprising greater than 60% 18: 1, and at least 0.20mg/g tocotrienol.

[0517] In one aspect, the present invention is directed to a fatty acid alkyl ester composition comprising greater than 60% 18: 1 ester, and at least 0.20mg/g tocotrienol.

[0518] Algal oil of the invention is prepared from concentrated, washed recombinant microalgal biomass by extraction. The cells in the biomass are lysed prior to extraction. Optionally, the microbial biomass may also be dried (oven dried, lyophilized, etc.) prior to lysis (cell disruption). Alternatively, cells can be lysed without separation from some or all of the fermentation broth when the fermentation is complete. For example, the cells can be at a ratio of less than 1 : 1 v:v cells to extracellular liquid when the cells are lysed.

[0519] Microalgae containing lipids can be lysed to produce a lysate. As detailed herein, the step of lysing a microorganism (also referred to as cell lysis) can be achieved by any convenient means, including heat-induced lysis, adding a base, adding an acid, using enzymes such as proteases and polysaccharide degradation enzymes such as amylases, using ultrasound, mechanical pressure-based lysis, and lysis using osmotic shock. Each of these methods for lysing a microorganism can be used as a single method or in combination simultaneously or sequentially. The extent of cell disruption can be observed by microscopic analysis. Using one or more of the methods above, typically more than 70% cell breakage is observed. Preferably, cell breakage is more than 80%, more preferably more than 90% and most preferred about 100%.

[0520] Lipids and oils generated by the microalgae in accordance with the present invention can be recovered by extraction. In some cases, extraction can be performed using an organic solvent or an oil, or can be performed using a solventless-extraction procedure.

[0521] For organic solvent extraction of the recombinant microalgal oil, the preferred organic solvent is hexane. Typically, the organic solvent is added directly to the lysate without prior separation of the lysate components. In one embodiment, the lysate generated by one or more of the methods described above is contacted with an organic solvent for a period of time sufficient to allow the lipid components to form a solution with the organic solvent. In some cases, the solution can then be further refined to recover specific desired lipid components. The mixture can then be filtered and the hexane removed by, for example, rotoevaporation. Hexane extraction methods are well known in the art. See, e.g. , Frenz et al., Enzyme Microb. Technol., 11:717 (1989).

[0522] Miao and Wu describe a protocol of the recovery of recombinant microalgal lipid from a culture of Chlorella protothecoides in which the cells were harvested by centrifugation, washed with distilled water and dried by freeze drying. The resulting cell powder was pulverized in a mortar and then extracted with n-hexane. Miao and Wu, Biosource Technology 97:841-846 (2006).

[0523] In some cases, recombinant microalgal oils can be extracted using liquefaction (see for example Sawayama et al., Biomass and Bioenergy 17:33-39 (1999) and Inoue et al., Biomass Bioenergy 6(4):269-274 (1993)); oil liquefaction (see for example Minowa et al., Fuel 74(12): 1735-1738 (1995)); or supercritical C0 2 extraction (see for example Mendes et al , Inorganica Chimica Acta 356:328-334 (2003)).

[0524] Oil extraction includes the addition of an oil directly to a lysate without prior separation of the lysate components. After addition of the oil, the lysate separates either of its own accord or as a result of centrifugation or the like into different layers. The layers can include in order of decreasing density: a pellet of heavy solids, an aqueous phase, an emulsion phase, and an oil phase. The emulsion phase is an emulsion of lipids and aqueous phase. Depending on the percentage of oil added with respect to the lysate (w/w or v/v), the force of centrifugation if any, volume of aqueous media and other factors, either or both of the emulsion and oil phases can be present. Incubation or treatment of the cell lysate or the emulsion phase with the oil is performed for a time sufficient to allow the lipid produced by the microorganism to become solubilized in the oil to form a heterogeneous mixture.

[0525] In various embodiments, the oil used in the extraction process is selected from the group consisting of oil from soy, rapeseed, canola, palm, palm kernel, coconut, corn, waste vegetable oil, Chinese tallow, olive, sunflower, cotton seed, chicken fat, beef tallow, porcine tallow, microalgae, macroalgae, Cuphea, flax, peanut, choice white grease (lard), Camelina sativa mustard seedcashew nut, oats, lupine, kenaf, calendula, hemp, coffee, linseed, hazelnut, euphorbia, pumpkin seed, coriander, camellia, sesame, safflower, rice, tung oil tree, cocoa, copra, pium poppy, castor beans, pecan, jojoba, jatropha, macadamia, Brazil nuts, and avocado. The amount of oil added to the lysate is typically greater than 5% (measured by v/v and/or w/w) of the lysate with which the oil is being combined. Thus, a preferred v/v or w/w of the oil is greater than 5%, 10%, 20%, 25%, 50%, 70%, 90%, or at least 95% of the cell lysate.

[0526] Lipids can also be extracted from a lysate via a solventless extraction procedure without substantial or any use of organic solvents or oils by cooling the lysate. Sonication can also be used, particularly if the temperature is between room temperature and 65°C. Such a lysate on centrifugation or settling can be separated into layers, one of which is an aqueous:lipid layer. Other layers can include a solid pellet, an aqueous layer, and a lipid layer. Lipid can be extracted from the emulsion layer by freeze thawing or otherwise cooling the emulsion. In such methods, it is not necessary to add any organic solvent or oil. If any solvent or oil is added, it can be below 5% v/v or w/w of the lysate.

IX. COMBINING RECOMBINANT MICROALGAL BIOMASS OR

MATERIALS DERIVED THEREFROM WITH OTHER FOOD INGREDIENTS

[0527] In one aspect, the present invention is directed to a food composition comprising at least 0.1% w/w algal biomass and one or more other edible ingredients, wherein the algal biomass comprises at least 10% triglyceride by dry weight, optionally wherein at least 90% of the oil is glycerolipid. The algal cells are cultivated heterotrophically and optionally in the absence of light. In some embodiments, the algal biomass contains at least 25%, 40%, 50% or 60% oil by dry weight. In some cases, the algal biomass contains 10-90%, 25-75%, 40- 75% or 50-70% oil by dry weight, optionally wherein at least 90% of the oil is glycerolipid. In at least one embodiment, at least 50% by weight of the oil is monounsaturated glycerolipid oil. In some cases, at least 50%, 60%, 70% 80% or 90% by weight of the oil is a C18:l lipid. In some embodiments, the lipid profile of the algal triglyceride oil is similar to a naturally occurring oil. Some of the naturally occurring oils are provided in table 5. In one embodiment, the algal triglycerides produced by the invention are similar to cocoa butter, coconut oil, palm oil, beef tallow or lard. In some cases, less than 5% by weight of the oil is docosahexanoic acid (DHA) (22:6). In at least one embodiment, less than 1% by weight of the oil is DHA. An algal lipid content with low levels of polyunsaturated fatty acids (PUFA) is preferred to ensure chemical stability of the biomass. In preferred embodiments, the algal biomass is grown under heterotrophic conditions and has reduced green pigmentation. In other embodiments, the microalgae is a color mutant that lacks or is reduced in pigmentation.

[0528] This invention also provide a food composition comprising at least 0.1% w/w algal triglyceride oil isolated from recombinant algal cells cultivated under heterotrophic conditions and one or more other ingredients. The recombinant algal cells can be optionally cultivated in the dark. In some embodiments, the triglyceride profile of the algal triglycerol oil is similar to the triglyceride profile of a naturally occurring oil. Some of the naturally occurring oils are provided in table 5. In an embodiment of the invention, the algal triglycerides produced by the invention are similar to cocoa butter, coconut oil, palm oil, beef tallow or lard. In at least one embodiment, at least 50% by weight of the oil is

monounsaturated glycerolipid oil. In some cases, at least 50%, 60%, 70% 80% or 90% by weight of the oil is a CI 8:1 lipid. [0529] In another aspect, the present invention is directed to a food composisiton comprising at least 0.1 % w/w algal biomass and one or more other edible ingredients, wherein the algal biomass comprises at least 30% protein by dry weight, at least 40% protein by dry weight, at least 45% protein by dry weight, at least 50% protein by dry weight, at least 55% protein by dry weight, at least 60% protein by dry weight or at least 75% protein by dry weight. In some cases, the algal biomass contains 30-75% or 40-60% protein by dry weight. In some embodiments, at least 40% of the crude protein is digestible, at least 50% of the crude protein is digestible, at least 60% of the crude protein is digestible, at least 70% of the crude protein is digestible, at least 80% of the crude protein is digestible, or at least 90% of the crude protein is digestible. In some cases, the algal biomass is grown under heterotrophic conditions. In at least one embodiment, the algal biomass is grown under nitrogen-replete conditions. In other embodiments, the microalgae is a color mutant that lacks or is reduced in pigmentation.

[0530] In some cases, the algal biomass comprises predominantly intact cells. In some embodiments, the food composition comprises oil which is predominantly or completely encapsulated inside cells of the biomass. In some cases, the food composition comprises predominantly intact recombinant microalgal cells. In some cases, the algal oil is

predominantly encapsulated in cells of the biomass. In other cases, the biomass comprises predominantly lysed cells (e.g., a homogenate). As discussed above, such a homogenate can be provided as a slurry, flake, powder, or flour.

[0531] In some embodiments of the food composition, the algal biomass further comprises at least 10 ppm selenium. In some cases, the biomass further comprises at least 15% w/w algal polysaccharide. In some cases, the biomass further comprises at least 5% w/w algal glycoprotein. In some cases, the biomass comprises between 0 and 115 mcg/g total carotenoids. In some cases, the biomass comprises at least 0.5% w/w algal phospholipids. In all cases, as just noted, these components are true cellular components and not extracellular.

[0532] In some cases, the algal biomass of the food composition contains components that have antioxidant qualities. The strong antioxidant qualities can be attributed to the multiple antioxidants present in the algal biomass, which include, but are not limited to carotenoids, essential minerals such as zinc, copper, magnesium, calcium, and manganese. Algal biomass has also been shown to contain other antioxidants such as tocotrienols and tocopherols. These members of the vitamin E family are important antioxidants and have other health benefits such as protective effects against stroke-induced injuries, reversal of arterial blockage, growth inhibition of breast and prostate cancer cells, reduction in cholesterol levels, a reduced-risk of type II diabetes and protective effects against glaucomatous damage. Natural sources of tocotrienols and tocopherols can be found in oils produced from palm, sunflower, corn, soybean and olive oil, however compositions provided herein have significantly greater levels of tocotrienols than heretofore known materials.

[0533] In some cases, food compositions of the present invention contain algal oil comprising at least 0.05 mg/g, at least 0.07 mg/g or at least 0.08 mg/g total tocopherol. In some cases, food compositions of the present invention contain algal oil comprising at least 0.15mg/g, at least 0.20mg/g or at least 0.25mg/g total tocotrienol.

[0534] In particular embodiments of the compositions and/or methods described above, the microalgae can produce carotenoids. In some embodiments, the carotenoids produced by the microalgae can be co-extracted with the lipids or oil produced by the microalgae (i.e., the oil or lipid will contain the carotenoids). In some embodiments, the carotenoids produced by the microalgae are xanthophylls. In some embodiments, the carotenoids produced by the microalgae are carotenes. In some embodiments, the carotenoids produced by the microalgae are a mixture of carotenes and xanthophylls. In various embodiments, the carotenoids produced by the microalgae comprise at least one carotenoid selected from the group consisting of astaxanthin, lutein, zeaxanthin, alpha-carotene, trans-beta carotene, cis-beta carotene, lycopene and any combination thereof.

[0535] In some embodiments of the food composition, the algal biomass is derived from algae cultured and dried under good manufacturing practice (GMP) conditions. In some cases, the algal biomass is combined with one or more other edible ingredients, including without limitation, grain, fruit, vegetable, protein, lipid, herb and/or spice ingredients. In some cases, the food composition is a salad dressing, egg product, baked good, bread, bar, pasta, sauce, soup drink, beverage, frozen dessert, butter or spread. In particular

embodiments, the food composition is not a pill or powder. In some cases, the food composition in accordance with the present invention weighs at least 50g, or at least lOOg.

[0536] Biomass can be combined with one or more other edible ingredients to make a food product. The biomass can be from a single algal source (e.g. , strain) or algal biomass from multiple sources (e.g., different strains). The biomass can also be from a single algal species, but with different composition profile. For example, a manufacturer can blend microalgae that is high in oil content with microalgae that is high in protein content to the exact oil and protein content that is desired in the finished food product. The combination can be performed by a food manufacturer to make a finished product for retail sale or food service use. Alternatively, a manufacturer can sell algal biomass as a product, and a consumer can incorporate the algal biomass into a food product, for example, by modification of a conventional recipe. In either case, the algal biomass is typically used to replace all or part of the oil, fat, eggs, or the like used in many conventional food products.

[0537] In one aspect, the present invention is directed to a food composition comprising at lest 0.1% w/w algal biomass and one or more other edible ingredients, wherein the algal biomass is formulated thorugh blending of algal biomass that contains at least 40% protein by dry weight with algal biomass that contains 40% lipid by dry weight to obtain a blend of a desired percent protein and lipid by dry weight. In some embodiments, the biomass is from the same strain of algae. Alternatively, algal biomass that contains at least 40% lipid by dry weight containing less than 1% of its lipid as DHA is blended with algal biomass that contains at lest 20% lipid by dry weight containing at least 5% of its lipid as DHA to obtain a blend of dry biomass that contains in the aggregate at least 10% lipid and 1% DHA by dry weight.

[0538] In one aspect, the present invention is directed to a method of preparing algal biomass by drying an algal culture to provide algal biomass comprising at least 15% oil by dry weight under GMP conditions, in which the algal oil is greater than 50%

monounsaturated lipid.

[0539] In one aspect, the present invention is directed to algal biomass containing at least 15% oil by dry weight manufactured under GMP conditions, in which the algal oil is greater than 50%, 60%, 70%, 80% or 90% C18: l lipid. In one aspect, the present invention is directed to algal biomass containing at least 40% oil by dry weight manufactured under GMP conditions. In one aspect, the present invention is directed to algal biomass containing at least 55% oil by dry weight manufactured under GMP conditions. In some cases, the algal biomass is packaged as a tablet for delivery of a unit dose of biomass. In some cases, the algal biomass is packaged with or otherwise bears a label providing directions for combining the algal biomass with other edible ingredients.

[0540] In one aspect, the present invention is directed to methods of combining

recombinant microalgal biomass and/or materials derived therefrom, as described above, with at least one other finished food ingredient, as described below, to form a food composition or foodstuff. In various embodiments, the food composition formed by the methods of the invention comprises an egg product (powdered or liquid), a pasta product, a dressing product, a mayonnaise product, a cake product, a bread product, an energy bar, a milk product, a juice product, a spread, or a smoothie. In some cases, the food composition is not a pill or powder. In various embodiments, the food composition weighs at least 10 g, at least 25 g, at least 50 g, at least 100 g, at least 250 g, or at least 500 g or more. In some embodiments, the food composition formed by the combination of recombinant microalgal biomass and/or product derived therefrom is an uncooked product. In other cases, the food composition is a cooked product.

[0541] In other cases, the food composition is a cooked product. In some cases, the food composition contains less than 25% oil or fat by weight excluding oil contributed by the algal biomass. Fat, in the form of saturated triglycerides (TAGs or trans fats), is made when hydrogenating vegetable oils, as is practiced when making spreads such as margarines. The fat contained in algal biomass has no trans fats present. In some cases, the food composition contains less than 10% oil or fat by weight excluding oil contributed by the biomass. In at least one embodiment, the food composition is free of oil or fat excluding oil contributed by the biomass. In some cases, the food composition is free of oil other than oil contributed by the biomass. In some cases, the food composition is free of egg or egg products.

[0542] In one aspect, the present invention is directed to a method of making a food composition in which the fat or oil in a conventional food product is fully or partially substituted with algal biomass containing at least 10% by weight oil. In one embodiment, the method comprises determining an amount of the algal biomass for substitution using the proportion of algal oil in the biomass and the amount of oil or fat in the conventional food product, and combining the algal biomass with at least one other edible ingredient and less than the amount of oil or fat contained in the conventional food product to form a food composition. In some cases, the amount of algal biomass combined with the at least one other ingredient is 1-4 times the mass or volume of oil and/or fat in the conventional food product.

[0543] In some embodiments, the method described above further includes providing a recipe for a conventional food product containing the at least one other edible ingredient combined with an oil or fat, and combining 1-4 times the mass or volume of the algal biomass with the at least one other edible ingredient as the mass or volume of fat or oil in the conventional food product. In some cases, the method further includes preparing the algal biomass under GMP conditions.

[0544] In some cases, the food composition formed by the combination of recombinant microalgal biomass and/or product derived therefrom comprises at least 0.1%, at least 0.5%, at least 1%, at least 5%, at least 10%, at least 25%, or at least 50% w/w or v/v recombinant microalgal biomass or recombinant microalgal oil. In some embodiments, food compositions formed as described herein comprise at least 2%, at least 5%, at least 10%, at least 25%, at least 50%, at least 75%, at least 90%, or at least 95% w/w recombinant microalgal biomass or product derived therefrom. In some cases, the food composition comprises 5-50%, 10-40%, or 15-35% algal biomass or product derived therefrom by weight or by volume.

[0545] As described above, recombinant microalgal biomass can be substituted for other components that would otherwise be conventionally included in a food product. In some embodiments, the food composition contains less than 50%, less than 40%, or less than 30% oil or fat by weight excluding recombinant microalgal oil contributed by the biomass or from recombinant microalgal sources. In some cases, the food composition contains less than 25%, less than 20%, less than 15%, less than 10%, or less than 5% oil or fat by weight excluding recombinant microalgal oil contributed by the biomass or from recombinant microalgal sources. In at least one embodiment, the food composition is free of oil or fat excluding recombinant microalgal oil contributed by the biomass or from recombinant microalgal sources. In some cases, the food composition is free of eggs, butter, or other fats/oils or at least one other ingredient that would ordinarily be included in a comparable conventional food product. Some food products are free of dairy products (e.g. , butter, cream and/or cheese).

[0546] The amount of algal biomass used to prepare a food composition depends on the amount of non-algal oil, fat, eggs, or the like to be replaced in a conventional food product and the percentage of oil in the algal biomass. Thus, in at least one embodiment, the methods of the invention include determining an amount of the algal biomass to combine with at least one other edible ingredient from a proportion of oil in the biomass and a proportion of oil and/or fat that is ordinarily combined with the at least one other edible ingredient in a conventional food product. For example, if the algal biomass is 50% w/w recombinant microalgal oil, and complete replacement of oil or fat in a conventional recipe is desired, then the oil can for example be replaced in a 2: 1 ratio. The ratio can be measured by mass, but for practical purposes, it is often easier to measure volume using a measuring cup or spoon, and the replacement can be by volume. In a general case, the volume or mass of oil or fat to be replaced is replaced by (100/100-X) volume or mass of algal biomass, where X is the percentage of recombinant microalgal oil in the biomass. In general, oil and fats to be replaced in conventional recipes can be replaced in total by algal biomass, although total replacement is not necessary and any desired proportion of oil and/or fats can be retained and the remainder replaced according to taste and nutritional needs. Because the algal biomass contains proteins and phospholipids, which function as emulsifiers, items such as eggs can be replaced in total or in part with algal biomass. If an egg is replaced in total with biomass, it is sometimes desirable or necessary to augment the emulsifying properties in the food composition with an additional emulsifying agent(s) and/or add additional water or other liquid(s) to compensate for the loss of these components that would otherwise be provided by the egg. Because an egg is not all fat, the amount of biomass used to replace an egg may be less than that used to replace pure oil or fat. An average egg weighs about 58 g and comprises about 11.2% fat. Thus, about 13 g of algal biomass comprising 50% recombinant microalgal oil by weight can be used to replace the total fat portion of an egg in total.

Replacing all or part of the eggs in a food product has the additional benefit of reducing cholesterol.

[0547] For simplicity, substitution ratios can also be provided in terms of mass or volume of oil, fat and/or eggs replaced with mass or volume of biomass. In some methods, the mass or volume of oil, fat and/or eggs in a conventional recipe is replaced with 5-150%, 25-100% or 25-75% of the mass or volume of oil, fat and/or eggs. The replacement ratio depends on factors such as the food product, desired nutritional profile of the food product, overall texture and appearance of the food product, and oil content of the biomass.

[0548] In cooked foods, the determination of percentages (i.e. , weight or volume) can be made before or after cooking. The percentage of algal biomass can increase during the cooking process because of loss of liquids. Because some algal biomass cells may lyse in the course of the cooking process, it can be difficult to measure the content of algal biomass directly in a cooked product. However, the content can be determined indirectly from the mass or volume of biomass that went into the raw product as a percentage of the weight or volume of the finished product (on a biomass dry solids basis), as well as by methods of analyzing components that are unique to the algal biomass such as genomic sequences or compounds that are delivered solely by the algal biomass, such as certain carotenoids.

[0549] In some cases, it may be desirable to combine algal biomass with the at least one other edible ingredient in an amount that exceeds the proportional amount of oil, fat, eggs, or the like that is present in a conventional food product. For example, one may replace the mass or volume of oil and/or fat in a conventional food product with 1, 2, 3, 4, or more times that amount of algal biomass. Some embodiments of the methods of the invention include providing a recipe for a conventional food product containing the at least one other edible ingredient combined with an oil or fat, and combining 1-4 times the mass or volume of algal biomass with the at least one other edible ingredient as the mass or volume of fat or oil in the conventional food product. [0550] Algal biomass (predominantly intact or homogenized or micronized) and/or algal oil are combined with at least one other edible ingredient to form a food product. In some food products, the algal biomass and/or algal oil is combined with 1-20, 2-10, or 4-8 other edible ingredients. The edible ingredients can be selected from all the major food groups, including without limitation, fruits, vegetables, legumes, meats, fish, grains (e.g., wheat, rice, oats, cornmeal, barley), herbs, spices, water, vegetable broth, juice, wine, and vinegar. In some food compositions, at least 2, 3, 4, or 5 food groups are represented as well as the algal biomass or algal oil.

[0551] Oils, fats, eggs and the like can also be combined into food compositions, but, as has been discussed above, are usually present in reduced amounts (e.g., less than 50%, 25%, or 10% of the mass or volume of oil, fat or eggs compared with conventional food products. Some food products of the invention are free of oil other than that provided by algal biomass and/or algal oil. Some food products are free of oil other than that provided by algal biomass. Some food products are free of fats other than that provided by algal biomass or algal oil. Some food products are free of fats other than that provided by algal biomass. Some food products are free of both oil and fats other than that provided by algal biomass or algal oil. Some food products are free of both oil and fats other than that provided by algal biomass. Some food products are free of eggs. In some embodiments, the oils produced by the microalgae can be tailored by culture conditions or strain selection to comprise a particular fatty acid component(s) or levels.

[0552] In some cases, the algal biomass used in making the food composition comprises a mixture of at least two distinct species of microalgae. In some cases, at least two of the distinct species of microalgae have been separately cultured. In at least one embodiment, at least two of the distinct species of microalgae have different glycerolipid profiles. In some cases, the method described above further comprises culturing algae under heterotrophic conditions and preparing the biomass from the algae. In some cases, all of the at least two distinct species of microalgae contain at least 10%, or at least 15% oil by dry weight. In some cases, a food composition contains a blend of two distinct preparations of biomass of the same species, wherein one of the preparations contains at least 30% oil by dry weight and the second contains less than 15% oil by dry weight. In some cases, a food composition contains a blend of two distinct preparations of biomass of the same species, wherein one of the preparations contains at least 50% oil by dry weight and the second contains less than 15% oil by dry weight, and further wherein the species is Chlorella protothecoides. [0553] As well as using algal biomass as an oil, fat or egg replacement in otherwise conventional foods, algal biomass can be used as a supplement in foods that do not normally contain oil, such as a smoothie. The combination of oil with products that are mainly carbohydrate can have benefits associated with the oil, and from the combination of oil and carbohydrate by reducing the glycemic index of the carbohydrate. The provision of oil encapsulated in biomass is advantageous in protecting the oil from oxidation and can also improve the taste and texture of the smoothie.

[0554] Oil extracted from algal biomass can be used in the same way as the biomass itself, that is, as a replacement for oil, fat, eggs, or the like in conventional recipes. The oil can be used to replace conventional oil and/or fat on about a 1 : 1 weight/weight or volume/volume basis. The oil can be used to replace eggs by substitution of about 1 teaspoon of algal oil per egg optionally in combination with additional water and/or an emulsifier (an average 58g egg is about 11.2% fat, algal oil has a density of about 0.915 g/ml, and a teaspoon has a volume of about 5 ml = 1.2 teaspoons of algal oil/egg). The oil can also be incorporated into dressings, sauces, soups, margarines, creamers, shortenings and the like. The oil is particularly useful for food products in which combination of the oil with other food ingredients is needed to give a desired taste, texture and/or appearance. The content of oil by weight or volume in food products can be at least 5, 10, 25, 40 or 50%.

[0555] In at least one embodiment, oil extracted from algal biomass can also be used as a cooking oil by food manufacturers, restaurants and/or consumers. In such cases, algal oil can replace conventional cooking oils such as safflower oil, canola oil, olive oil, grape seed oil, corn oil, sunflower oil, coconut oil, palm oil, or any other conventionally used cooking oil. The oil obtained from algal biomass as with other types of oil can be subjected to further refinement to increase its suitability for cooking (e.g. , increased smoke point). Oil can be neutralized with caustic soda to remove free fatty acids. The free fatty acids form a removable soap stock. The color of oil can be removed by bleaching with chemicals such as carbon black and bleaching earth. The bleaching earth and chemicals can be separated from the oil by filtration. Oil can also be deodorized by treating with steam.

[0556] Predominantly intact biomass, homogenized or micronized biomass (as a slurry, flake, powder or flour) and purified algal oil can all be combined with other food ingredients to form food products. All are a source of oil with a favorable nutritional profile (relatively high monounsaturated content). Predominantly intact, homogenized, and micronized biomass also supply high quality protein (balanced amino acid composition), carbohydrates, fiber and other nutrients as dicussed above. Foods incorporating any of these products can be made in vegan or vegetarian form. Another advantage in using recombinant microalgal biomass (either predominantly intact or homogenized (or micronized) or both) as a protein source is that it is a vegan/vegetarian protein source that is not from a major allergen source, such as soy, eggs or dairy.

[0557] Other edible ingredients with which algal biomass and/or algal oil can be combined in accordance with the present invention include, without limitation, grains, fruits, vegetables, proteins, meats, herbs, spices, carbohydrates, and fats. The other edible ingredients with which the algal biomass and/or algal oil is combined to form food compositions depend on the food product to be produced and the desired taste, texture and other properties of the food product.

[0558] Although in general any of these sources of algal oil can be used in any food product, the preferred source depends in part whether the oil is primarily present for nutritional or caloric purposes rather than for texture, appearance or taste of food, or alternatively whether the oil in combination with other food ingredients is intended to contribute a desired taste, texture or appearance of the food as well as or instead of improving its nutritional or caloric profile.

[0559] The food products can be cooked by conventional procedures as desired.

Depending on the length and temperature, the cooking process may break down some cell walls, releasing oil such that it combines with other ingredients in the mixture. However, at least some algal cells often survive cooking intact. Alternatively, food products can be used without cooking. In this case, the algal wall remains intact, protecting the oil from oxidation.

[0560] The algal biomass, if provided in a form with cells predominantly intact, or as a homogenate powder, differs from oil, fat or eggs in that it can be provided as a dry ingredient, facilitating mixing with other dry ingredients, such as flour. In one embodiment the algal biomass is provided as a dry homogenate that contains between 25 and 40% oil by dry weight. A biomass homogenate can also be provided as slurry. After mixing of dry ingredients (and biomass homogenate slurry, if used), liquids such as water can be added. In some food products, the amount of liquid required is somewhat higher than in a conventional food product because of the non-oil component of the biomass and/or because water is not being supplied by other ingredients, such as eggs. However, the amount of water can readily be determined as in conventional cooking.

[0561] In one aspect, the present invention is directed to a food ingredient composition comprising at least 0.5% w/w algal biomass containing at least 10% algal oil by dry weight and at least one other edible ingredient, in which the food ingredient can be converted into a reconstituted food product by addition of a liquid to the food ingredient composition. In one embodiment, the liquid is water.

[0562] Homogenized or micronized high-oil biomass is particularly advantageous in liquid,and/or emulsified food products (water in oil and oil in water emulsions), such as sauces, soups, drinks, salad dressings, butters, spreads and the like in which oil contributed by the biomass forms an emulsion with other liquids. Products that benefit from improved rheology, such as dressings, sauces and spreads are described below in the Examples. Using homogenized biomass an emulsion with desired texture (e.g., mouth-feel), taste and appearance (e.g., opacity) can form at a lower oil content (by weight or volume of overall product) than is the case with conventional products employing conventional oils, thus can be used as a fat extender. Such is useful for low-calorie (i. e., diet) products. Purified algal oil is also advantageous for such liquid and/or emulsified products. Both homogenized or micronized high-oil biomass and purified algal oil combine well with other edible ingredients in baked goods achieving similar or better taste, appearance and texture to otherwise similar products made with conventional oils, fats and/or eggs but with improved nutritional profile (e.g. , higher content of monosaturated oil, and/or higher content or quality of protein, and/or higher content of fiber and/or other nutrients).

[0563] Predominantly intact biomass is particularly useful in situations in which it is desired to change or increase the nutritional profile of a food (e.g. , higher oil content, different oil content (e.g. , more monounsaturated oil), higher protein content, higher calorie content, higher content of other nutrients). Such foods can be useful for example, for athletes or patients suffering from wasting disorders. Predominantly intact biomass can be used as a bulking agent. Bulking agents can be used, for example, to augment the amount of a more expensive food (e.g., meat helper and the like) or in simulated or imitation foods, such as vegetarian meat substitutes. Simulated or imitation foods differ from natural foods in that the flavor and bulk are usually provided by different sources. For example, flavors of natural foods, such as meat, can be imparted into a bulking agent holding the flavor. Predominantly intact biomass can be used as a bulking agent in such foods. Predominantly intact biomass is also particularly useful in dried food, such as pasta because it has good water binding properties, and can thus facilitate rehydration of such foods. Predominantly intact biomass is also useful as a preservative, for example, in baked goods. The predominantly intact biomass can improve water retention and thus shelf-life.

[0564] Algal biomass that has been disrupted or micronized can also improve water retention and thus shelf-life. Increased moisture retention is especially desirable in gluten- free products, such as gluten-free baked goods. A detailed description of formulation of a gluten-free cookie using disrupted algal biomass and subsequent shelf-life study is described in the Examples below.

[0565] In some cases, the algal biomass can be used in egg preparations. In some embodiments, algal biomass (e.g., algal flour) added to a conventional dry powder egg preparation to create scrambled eggs that are creamier, have more moisture and a better texture than dry powdered eggs prepared without the algal biomass. In other embodiments, algal biomass is added to whole liquid eggs in order to improve the overall texture and moisture of eggs that are prepared and then held on a steam table. Specific examples of the foregoing preparations are described in the Examples below.

[0566] Algal biomass (predominantly intact and/or homogenized or micronized) and/or algal oil can be incorporated into virtually any food composition. Some examples include baked goods, such as cakes, brownies, yellow cake, bread including brioche, cookies including sugar cookies, biscuits, and pies. Other examples include products often provided in dried form, such as pastas or powdered dressing, dried creamers, commuted meats and meat substitutes. Incorporation of predominantly intact biomass into such products as a binding and/or bulking agent can improve hydration and increase yield due to the water binding capacity of predominantly intact biomass. Re-hydrated foods, such as scrambled eggs made from dried powdered eggs, may also have improved texture and nutritional profile. Other examples include liquid food products, such as sauces, soups, dressings (ready to eat), creamers, milk drinks, juice drinks, smoothies, creamers. Other liquid food products include nutritional beverages that serve as a meal replacement or algal milk. Other food products include butters or cheeses and the like including shortening, margarine/spreads, nut butters, and cheese products, such as nacho sauce. Other food products include energy bars, chocolate confections-lecithin replacement, meal replacement bars, granola bar-type products. Another type of food product is batters and coatings. By providing a layer of oil surrounding a food, predominantly intact biomass or a homogenate repel additional oil from a cooking medium from penetrating a food. Thus, the food can retain the benefits of high monounsaturated oil content of coating without picking up less desirable oils (e.g., trans fats, saturated fats, and by products from the cooking oil). The coating of biomass can also provide a desirable (e.g. , crunchy) texture to the food and a cleaner flavor due to less absorption of cooking oil and its byproducts.

[0567] In uncooked foods, most algal cells in the biomass remain intact. This has the advantage of protecting the algal oil from oxidation, which confers a long shelf-life and minimizes adverse interaction with other ingredients. Depending on the nature of the food products, the protection conferred by the cells may reduce or avoid the need for refrigeration, vacuum packaging or the like. Retaining cells intact also prevents direct contact between the oil and the mouth of a consumer, which reduces the oily or fatty sensation that may be undesirable. In food products in which oil is used more as nutritional supplement, such can be an advantage in improving the organoleptic properties of the product. Thus,

predominantly intact biomass is suitable for use in such products. However, in uncooked products, such as a salad dressing, in which oil imparts a desired mouth feeling (e.g., as an emulsion with an aqueous solution such as vinegar), use of purified algal oil or micronized biomass is preferred. In cooked foods, some algal cells of original intact biomass may be lysed but other algal cells may remain intact. The ratio of lysed to intact cells depends on the temperature and duration of the cooking process. In cooked foods in which dispersion of oil in a uniform way with other ingredients is desired for taste, texture and/or appearance (e.g., baked goods), use of micronized biomass or purified algal oil is preferred. In cooked foods, in which algal biomass is used to supply oil and/or protein and other nutrients, primarily for their nutritional or caloric value rather than texture.

[0568] Algal biomass can also be useful in increasing the satiety index of a food product (e.g., a meal-replacement drink or smoothie) relative to an otherwise similar conventional product made without the algal biomass. The satiety index is a measure of the extent to which the same number of calories of different foods satisfy appetite. Such an index can be measured by feeding a food being tested and measuring appetite for other foods at a fixed interval thereafter. The less appetite for other foods thereafter, the higher the satiety index. Values of satiety index can be expressed on a scale in which white bread is assigned a value of 100. Foods with a higher satiety index are useful for dieting. Although not dependent on an understanding of mechanism, algal biomass is believed to increase the satiety index of a food by increasing the protein and/or fiber content of the food for a given amount of calories.

[0569] Algal biomass (predominantly intact and homogenized or micronized) and/or algal oil can also be manufactured into nutritional or dietary supplements. For example, algal oil can be encapsulated into digestible capsules in a manner similar to fish oil. Such capsules can be packaged in a bottle and taken on a daily basis (e.g., 1-4 capsules or tablets per day). A capsule can contain a unit dose of algal biomass or algal oil. Likewise, biomass can be optionally compressed with pharmaceutical or other excipients into tablets. The tablets can be packaged, for example, in a bottle or blister pack, and taken daily at a dose of, e.g. , 1-4 tablets per day. In some cases, the tablet or other dosage formulation comprises a unit dose of biomass or algal oil. Manufacturing of capsule and tablet products and other supplements is preferably performed under GMP conditions appropriate for nutritional supplements as codified at 21 C.F.R. I l l, or comparable regulations established by foreign jurisdictions. The algal biomass can be mixed with other powders and be presented in sachets as a ready- to-mix material (e.g., with water, juice, milk or other liquids). The algal biomass can also be mixed into products such as yogurts.

[0570] Although algal biomass and/or algal oil can be incorporated into nutritional supplements, the functional food products discussed above have distinctions from typical nutritional supplements, which are in the form of pills, capsules, or powders. The serving size of such food products is typically much larger than a nutritional supplement both in terms of weight and in terms of calories supplied. For example, food products often have a weight of over lOOg and/or supply at least 100 calories when packaged or consumed at one time. Typically food products contain at least one ingredient that is either a protein, a carbohydrate or a liquid and often contain two or three such other ingredients. The protein or carbohydrate in a food product often supplies at least 30%, 50%, or 60% of the calories of the food product.

[0571] As discussed above, algal biomass can be made by a manufacturer and sold to a consumer, such as a restaurant or individual, for use in a commercial setting or in the home. Such algal biomass is preferably manufactured and packaged under Good Manufacturing Practice (GMP) conditions for food products. The algal biomass in predominantly intact form or homogenized or micronized form as a powder is often packaged dry in an airtight container, such as a sealed bag. Homogenized or micronized biomass in slurry form can be conveniently packaged in a tub among other containers. Optionally, the algal biomass can be packaged under vacuum to enhance shelf life. Refrigeration of packaged algal biomass is not required. The packaged algal biomass can contain instructions for use including directions for how much of the algal biomass to use to replace a given amount of oil, fat or eggs in a conventional recipe, as discussed above. For simplicity, the directions can state that oil or fat are to be replaced on a 2: 1 ratio by mass or volume of biomass, and eggs on a ratio of 1 lg biomass or 1 teaspoon of algal oil per egg. As discussed above, other ratios are possible, for example, using a ratio of 10- 175% mass or volume of biomass to mass or volume of oil and/or fat and/or eggs in a conventional recipe. Upon opening a sealed package, the instructions may direct the user to keep the algal biomass in an airtight container, such as those widely commercially available (e.g., Glad), optionally with refrigeration. [0572] Algal biomass (predominantly intact or homogenized or micronized powder) can also be packaged in a form combined with other dry ingredients (e.g., sugar, flour, dry fruits, flavorings) and portioned packed to ensure uniformity in the final product. The mixture can then be converted into a food product by a consumer or food service company simply by adding a liquid, such as water or milk, and optionally mixing, and/or cooking without adding oils or fats. In some cases, the liquid is added to reconstitute a dried algal biomass composition. Cooking can optionally be performed using a microwave oven, convection oven, conventional oven, or on a cooktop. Such mixtures can be used for making cakes, breads, pancakes, waffles, drinks, sauces and the like. Such mixtures have advantages of convenience for the consumer as well as long shelf life without refrigeration. Such mixtures are typically packaged in a sealed container bearing instructions for adding liquid to convert the mixture into a food product.

[0573] Algal oil for use as a food ingredient is likewise preferably manufactured and packaged under GMP conditions for a food. The algal oil is typically packaged in a bottle or other container in a similar fashion to conventionally used oils. The container can include an affixed label with directions for using the oil in replacement of conventional oils, fats or eggs in food products, and as a cooking oil. When packaged in a sealed container, the oil has a long shelf-life (at least one year) without substantial deterioration. After opening, algal oil comprised primarily of monounsaturated oils is not acutely sensitive to oxidation. However, unused portions of the oil can be kept longer and with less oxidation if kept cold and/or out of direct sunlight (e.g., within an enclosed space, such as a cupboard). The directions included with the oil can contain such preferred storage information.

[0574] Optionally, the algal biomass and/or the algal oil may contain a food approved preservative/antioxidant to maximize shelf-life, including but not limited to, carotenoids (e.g. , astaxanthin, lutein, zeaxanthin, alpha-carotene, beta-carotene and lycopene), phospholipids (e.g. , N-acylphosphatidylethanolamine, phosphatidic acid, phosphatidylethanolamine, phosphatidylcholine, phosphatidylinositol and lysophosphatidylcholine), tocopherols (e.g. , alpha tocopherol, beta tocopherol, gamma tocopherol and delta tocopherol), tocotrienols (e.g., alpha tocotrienol, beta tocotrienol, gamma tocotrienol and delta tocotrienol), Butylated hydroxytoluene, Butylated hydroxyanisole, polyphenols, rosmarinic acid, propyl gallate, ascorbic acid, sodium ascorbate, sorbic acid, benzoic acid, methyl parabens, levulinic acid, anisic acid, acetic acid, citric acid, and bioflavonoids. [0575] The description of incorporation of predominantly intact biomass, homogenized, or micronized biomass (slurry, flake, powder, or flour) or algal oil into food for human nutrition is in general also applicable to food products for non-human animals.

[0576] The biomass imparts high quality oil or proteins or both in such foods. The content of algal oil is preferably at least 10 or 20% by weight as is the content of algal protein.

Obtaining at least some of the algal oil and/or protein from predominantly intact biomass is sometimes advantageous for food for high performance animals, such as sport dogs or horses. Predominantly intact biomass is also useful as a preservative. Algal biomass or oil is combined with other ingredients typically found in animal foods (e.g., a meat, meat flavor, fatty acid, vegetable, fruit, starch, vitamin, mineral, antioxidant, probiotic) and any combination thereof. Such foods are also suitable for companion animals, particularly those having an active life style. Inclusion of taurine is recommended for cat foods. As with conventional animal foods, the food can be provided in bite-size particles appropriate for the intended animal.

[0577] Delipidated meal is useful as animal feed for farm animals, e.g., ruminants, poultry, swine, and aquaculture. Delipidated meal is a byproduct of preparing purified algal oil either for food or other purposes. The resulting meal although of reduced oil content still contains high quality proteins, carbohydrates, fiber, ash and other nutrients appropriate for an animal feed. Because the cells are predominantly lysed, delipidated meal is easily digestible by such animals. Delipidated meal can optionally be combined with other ingredients, such as grain, in an animal feed. Because delipidated meal has a powdery consistency, it can be pressed into pellets using an extruder or expanders, which are commercially available.

[0578] The tailored oils of the present invention can be used in place of conventional oils such as palm oil, palm kernel oil, coconut oil, cocoa butter, tallow, or lard.

[0579] Palm Oih_Palm oil is used around the world in such foods as margarine, shortening, baked goods, and confections. Palm oil is comprised of approximately 50% saturated fat and 50% unsaturated fat, and can therefore be separated into (solid) palm stearin containing C18:0 and lower chain saturated fatty acids and (liquid) palm olein containing C18: 1 and higher chain unsaturated fatty acids. Palm stearin can be used to form solid trans- free fats. A microbial triglyceride composition of the present invention in which the oil comprises higher amounts of C 18: 1 is an excellent healthier substitute for partially hydrogenated vegetable oils that are high in trans-fats that are commonly used today. Foods containing trans-fats, including hydrogenated vegetable oils, are widely believed to be an unhealthy food. The invention provides a tailored oil, higher in C18: l, that is healthier than the partially hydrogenated vegetable oils. Thus, the present invention provides a healthier oil and meets the demands placed by the public on the food industry to supply healthier choices. The tailored oils of the present invention can be used as a replacement of the unhealthy partially hydrogenated vegetable oils.

[0580] In addition to food products, palm oil, with its moderate linoleic acid content and high level of natural antioxidants, is suitable for direct use in most cooking ad frying applications. The use of palm oil as a frying oil is a major use of palm oil worldwide. Potato chips, French fries, doughnuts, ramen noodles, nuts, etc. are typically fried in palm oil.

[0581] For the large scale frying of potato chips, palm olein or a blend of palm olein with soya or rapeseed oil is preferred. This is because the surface appearance of the finished product is improved. French fries are often purchased as part fried and deep frozen products.

[0582] In addition to its use in baking and frying, palm oil is also extensively used as an ingredient in numerous types of foods, including biscuits, crackers, bread, cereals, chips, chocolate, ice cream, soup, sauces, mayonnaise and many others.

[0583] The palm oil mimetic of the present invention are a healthy replacement of palm oil in the human food supply.

[0584] Palm Kernel Oil & Coconut Oil:_Whole palm kernel oil and whole coconut oil as well as fractionated parts are used alone or in blends with other oils for the manufacture of cocoa butter substitutes and other confectionary fats (toffees and caramels), biscuit dough and filling creams, cake icings, ice cream, imitation whipping cream, non-dairy creamers (coffee whiteners), filled milk and table margarines and spreads. These oils are also used widely in making bar and liquid soap. Palm kernel oil and coconut oil are high in C12:0 and C14:0. Table 5 discloses that the total amount of C12:0 and C14:0 of coconut oil is approximately 50 -75 . Similarly, the total amount of C12:0 and C14:0 of palm kerne oil is

approximately 50 -70 . The present invention provides a palm oil mimetic, a microbial oil comprising about 50 -75 C12:0 and C14:0.

[0585] Cocoa Butter:_Cocoa butter, is a pale-yellow, pure edible vegetable fat extracted from the cocoa bean. It is used to make chocolate, biscuits, baked goods, pharmaceuticals, ointments and toiletries. Cocoa butter historically has served as a major ingredient in the commercial production of both white chocolate and milk chocolate.

[0586] Additionally, pharmaceutical companies have made use of cocoa butter's specific physical properties. As an edible oil, solid at room temperature, but melts at body temperature, it is considered an ideal base for delivering medicinal ingredients, for example as a suppository. [0587] Cocoa butter is also one of the most stable fats known, a quality that coupled with natural antioxidant that prevents rancidity, grants it a storage life of two to five years. The velvety texture, pleasant fragrance and emollient properties of cocoa butter have made it a popular ingredient in products for the skin, such as cosmetics, soaps and lotions.

[0588] The moisturizing abilities of cocoa butter are frequently recommended for prevention of stretch marks in pregnant women, treatment of chapped skin and lips, and as a daily moisturizer to prevent dry, itchy skin.

[0589] Example 14 provides a microbial triglyceride composition that is very similar to cocoa butter. The cocoa butter mimetic of the invention is useful in all applications that use cocoa butter.

[0590] Tallow: The USA is by far the biggest producer and exporter of beef tallow, with production accounting for approx. 25 to 30% of global oil and fat production. In the past, beef tallow was used in early cultures for lighting (woodchips soaked in beef tallow) and used for making soaps and ointments. Today, beef tallow is a major raw material used in the production of chemical intermediate products, such as fatty acids and fat alcohols. In addition to its principal uses in edible fats and oils, beef tallow is also used as baking and cooking fat, as well as in margarine production. With its similar fatty acid spectrum, beef tallow was formerly used to stretch cocoa butter.

[0591] In food and cooking, tallow may be used as oil for frying and as ingredient for making pemmican, a Native American dish. Tallow may also be used as shortening for breads and pastries and as part of the ingredients for margarine. Aside from beef fat, tallow may also be sourced from horses, sheep, and pigs. People who don't eat meat and are vegetarians have also their own version of tallow using wax isolated from the seeds of the tallow tree, Triadica sebiferum. Tallow is also used as part of the ingredients of various animal feeds like those for chickens and pigs.

[0592] The tallow mimetic as described herein is useful as a replacement of tallow.

[0593] Lard: Lard is isolated from pigs and is one of the few edible oils with a relatively high smoke point, attributable to its high saturated fatty acids content. Pure lard is especially useful for cooking since it produces little smoke when heated and has a distinct taste when combined with other foods. Many chefs and bakers deem lard a superior cooking fat over shortening because of lard's range of applications and taste.

[0594] Because of the relatively large fat crystals found in lard, it is extremely effective as a shortening in baking. Pie crusts made with lard tend to be more flaky than those made with butter. Many cooks employ both types of fat in their pastries to combine the shortening properties of lard with the flavor of butter.

[0595] Example 14 provides a microbial triglyceride composition that is similar lard. The lard mimetic of the invention is useful in all applications that use lard.

X. EXAMPLES

EXAMPLE 1: Methods for Culturing Prototheca

[0596] Prototheca strains were cultivated to achieve a high percentage of oil by dry cell weight. Cryopreserved cells were thawed at room temperature and 500 ul of cells were added to 4.5 ml of medium (4.2 g/L K 2 HP0 4 , 3.1 g/L NaH 2 P0 4 , 0.24 g/L MgS0 4 -7H 2 0, 0.25 g/L Citric Acid monohydrate, 0.025 g/L CaCl 2 2H 2 0, 2g/L yeast extract) plus 2% glucose and grown for 7 days at 28 °C with agitation (200 rpm) in a 6-well plate. Dry cell weights were determined by centrifuging 1 ml of culture at 14,000 rpm for 5 min in a pre-weighed Eppendorf tube. The culture supernatant was discarded and the resulting cell pellet washed with 1 ml of deionized water. The culture was again centrifuged, the supernatant discarded, and the cell pellets placed at -80°C until frozen. Samples were then lyophilized for 24 hrs and dry cell weights calculated. For determination of total lipid in cultures, 3 ml of culture was removed and subjected to analysis using an Ankom system (Ankom Inc., Macedon, NY) according to the manufacturer's protocol. Samples were subjected to solvent extraction with an Amkom XT 10 extractor according to the manufacturer's protocol. Total lipid was determined as the difference in mass between acid hydrolyzed dried samples and solvent extracted, dried samples. Percent oil dry cell weight measurements are shown in Table 10.

[0597] Table 10. Percent oil by dry cell weight

[0598] Microalgae samples from multiple strains from the genus Prototheca were genotyped. Genomic DNA was isolated from algal biomass as follows. Cells (approximately 200 mg) were centifuged from liquid cultures 5 minutes at 14,000 x g. Cells were then resuspended in sterile distilled water, centrifuged 5 minutes at 14,000 x g and the supernatant discarded. A single glass bead ~2mm in diameter was added to the biomass and tubes were placed at -80°C for at least 15 minutes. Samples were removed and 150 μΐ of grinding buffer (1% Sarkosyl, 0.25 M Sucrose, 50 mM NaCl, 20 mM EDTA, 100 mM Tris-HCl, pH 8.0, RNase A 0.5 ug/ul) was added. Pellets were resuspended by vortexing briefly, followed by the addition of 40 ul of 5M NaCl. Samples were vortexed briefly, followed by the addition of 66 μΐ of 5% CTAB (Cetyl trimethylammonium bromide) and a final brief vortex. Samples were next incubated at 65 °C for 10 minutes after which they were centrifuged at 14,000 x g for 10 minutes. The supernatant was transferred to a fresh tube and extracted once with 300 μΐ of Phenol:Chloroform:Isoamyl alcohol 12: 12: 1, followed by centrifugation for 5 minutes at 14,000 x g. The resulting aqueous phase was transferred to a fresh tube containing 0.7 vol of isopropanol (-190 μΐ), mixed by inversion and incubated at room temperature for 30 minutes or overnight at 4°C. DNA was recovered via centrifugation at 14,000 x g for 10 minutes. The resulting pellet was then washed twice with 70% ethanol, followed by a final wash with 100% ethanol. Pellets were air dried for 20-30 minutes at room temperature followed by resuspension in 50 μΐ of lOmM TrisCl, ImM EDTA (pH 8.0).

[0599] Five μΐ of total algal DNA, prepared as described above, was diluted 1 :50 in 10 mM Tris, pH 8.0. PCR reactions, final volume 20 μΐ, were set up as follows. Ten μΐ of 2 x iProof HF master mix (BIO-RAD) was added to 0.4 μΐ primer SZ02613 (5'-

TGTTGAAGAATGAGCCGGCGAC-3 ' (SEQ ID NO: 9) at lOmM stock concentration). This primer sequence runs from position 567-588 in Gen Bank accession no. L43357 and is highly conserved in higher plants and algal plastid genomes. This was followed by the addition of 0.4 μΐ primer SZ02615 (5 ' -CAGTGAGCTATTACGCACTC-3 ' (SEQ ID NO: 10) at 10 mM stock concentration). This primer sequence is complementary to position 1112-1093 in Gen Bank accession no. L43357 and is highly conserved in higher plants and algal plastid genomes. Next, 5 μΐ of diluted total DNA and 3.2 μΐ d¾0 were added. PCR reactions were run as follows: 98°C, 45" ; 98°C, 8" ; 53°C, 12" ; 72°C, 20" for 35 cycles followed by 72°C for 1 min and holding at 25 °C. For purification of PCR products, 20 μΐ of 10 mM Tris, pH 8.0, was added to each reaction, followed by extraction with 40 μΐ of

Phenol:Chloroform:isoamyl alcohol 12: 12: 1, vortexing and centrifuging at 14,000 x g for 5 minutes. PCR reactions were applied to S-400 columns (GE Healthcare) and centrifuged for 2 minutes at 3,000 x g. Purified PCR products were subsequently TOPO cloned into

PCR8/GW/TOPO and positive clones selected for on LB/Spec plates. Purified plasmid DNA was sequenced in both directions using Ml 3 forward and reverse primers. In total, twelve Prototheca strains were selected to have their 23 S rRNA DNA sequenced and the sequences are listed in the Sequence Listing. A summary of the strains and Sequence Listing Numbers is included below. The sequences were analyzed for overall divergence from the UTEX 1435 (SEQ ID NO: 15) sequence. Two pairs emerged (UTEX 329/UTEX 1533 and UTEX

329/UTEX 1440) as the most divergent. In both cases, pairwise alignment resulted in 75.0% pairwise sequence identity. The percent sequence identity to UTEX 1435 is also included below:

Species Strain % nt identity SEO ID NO.

Prototheca kruegani UTEX 329 75.2 SEQ ID NO 11

Prototheca wickerhamii UTEX 1440 99 SEQ ID NO 12

Prototheca stagnora UTEX 1442 75.7 SEQ ID NO 13

Prototheca moriformis UTEX 288 75.4 SEQ ID NO 14

Prototheca moriformis UTEX 1439; 1441; 100 SEQ ID NO 15

1435; 1437

Prototheca wikerhamii UTEX 1533 99.8 SEQ ID NO 16

Prototheca moriformis UTEX 1434 75.9 SEQ ID NO 17

Prototheca zopfii UTEX 1438 75.7 SEQ ID NO 18

Prototheca moriformis UTEX 1436 88.9 SEQ ID NO 19

[0600] Lipid samples from a subset of the above-listed strains were analyzed for lipid profile using HPLC. Results are shown below in Table 11.

[0601] Table 11. Diversity of lipid chains in Prototheca species

[0602] Oil extracted from Prototheca moriformis UTEX 1435 (via solvent extraction or using an expeller press was analyzed for carotenoids, chlorophyll, tocopherols, other sterols and tocotrienols. The results are summarized below in Table 12.

[0603] Table 12. Carotenoid, chlorophyll, tocopherol/sterols and tocotrienol analysis in oil extracted from Prototheca moriformis (UTEX 1435).

Total Tocotrienols 0.36 0.36

[0604] Oil extracted from Prototheca moriformis, from four separate lots, were refined and bleached using standard vegetable oil processing methods. Briefly, crude oil extracted from Prototheca moriformis was clarified in a horizontal decanter, where the solids were separated from the oil. The clarified oil was then transferred to a tank with citric acid and water and left to settle for approximately 24 hours. After 24 hours, the mixture in the tank formed 2 separate layers. The bottom layer was composed of water and gums that were then removed by decantation prior to transferring the degummed oil into a bleaching tank. The oil was then heated along with another dose of citric acid. Bleaching clay was then added to the bleaching tank and the mixture was further heated under vacuum in order to evaporate off any water that was present. The mixture was then pumped through a leaf filter in order to remove the bleaching clay. The filtered oil was then passed through a final 5μιη polishing filter and then collected for storage until use. The refined and bleached (RB) oil was then analyzed for carotenoids, chlorophyll, sterols, tocotrienols and tocopherols. The results of these analyses are summarized in Table 13 below. "Nd" denotes none detected and the sensitivity of detection is listed below:

Sensitivity of Detection

Carotenoids (mcg/g) nd = <0.003 mcg/g

Chlorophyll (mcg/g) nd = <0.03 mcg/g

Sterols (%) nd = 0.25

Tocopherols (mcg/g); nd = 3 mcg/g

[0605] Table 13. Carotenoid, chlorophyll, sterols, tocotrienols and tocopherol analysis from refined and bleached Prototheca moriformis oil.

trans-beta-Cryptoxanthin nd nd nd nd trans-alpha-Carotene nd nd nd nd cis-alpha-Carotene nd nd nd nd trans-beta-Carotene nd nd nd nd cis-beta-Carotene nd nd nd nd

Lycopene nd nd nd nd

Unidentified 0.219 0.066 0.050 0.026

Total Carotenoids 0.244 0.069 0.050 0.065

Chlorophyll (mcg/g)

Chlorophyll A 0.268 0.136 0.045 0.166

Chlorophyll B nd nd nd nd

Total Chlorophyll 0.268 0.136 0.045 0.166

Sterols (% )

Brassicasterol nd nd nd nd

Campesterol nd nd nd nd

Stigmasterol nd nd nd nd beta-Sitosterol nd nd nd nd

Total Sterols nd nd nd nd

Tocopherols (mcg/g)

alpha- Tocopherol 23.9 22.8 12.5 8.2 beta-Tocopherol 3.72 nd nd nd gamma-Tocopherol 164 85.3 43.1 38.3 delta-Tocopherol 70.1 31.1 18.1 14.3

Total Tocopherols 262 139.2 73.7 60.8

Tocotrienols (mcg/g)

alpha- Tocotrienol 190 225 253 239 beta-Tocotrienol nd nd nd nd gamma-Tocotrienol 47.3 60.4 54.8 60.9 delta-Tocotrienol 12.3 16.1 17.5 15.2

Total Tocotrienols 250 302 325 315

[0606] The same four lots of Prototheca moriformis oil was also analyzed for trace elements and the results are summarized below in Table 14.

[0607] Table 14. Elemental analysis of refined and bleached Prototheca moriformis oil.

Vanadium < 0.05 < 0.05 <0.05 < 0.05

Lovibond Color (°L)

Red 5.0 4.3 3.2 5.0

Yellow 70.0 70.0 50.0 70.0

Mono & Digl cerides by HPLC (% )

Diglycerides 1.68 2.23 1.25 1.61

Monoglycerides 0.03 0.04 0.02 0.03

Free fatty acids (FFA) 1.02 1.72 0.86 0.83

Soaps 0 0 0

Oxidized and Polymerized Triglycerides

Oxidized Triglycerides (%) 3.41 2.41 4.11 1.00

Polymerized Triglycerides 1.19 0.45 0.66 0.31 (%)

Peroxide Value (meg/kg) 0.75 0.80 0.60 1.20 p-Anisidine value 5.03 9.03 5.44 20.1 (dimensionless)

Water and Other Impurities (%)

Karl Fisher Moisture 0.8 0.12 0.07 0.18

Total polar compounds 5.02 6.28 4.54 5.23

Unsaponificable matter 0.92 1.07 0.72 1.04

Insoluble impurities <0.01 <0.01 0.01 < 0.01

Total oil (%)

Neutral oil 98.8 98.2 99.0 98.9

EXAMPLE 2: General Methods for Biolistic Transforation of Prototheca

[0608] Seashell Gold Microcarriers 550 nanometers were prepared according to the protocol from manufacturer. Plasmid (20 μg) was mixed with 50 μΐ of binding buffer and 60 μΐ (30 mg) of S550d gold carriers and incubated in ice for 1 min. Precipitation buffer (100 μΐ) was added, and the mixture was incubated in ice for another 1 min. After vortexing, DNA- coated particles were pelleted by spinning at 10,000 rpm in an Eppendorf 5415C microfuge for 10 seconds. The gold pellet was washed once with 500 μΐ of cold 100% ethanol, pelleted by brief spinning in the microfuge, and resuspended with 50 μΐ of ice-cold ethanol. After a brief (1-2 sec) sonication, 10 μΐ of DNA-coated particles were immediately transferred to the carrier membrane.

[0609] Prototheca strains were grown in proteose medium (2g/L yeast extract, 2.94mM NaN03, 0.17mM CaC12 » 2H20, 0.3mM MgS04 » 7H20, 0.4mM K2HP04, 1.28mM

KH2P04, 0.43mM NaCl) with 2% glucose on a gyratory shaker until it reaches a cell density of 2xl0 6 cells/ml. The cells were harvested, washed once with sterile distilled water, and resuspended in 50 μΐ of medium. 1 x 10 7 cells were spread in the center third of a nonselective proteose media plate. The cells were bombarded with the PDS-1000/He Biolistic Particle Delivery system (Bio-Rad). Rupture disks (1350 psi) were used, and the plates are placed 6 cm below the screen/macrocarrier assembly. The cells were allowed to recover at 25 °C for 12-24 h. Upon recovery, the cells were scraped from the plates with a rubber spatula, mixed with 100 μΐ of medium and spread on plates containing the appropriate antibiotic selection. After 7-10 days of incubation at 25°C, colonies representing transformed cells were visible on the plates. Colonies were picked and spotted on selective (either antibiotic or carbon source) agar plates for a second round of selection.

EXAMPLE 3: Transformation of Chlorella

Vector construction

[0610] A BamHI-SacII fragment containing the CMV promoter, a hygromycin resistance cDNA, and a CMV 3' UTR (SEQ ID NO: 152, a subsequence of the pCAMBIA1380 vector, Cambia, Canberra, Australia) was cloned into the BamHI and SacII sites of pBluescript and is referred to herein as pHyg.

Biolistic transformation of Chlorella

[0611] S550d gold carriers from Seashell Technology were prepared according to the protocol from manufacturer. Linearized pHyg plasmid (20 μg) was mixed with 50 μΐ of binding buffer and 60 μΐ (30 mg) of S550d gold carriers and incubated in ice for 1 min. Precipitation buffer (100 μΐ) was added, and the mixture was incubated in ice for another 1 min. After vortexing, DNA-coated particles were pelleted by spinning at 10,000 rpm in an Eppendorf 5415C microfuge for 10 seconds. The gold pellet was washed once with 500 μΐ of cold 100% ethanol, pelleted by brief spinning in the microfuge, and resuspended with 50 μΐ of ice-cold ethanol. After a brief (1-2 sec) sonication, 10 μΐ of DNA-coated particles were immediately transferred to the carrier membrane.

[0612] Chlorella protothecoides culture (Univeristy of Texas Culture Collection 250) was grown in proteose medium (2g/L yeast extract, 2.94mM NaN03, 0.17mM CaC12 » 2H20, 0.3mM MgS04 » 7H20, 0.4mM K2HP04, 1.28mM KH2P04, 0.43mM NaCl) on a gyratory shaker under continuous light at 75 μιηοΐ photons m "2 sec "1 until it reached a cell density of 2xl0 6 cells/ml. The cells were harvested, washed once with sterile distilled water, and resuspended in 50 μΐ of medium. 1 x 10 7 cells were spread in the center third of a nonselective proteose media plate. The cells were bombarded with the PDS-1000/He Biolistic Particle Delivery system (Bio-Rad). Rupture disks (1100 and 1350 psi) were used, and the plates were placed 9 and 12 cm below the screen/macrocarrier assembly. The cells were allowed to recover at 25°C for 12-24 h. Upon recovery, the cells were scraped from the plates with a rubber spatula, mixed with 100 μΐ of medium and spread on hygromycin contained plates (200 μg/ml). After 7-10 days of incubation at 25°C, colonies representing transformed cells were visible on the plates from 1100 and 1350 psi rupture discs and from 9 and 12 cm distances. Colonies were picked and spotted on selective agar plates for a second round of selection.

Transformation of Chlorella by Electroporation

[0613] Chlorella protothecoides culture was grown in proteose medium on a gyratory shaker under continuous light at 75 μιηοΐ photons m "2 sec "1 until it reached a cell density of of 2xl0 6 cells/ml. The cells were harvested, washed once with sterile distilled water, and resuspended in a tris-phosphate buffer (20m M Tris-HCl, pH 7.0; 1 mM potassium phosphate) containing 50 mM sucrose to a density of 4xl0 8 cells/ml. About 250 μΐ cell suspension (lxl0 8 cells) was placed in a disposable electroporation cuvette of 4 mm gap. To the cell suspension, 5 μg of linearized pHyg plasmid DNA and 200 μg of carrier DNA (sheared salmon sperm DNA) was added. The electroporation cuvette was then incubated in a water bath at 16°C for 10 minutes. An electrical pulse (1100 V/cm) was then applied to the cuvette at a capacitance of 25 μΕ (no shunt resistor was used for the electroporation) using a Gene Pulser II (Bio-Rad Labs, Hercules, CA) electroporation apparatus. The cuvette was then incubated at room temperature for 5 minutes, following which the cell suspension was transferred to 50 ml of proteose media, and shaken on a gyratory shaker for 2 days.

Following recovery, the cells were harvested by centrifugation at low speed, resuspended in proteose media, and plated at low density on plates supplemented with 200 μg/ ml hygromycin. The plates were incubated under continuous light at 75 μιηοΐ photons m "2 sec "1 . Transformants appeared as colonies in 1 - 2 weeks. Colonies were picked and spotted on selective agar plates for a second round of selection.

Genotyping

[0614] A subset of colonies that survived a second round of selection were cultured in small volume and harvested. Pellets of approximately 5-10 uL volume were resuspended in 50 uL of lOmM NaEDTA by vortexing and then incubated at 100°C for 10. The tubes were then vortexed briefly and sonicated for 10 seconds, then centifuged at 12,000 x g for 1 minute. 2 uL of supernatant as template was used in a 50 uL PCR reaction. Primers used for genotyping were SEQ ID NO: 153 and SEQ ID NO: 154. PCR conditions were as follows: 95°C 5 min x 1 cycle; 95°C 30 sec - 58°C 30 sec - 72°C 1 min 30 sec x 35 cycles; 72°C 10 min x 1 cycle. The expected 992 bp fragment was found in 6 of 10 colonies from the biolistic method and from a single electroporation colony. A lower sized, nonspecific band was present in all lanes. To confirm the identity of the amplified 992bp fragment, two biolistic bands and the electroporation band were excised from the gel and individually sequenced. The sequence of all three bands corresponded to the expected 992 bp fragment. (DNA ladder: Bionexus ® All Purpose Hi-Lo ® DNA ladder catalog # BN2050).

EXAMPLE 4: Algal-derived Promoters and Genes for Use in Microalgae

A. 5'UTR and Promoter Sequences from Chlorella protothecoides

[0615] A cDNA library was generated from mixotrophically grown Chlorella

protothecoides (UTEX 250) using standard techniques. Based upon the cDNA sequences, primers were designed in certain known housekeeping genes to "walk" upstream of the coding regions using Seegene's DNA Walking kit (Rockville, MD). Sequences isolated include an actin (SEQ ID NO: 155) and elongation factor-la (EFla) (SEQ ID NO: 156) promoter/UTR, both of which contain introns (as shown in the lower case) and exons (upper case italicized) and the predicted start site (in bold) and two beta-tubulin promoter/UTR elements: Isoform A (SEQ ID NO: 157) and Isoform B (SEQ ID NO: 158).

B. Lipid Biosynthesis Enzyme and Plastid Tar2etin2 Sequences from C. protothecoides

[0616] From the cDNA library described above, three cDNAs encoding proteins functional in lipid metabolism in Chlorella protothecoides (UTEX 250) were cloned using the same methods as described above. The nucleotide and amino acid sequences for an acyl ACP desaturase (SEQ ID NOs: 159 and 160) and two geranyl geranyl diphosphate synthases (SEQ ID NOs: 161-164) are included in the Sequence Listing below. Additionally, three cDNAs with putative signal sequences targeting to the plastid were also cloned. The nucleotide and amino acid sequences for a glyceraldehyde-3 -phosphate dehydrogenase (SEQ ID NOs: 165 and 166), an oxygen evolving complex protein OEE33 (SEQ ID NOs: 167 and 168) and a Clp protease (SEQ ID NOs: 169 and 170) are included in the Sequence Listing below. The putative plastid targeting sequence has been underlined in both the nucleotide and amino acid sequence. The plastid targeting sequences can be used to target the producs of transgenes to the plastid of microbes, such as lipid modification enzymes.

EXAMPLE 5: Genetic Engineering of Chlorella protothecoides to Express an

Exogenous Sucrose Invertase

[0617] Strains and Media: Chlorella protothecoides (UTEX 250) was obtained from the Culture Collection of Alga at the University of Texas (Austin, TX, USA). The stock cultures were maintained on modified Proteose medium. Modified Proteose medium consists of 0.25 g NaN0 3 , 0.09 g K 2 HP0 4 , 0.175 g KH 2 P0 4 0.025 g, 0.025 g CaCl r 2H 2 0, 0.075 g

MgS0 4 -7H 2 0, and 2 g yeast extract per liter (g/L).

[0618] Plasmid Construction: To express the secreted form of invertase in Chlorella protothecoides, a Saccharomyces cerevisiae SUC2 gene was placed under the control of three different promoters: Cauliflower mosaic virus 35 S promoter (CMV), Chlorella virus promoter (NC-1A), and Chlorella HUP1 promoter. A yeast SUC2 gene was synthesized to accommodate codon usage optimized for C. protothecoides and includes a signal sequence required for directing extracellular secretion of invertase. Each construct was built in pBluescript KS+, and EcoRI/AscI, Ascl/Xhol, and XhoI/BamHI sites were introduced to each promoter, invertase gene, and CMV 3'UTR, respectively, by PCR ampilication using specific primers. Purified PCR products were cloned sequentially.

[0619] Transformation of Chlorella protothecoides: A Chlorella protothecoides culture was grown in modified Proteose medium on a gyratory shaker under continuous light at 75 μιηοΐ photons m "2 sec "1 till it reached a cell density of of 6xl0 6 cells/ml.

[0620] For biolistic transformation, S550d gold carriers from Seashell Technology were prepared according to the protocol from the manufacturer. Briefly, a linearized construct (20 μg) by Bsal was mixed with 50 μΐ of binding buffer and 60 μΐ (3 mg) of S550d gold carriers and incubated in ice for 1 min. Precipitation buffer (100 μΐ) was added, and the mixture was incubated in ice for another 1 min. After mild vortexing, DNA-coated particles were pelleted by spinning at 10,000 rpm in an Eppendorf microfuge for 10 seconds. The gold pellet was washed once with 500 μΐ of cold 100% ethanol, pelleted by brief spinning in the microfuge, and resuspended with 50 μΐ of ice-cold ethanol. After a brief (1-2 sec) sonication, 10 μΐ of DNA-coated particles were immediately transferred to the carrier membrane. The cells were harvested, washed once with sterile distilled water, resuspended in 50 μΐ of medium (1 x 10 7 cells), and were spread in the center third of a non-selective Proteous plate. The cells were bombarded with the PDS-1000/He Biolistic Particle Delivery system (Bio-Rad). Rupture disks (1100 and 1350 psi) were used, and the plates were placed 9-12 cm below the screen/macrocarrier assembly. The cells were allowed to recover at 25°C for 12-24 hours. Upon recovery, the cells were scraped from the plates with a rubber spatula, mixed with 100 μΐ of medium and spread on modified Proteose plates with 1% sucrose. After 7-10 days of incubation at 25 °C in the dark, colonies representing transformed cells were visible on the plates.

[0621] For transformation with electroporation, cells were harvested, washed once with sterile distilled water, and resuspended in a Tris- phosphate buffer (20m M Tris-HCl, pH 7.0; 1 mM potassium phosphate) containing 50 mM sucrose to a density of 4x10 cells/ml. About 250 μΐ cell suspension (lxl0 8 cells) was placed in a disposable electroporation cuvette of 4 mm gap. To the cell suspension, 5 μg of linearized plasmid DNA and 200 μg of carrier DNA (sheared salmon sperm DNA) were added. The electroporation cuvette was then incubated in an ice water bath at 16 °C for 10 min. An electrical pulse (1100 V/cm) was then applied to the cuvette at a capacitance of 25 μΡ (no shunt resistor was used for the electroporation) using a Gene Pulser II (Bio-Rad Labs, Hercules, CA) electroporation apparatus. The cuvette was then incubated at room temperature for 5 minutes, following which the cell suspension was transferred to 50 ml of modified Proteose media, and shaken on a gyratory shaker for 2 days. Following recovery, the cells were harvested at low speed (4000 rpm), resuspended in modified Proteose media, and plated out at low density on modified Proteose plates with 1% sucrose. After 7-10 days of incubation at 25 °C in the dark, colonies representing transformed cells were visible on the plates.

[0622] Screening Transformants and Genotyping: The colonies were picked from dark grown-modified Proteose plates with 1% sucrose, and approximately the same amount of cells were transferred to 24 well-plates containing 1 ml of modified Proteose liquid media with 1 % sucrose. The cultures were kept in dark and agitated by orbital shaker from Labnet (Berkshire, UK) at 430 rpm for 5 days.

[0623] To verify the presence of the invertase gene introduced in Chlorella transformants, DNA of each transformant was isolated and amplified with a set of gene-specific primers (CMV construct: forward primer (CAACCACGTCTTCAAAGCAA) (SEQ ID NO: 153)/ reverse primer (TCCGGTGTGTTGTAAGTCCA) (SEQ ID NO: 171), CV constructs: forward primer (TTGTCGGAATGTCATATCAA) (SEQ ID NO: 172)/ reverse primer (TCCGGTGTGTTGTAAGTCCA) (SEQ ID NO: 171), and HUP1 construct: forward primer (AACGCCTTTGTACAACTGCA) (SEQ ID NO: 173)/ reverse primer

(TCCGGTGTGTTGTAAGTCCA) (SEQ ID NO: 171)). For quick DNA isolation, a volume of cells (approximately 5-10 uL in size) were resuspended in 50 uL of 10 mM Na-EDTA. The cell suspension was incubated at 100°C for 10 min and sonicated for 10 sec. After centrifugation at 12000g for 1 min, 3 uL of supernatant was used for the PCR reaction. PCR amplification was performed in the DNA thermal cycler (Perkin-Elmer Gene Amp 9600). The reaction mixture (50 uL) contained 3 uL extracted DNA, 100 pmol each of the respective primers described above, 200 uM dNTP, 0.5 units of Taq DNA polymerase (NEB), and Taq DNA polymerase buffer according to the manufacturer's instructions. Denaturation of DNA was carried out at 95°C for 5 min for the first cycle, and then for 30 sec. Primer annealing and extension reactions were carried out at 58°C for 30 sec and 72°C for 1 min respectively. The PCR products were then visualized on 1 % agarose gels stained with ethidium bromide.

[0624] Growth in Liquid Culture: After five days growth in darkness, the genotype- positive transformants showed growth on minimal liquid Proteose media + 1 % sucrose in darkness, while wild-type cells showed no growth in the same media in darkness.

EXAMPLE 6: Transformation of algal strains with a secreted invertase derived from S. cerevisiae

[0625] Secreted Invertase: A gene encoding a secreted sucrose invertase (Gen Bank

Accession no. NP_012104 from Saccharomyces cerevisiae) was synthesized de-novo as a 1599 bp Asc I-Xho fragment that was subsequently sub-cloned into a pUC19 derivative possessing the Cauliflower Mosaic Virus 35s promoter and 3' UTR as EcoR I/Asc I and Xho/Sac I cassettes, respectively.

[0626] Growth of Algal Cells: Media used in these experiments was liquid base media (2g/L yeast extract, 2.94mM NaN0 3 , 0.17mM CaCl 2 » 2H 2 0, 0.3mM MgS0 4 » 7H 2 0, 0.4mM K 2 HP0 4 , 1.28mM KH 2 P0 4 , 0.43mM NaCl) and solid base media (+ 1.5% agarose) containing fixed carbon in the form of sucrose or glucose (as designated) at 1% final concentration. The strains used in this experiment did not grow in the dark on base media in the absence of an additional fixed carbon source. Species were struck out on plates, and grown in the dark at 28°C. Single colonies were picked and used to inoculate 500 mL of liquid base media containing 1 % glucose and allowed to grow in the dark until mid-log phase, measuring cell counts each day. Each of the following strains had been previously tested for growth on sucrose in the dark as a sole carbon source and exhibited no growth, and were thus chosen for transformation with a secreted invertase: (1) Chlorella protothecoides (UTEX 31); (2) Chlorella minutissima (UTEX 2341); and (3) Chlorella emersonii (CCAP 211/15).

[0627] Transformation of Algal Cells via Particle Bombardment: Sufficient culture was centrifuged to give approximately 1-5 x 10 8 total cells. The resulting pellet was washed with base media with no added fixed carbon source. Cells were centrifuged again and the pellet was resuspended in a volume of base media sufficient to give 5 x 10 7 to 2 x 10 8 cells/ml. 250-1000 μΐ of cells were then plated on solid base media supplemented with 1 % sucrose and allowed to dry onto the plate in a sterile hood. Plasmid DNA was precipitated onto gold particles according to the manufacturer's recommendations (Seashell Technology, La Jolla, CA). Transformations were carried out using a BioRad PDS He-1000 particle delivery system using 1350 psi rupture disks with the macrocarrier assembly set at 9cm from the rupture disk holder. Following transformations, plates were incubated in the dark at 28 °C. All strains generated multiple transformant colonies. Control plates transformed with no invertase insert, but otherwise prepared in an identical fashion, contained no colonies.

[0628] Analysis of Chlorella protothecoides transformants: Genomic DNA was extracted from Chlorella protothecoides wild type cells and transformant colonies as follows: Cells were resuspended in 100 ul extraction buffer (87.5 mM Tris CI, pH 8.0, 50 mM NaCl, 5 mM EDTA, pH 8.0, 0.25% SDS) and incubated at 60°C, with occasional mixing via inversion, for 30 minutes. For PCR, samples were diluted 1: 100 in 20 mM Tris CI, pH 8.0.

[0629] Genotyping was done on genomic DNA extracted from WT, the transformants and plasmid DNA. The samples were genotyped for the marker gene. Primers 2383 (5' CTGACCCGACCTATGGGAGCGCTCTTGGC 3') (SEQ ID NO: 174) and 2279 (5' CTTGACTTCCCTCACCTGGAATTTGTCG 3') (SEQ ID NO: 175) were used in this genotyping PCR. The PCR profile used was as follows: 94°C denaturation for 5 min; 35 cycles of 94°C -30 sec, 60°C - 30 sec, 72°C -3 min; 72°C -5 min. A band of identical size was amplified from the positive controls (plasmid) and two transformants of Chlorella protothecoides (UTEX 31).

[0630] Analysis of Chlorella minutissima and Chlorella emersonii transformants:

Genomic DNA was extracted from Chlorella WT and the tranformants as follows: Cells were resuspended in 100 ul extraction buffer (87.5 mM Tris CI, pH 8.0, 50 mM NaCl, 5 mM EDTA, pH 8.0, 0.25% SDS) and incubated at 60°C, with occasional mixing via inversion, for 30 minutes. For PCR, samples were diluted 1: 100 in 20 mM Tris CI, pH 8.0. Genotyping was done on genomic DNA extracted from WT, the transformants and plasmid DNA. The samples were genotyped for the marker gene. Primers 2336 (5'

GTGGCCATATGGACTTACAA 3') (SEQ ID NO: 176) and 2279

(5' CTTGACTTCCCTCACCTGGAATTTGTCG 3') (SEQ ID NO: 175) were designated primer set 2 (1215 bp expected product), while primers 2465

(5' CAAGGGCTGGATGAATGACCCCAATGGACTGTGGTACGACG 3') (SEQ ID NO:

177) and 2470 (5' CACCCGTCGTCATGTTCACGGAGCCCAGTGCG 3') (SEQ ID NO:

178) were designated primer set 4 (1442 bp expected product). The PCR profile used was as follows: 94°C denaturation for 2 min; 29 cycles of 94°C -30 sec, 60°C - 30 sec, 72°C - 1 min, 30 sec; 72°C -5 min. A plasmid control containing the secreted invertase was used as a PCR control.

[0631] The sequence of the invertase construct corresponds to SEQ ID NO: 8. [0632] EXAMPLE 7: Homologous Recombination in Prototheca species

[0633] Homologous recombination of transgenes has several advantages. First, the introduction of transgenes without homologous recombination can be unpredictable because there is no control over the number of copies of the plasmid that gets introduced into the cell. Also, the introduction of transgenes without homologous recombination can be unstable because the plasmid may remain episomal and is lost over subsequent cell divisions. Another advantage of homologous recombination is the ability to "knock-out" gene targets, introduce epitope tags, switch promoters of endogenous genes and otherwise alter gene targets (e.g., the introduction of point mutations.

[0634] Two vectors were constructed using a specific region of the Prototheca moriformis (UTEX 1435) genome, designated KE858. KE858 is a 1.3 kb, genomic fragment that encompasses part of the coding region for a protein that shares homology with the transfer RNA (tRNA) family of proteins. Southern blots have shown that the KE858 sequence is present in a single copy in the Prototheca moriformis (UTEX 1435) genome. The first type of vector that was constructed, designated SZ725 (SEQ ID NO: 179), consisted of the entire 1.3 kb KE858 fragment cloned into a pUC19 vector backbone that also contains the optimized yeast invertase (suc2) gene. The KE858 fragment contains a unique SnaBl site that does not occur anywhere else in the targeting construct. The second type of vector that was constructed, designated SZ726 (SEQ ID NO: 180), consisted of the KE858 sequence that had been disrupted by the insertion of the yeast invertase gene (suc2) at the SnaB l site within the KE858 genomic sequence. The entire DNA fragment containing the KE858 sequences flanking the yeast invertase gene can be excised from the vector backbone by digestion with EcoRI, which cuts at either end of the KE858 region.

[0635] Both vectors were used to direct homologous recombination of the yeast invertase gene (suc2) into the corresponding KE858 region of the Prototheca moriformis (UTEX 1435) genome. The linear DNA ends homologous to the genomic region that was being targeted for homologous recombination were exposed by digesting the vector construct SZ725 with SnaB 1 and vector construct SZ726 with EcoRI. The digested vector constructs were then introduced into Prototheca moriformis cultures using methods described above.

Transformants from each vector construct were then selected using sucrose plates. Ten independent, clonally pure transformants from each vector transformation were analyzed for successful recombination of the yeast invertase gene into the desired genomic location (using Southern blots) and for transgene stability. [0636] Southern blot analysis of the SZ725 transformants showed that 4 out of the 10 transformants picked for analysis contained the predicted recombinant bands, indicating that a single crossover event had occurred between the KE858 sequences on the vector and the KE858 sequences in the genome. In contrast, all ten of the SZ726 transformants contained the predicted recombinat bands, indicating that double crossover events had occurred between the EcoRI fragment of pSZ726 carrying KE858 sequence flanking the yeast invertase transgene and the corresponding KE858 region of the genome.

[0637] Sucrose invertase expression and transgene stability were assessed by growing the transformants for over 15 generations in the absence of selection. The four SZ725 transformants and the ten SZ276 transformants that were positive for the transgene by Southern blotting were selected and 48 single colonies from each of the transformants were grown serially: first without selection in glucose containing media and then with selection in media containing sucrose as the sole carbon source. All ten SZ276 transformants (100%) retained their ability to grow on sucrose after 15 generations, whereas about 97% of the SZ725 transformants retained their ability to grow on sucrose after 15 generations.

Transgenes introduced by a double crossover event (SZ726 vector) have extremely high stability over generation doublings. In contrast, transgenes introduced by a single cross over event (SZ725 vector) can result in some instability over generation doublings because is tandem copies of the transgenes were introduced, the repeated homologous regions flanking the transgenes may recombine and excise the transgenic DNA located between them.

[0638] These experiments demonstrate the successful use of homologous recombination to generate Prototheca transformants containing a heterologous sucrose invertase gene that is stably integrated into the nuclear chromosomes of the organism. The success of the homologous recombination enables other genomic alterations in Prototheca, including gene deletions, point mutations and epitope tagging a desired gene product. These experiments also demonstrate the first documented system for homologous recombination in the nuclear genome of a eukaryotic microalgae.

[0639] Use of Homologous Recombination to Knock-out an Endogenous Prototheca moriformis gene: In a Prototheca moriformis cDNA/genomic screen, like that described above in Example 4, an endogenous stearoyl ACP desaturase (SAPD) cDNA was identified. Stearoyl ACP desaturase enzymes are part of the lipid synthesis pathway and they function to introduce double bonds into the fatty acyl chains. In some cases, it may be advantages to knock-out or reduce the expression of lipid pathway enzymes in order to alter a fatty acid profile. A homologous recombination construct was created to assess whether the expression of an endogenous stearoyl ACP desaturase enzyme can be reduced (or knocked out) and if a corresponding reduction in unsaturated fatty acids can be observed in the lipid profile of the host cell. An approximately 1.5kb coding sequence of a stearoyl ACP desaturase gene from Prototheca moriformis (UTEX 1435) was identified and cloned (SEQ ID NO: 181). The homologous recombination construct was constructed using 0.5kb of the SAPD coding sequence at the 5 'end (5' targeting site), followed by the Chlamydomonas reinhardtii β-tublin promoter driving a codon-optimized yeast sucrose invertase suc2 gene with the Chlorella vulgaris 3'UTR. The rest (~lkb) of the Prototheca moriformis SAPD coding sequence was then inserted after the C. vulgaris 3'UTR to make up the 3' targeting site. The sequence for this homologous recombination cassette is listed in SEQ ID NO: 182. As shown above, the success-rate for integration of the homologous recombination cassette into the nuclear genome can be increased by linearizing the cassette before transforming the microalgae, leaving exposed ends. The homologous recombination cassette targeting an endogenous SAPD enzyme in Prototheca moriformis is linearized and then transformed into the host cell {Prototheca moriformis, UTEX 1435). A successful integration will eliminate the endogenous SAPD enzyme coding region from the host genome via a double reciprocal recombination event, while expression of the newly inserted suc2 gene will be regulated by the C. reinhardtii β-tubulin promoter. The resulting clones can be screened using

plates/media containing sucrose as the sole carbon source. Clones containing a successful integration of the homologous recombination cassette will have the ability to grow on sucrose as the sole carbon source and changes in overall saturation of the fatty acids in the lipid profile will serve as a secondary confirmation factor. Additionally, Southern blotting assays using a probe specific for the yeast sucrose invertase suc2 gene and RT-PCR can also confirm the presence and expression of the invertase gene in positive clones. As an alternative, the same construct without the β-tubulin promoter can be used to excise the endogenous SAPD enzyme coding region. In this case, the newly inserted yeast sucrose invertase suc2 gene will be regulated by the endogenous SAPD promoter/5 'UTR.

EXAMPLE 8: Expression of various thioesterases in Prototheca

[0640] Methods and effects of expressing a heterologous thioesterase gene in Prototheca species have been previously described in PCT Application No. PCT/US2009/66142, hereby incorporated by reference. The effect of other thioesterase genes/gene products from higher plants species was further investigated. These thioesterases include thioesterases from the following higher plants: GenBank

Species Accession No. Specificity SEO ID NO:

Cinnamomum camphora Q39473 C14 SEQ ID NOs: 30- 31

Umbellularia californica Q41635 C10-C12 SEQ ID NOs: 34- -35

Cuphea hookeriana AAC49269 C8-C10 SEQ ID NOs: 32- -33

Cuphea palustris AAC49179 C8 SEQ ID NOs: 36- -37

Cuphea lanceolata CAB 60830 CIO SEQ ID NOs: 38- -39

Iris germanica AAG43858.1 C14 SEQ ID NOs: 40- -41

Myristica fragrans AAB717291.1 C14 SEQ ID NOs: 42- -43

Cuphea palustris AAC49180 C14 SEQ ID NOs: 44- -45

Ulmus americana AAB71731 broad SEQ ID NOs: 46- -47

[0641] In all cases, each of the above thioesterase constructs was transformed in to Prototheca moriformis (UTEX 1435) using biolistic particle bombardment. Other transformation methods including homologous recombination as disclosed in PCT

Application No. PCT/US2009/66142, would also be suitable for heterologous expression of genes of interest. Transformation of Prototheca moriformis (UTEX 1435) with each of the above thioesterase constructs was performed using the methods described in Example 2. Each of the constructs contained a NeoR gene and selection for positive clones was carried out using 100 μg/ml G418. All coding regions were codon optimized to reflect the codon bias inherent in Prototheca moriformis UTEX 1435 (see Table 2) nuclear genes. Both amino acid sequences and the cDNA sequences for the construct used are listed in the sequence identity listing. The transit peptide for each of the higher plant thioesterase was replaced with an algal codon optimized transit peptide from Prototheca moriformis delta 12 fatty acid desaturase (SEQ ID NO: 48)) or from Chlorella protothecoides stearoyl ACP desaturase (SEQ ID NO: 49). All thioesterase constructs were driven by the Chlamydomanas reinhardtii beta-tubulin promoter/5 'UTR. Growth and lipid production of selected positive clones were compared to wildtype (untransformed) Prototheca moriformis (UTEX 1435). Wildtype and selected positive clones were grown on 2% glucose G418 plates. Lipid profiles analysis on selected positive clones for each construct is summarized below (expressed in Area ) in Table 15.

[0642] Table 15. Lipid profiles of Prototheca moriformis expressing various heterologous thioesterases.

Fatty UTEX Thioesterase C8:0 0 0 0 0 3.1 1.8 0 0 .09

C10:0 0.02 .07 .02 .01 .09 .56 6.85 1.91 .01 2.85

C12:0 0.05 14 1.82 .09 .05 .25 .2 .29 .06 .74

C14:0 1.65 3 17.3 2.59 5.31 1.45 1.8 1.83 2.87 10.45

f ca li orn i ca

C16:0 28.0 21.4 24.3 26.52 31.08 22.84 23.9 25.55 27.23 33.3

U.

C18:0 2.9 2.9 2.7 3.11 2.71 3.24 2.8 3.26 3.62 3.47

C18: l 53.8 45.2 p cam h ora 41.3 49.96 39.77 56.62 49.8 55.43 51.04 38.71

C.

C18:2 10.95 10 9.7 11.86 14.17 8.24 9.7 8.17 10.81 7.38

C18:3 a 0.8 .86 .8 .40 .64 .61 .9 .58 .97 .52

german i ca

Total 32.62 44.97 46.14 32.32 39.24 31.44 37.35 32.84 33.79 50.9 saturates

(area %)

fg M.rarans

[0643] The results show that all of the thioesterases C 8:0 expressed impacted fatty acid profiles

p C.a l us t r i s

to some level. Looking at the "Total saturates" row, the degree of saturation was profoundly

hoo k er i ana

impacted by the expression of several of the thioesterases, inclu C.ding those from U.

californica, C. camphora, and most notably, U. americana. These ch l anceo l a t aanges in the percentage

C.

of total saturates were unexpected in that the heterologous expression of thioesterases from higher plants can apparently impact more than just lipid chain lengths; it ca C 14:0 n also impact

p C.a l us t r i s

other attributes of lipid profiles produced by microalgae, namely the degree of saturation of

amer i cana

the fatty acids. U.

[0644] Selected clones transformed with C. palustris C8 thioesterase, C. hookeriana thioesterase, U. californica and C. camphora thioesterase were further grown in varing amounts of G418 (from 25 mg/L to 50 mg/L) and at varying temperatures (from 22°C to 25 °C) and the lipid profile was determined for these clones. Table 16 summarizes the lipid profile (in Area ) of representative clones containing each thioesterase. A second construct containing the U. americana thioesterase was constructed and transformed into Prototheca moriformis (UTEX 1435) using the biolistic methods described above. This second construct was introduced into the cell via homologous recombination. Methods of homologous recombination in Prototheca species were described previously in PCT Application No. PCT/US2009/66142. The homologous DNA that was used was from genomic DNA sequence of 6S rRNA from Prototheca moriformis UTEX 1435. The selection agent was the ability to grow on sucrose, using a codon optimized sucl gene from S. cereveisiae driven by the C. reinhardtii beta tubulin promoter. The native U. americana transit peptide was replaced by the Chlorella protothecoides (UTEX 250) stearoyl ACP desaturase transit peptide. The cDNA of this construct is listed in the Sequence Listing as SEQ ID NO: 50. Selection of positive clones was performed on 2% sucrose plates and the resulting cultures for lipid profile determination was also grown on 2% sucrose containing medium. A representative lipid profile for this Prototheca moriformis strain containing a homologously recombined heterologous U. americana thioesterase is summarized in Table 16.

[0645] Table 16. Lipid profiles of Prototheca moriformis strains containing heterologous thioesterase genes.

[0646] As with the clones described above, all transformants containing a heterologous thioesterase gene showed impacted fatty acid profiles to some level, and the total percent of saturated fatty acids were also changed, as compared to wildtype (untransformed) Prototheca moriformis. The Prototheca moriformis containing the U. americana thioesterase introduced by homologous recombination had the greatest increase in total saturates.

[0647] Additionally, transgenic clones containing the exogenous C. hookeriana, C.

camphora, U. californica or U. americana thioesterase were assessed for novel lipid profiles. The C. hookeriana thioesterase containing clone achieved the following lipid profile when grown in 2% glucose, 25mg/ml G418 at 22°C: 5.10% C8:0; 18.28% C10:0; 0.41% C12:0; 1.76% C14:0; 16.31% C16:0; 1.40% C18:0; 40.49% C18: l; and 13.16% C18:2. The C. camphora thioesterase-containing clone (also containing an exogenous sucrose invertase) achieved the following lipid profile when grown in 2% sucrose at 25°C: 0.04% C10:0; 6.01% C12:0; 35.98% C14:0; 19.42 C16:0; 1.48% C18:0; 25.44% C18: l ; and 9.34% C18:2. The U. calfornica thioesterase containing clone achieved the following lipid profile when grown in 2% glucose, 25-100 mg/ml G418 at 22°C: 0% C8:0; 0.11% C10:0; 34.01% C12:0; 5.75% C14:0; 14.02% C16:0; 1.10% C18:0; 28.93% C18: l; and 13.01% C18:2. The U. americana thioesterase containing clone achieved the following lipid profile when grown in 2% glucose at 28°C: 1.54% C10:0; 0.43% C12:0; 7.56% C14:0; 39.45% C16:0; 2.49% C18:0; 38.49% C18: l; and 7.88% C18:2.

EXAMPLE 9: Transformation of Prototheca with multiple exogenous heterologous thioesterase genes

[0648] Microalgae strain Prototheca moriformis (UTEX 1435) was transformed using the above disclosed methods to express multiple thioesterases in a single clone. The expression of multiple thioesterases in a single clone allows the microaglae to produce oils with fatty acid profiles completely different from those elaborated when any single thioesterase is expressed alone (as demonstrated in the preceding Examples). Prototheca moriformis (UTEX 1435) was first transformed with the Cinnamomum camphora thioesterase (a C14 preferring thioesterase) along with a sucrose invertase gene, the sucl from S. cerevisiae (selection was the ability to grow on sucrose) using homologous recombination. The DNA used for this homologous recombination construct is from the KE858 region of Prototheca moriformis genomic DNA as described in the Section III above. The relevant portion of this construct is listed in the Sequence Listing as SEQ ID NO: 51. Positive clones were screened on sucrose-containing plates. A positive clone was then re-transformed with one of three cassettes, each encoding resistence to the antibiotic G418 as well as an additional thioesterase: (1) thioesterase gene from Cuphea hookeriana (C8-10 preferring), SEQ ID NO: 52; (2) thioesterase gene from Umbellularia californica (C12 preferring), SEQ ID NO: 53; or thioesterase from Ulmus americana (broad; C10-C16 preferring), SEQ ID NO: 54. Included in the Sequence Listing is the sequence of the relevant portion of each construct. Clones expressing both thioesterase genes were screened on sucrose containing medium with 50 μg/ml G418. Positive clones were selected and growth and lipid profile were assayed. Table 17 summarizes the lipid profile of representative positive clones (expressed in Area %).

[0649] Table 17. Lipid profiles of Prototheca moriformis transformed with multiple thioesterases.

C18:l 53.8 47.78 48.54 42.55 37.3

C18:2 10.95 12.3 11.76 10.13 8.9

C18:3 a 0.8 0.93 0.91 0.91 0.76

Total 32.62 40.43 36.06 43.84 50.58 saturates

(Area %)

[0650] Additionally, a double thioesterase clone with C. camphora and U. californica thioesterases was grown in 2% sucrose containing medium with 50 mg/L G418 at 22°C. The fatty acid profile obtained from this strain under these growth conditions was: C8:0 (0); C10:0 (0.10); C12:0 (31.03); C14:0 (7.47); C16:0 (15.20); C18:0 (0.90); C18: l (30.60); C18:2 (12.44); and C18:3a (1.38), with a total saturates of 54.7.

[0651] Double thioesterase clones with two homologous recombination constructs (one targeting the 6S region and the other targeting the KE858 region) containing the C. camphora thioestease were produced. A positive representative clone had a fatty acid profile of: 0% C8:0; 0.06% C10:0; 5.91% C12:0; 43.27% C14:0; 19.63% C16:0; 0.87% C18:0; 13.96% C18: l; and 13.78% C18:2, with a total saturates at 69.74%. This clone had a C12-C14 level at over 49%, which is over 37 times the C12-C14 level in wildtype cells.

[0652] The above data shows that multiple thioesterases can be successfully co-expressed in microalgae. The co-expression of multiple thioesterases results in altered fatty acid profiles that differ significantly not only from the wild type strain, but also from the fatty acid profile obtained by the expression of any one of the individual thioesterases. The expression of multiple thioesterases with overlapping chain length specificity can result in cumulative increases in those specific fatty acids.

[0653] The expression of heterologous thioesterases (either alone or in combination) in Prototheca moriformis not only alters the fatty acid/lipid profiles in the host strain, but when compared to oils currently available from a variety of seed crops (Table 5), these profiles are of truly unique oils found in no other currently available system. Not only do the transgenic strains show significant differences from the untransformed wildtype strain, they have remarkably different profiles from any of the commercial oils that are shown in Table 5. As an example, both coconut and palm kernel oils have levels of C8-C10 fatty acids ranging from 5.5-17%. Transgenic strain expressing the C. palustris C8-preferring thioesterase or the C. hookeriana ClO-preferring thioesterase accumulates anywhere from 3.66 to 8.65%, respectively. These C8-C10 fatty acid levels are similar to coconut oil and palm kernel, however, the transgenic algal strains lack the significantly higher C12:0 fatty acids, and they have extremely high C16:0 (23% in transgenics versus 11-16% in coconut or palm kernel oil, respectively and/or 18: 1 (50-57% in transgenics versus 8-19% in coconut or palm kernel oil, respectively.

EXAMPLE 10: Identification of endogenous nitrogen-dependent Prototheca promoters A. Identification and characterization of endo2enous nitro2en-dependent promoters.

[0654] A cDNA library was generated from Prototheca moriformis (UTEX 1435) using standard techniques. The Prototheca moriformis cells were grown for 48 hours under nitrogen replete conditions. Then a 5% innoculum (v/v) was then transferred to low nitrogen and the cells were harvested every 24 hours for seven days. After about 24 hours in culture, the nitrogen supply in the media was completely depleted. The collected samples were immediately frozen using dry ice and isopropanol. Total RNA was subsequently isolated from the frozen cell pellet samples and a portion from each sample was held in reserve for RT-PCR studies. The rest of the total RNA harvested from the samples was subjected to polyA selection. Equimolar amounts of polyA selected RNA from each condition was then pooled and used to generate a cDNA library in vector pcDNA 3.0 (Invitrogen). Roughly 1200 clones were randomly picked from the resulting pooled cDNA libray and subjected to sequencing on both strands. Approximately 68 different cDNAs were selected from among these 1200 sequences and used to design cDNA-specific primers for use in real-time RT-PCR studies.

[0655] RNA isolated from the cell pellet samples that were held in reserve was used as substrate in the real time RT-PCR studies using the cDNA-specific primer sets generated above. This reserved RNA was converted into cDNA and used as substrate for RT-PCR for each of the 68 gene specific primer sets. Threshold cylcle or CT numbers were used to indicate relative transcript abundance for each of the 68 cDNAs within each RNA sample collected throughout the time course. cDNAs showing significant increase (greater than three fold) between nitrogen replete and nitrogen-depleted conditions were flagged as potential genes whose expression was up-regulated by nitrogen depletion. As discussed in the specification, nitrogen depletion/limitation is a known inducer of lipogenesis in oleaginous microorganisms.

[0656] In order to identify putative promoters/5 'UTR sequences from the cDNAs whose expression was upregulated during nitrogen depletion/limitation, total DNA was isolated from Prototheca moriformis (UTEX 1435) grown under nitrogen replete conditions and were then subjected to sequencing using 454 sequencing technology (Roche). cDNAs flagged as being up-regulated by the RT-PCR results above were compared using BLAST against assembled contigs arising from the 454 genomic sequencing reads. The 5' ends of cDNAs were mapped to specific contigs, and where possible, greater than 500bp of 5' flanking DNA was used to putatively identify promoters/UTRs. The presence of promoters/5 'UTR were subsequently confirmed and cloned using PCR amplification of genomic DNA. Individual cDNA 5' ends were used to design 3' primers and 5' end of the 454 contig assemblies were used to design 5' gene- specific primers.

[0657] As a first screen, one of the putative promoters, the 5'UTR/promoter isolated from Aat2 (Ammonium transporter, SEQ ID NO: 63), was cloned into the Cinnamomum camphor a C14 thioesterase construct with the Chlorella protothecoides stearoyl ACP desaturase transit peptide, replacing the C.sorokinana glutamate dehydrogenase promoter. This construct is listed as SEQ ID NO: 81. To test the putative promoter, the thioesterase construct is transformed into Prototheca moriformis cells to confirm actual promoter activity by screening for an increase in C14/C12 fatty acids under low/no nitrogen conditions, using the methods described above. Similar testing of the putative nitrogen-regulated promoters isolated from the cDNA/genomic screen can be done using the same methods.

[0658] Other putative nitrogen-regulated promoters/5 'UTRs that were isolated from the cDNA/genomic screen were:

Promoter/5 'UTR SEQ ID NO. Fold increased

FatB/A promoter/5 'UTR SEQ ID NO: 55 n/a

NRAMP metal transporter promoter/5 'UTR SEQ ID NO: 56 9.65

Flap Flagellar-associated protein promoter/5 'UTR SEQ ID NO: 57 4.92

SulfRed Sulfite reductase promoter/5 'UTR SEQ ID NO: 58 10.91

SugT Sugar transporter promoter/5 'UTR SEQ ID NO: 59 17.35

Amt03 -Ammonium transporter 03 promoter/5 'UTR SEQ ID NO: 60 10.1

Amt02- Ammonium transporter 02 promoter/5 'UTR SEQ ID NO: 61 10.76

AatOl -Amino acid transporter 01 promoter/5 'UTR SEQ ID NO: 62 6.21

Aat02- Amino acid transporter 02 promoter/5 'UTR SEQ ID NO: 63 6.5

Aat03- Amino acid transporter 03 promoter/5 'UTR SEQ ID NO: 64 7.87

Aat04- Amino acid transporter 04 promoter/5 'UTR SEQ ID NO: 65 10.95

Aat05- Amino acid transporter 05 promoter/5 'UTR SEQ ID NO: 66 6.71

Fold increase refers to the fold increase in cDNA abundance after 24 hours of in low nitrogen medium. [0660] To gain further insight into potential regulation of these putative promoter/5 'UTRs, eight of the sequences were selected for further testing: (1) FatB/A; (2) SulfRed Sulfite reductase; (3) SugT Sugar transporter; (4) Amt02-Ammonium transporter 02; (5) AatOl- Amino acid transporter 01 ; (6) Aat03- Amino acid transporter 03; (7) Aat04- Amino acid transporter 04; and (8) Aat05-Amino acid transporter 05. Higher resolution transcriptome analysis utilizing Illumina sequencing reads were carried out on RNA isolated from

Prototheca moriformis cells various time points: TO (seed); 20 hours; 32 hours; 48 hours; 62 hours; and 114 hours post inoculation from seed. The medium at TO (seed) was nitrogen replete, while at the time points 20 hours and longer, the medium contained little to no nitrogen. Assembled transcript contigs generated from RNA isolated from each of the time points were then blasted independently with each of the eight previously identified transcripts. The results are summarized in Table 18 below.

[0661] Table 18. Transcriptome expression profiles for eight putative promoters/5 'UTRs.

cDNA TS T20 T32 T48 T62 T114

aa trans_01 absolute 98 96 321 745 927 1300

relative 1 0.98 3.28 7.61 9.47 13.28

aa trans_03 absolute 7 21 51 137 102 109

relative 1 2.95 7.2 19.42 14.47 15.45

aa trans_04 absolute 1 6 25 90 131 160

relative 1 5.16 21.29 74.97 109.35 133.31

aa trans_05 absolute 109 88 123 210 214 273

relative 1 0.81 1.13 1.93 1.97 2.51

ammon trans_02 absolute 683 173 402 991 1413 1397

relative 1 0.25 0.59 1.45 2.07 2.04

fatA/B-l_cDNA absolute 13 36 654 617 544 749

relative 1 2.8 51.57 48.65 42.9 59.1

sug trans_01 absolute 25 25 106 261 266 251

relative 1 1 4.22 10.4 10.63 10

sulfite reductase_01 absolute 634 238 138 145 163 155

relative 1 0.38 0.22 0.22 0.26 0.24

[0662] From the above- summarized results, several of the transcripts show increased accumulation over time, although interestingly, the sulfite reductase mRNA shows a distinct decrease in mRNA accumulation over time.

[0663] These eight putative promoter/5 'UTR regions were cloned upstream of the C.

camphora thioesterase coding region with its native transit peptide taken out and substituted with the transit peptide from Chlorella protothecoides (UTEX 250) stearoyl ACP desaturase. Each putative promoter/5 'UTR region construct was introduced into Prototheca moriformis UTEX 1435 via homologous recombination using DNA from the genomic sequence of 6S rRNA. Also contained within the construct is a sucl sucrose invertase gene from S. cerevisiae for selection of positive clones on sucrose containing media/plates. The cDNA sequence for the relevant portions of the construct for AatOl is listed in the Sequence Listing as SEQ ID NO: 67. For the other constructs, the same backbone was use, the only variable was the putative promoter/5 'UTR sequence. An additional control transgenic strain was generated in which the C. reinhardtii beta tubulin promoter was used to drive expression of the C.

camphora thioesterase gene. This promoter have shown to drive constitutive expression of the gene of interest, and thus provides a useful control against which to measure expression of the same thioesterase message when driven by the various putative N-regulated promoters/5 'UTRs tested.

[0664] Once the transgenic clones were generated, three separate experiments were carried out. The first two experiments assess the potential nitrogen regulatability of all eight putative promoters by measuring steady state thioesterase mRNA levels via RT-PCR, fatty acid profiles and ammonia levels in the culture supematants. Clones were initially grown at 28 °C with agitation (200rpm) in nitrogen rich seed medium (lg/L ammonium nitrate— 15mM nitrogen as ammonia, 4g/L yeast extract) for 24 to 48 hours, at which point 20 OD units (A750) were used to inoculate 50 ml of low nitrogen media (0.2 g/L ammonium sulfate— 3mM nitrogen as ammonia, 0.2 g/L yeast extract). Cells were sampled every 24 hours for 6 days and a sample was also collected right before switching to low nitrogen conditions. A portion of the cells from each sample was then used for total RNA extraction using Trizol reagent (according to manufacturer's suggested methods). Ammonia assays revealed that ammonia levels in the supematants fell below the limits of detection (~ ΙΟΟμΜ) after 24 hours in low nitrogen medium.

[0665] For real-time RT-PCR, all RNA levels were normalized to levels of an internal control RNA expressed in Prototheca moriformis (UTEX 1435) for each time point. The internal control RNA, termed cdl89, is a product of the ARG9 gene which encodes N-acetyl ornithine aminotransferase. Primers sets used for real-time RT-PCR in these experiments were:

Gene specific to Primer sequence 5'-3' SEQ ID NO:

C. camphora TE forward TACCCCGCCTGGGGCGACAC SEQ ID NO: 68

C. camphora TE reverse CTTGCTCAGGCGGCGGGTGC SEQ ID NO: 69 cdl89 forward CCGGATCTCGGCCAGGGCTA SEQ ID NO: 70 cdl89 reverse TCGATGTCGTGCACCGTCGC SEQ ID NO: 71 [0666] Lipid profiles from each of the transformants from each time point were also generated and compared to the RT-PCR results. Based on the ammonia levels, RT-PCR results and changes in C12-C14 fatty acid levels, it was concluded that the Amino acid transporter 01 (Aat-01), Amino acid transporter 04 (Aat-04), and Ammonium transporter 02 (Amt-02) sequences do contain a functional nitrogen-regulatable promoter/5 'UTR.

[0667] From the RT-PCR results, Aat-01 demonstrated the ability to drive steady state C. camphora thioesterase mRNA levels up to four times higher than control (C. reinhardtii beta tubulin promoter). The mRNA levels also correlated with nitrogen limitation and a marked increase in C12-C14 fatty acid levels. These results demonstrate that the 5'UTR associated with the Aat-01 promoter is likely more efficient at driving protein synthesis under lipid biosynthesis than the control C. reinhardtii promoter. Like the Aat-01 promoter, the Aat-04 promoter was able to drive mRNA accumulation up to five times higher than that of the C. reinhardtii control promoter. However, the Aat-04 promoter construct only produced a modest ability to impact C12-C14 fatty acid levels. These data demonstrate that the Aat-04 promoter is clearly regulatable by nitrogen depletion, but the UTR associated with the promoter likely functions poorly as a translational enhancer. Finally, the Amt-02 promoter was similar to the Aat-01 promoter, in that it was able to drive mRNA accumulation up to three times higher than that of the control promoter. The mRNA levels also correlated with nitrogen limitation and a marked increase in C12-C14 fatty acid levels. Taken together, all three of these promoters were demonstrated to be nitrogen-regulated.

B. Further characterization of the ammonium transporter 3 (amt03)

promoter and expression of various thioesterases.

[0668] As described above, partial cDNAs termed ammonium transporter 02 and 03 (amt02 and amt03) were identified. Along with these two partial cDNAs, a third partial cDNA termed ammonium transporter 01 (amtOl) was also identified. Alignment of the partial cDNA and the putative translated amino acid sequences were compared. Results show amtOl to be more distantly related of the three sequences, while amt02 and amt03 differ by only a single amino acid.

[0669] Promoters/5 'UTRs were generated initially in silico by blasting the partial cDNA sequences against Roche 454 genomic DNA assemblies and Illumina transcriptome assemblies as described above. Transcript contigs showing identity to the cDNA encoding amtOl, amt02, and amt03 were identified, however, the transcript contigs could not differentiate between the three mRNAs as the contigs contained sequences shared by all three. Roche 454 genomic DNA assemblies gave hits to amt02 and amt03 cDNA sequences and contained N-terminal protein sequences. PCR was carried out to clone the 5' flanking regions. The PCR primers used to validate the clone amt02 and amt03 promoter/UTR were: Amt03 forward: 5 ' -GGAGGAATTCGGCCGACAGGACGCGCGTCA-3 ' (SEQ ID NO: 85)

Amt03 reverse: 5 ' -GGAGACTAGTGGCTGCGACCGGCCTGTG-3 ' (SEQ ID NO: 86)

Amt02 forward: 5 ' -GGAGGAATTCTCACCAGCGGACAAAGCACCG-3 ' (SEQ ID NO: 87)

Amt02 reverse: 5 ' -GGAGACTAGTGGCTGCGACCGGCCTCTGG-3 ' (SEQ ID NO: 88)

In both cases, the 5' and 3' primers contained useful restriction sites for the anticipated cloning into expression vectors to validate the the functionality of these promoter/5 'UTR regions.

[0670] Pair wise alignments between the DNAs cloned through this combined in silico and PCR-based method and the original cDNA encoding amt02 (SEQ ID NO: 61) and amt03 (SEQ ID NO: 60) were performed. Results of these alignments showed significant differences between the original cDNAs and the cloned genomic sequences, indicating that ammonium transporters likely represent a diverse gene family. Additionally, the

promoter/5 'UTR clone based on the combined method for amt03 was different than the original amt03 sequence, whereas the amt02 sequences were identical. Further experiments to characterize the amt03 promoter/UTR sequence (SEQ ID NO: 89) was carried out and described below.

[0671] The above identified amt03 promoter/UTR sequence (SEQ ID NO: 89) was tested by cloning this putative promoter/UTR sequence to drive the expression of four different thioesterases. The expression cassette contained upstream and downstream homologous recombination sequences to the 6S locus of the genome (SEQ ID NOs: 82 and 84, respectively). The cassette also contains a S. cerevisiae SUC2 sucrose invertase cDNA to enable the selection for positive clones on sucrose containing medium. The sucrose invertase expression was driven by the C. reinhardtii beta tubulin promoter and also contained a C. vulgaris nitrate reductase 3 'UTR . The amt03 promoter/UTR sequence was then cloned downstream of the sucrose invertase cassette followed by in- frame thioesterase cDNA sequence from one of four thioesterase genes: (1) C14 thioesterase from C. camphora; (2) C12 thioesterase from U. californica; (3) C10-C16 thioesterase from U. americana; or (4) CIO thioesterase from C. hookeriana and also contained a C. vulgaris nitrate reductase 3 'UTR. The C14 C. camphora thioesterase, C12 U. californica thioesterase, and the C10- C16 U. americana all contained the transit peptide from a Chlorella protothecoides stearoyl ACP desaturase. The CIO C. hookeriana thioesterase contained the transit peptide from a Prototheca moriformis delta 12 fatty acid desaturase (FAD). In all cases, the sequences were codon optimized for expression in Prototheca moriformis. The sequences to the foregoing thioesterase constructs are described in the Sequence Listing: amt03 promoter/UTR: :C. camphora thioesterase construct SEQ ID NO 90 C. camphora thioesterase construct SEQ ID NO 91 U. californica thioesterase construct SEQ ID NO 92

U. americana thioesterase construct SEQ ID NO 93

C. hookeriana thioesterase construct SEQ ID NO 94

[0672] Transgenic lines were generated via biolistic transformation methods as described above in Example 2 into wild type Prototheca moriformis cells and selection was carried out on sucrose containing plates/medium. Positive lines were then screened for the degree to which their fatty acid profiles were altered. Four lines, one resulting from the transformation with each of the four above-described constructs, were then subjected to additional analysis. Line 76 expressed the C. camphora C14 thioesterase, line 37 expressed the U. californica C12 thioesterase, line 60 expressed the U. americana C10-C16 thioesterase, and line 56 expressed the C. hookeriana CIO thioesterase. Each line was grown for 48 hours in medium containing sucrose as the sole carbon source and samples of cells were removed at 14, 24, 36 and 48 hours (seed culture) for determination of fatty acid profile via direct transesterification to fatty acid methyl esters and subsequent analysis by GC-FID (described above) and for isolation of total RNA. At the end of 48 hours, these cells were used to inoculate cultures with no or low levels of nitrogen (containing sucrose as the sole carbon source) maintained at either pH 5.0 (citrate buffered, 0.05M final concentration) or pH 7.0 (HEPES buffered, 0.1M final concentration). Culture samples were removed at 12, 24, 72 and 108 hours (lipid production) for fatty acid profiling and isolation of total RNA. Ammonia assays of these cultures revealed that ammonia levels fell below the limits of detection (ca. 100 μΜ) after 24 hours in low nitrogen medium.

[0673] Real-time RT-PCR assays on the mRNA levels of the thioesterases were performed on total RNA from each of the time points collected above and all mRNA levels were normalized to the levels of an internal control RNA (cdl89). Primer sets used in real-time PCR are shown in Table 19 below:

[0674] Table 19. Primer sets for real-time PCR. Gene specific to Primer sequence 5'-3' SEQ ID NO:

C. camphora TE forward TACCCCGCCTGGGGCGACAC SEQ ID NO: 68

C. camphora TE reverse CTTGCTCAGGCGGCGGGTGC SEQ ID NO: 69

U. californica TE forward CTGGGCGACGGCTTCGGCAC SEQ ID NO: 95

U. californica TE reverse AAGTCGCGGCGCATGCCGTT SEQ ID NO: 96

U. americana TE forward CCCAGCTGCTCACCTGCACC SEQ ID NO: 97

U. americana TE reverse CACCCAAGG CCAACG GC AG CGCCGTG SEQ ID NO: 98

C. hookeriana TE forward TACCCCGCCTGGGGCGACAC SEQ ID NO: 99

C. hookeriana TE reverse AGCTTGGACAGGCGGCGGGT SEQ ID NO: 100

cdl89 reverse TCGATGTCGTGCACCGTCGC SEQ ID NO: 71

cdl89 forward CCGGATCTCGGCCAGGGCTA SEQ ID NO: 70

[0675] The results from the fatty acid profiles at each of the time points in the seed culture phase showed very little impact from the thioesterases. With the commencement of the lipid production phase, the fatty acid profiles were significantly impacted, with the increases that are far more dramatic for the cultures maintained at pH 7.0 as compared to the cultures at pH 5.0. While the magnitude of the difference between pH 7.0 and 5.0 target fatty acid accumulation varied with each thioesterase tested, the overall effect was the same: that the cells grown at pH 5.0 showed significantly lower levels of the target fatty acids accumulated, but more than compared to control wild type cells.

[0676] Analysis of the RNA isolated from these same samples correlated very will with the fatty acid profile data, in that there was a clear impact of culture pH on the steady state mRNA levels for each of the thioesterases. Taking the fatty acid accumulation data and the mRNA data together, the pH regulation of thioesterase gene expression driven by the amt03 promoter/UTR was clearly mediated either at the level of transcription, mRNA stability or both. Additionally, it was observed that the steady state levels of U. californica mRNA were four logs lower as compared to the steady state levels of C. hookeriana mRNA. This observation is consistent with the hypothesis that the individual mRNA sequences may play a role in controlling expression. These data imply that ammonium uptake in Prototheca moriformis by the amt03 family of transporters is coupled directly to pH.

[0677] Additional fatty acid profile analysis was performed on twelve lines generated from the transformation of Prototheca moriformis cells with the construct amt03 promoter/UTR driving the expression of the U. americana C10-C16 thioesterase. Line 60, described above, was a part of the following analysis. Table 20 below shows the lipid profiles of three of the twelve lines that were analyzed along with the wild type control.

[0678] Table 20. Fatty acid profiles of transformants containing the U. americana TE driven by the amt03 promoter/UTR. Saturates wild type 0.00 0.01 0.04 1.27 27.20 3.85 58.70 7.18 32.36

Line 40 2.38 20.61 3.41 28.41 29.92 1.91 8.57 3.74 86.64

Line 44 1.50 20.16 4.44 31.88 26.66 1.88 6.95 5.42 86.50

Line 60 0.98 14.56 3.15 27.49 31.76 2.14 12.23 6.36 80.06

[0679] As shown in the table above, the levels of total saturates was increased dramatically over that of wild type with over 2.6 fold in the case of line 40 compared to wildtype (total saturates from the twelve lines analyzed ranged from about 63% to over 86%). Additionally, the U. americana thioesterase, when expressed at these levels, dramatically reduces the level of unsaturates, especially C18: l and C18:2 (see lines 40 and 44), where in line 44, C18: l levels are reduced by over 8 fold compared to the wild type. Also, the U. americana thioesterase (driven by the amt03 promoter) greatly increases the levels of mid-chain fatty acids. Line 44 shows 00:0-04:0 levels at greater than 56%, approximately 42 fold higher than the levels seen in the wildtype strain and C8: 0-04:0 levels at greater than 57%.

Additional strains transformed with a construct of the Amt03 promoter driving the expression of the U. americana thioesterase had representative lipid profile of: 0.23% C8:0; 9.64% 00:0; 2.62% 02:0; 31.52% 04:0; 37.63% 06:0; 5.34% 08:0; 7.05% C18: l ; and 5.03% C18:2, with a total saturates percentage at 86.98%.

[0680] Additional lipid profiles generated from the transformation of Prototheca moriformis cells with the construct amt03 promoter/UTR (SEQ ID NO: 89) driving the expression of the C. hookeriana CIO thioesterase (SEQ ID NO: 94). Positive clones expressing this construct were selected and grown at pH 7.0 conditions. Representative lipid profile from a positive clone was: 9.87% C8:0; 23.97% 00:0; 0.46% 02:0; 1.24% 04:0; 10.24% 06:0; 2.45% 08:0; 42.81% C18: l ; and 7.32% C18:2. This clone had a C8-C10 percentage of 33.84

[0681] Taken together, the data suggest that the amt03 promoter/UTR, and other promoters like it, can be used as a tightly regulated promoter, which may be particularly useful for expressing a potentially toxic compound and strict enforcement of gene expression is required. The ability of Prototheca moriformis to grow under a wide range (at least pH 5.0 to 7.0) of pH regimes makes this organism particularly useful in combination with regulatory elements such as the amt03 promoter/UTR. Additionally, the lipid profile data above demonstrates the impressive ability of the amt03 promoter/UTR to drive gene expression. EXAMPLE 11: Altering the Levels of Saturated Fatty Acids in the Microalgae

Prototheca moriformis

[0682] As part of a genomics screen using a bioinformatics based approach based on cDNAs, Illumia transcriptome and Roche 454 squencing of genomic DNA from Prototheca moriformis (UTEX 1435), two specific groups of genes involved in fatty acid desaturation were identified: stearoyl ACP desaturases (SAD) and delta 12 fatty acid desaturases (Δ12 FAD). Stearoyl ACP desaturase enzymes are part of the lipid synthesis pathway and they function to introduce double bonds into the fatty acyl chains, for example, the synthesis of C18: l fatty acids from C18:0 fatty acids. Delta 12 fatty acid desaturases are also part of the lipid synthesis pathway and they function to introduce double bonds into already unsaturated fatty acids, for example, the synthesis of C18:2 fatty acids from C18:l fatty acids. Southern blot analysis using probes based on the two classes of fatty acid desaturase genes identified during the bioinformatics efforts indicated that each class of desaturase genes was likely comprised of multiple family members. Additionally the genes encoding stearoyl ACP desaturases fell into two distinct families. Based on these results, three gene disruption constructs were designed to potentially disrupt multiple gene family members by targeting more highly conserved coding regions within each family of desaturase enzymes.

[0683] Three homologous recombination targeting constructs were designed using: (1) highly conserved portions of the coding sequence of delta 12 fatty acid desaturase (dl2FAD) family members and (2) two constructs targeting each of the two distinct families of SAD , each with conserved regions of the coding sequences from each family. This strategy would embed a selectable marker gene (the sucl sucrose invertase cassette from S. cerevisiae conferring the ability to hydrolyze sucrose) into these highly conserved coding regions (targeting multiple family members) rather than a classic gene replacement strategy where the homologous recombination would target flanking regions of the targeted gene.

[0684] All constructs were introduced into the cells by biolistic transformation using the methods described above and constructs were linearized before being shot into the cells. Transformants were selected on sucrose containing plates/media and changes in lipid profile were assayed using the above-described method. Relevant sequences from each of the three targeting constructs are listed below.

Description SEP ID NO:

5' sequence from coding region of dl2FAD from targeting construct SEQ ID NO: 72

3' sequence from coding region of dl2FAD from targeting construct SEQ ID NO: 73 dl2FAD targeting construct cDNA sequence SEQ ID NO: 74

5' sequence from coding region of SAD2A SEQ ID NO: 75 3 ' sequence from coding region of S AD2A SEQ ID NO: 76 SAD2A targeting construct cDNA sequence SEQ ID NO: 77 5 ' sequence from coding region os S AD2B SEQ ID NO: 78 3 ' sequence from coding region of S AD2B SEQ ID NO: 79 SAD2B targeting construct cDNA sequence SEQ ID NO: 80

[0685] Representative positive clones from transformations with each of the constructs were picked and the lipid profiles for these clones were determined (expressed in Area ) and summarized in Table 21 below.

[0686] Table 21. Lipid profiles for desaturase knockouts.

Fatty Acid (J12FAD KO SAD2A KO SAD2B KO wt UTEX 1435

C8:0 0 0 0 0

C10:0 0.01 0.01 0.01 0.01

C12:0 0.03 0.03 0.03 0.03

C14:0 1.08 0.985 0.795 1.46

C16:0 24.42 25.335 23.66 29.87

C18:0 6.85 12.89 19.555 3.345

C18:l 58.35 47.865 43.115 54.09

C18:2 7.33 10.27 9.83 9.1

C18:3 alpha 0.83 0.86 1 0.89

C20:0 0.48 0.86 1.175 0.325

[0687] Each of the construct had a measurable impact on the desired class of fatty acid and in all three cases C18:0 levels increased markedly, particularly with the two SAD knockouts. Further comparison of multiple clones from the SAD knockouts indicated that the SAD2B knockout lines had significantly greater reductions in C 18: 1 fatty acids than the CI 8: 1 fatty acid levels observed with the SAD2A knockout lines.

[0688] Additional Δ12 fatty acid desaturase (FAD) knockouts were generated in a

Prototheca moriformis background using the methods described above. In order to identify potential homologous of A12FADs, the following primers were used in order to amplify a genomic region encoding a putative FAD:

Primer 1 5'-TCACTTCATGCCGGCGGTCC-3' SEQ ID NO: 101

Primer 2 5'- GCGCTCCTGCTTGGCTCGAA-3 ' SEQ ID NO : 102

The sequences resulting from the genomic amplification of Prototheca moriformis genomic DNA using the above primers were highly similar, but indicated that multiple genes or alleles of A12FADs exist in Prototheca. [0689] Based on this result, two gene disruption constructs were designed that sought to inactivate one or more A12FAD genes. The strategy would to embed a sucrose invertase (suc2 from S. cerevisiae) cassette, thus conferring the ability to hydrolyze sucrose as a selectable marker, into highly conserved coding regions rather than use a classic gene replacement strategy. The first construct, termed pSZl 124, contained 5' and 3' genomic targeting sequences flanking a C. reinhardtii β-tubulin promoter driving the expression of the S. cerevisiae suc2 gene and a Chlorella vulgaris nitrate reductase 3'UTR (S. cerevisiae suc2 cassette). The second construct, termed pSZ1125, contained 5' and 3' genomic targeting sequences flanking a C. reinhardtii β-tubulin promoter driving the expression of the S.

cerevisiae suc2 gene and a Chlorella vulgaris nitrate reductase 3'UTR. The relevant sequences of the constructs are listed in the Sequence Listing:

pSZ1124 (FAD2B) 5' genomic targeting sequence SEQ ID NO 103

pSZ1124 (FAD2B) 3' genomic targeting sequence SEQ ID NO 104

S. cerevisiae suc2 cassette SEQ ID NO 105

pSZ1125 (FAD2C) 5' genomic targeting sequence SEQ ID NO 106

pSZ1125 (FAD2C) 3' genomic targeting sequence SEQ ID NO 107

[0690] pSZl 124 and pSZl 125 were each introduced into a Prototheca moriformis background and positive clones were selected based on the ability to hydrolyze sucrose. Table 22 summarizes the lipid profiles (in Area , generated using methods described above) obtained in two transgenic lines in which pSZ1124 and pSZ1125 targeting vectors were utilized.

[0691] Table 22. Lipid profiles of Δ12 FAD knockouts

[0692] The transgenic containing the FAD2B (pSZl 124) construct gave a very interesting and unexpected result in lipid profile, in that the C18:2 levels, which would be expected to decrease, only decreased by about one area . However, the C18: l fatty acid levels increased significantly, almost exclusively at the expense of the CI 6:0 levels, which decreased significantly. The transgenic containing the FAD2C (pSZ1125) construct also gave a change in lipid profile: the levels of C18:2 are reduced significantly along with a corresponding increase in CI 8: 1 levels.

Beef Tallow Mimetic [0693] One positive clone generated from the above SAD2B knockout experiment as described above was selected to be used as the background for the further introduction of a C14-pref erring fatty acyl-ACP thioesterase gene. The construct introducing the C. camphora C14-pref erring thioesterase contained targeting sequence to the 6S rRNA genomic region (allowing for targeted integration of the transforming DNA via homologous recombination) and the expression construct contained the C. reinhardtii β-tubulin promoter driving the expression of the neoR gene with the Chlorella vulgaris nitrate reductase 3 'UTR, followed by a second C. reinhardtii β-tubulin promter driving the expression of a codon-optimized C. camphora thioesterase with a Chlorella protothecoides stearoyl ACP desaturase transit peptide with a second Chlorella vulgaris nitrate reductase 3 'UTR. The 5' 6S rRNA genomic donor sequence is listed in SEQ ID NO: 82; the 3' 6S rRNA genomic donor sequence is listed in SEQ ID NO: 84; and the relevant expression construct for the C. camphora thioesterase is listed in SEQ ID NO: 83.

[0694] Transformation was carried out using biolistic methods as decribed above and the cells were allowed to recover for 24 hours on plates containing 2% sucrose. After this time, the cells were re-suspended and re-plated on plates containing 2% sucrose and 50 μg/ml G418 for selection. Nine clones out of the positive clones generated were selected for lipid production and lipid profile. The nine transgenic clones (with the SAD2B KO and expressing C. camphora C14-pref erring thioesterase) were cultured as described above and analyzed for lipid profile. The results are summarized below in Table 23. The lipid profile for tallow is also included in Table 23 below (National Research Council 1976: Fat Content and Composition of Animal Product).

[0695] Table 23. Lipid profile of thioesterase transformed clones.

C10:0 C12:0 C14:0 C16:0 C16:l C18:0 C18:l C18:2 C18:3 C20

SAD2BKO 0.01 0.33 6.13 24.24 0.19 11.08 42.03 13.45 0.98 0.73 C. camphora

TE clone 1

SAD2BKO 0.01 0.16 3.42 23.80 0.40 9.40 50.62 10.2 0.62 0.70 C. camphora

TE clone 2

SAD2BKO 0.01 0.20 4.21 25.69 0.40 7.79 50.51 9.37 0.66 0.63

C. camphora

TE clone 3

SAD2BKO 0.01 0.21 4.29 23.57 0.31 9.44 50.07 10.07 0.70 0.70 C. camphora

TE clone 4

SAD2BKO 0.01 0.18 3.87 24.42 0.32 9.24 49.75 10.17 0.71 0.71 C. camphora

TE clone 5

SAD2BKO 0.01 0.28 5.34 23.78 0.33 9.12 49.12 10.00 0.68 0.70 C. camphora

TE clone 6

SAD2BKO 0.01 0.15 3.09 23.07 0.32 10.08 51.21 10.00 0.66 0.74 C. camphora TE clone 7

SAD2BKO 0.01 0.29 5.33 24.62 0.37 7.02 49.67 10.74 0.69 0.70

C.camphora

TE clone 8

SAD2BKO 0.01 0.12 2.74 25.13 0.30 10.17 50.18 9.42 0.71 0.71

C.camphora

TE clone 9

wt UTEX 0.01 0.02 0.96 23.06 0.79 3.14 61.82 9.06 0.46 0.27

1435

SAD2BKO 0.01 0.03 0.80 23.66 0.13 19.56 43.12 9.83 1.00 1.18

Tallow 0.00 0.00 4.00 26.00 3.00 14.00 41.00 3.00 1.00 0.00

[0696] As can be seen in Table 23, the lipid profiles of the transgenic lines are quite similar to the lipid profile of tallow. Taken collectively, the data demonstrate the utility of combining specific transgenic backgrounds, in this case, a SAD2B knockout with a C14- pref erring thioesterase (from C. camphor a), to generate an transgenic algal strain that produce oil similar to the lipid profile of tallow.

Construct used to down regulate the expression of β-Ketoacyl Synthase II (KASII) by targeted knock-out approach

[0697] Vector down-regulating KASII gene expression by targeted knock-out approach was introduced into a classically mutagenized derivative of UTEX 1435, S1331. The Saccharomyces cerevisiae invertase gene was utilized as a selectable marker, conferring the ability to grow on sucrose. The invertase expression cassette under control of C. reinhardtii B -tubulin promoter was inserted in the middle of the 315bp long KASII genomic region to permit targeted integration (pSZ1503).

[0698] Relevant restriction sites in pSZ1503 are indicated in lowercase, bold and underlining and are 5 '-3' BspQ 1, Kpn I, AscI, Xho I, Sac I, BspQ I, respectively. BspQI sites delimit the 5' and 3' ends of the transforming DNA. Bold, lowercase sequences represent genomic DNA from S1331 that permit targeted integration at KASII locus via homologous recombination. Proceeding in the 5' to 3' direction, the C. reinhardtii B-tubulin promoter driving the expression of the yeast sucrose invertase gene (conferring the ability of SI 331 to metabolize sucrose) is indicated by boxed text. The initiator ATG and terminator TGA for invertase are indicated by uppercase, bold italics while the coding region is indicated in lowercase italics. The Chlorella vulgaris nitrate reductase 3' UTR is indicated by lowercase underlined text.

[0699] Nucleotide sequence of transforming DNA contained in pSZ1503_[KASII_btub- y.inv-nr_KASII]:

gctctteccgcaccggctggctccaccccaacttgaacctcgagaaccccgcgcctg gcgtcgaccccgtcgtgctcgtggggc cgcggaaggagcgcgccgaagacctggacgtcgtcctctccaactcctttggctttggcg ggcacaattcgtgc2tc22tacc ctttcttgcgctatgacacttccagcaaaaggtagggcgggctgcgagacggcttcccgg cgctgcatgcaacaccgatgatgcttcg accccccgaagctccttcggggctgcatgggcgctccgatgccgctccagggcgagcgct gtttaaatagccaggcccccgattgc aaagacattatagcgagctaccaaagccatattcaaacacctagatcactaccacttcta cacaggccactcgagcttgtgatcgcactc cgctaagggggcgcctcttcctcttcgtttcagtcacaacccgcaaac ££ £ .ccATGctgctgcaggccttcctgttcctgctgg

ccggcttcgccgccaagatcagcgcctccatgacgaacgagacgtccgaccgccccc tggtgcacttcacccccaacaagggct ggatgaacgaccccaacggcctgtggtacgacgagaaggacgccaagtggcacctgtact tccagtacaacccgaacgacac cgtctgggggacgcccttgttctggggccacgccacgtccgacgacctgaccaactggga ggaccagcccatcgccatcgcccc gaagcgcaacgactccggcgccttctccggctccatggtggtggactacaacaacacctc cggcttcttcaacgacaccatcgac ccgcgccagcgctgcgtggccatctggacctacaacaccccggagtccgaggagcagtac atctcctacagcctggacggcgg ctacaccttcaccgagtaccagaagaaccccgtgctggccgccaactccacccagttccg cgacccgaaggtcttctggtacgag ccctcccagaagtggatcatgaccgcggccaagtcccaggactacaagatcgagatctac tcctccgacgacctgaagtcctgg aagctggagtccgcgttcgccaacgagggcttcctcggctaccagtacgagtgccccggc ctgatcgaggtccccaccgagcag gaccccagcaagtcctactgggtgatgttcatctccatcaaccccggcgccccggccggc ggctccttcaaccagtacttcgtcgg cagcttcaacggcacccacttcgaggccttcgacaaccagtcccgcgtggtggacttcgg caaggactactacgccctgcagacc ttcttcaacaccgacccgacctacgggagcgccctgggcatcgcgtgggcctccaactgg gagtactccgccttcgtgcccacca acccctggcgctcctccatgtccctcgtgcgcaagttctccctcaacaccgagtaccagg ccaacccggagacggagctgatcaa cctgaaggccgagccgatcctgaacatcagcaacgccggcccctggagccggttcgccac caacaccacgttgacgaaggcc aacagctacaacgtcgacctgtccaacagcaccggcaccctggagttcgagctggtgtac gccgtcaacaccacccagacgatc tccaagtccgtgttcgcggacctctccctctggttcaagggcctggaggaccccgaggag tacctccgcatgggcttcgaggtgtc cgcgtcctccttcttcctggaccgcgggaacagcaaggtgaagttcgtgaaggagaaccc ctacttcaccaaccgcatgagcgtg aacaaccagcccttcaagagcgagaacgacctgtcctactacaaggtgtacggcttgctg gaccagaacatcctggagctgtact tcaacgacggcgacgtcgtgtccaccaacacctacttcatgaccaccgggaacgccctgg gctccgtgaacatgacgacgggg gtggacaacctgttctacatcgacaagttccaggtgcgcgaggtcaagTGAcaatt Sca.zca.zca.zctczs&ta.ztSLtcg,SiC

acactctggacgctggtcgtgtgatggactgttgccgccacacttgctgccttgacc tgtgaatatccctgccgcttttatcaaacagcct cagtgtgtttgatcttgtgtgtacgcgcttttgcgagttgctagctgcttgtgctatttg cgaataccacccccagcatccccttccctcgttt catatcgcttgcatcccaaccgcaacttatctacgctgtcctgctatccctcagcgctgc tcctgctcctgctcactgcccctcgcacagc cttggtttgggctccgcctgtattctcctggtactgcaacctgtaaaccagcactgcaat gctgatgcacgggaagtagtgggatggga

acacaaatggaggatcgta2a2ctcatcttcc2aaa2tac2ac2a2t2a2C2a2ct2 attctcttt2a2C2222tc222t22ttc ggggagagtgcgcggaaaggcgcagagacgtgcggccggccgtgtccctccgtcttcccc tggttggtgctatagtaacctgc ct2t2tc2C2t2C2C2tc222aa2a2c (SEP ID NO: 149)

[0700] The cDNAs of the KAS II allele 1 and allele 2 are identified in SEQ ID NOs: 279 and 280, respectively. The amino acid sequences of alleles 1 and 2 are identified in SEQ ID

NOs: 281 and 282, respectively.

[0701] To determine the impact of KASII inactivation on lipid composition, pSZ1503

vector DNA was transformed into S1331 to generate a targeted KASII knock-out phenotype.

Initial single clones were isolated and grown under standard lipid production conditions at

pH5.0. The resulting profiles of the best representative clone and the wild-type cells are

shown below in Table 31

[0702] Table 31. Fatty acid profiles in S 1331 and a derivative transgenic line transformed with pSZ1503 DNA.

Sample ID C10:0 C12:0 C14:0 C16:0 C16:1 C18:0 C18:1 C18:2 C18:3 a

1331-5 0.01 0.03 0.96 24.28 0.64 3.94 62.69 6.21 0.49

D698-2 0.01 0.01 0.83 38.36 1.38 2.21 48.31 7.60 0.55 EXAMPLE 12: Engineering Prototheca with Alternative Selectable Markers

A. Expression of a secretable a-galactosidase in Prototheca moriformis

[0703] Methods and effects of expressing a heterologous sucrose invertase gene in

Prototheca species have been previously described in PCT Application No.

PCT/US2009/66142, hereby incorporated by reference. The expression of other heterologous polysaccharide degrading enzymes was examined in this Example. The ability to grow on melibiose (a-D-gal-glu) by Prototheca moriformis UTEX 1435 with one of the following exogenous gene encoding a a-galactosidase was tested: MEL1 gene from Saccharomyces carlbergensis (amino acid sequence corresponding to NCBI accession number P04824 (SEQ ID NO: 108)), AglC gene from Aspergillus niger (amino acid sequence corresponding to NCBI accession number Q9UUZ4 (SEQ ID NO: 116)), and the α-galactosidase from the higher plant Cyamopsis tetragobobola (Guar bean) (amino acid sequence corresponding to NCBI accession number P14749 (SEQ ID NO: 120). The above accession numbers and corresponding amino acid sequences are hereby incorporated by reference. In all cases, genes were optimized according to the preferred codon usage in Prototheca moriformis. The relevant portions of the expression cassette are listed below along with the Sequence Listing numbers. All expression cassettes used the 5' and 3' Clp homologous recombination targeting sequences for stable genomic integration, the Chlamydomonas reinhardtii TUB2 promoter/5 'UTR, and the Chlorella vulgaris nitrate reductase 3'UTR.

S. carlbergensis MEL1 amino acid sequence SEQ ID NO 108

S. carlbergensis MEL1 amino acid sequence signal peptide SEQ ID NO 109

S. carlbergensis MEL1 transformation cassette SEQ ID NO 110

S. carlbergensis MEL1 sequence (codon optimized) SEQ ID NO 111

5 ' Clp homologous recombination targeting sequence SEQ ID NO 112

3 ' Clp homologous recombination targeting sequence SEQ ID NO 113

Chlamydomonas reinhardtii TUB2 promoter/5 'UTR SEQ ID NO 114

Chlorella vulgaris nitrate reductase 3'UTR SEQ ID NO 115

A. niger AlgC amino acid sequence SEQ ID NO 116

A. niger AlgC amino acid sequence signal peptide SEQ ID NO 117

A. niger AlgC sequence (codon optimized) SEQ ID NO 118

A. niger AlgC transformation cassette SEQ ID NO 119

C. tetragonobola α-galactosidase amino acid sequence SEQ ID NO 120

C. tetragonobola α-galactosidase sequence (codon optimized) SEQ ID NO 121

C. tetragonobola α-galactosidase transformation cassette SEQ ID NO 122 [0704] Prototheca moriformis cells were transformed with each of the three expression cassettes containing S. carlbergensis MEL1, A. niger AlgC, or C. tetragonobola - galactosidase gene using the biolistic transformation methods as described in Example 2 above. Positive clones were screened using plates containing 2% melibiose as the sole carbon source. No colonies appeared on the plates for the C. tetragonobola expression cassette transformants. Positive clones were picked from the plates containing the S. carlbergensis MEL1 transformants and the A. niger AlgC transformants. Integration of the transforming DNA was confirmed using PCR with primers targeting a portion of the C. vulgaris 3'UTR and the 3 ' Clp homologous recombination targeting sequence.

5' primer C.vulgaris 3'UTR: downstream Clp sequence (SEQ ID NO: 123)

ACTGCAATGCTGATGCACGGGA

3' primer C.vulgaris 3'UTR: downstream Clp sequence (SEQ ID NO: 124)

TCCAGGTCCTTTTCGCACT

[0705] As a negative control, genomic DNA from untransformed Prototheca moriformis cells were also amplified with the primer set. No products were amplified from genomic DNA from the wild type cells.

[0706] Several positive clones from each of the S. carlbergensis MEL1 transformants and the A. niger AlgC transformants (as confirmed by PCR) were tested for their ability to grow on melibiose as the sole carbon source in liquid media. These selected clones were grown for 3 days in conditions and base medium described in Example 1 above with melibiose as the sole carbon source. All clones containing either a-galactosidase-encoding genes grew robustly during this time, while the untransformed wild type strain and Prototheca moriformis expressing a Saccharomyces cerevisiae SUC2 sucrose invertase both grew poorly on the melibiose media. These results suggest that the a-galactosidase encoding genes may be used as a selectable marker for transformation. Also, these data indicate that the native signal peptides present in the 5". carlbergensis MEL1 (SEQ ID NO: 109) or A. niger AlgC (SEQ ID NO: 117) are useful for targeting proteins to the periplasm in Prototheca moriformis cells.

B. THIC 2enes complements thiamine auxotrophv in Prototheca

[0707] Thiamine prototrophy in Prototheca moriformis cells was examined using expression of exogenous THIC genes. Thiamine biosynthesis in plants and algae is typically carried out in the plastid, hence most nuclear encoded proteins involved in its production will need to be efficiently targeted to the plastid. DNA sequencing and transcriptome sequencing of Prototheca moriformis cells revealed that all of the genes encoding the thiamine biosynthetic enzymes were present in the genome, with the exception of THIC. To dissect the lesion responsible for thiamine auxotrophy at the biochemical level, the growth of Prototheca moriformis cells under five different regimes were examined: (1) in the presence of 2 μΜ thiamine hydrochloride; (2) without thiamine; (3) without thiamine, but with 2 μΜ hydroxyethyl thiazole (THZ); (4) without thiamine, but with 2 μΜ 2-methyl-4-amino-5- (aminomethyl)pyrimidine (PYR); and (5) without thiamine, but with 2 μΜ THZ and 2μΜ PYR. Results from the growth experiments under these 5 different conditions indicated that Prototheca moriformis cells are capable of de novo synthesis, but can only produce thiamine pyrophosphate (TPP) if the PYR precursor is provided. This result is consistent with the hypothesis that the thiamine auxotrophy of Prototheca moriformis is due to the inability to synthesize hydroxymethylpyrimidine phosphate (HMP-P) from aminoimidazole ribonucleotide, which is the conversion catalyze by THIC enzyme.

[0708] Prototheca moriformis cells were transformed using the biolistic transformation methods described above in Example 2, expressing the Coccomyxa C-169 THIC (amino acid sequence corresponding to JGI Protein ID 30481, and hereby incorporated by reference) and a S. cerevisiae SUC2 sucrose invertase as the selective marker. This expression construct contained the native transit peptide sequence from Coccomyxa C-169 THIC, upstream and downstream homologous recombination targeting sequences to the 6S region of genomic DNA, a C reinhardtii TUB2 promoter/5 'UTR region (SEQ ID NO: 104), and a Chlorella vulgaris nitrate reductase 3'UTR (SEQ ID NO: 115). The S. cerevisiae SUC2 expression was also driven by a C. reinhardtii TUB2 promoter/5 'UTR region (SEQ ID NO: 114) and contained a Chlorella vulgaris nitrate reductase 3'UTR (SEQ ID NO: 115). Genes were optimized according to the preferred codon usage in Prototheca moriformis. The relevant expression cassette sequences are listed in the Sequence Listing and detailed below:

Coccomyxa C-169 THIC amino acid sequence SEQ ID NO 125

Coccomyxa C-169 THIC amino acid sequence native transit peptide SEQ ID NO 126

Coccomyxa C-169 THIC transformation cassette SEQ ID NO 127

Coccomyxa C-169 THIC sequence (codon optimized) SEQ ID NO 128

S. cerevisiae SUC2 sequence (codon optimized) SEQ ID NO 129

5' 6S homologous recombination targeting sequence SEQ ID NO 82

3' 6S homologous recombination targeting sequence SEQ ID NO 84

Selection of positive clones were performed on plates without thiamine and containing sucrose as the sole carbon source. Positive clones were confirmed using PCR with a 5' primer that binds within the Coccomyxa C-169 THIC gene and a 3' primer that anneals downsteam of the transforming DNA in the 6S locus. PCR confirmed positive clones were also confirmed using Southern blot assays.

[0709] To observe the thiamine auxotrophy of wildtype Prototheca moriformis cells, it was necessary to first deplete cells of internal thiamine reserves. To test growth in medium without thiamine, cells were first grown to stationary phase in medium containing 2 μΜ thiamine and then the cells were diluted to an optical density at 750 nm (OD750) of approximately 0.05 in medium without thiamine. The diluted cells were then grown once more to stationary phase in medium without thiamine (about 2-3 days). These thiamine- depleted cells were used to inoculate cultures for growth studies in medium without thiamine. Wildtype cells were grown in medium with glucose as the carbon source (with or without thiamine) and positive clones with the native transit peptide Coccomyxa C-169 THIC construct were grown in medium with sucrose as the sole carbon source. Growth was measured by monitoring the absorbance at 750nm. Results of the growth experiments showed substantial greater growth in thiamine-free medium of strains expressing the transgene compared to wildtype cells in thiamine-free medium. However, the transformants failed to achieve the growth rate and cell densities of wildtype cells in thiamine-containing media. There was also a strong correlation between the amount of growth in the transformant clones in thiamine-free medium and the copy number of the integrated Coccomyxa enzyme (i.e., the more copy numbers of the transgene, the better the growth of the cells in thiamine- free medium).

[0710] Additional transformants were generated using expression constructs containing the Coccomyxa THIC , the Arabidopsis thaliana THIC gene, and the Synechocystis sp. PCC 6803 thiC gene. In the case of the Coccomyxa and the A. thaliana THIC gene, the native transit peptide sequence was replaced with the transit peptide sequence from a Chlorella

protothecoides stearoyl-ACP desaturase (SAD) gene. Synechocystis sp. is a cyanobacterium and the thiC protein does not contain a native transit peptide sequence. In the Synechocystis sp thiC construct, the transit peptide sequence from a Chlorella protothecoides SAD gene was fused to the N-terminus of the Synechocystis sp. thiC. In all cases, the sequences were codon optimized for expression in Prototheca moriformis. All three of the foregoing constructs contained a upstream and downstream homologous recombination targeting sequence to the 6S region of the genome (SEQ ID NOs: 82 and 84), a Chlorella

protothecoides actin promoter/5' UTR, and a Chlorella protothecoides EF1A gene 3'UTR. All three constructs contained a neoR gene driven by the C. reinhardtii TUB2

promoter/5 'UTR (SEQ ID NO: 114) and contained the C. vulgaris 3'UTR (SEQ ID NO: 115), conferring the selection by G418. The amino acid sequence of the A. thaliana THIC corresponded to NCBI accession number NP_180524 and the amino acid sequence of the Synechocystis sp. thiC corresponded to NCBI accession number NP_442586, both sequences hereby incorporated by reference. The relevant expression cassette sequences are listed in the Sequence Listing and detailed below:

Coccomyxa THIC expression construct with C. protothecoides

transit peptide SEQ ID NO 130

Coccomyxa THIC with C. protothecoides transit peptide SEQ ID NO 131

C. protothecoides actin promoter/5 ' UTR SEQ ID NO 132

C. protothecoides EF1A 3 'UTR SEQ ID NO 133

A. thaliana THIC expression construct SEQ ID NO 134

A. thaliana THIC with C. protothecoides transit peptide SEQ ID NO 135

A. thaliana THIC amino acid sequence with native transit peptide SEQ ID NO 136

Synechocystis sp. thiC expression construct SEQ ID NO 137

Synechocystis sp. thiC with C. protothecoides transit peptide SEQ ID NO 138

Synechocystis sp. thiC amino acid sequence SEQ ID NO 139 neoR gene SEQ ID NO 140

[0711] Positive clones were screened on plates containing G418 and several clones from each transformation were picked for verification by PCR. Integration of the transforming DNA constructs containing the Coccomyxa C-169 (with C. protothecoides transit peptide), A. thaliana and Synechocystis sp. PCC 6803 THIC genes, respectively into the 6S locus of the genome was confirmed using PCR analysis with the following primers:

5' THIC Coccomyxa confirmation primer sequence (SEQ ID NO: 141)

ACGTCGCGACCCATGCTTCC

3' THIC confirmation primer sequence (SEQ ID NO: 142)

GGGTGATCGCCTACAAGA

5' THIC A. thaliana confirmation primer sequence (SEQ ID NO: 143)

GCGTCATCGCCTACAAGA

5' thiC Synechocystis sp. confirmation primer sequence (SEQ ID NO: 144)

CGATGCTGTGCTACGTGA

[0712] Growth experiments on thiamine depleted cells (as described above) were performed using selected confirmed positive clones from transformants of each of the different constructs in medium containing G418. All transformants were able to grow (with varying degrees of robustness) in thiamine-free medium. Comparison of the growth of the transformants in thiamine-free medium to wild type cells on thiamine-containing medium showed the following ranking with respect to their ability to support growth in thiamine-free medium: (1) A. thaliana transformants; (2) Coccomyxa C-169 (with C. protothecoides transit peptide) transformants; and (3) Synechocystis sp. transformants. These results suggest that while a single copy of A. thaliana THIC was able to complement thiamine auxotrophy in Prototheca moriformis cells, multiple copies of Coccomyxa C-169 (with either the native transit peptide sequence or a transit peptide sequence from C. protothecoides) and

Synechocystis sp. THIC was required to enable rapid growth in the absence of thiamine. Given the variability in results of the different THIC from the different sources, the ability of any particular THIC gene to fully complement the lesion present in Prototheca species is not predictable.

[0713] An alignment of the three THIC amino acid sequences was performed. While there exist significant sequence conservation between thiC from Synechocystis sp. compared to the THICs from Coccomyxa and A. thaliana (41 identity at the amino acid level), the cyanobacterial protein is missing a domain at the N-terminus that is well-conserved in the algal and plant proteins. Despite the missing domain (and presumably resulting in structural differences), the construct expressing the Synechocystis sp. thiC was able to at least partially restore thiamine prototrophic in Prototheca moriformis cells.

EXAMPLE 13: Fuel Production

A. Extraction of oil from mieroalgae using an expeller press and a press aid

[0714] Microalgal biomass containing 38% oil by DCW was dried using a drum dryer resulting in resulting moisture content of 5-5.5%. The biomass was fed into a French L250 press. 30.4 kg (67 lbs.) of biomass was fed through the press and no oil was recovered. The same dried microbial biomass combined with varying percentage of switchgrass as a press aid was fed through the press. The combination of dried microbial biomass and 20% w/w switchgrass yielded the best overall percentage oil recovery. The pressed cakes were then subjected to hexane extraction and the final yield for the 20% switchgrass condition was 61.6% of the total available oil (calculated by weight). Biomass with above 50% oil dry cell weight did not require the use of a pressing aid such as switchgrass in order to liberate oil. Other methods of extraction of oil from mieroalgae using an expeller press are described in PCT Application No. PCT/US2010/31108 and is hereby incorporated by reference.

B. Production of biodiesel from Prototheca oil [0715] Degummed oil from Prototheca moriformis UTEX 1435, produced according to the methods described above, was subjected to transesterification to produce fatty acid methyl esters. Results are shown in Table 24 below.

[0716] The lipid profile of the oil was:

CIO: :0 0.02

C12: :0 0.06

C14: :0 1.81

C14. 1 0.07

C16: :0 24.53

C16: : 1 1.22

C18: :0 2.34

C18: : 1 59.21

C18: :2 8.91

C18: :3 0.28

C20: :0 0.23

C20: : 1 0.10

C20: : 1 0.08

C21: :0 0.02

C22: :0 0.06

C24: :0 0.10

[0717] Table 24. Biodiesel profile from Prototheca moriformis triglyceride oil.

ASTM Cloud Point Cloud Point 6 °C

D2500

ASTM Micro Carbon Residue Average Micro < 0.10 Wt %

D4530 Method Carbon

Residue

Acid Number of Petroleum Procedure Used A

ASTM D664 Products by Potentiometric Acid Number 0.20 mg

Titration KOH/g

Determination of Free and Free Glycerin < 0.005 Wt %

ASTM Total Glycerin in B- 100 Total Glycerin 0.123 Wt %

D6584 Biodiesel Methyl Esters By

Gas Chromatography

ASTM Additive Elements in Phosphorus 0.000200 Wt %

D4951 Lubricating Oils by ICP-AES

IBP 248 °c

AET @ 5% 336 °c

Recovery

AET @ 10% 338 °c

Recovery

AET @ 20% 339 °c

Recovery

AET @ 30% 340 °c

Recovery

AET @ 40% 342 °c

Recovery

AET @ 50% 344 °c

Recovery

AET @ 60% 345

ASTM Distillation of Petroleum °c

Recovery

D1160 Products at Reduced Pressure

AET @ 70% 347 °c Recovery

AET @ 80% 349 °c

Recovery

AET @ 90% 351 °c

Recovery

AET @ 95% 353 °c

Recovery

FBP 362 °c

% Recovered 98.5 %

% Loss 1.5 %

% Residue 0.0 %

Cold Trap Volume 0.0 ml

IBP 248 °c

Determination of Oxidation Oxidation Stability > 12 hr

EN 14112 Stability (Accelerated Operating Temp 110 °C

Oxidation Test) (usually 110 deg C)

ASTM Density of Liquids by Digital API Gravity @ 60°F 29.5 °API

D4052 Density Meter ASTM D Determination of Ignition Derived Cetane > 61.0

6890 Delay (ID) and Derived Number (DCN)

Cetane Number (DCN)

[0718] The lipid profile of the biodiesel was highly similar to the lipid profile of the feedstock oil. Other oils provided by the methods and compositions of the invention can be subjected to transesterification to yield biodiesel with lipid profiles including (a) at least 4% C8-C14; (b) at least 0.3% C8; (c) at least 2% CIO; (d) at least 2% C12; and (3) at least 30% C8-C14.

[0719] The Cold Soak Filterability by the ASTM D6751 Al method of the biodiesel produced was 120 seconds for a volume of 300ml. This test involves filtration of 300 ml of B 100, chilled to 40°F for 16 hours, allowed to warm to room temp, and filtered under vacuum using 0.7 micron glass fiber filter with stainless steel support. Oils of the invention can be transesterified to generate biodiesel with a cold soak time of less than 120 seconds, less than 100 seconds, and less than 90 seconds.

C. Production of Renewable Diesel

[0720] Degummed oil from Prototheca moriformis UTEX 1435, produced according to the methods described above and having the same lipid profile as the oil used to make biodiesel in this Example, above, was subjected to transesterification to produce renewable diesel.

[0721] The oil was first hydrotreated to remove oxygen and the glycerol backbone, yielding n-paraffins. The n-parrafins were then subjected to cracking and isomerization. A chromatogram of the material is shown in Figure 1. The material was then subjected to cold filtration, which removed about 5% of the C 18 material. Following the cold filtration the total volume material was cut to flash point and evaluated for flash point, ASTM D-86 distillation distribution, cloud point and viscosity. Flash point was 63°C; viscosity was 2.86 cSt (centistokes); cloud point was 4°C. ASTM D86 distillation values are shown in Table 25:

[0722] Table 25. ASTM D86 distillation values.

Readings in °C:

Volume Temperature

IBP 173

5 217.4

10 242.1

15 255.8

20 265.6

30 277.3

40 283.5

50 286.6

60 289.4 70 290.9

80 294.3

90 300

95 307.7

FBP 331.5

[0723] The T10-T90 of the material produced was 57.9°C. Methods of hydrotreating, isomerization, and other covalent modification of oils disclosed herein, as well as methods of distillation and fractionation (such as cold filtration) disclosed herein, can be employed to generate renewable diesel compositions with other T10-T90 ranges, such as 20, 25, 30, 35, 40, 45, 50, 60 and 65°C using triglyceride oils produced according to the methods disclosed herein.

[0724] The T10 of the material produced was 242.1 °C. Methods of hydrotreating, isomerization, and other covalent modification of oils disclosed herein, as well as methods of distillation and fractionation (such as cold filtration) disclosed herein, can be employed to generate renewable diesel compositions with other T10 values, such as T10 between 180 and 295, between 190 and 270, between 210 and 250, between 225 and 245, and at least 290.

[0725] The T90 of the material produced was 300°C. Methods of hydrotreating, isomerization, and other covalent modification of oils disclosed herein, as well as methods of distillation and fractionation (such as cold filtration) disclosed herein can be employed to generate renewable diesel compositions with other T90 values, such as T90 between 280 and 380, between 290 and 360, between 300 and 350, between 310 and 340, and at least 290.

[0726] The FBP of the material produced was 300°C. Methods of hydrotreating, isomerization, and other covalent modification of oils disclosed herein, as well as methods of distillation and fractionation (such as cold filtration) disclosed herein, can be employed to generate renewable diesel compositions with other FBP values, such as FBP between 290 and 400, between 300 and 385, between 310 and 370, between 315 and 360, and at least 300.

[0727] Other oils provided by the methods and compositions of the invention can be subjected to combinations of hydrotreating, isomerization, and other covalent modification including oils with lipid profiles including (a) at least 4% C8-C14; (b) at least 0.3% C8; (c) at least 2% CIO; (d) at least 2% C12; and (3) at least 30% C8-C14.

EXAMPLE 14: Production of Tailored Oils

[0728] Using the methods and materials as disclosed herein, various tailored oils were produced. Table 32 shows the strain, the gene and the genbank accession numbers of the genes conferring the phenotype and the various fatty acid profiles produced by the indicated strain. Strains A and B are both Prototheca moriformis (UTEX 1435) strains, both of which were classically mutagenized by a fee-for-service laboratory to improve oil yield. Strains A and B were then genetically engineered as described herein with the appropriate DNA constructs to express the desired genes. The strains were also engineered to inactivate endogenous desaturases, as indicated. The nucleotide sequences of the thioesterases were codon optimized for expression and use in Prototheca.

[0729] The fatty acid profile of wild type, un-engineered Prototheca is shown in the first line of Table 32. As can be seen, the fatty acid profile has been dramatically altered in different ways in the different strains. For example, the percentage of C8:0 produced by non- genetically engineered P. moriformis cells is 0%. However, P. moriformis cells engineered to express a C. hookeriana thioesterase increased C8:0 production from 0% to 13.2 % of the total triglycerides. As another example, the total combined amount of C8:0 and C10:0 in the engineered strains was about 39% of the total fatty acids. In contrast, the total combined amount of C8:0 and C10:0 in the wild type cells is 0.01%. In another example, the total amount of saturated fatty acids was increased from about 32% to about 90% by the expression of an U. americana thioesterase in cells in which expression of endogenous SAD2b was disrupted. This is an increase of almost 300%.

[0730] The various fatty acid profiles as disclosed below are useful in myriad applications involving triglyceride oils. For example, high levels of lower carbon chain length saturated fatty acids comprising triglyceride (C12:0, C14:0, C16:0) are particularly useful in renewable jet fuel production. For biodiesel production, high amounts of C18: l are desirable. For bar soap production, controlling and achieving the appropriate balance between the levels of saturation and shorter chain fatty acids is desirable. As an example, high amounts of C12:0 are desirable for lathering properties while longer chain lengths provide more structure, while linoleic and linolenic containing triglycerides are less desirable as they contribute to oxidative instability. For liquid soaps, high amounts of C12:0 and C14:0 are desirable. Additionally, for both bar soap and liquid soap production, low amounts of C6:0, C8:0 and C10:0 are desirable as these lower chain triglycerides are skin irritants.

[0731] Table 32. Genes and accession numbers conferring phenotypes of various triglyceride profiles.

Disruption NO: 242)

Palm Kernel Oil [0732] We produced a microbial palm kernel oil mimetic that was similar to palm kernel oil (PKO). To produce the palm kernel oil mimetic, a plasmid was constructed and used to transform Strain A and oil production was carried out. The construct, pSZ1413 (SEQ ID NO: 231), comprised codon optimized Cuphea wrightii FATB2 gene (SEQ ID NO: 284) (Gen bank accession no. U56106) and SAD2B (stearoyl ACP desaturase) gene disruption.

[0733] As shown in Table 33 below, the palm kernel oil mimetic was similar to palm kernel oil. The percentages of the three most abundant fatty acids of the PKO mimetic (C12:0, C14:0 and C18: l) were identical to or within 10% of the palm kernel oil.

[0734] Table 33. Triglyceride profile of palm kernel oil mimetic.

C8:0 C10:0 C12:0 C14:0 C16:0 C18:0 C18:l C18:2

E. 3.0-5.0 2.5-6.0 40-52 14.0-18.0 7.0-10.0 1.0-3.0 11.0-19.0 0.5-4.0 guineensis

(Palm

kernel)

pSZ1413 8.33 37.45 18.22 13.52 1.25 15.29 4.95

Palm Oil

[0735] We produced a microbial palm oil mimetic that was similar to palm oil. Several different plasmids were constructed and transformed individually into Strain A and oil production was carried out. The construct, pSZ1503 (SEQ ID NO: 283), was designed to disrupt an endogenous KASII gene. The construct, pSZ1439 (SEQ ID NO: 237), comprised a codon optimized Elaeis guiniensis TE gene (SEQ ID NO: 205) (Gen bank accession no. AAD42220.2). The construct, pSZ1420 (SEQ ID NO: 225), comprised a codon optimized Cuphea hookeriana TE gene (SEQ ID NO: 201) (Gen Bank Accession no. Q39513). The construct, pSZ1119 (SEQ ID NO: 227), comprised a codon optimized Cuphea hookeriana KAS IV gene (SEQ ID NO: 186) (Gen Bank Accession no. AF060519) as well as a Cuphea wrightii FATB2 gene (SEQ ID NO: 184) (Gen Bank Accession no. U56104).

[0736] As shown in Table 34 below, the palm oil mimetic was similar to palm oil. The percentages of the three most abundant fatty acids of the palm oil mimetic (C16:0, C18: l and C18:2) were identical to or within 10% of palm oil.

[0737] Table 34. Triglyceride profile of palm oil mimetic.

C10:0 C12:0 C14:0 C16:0 C18:0 C18:l C18:2

E. 0 0 0.5-5.9 32.0-47.0 2.0-8.0 34-44 7.2-12.0 guineensis

(Palm)

pSZ1503 0.01 0.01 0.83 38.36 2.21 48.31 7.60 pSZ1439 0.01 0.04 1.88 43.50 3.32 39.95 9.16 pSZ1420 0.02 0.04 2.44 48.04 2.76 35.62 8.91 pSZ1119 1.77 0.40 7.85 35.45 2.47 42.85 8.15 Cocoa Butter

[0738] We produced a microbial cocoa butter mimetic that was similar to cocoa butter.

The construct, pSZ1451, was constructed and transformed into Strain A and oil production was carried out. The construct, pSZ1451 (SEQ ID NO: 239), comprised codon optimized

Carthamus tinctorus TE gene (SEQ ID NO: 187) (Gen Bank Accession no. AAA33019.1).

[0739] As shown in Table 35 below, the cocoa butter oil mimetic was similar to cocoa butter. The percentages of the three most abundant fatty acids of the cocoa butter mimetic

(C16:0, C18:0 and C18: l) were identical to or within 10% of cocoa butter.

[0740] Table 35. Triglyceride profile of cocoa butter mimetic.

C8:0 C10:0 C12:0 C14:0 C16:0 C18:0 C18:l C18:2 Cocoa Butter 0 0-1 0-1 0-4 22-38 24-37 29-38 0-3 pSZ1451 0.05 0.14 0.99 28.34 27.39 29.40 10.26

Lard

[0741] We produced a microbial lard mimetic that was similar to lard. Several different plasmids were constructed and transformed individually into Strain A and oil production was carried out. The construct, pSZ1493 (SEQ ID NO: 241), was designed to disrupt the endogenous SAD 2B gene and simultaneously express a codon optimized Umbellularia californica TE gene (SEQ ID NO: 285) (Gen Bank Accession no. M94159). The construct, pSZ1452 (SEQ ID NO: 240), was designed to disrupt the endogenous SAD 2B gene and express a codon optimized Garcinia mangostana TE gene (SEQ ID NO: 196) (Gen Bank Accession no. AAB51525.1). The construct, pSZ1449 (SEQ ID NO: 238), was designed to express the codon optimized Brassica napus TE gene (SEQ ID NO: 195) (Gen Bank Accession no. CAA52070.1). The polynucleotide sequence of the construct pSZ1458 was identical to pSZ1449 except that a codon optimized polynucleotide sequence encoding a Cuphea hookeriana thioesterase (Gen Bank accession No. U39834) replaced the

polynucleotide sequence encoding Brassica napus TE gene (SEQ ID NO: 195) (Gen Bank Accession no. CAA52070.1).

[0742] As shown in Table 36 below, the lard mimetic was similar to lard. The percentages of the three most abundant fatty acids of the lard mimetic (C16:0, C18:0 and C18: l) were identical to or within 10% of lard.

[0743] Table 36. Triglyceride profile of lard mimetic.

C14:0 C16:0 C18:0 C18:l C18:2

Lard 3-4 22-26 13-18 39-45 8-15 pSZ1493 1.32 24.79 17.49 41.87 10.01 pSZ1452 1.16 24.49 17.94 45.49 8.05 pSZ1449 1.16 23.98 15.79 47.88 8.29

[0744] Although this invention has been described in connection with specific

embodiments thereof, it will be understood that it is capable of further modifications. This application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains and as may be applied to the essential features hereinbefore set forth.

[0745] All references cited herein, including patents, patent applications, and publications, including Genbank Accession numbers, are hereby incorporated by reference in their entireties, whether previously specifically incorporated or not. The publications mentioned herein are cited for the purpose of describing and disclosing reagents, methodologies and concepts that may be used in connection with the present invention. Nothing herein is to be construed as an admission that these references are prior art in relation to the inventions described herein. In particular, the following patent applications are hereby incorporated by reference in their entireties for all purposes: PCT Application No. PCT/US2008/065563, filed June 2, 2008, entitled "Production of Oil in Microorganisms", PCT Application No.

PCT/US2010/31108, filed April 14, 2010, entitled "Methods of Microbial Oil Extraction and Separation", and PCT Application No. PCT/US2009/066142, filed November 30, 2009, entitled "Production of Tailored Oils in Heterotrophic Microorganisms".

SEQUENCE LISTING

SEQ ID NO: 1

HUP promoter from Chlorella (subsequence of GenBank accession number X55349)

GATCAGACGGGCCTGACCTGCGAGATAATCAAGTGCTCGTAGGCAACCAACTCA GCAGCTGCTTGGTGTTGGGTCTGCAGGATAGTGTTGCAGGGCCCCAAGGACAGC

GCCTAGCCGGAGCGCTAGGCTACATACTTGCCGCACCGGTATGAGGGGATATAG

TACTCGCACTGCGCTGTCTAGTGAGATGGGCAGTGCTGCCCATAAACAACTGGCT

GCTCAGCCATTTGTTGGCGGACCATTCTGGGGGGGCCAGCAATGCCTGACTTTCG

GGTAGGGTGAAAACTGAACAAAGACTACCAAAACAGAATTTCTTCCTCCTTGGA

GGTAAGCGCAGGCCGGCCCGCCTGCGCCCACATGGCGCTCCGAACACCTCCATA

GCTGTAAGGGCGCAAACATGGCCGGACTGTTGTCAGCACTCTTTCATGGCCATAC

AAGGTCATGTCGAGATTAGTGCTGAGTAAGACACTATCACCCCATGTTCGATTGA

AGCCGTGACTTCATGCCAACCTGCCCCTGGGCGTAGCAGACGTATGCCATCATGA

CCACTAGCCGACATGCGCTGTCTTTTGCCACCAAAACAACTGGTACACCGCTCGA

AGTCGTGCCGCACACCTCCGGGAGTGAGTCCGGCGACTCCTCCCCGGCGGGCCG

CGGCCCTACCTGGGTAGGGTCGCCATACGCCCACGACCAAACGACGCAGGAGGG

GATTGGGGTAGGGAATCCCAACCAGCCTAACCAAGACGGCACCTATAATAATAG

GTGGGGGGACTAACAGCCCTATATCGCAAGCTTTGGGTGCCTATCTTGAGAAGCA

CGAGTTGGAGTGGCTGTGTACGGTCGACCCTAAGGTGGGTGTGCCGCAGCCTGA

AACAAAGCGTCTAGCAGCTGCTTCTATAATGTGTCAGCCGTTGTGTTTCAGTTAT

ATTGTATGCTATTGTTTGTTCGTGCTAGGGTGGCGCAGGCCCACCTACTGTGGCG

GGCCATTGGTTGGTGCTTGAATTGCCTCACCATCTAAGGTCTGAACGCTCACTCA

AACGCCTTTGTACAACTGCAGAACTTTCCTTGGCGCTGCAACTACAGTGTGCAAA

CCAGCACATAGCACTCCCTTACATCACCCAGCAGTACAACA

SEQ ID NO: 2

Chlorella ellipsoidea nitrate reductase promoter from AY307383

CGCTGCGCACCAGGGCCGCCAGCTCGCTGATGTCGCTCCAAATGCGGTCCCCCGA

TTTTTTGTTCTTCATCTTCTCCACCTTGGTGGCCTTCTTGGCCAGGGCCTTCAGCTG

CATGCGCACAGACCGTTGAGCTCCTGATCAGCATCCTCAGGAGGCCCTTTGACAA

GCAAGCCCCTGTGCAAGCCCATTCACGGGGTACCAGTGGTGCTGAGGTAGATGG

GTTTGAAAAGGATTGCTCGGTCGATTGCTGCTCATGGAATTGGCATGTGCATGCA

TGTTCACAATATGCCACCAGGCTTTGGAGCAAGAGAGCATGAATGCCTTCAGGC

AGGTTGAAAGTTCCTGGGGGTGAAGAGGCAGGGCCGAGGATTGGAGGAGGAAA

GCATCAAGTCGTCGCTCATGCTCATGTTTTCAGTCAGAGTTTGCCAAGCTCACAG

GAGCAGAGACAAGACTGGCTGCTCAGGTGTTGCATCGTGTGTGTGGTGGGGGGG

GGGGGGTTAATACGGTACGAAATGCACTTGGAATTCCCACCTCATGCCAGCGGA

CCCACATGCTTGAATTCGAGGCCTGTGGGGTGAGAAATGCTCACTCTGCCCTCGT

TGCTGAGGTACTTCAGGCCGCTGAGCTCAAAGTCGATGCCCTGCTCGTCTATCAG

GGCCTGCACCTCTGGGCTGACCGGCTCAGCCTCCTTCGCGGGCATGGAGTAGGCG

CCGGCAGCGTTCATGTCCGGGCCCAGGGCAGCGGTGGTGCCATAAATGTCGGTG

ATGGTGGGGAGGGGGGCCGTCGCCACACCATTGCCGTTGCTGGCTGACGCATGC

ACATGTGGCCTGGCTGGCACCGGCAGCACTGGTCTCCAGCCAGCCAGCAAGTGG

CTGTTCAGGAAAGCGGCCATGTTGTTGGTCCCTGCGCATGTAATTCCCCAGATCA

AAGGAGGGAACAGCTTGGATTTGATGTAGTGCCCAACCGGACTGAATGTGCGAT GGCAGGTCCCTTTGAGTCTCCCGAATTACTAGCAGGGCACTGTGACCTAACGCAG

CATGCCAACCGCAAAAAAATGATTGACAGAAAATGAAGCGGTGTGTCAATATTT

GCTGTATTTATTCGTTTTAATCAGCAACCAAGTTCGAAACGCAACTATCGTGGTG

ATCAAGTGAACCTCATCAGACTTACCTCGTTCGGCAAGGAAACGGAGGCACCAA

ATTCCAATTTGATATTATCGCTTGCCAAGCTAGAGCTGATCTTTGGGAAACCAAC

TGCCAGACAGTGGACTGTGATGGAGTGCCCCGAGTGGTGGAGCCTCTTCGATTCG

GTTAGTCATTACTAACGTGAACCCTCAGTGAAGGGACCATCAGACCAGAAAGAC

CAGATCTCCTCCTCGACACCGAGAGAGTGTTGCGGCAGTAGGACGACAAG

SEQ ID NO: 3

Yeast sucrose invertase

MTNETSDRPLVHFTPNKGWMNDPNGLWYDEKDAKWHLYFQYNPNDTVWGTPLF

WGHATSDDLTNWEDQPIAIAPKRNDSGAFSGSMVVDYNNTSGFFNDTIDPRQRCVAI

WTYNTPESEEQYISYSLDGGYTFTEYQKNPVLAANSTQFRDPKVFWYEPSQKWIMT

AAKSQDYKIEIYSSDDLKSWKLESAFANEGFLGYQYECPGLIEVPTEQDPSKSYWVM

FISINPGAPAGGSFNQYFVGSFNGTHFEAFDNQSRVVDFGKDYYALQTFFNTDPTYG S

ALGIAWASNWEYSAFVPTNPWRSSMSLVRKFSLNTEYQANPETELINLKAEPILNIS N

AGPWSRFATNTTLTKANSYNVDLSNSTGTLEFELVYAVNTTQTISKSVFADLSLWFK

GLEDPEEYLRMGFEVSASSFFLDRGNSKVKFVKENPYFTNRMSVNNQPFKSENDLSY

YKVYGLLDQNILELYFNDGDVVSTNTYFMTTGNALGSVNMTTGVDNLFYIDKFQVR

EVK

SEQ ID NO: 4

Yeast secretion signal

MLLQAFLFLLAGFAAKISAS

SEQ ID NO: 5

Higher plants secretion signal

MANKSLLLLLLLGSLASG SEQ ID NO: 6

Consensus eukaryotic secretion signal

MARLPLAALG

SEQ ID NO: 7

Combination higher plant/eukaryotic secretion signal

M ANKLLLLLLLLLLPL AAS G

SEQ ID NO: 8

S. cerevisiae sucrose invertase NP_012104

GAATTCCCCAACATGGTGGAGCACGACACTCTCGTCTACTCCAAGAATATCAAAG

ATACAGTCTCAGAAGACCAAAGGGCTATTGAGACTTTTCAACAAAGGGTAATAT

CGGGAAACCTCCTCGGATTCCATTGCCCAGCTATCTGTCACTTCATCAAAAGGAC

AGTAGAAAAGGAAGGTGGCACCTACAAATGCCATCATTGCGATAAAGGAAAGGC

TATCGTTCAAGATGCCTCTGCCGACAGTGGTCCCAAAGATGGACCCCCACCCACG

AGGAGCATCGTGGAAAAAGAAGACGTTCCAACCACGTCTTCAAAGCAAGTGGAT TGATGTGAACATGGTGGAGCACGACACTCTCGTCTACTCCAAGAATATCAAAGAT

ACAGTCTCAGAAGACCAAAGGGCTATTGAGACTTTTCAACAAAGGGTAATATCG

GGAAACCTCCTCGGATTCCATTGCCCAGCTATCTGTCACTTCATCAAAAGGACAG

TAGAAAAGGAAGGTGGCACCTACAAATGCCATCATTGCGATAAAGGAAAGGCTA

TCGTTCAAGATGCCTCTGCCGACAGTGGTCCCAAAGATGGACCCCCACCCACGAG

GAGCATCGTGGAAAAAGAAGACGTTCCAACCACGTCTTCAAAGCAAGTGGATTG

ATGTGATATCTCCACTGACGTAAGGGATGACGCACAATCCCACTATCCTTCGCAA

GACCCTTCCTCTATATAAGGAAGTTCATTTCATTTGGAGAGGACACGCTGAAATC

ACCAGTCTCTCTCTACAAATCTATCTCTGGCGCGCCATATCAATGCTTCTTCAGGC

CTTTCTTTTTCTTCTTGCTGGTTTTGCTGCCAAGATCAGCGCCTCTATGACGAACG

AAACCTCGGATAGACCACTTGTGCACTTTACACCAAACAAGGGCTGGATGAATG

ACCCCAATGGACTGTGGTACGACGAAAAAGATGCCAAGTGGCATCTGTACTTTC

AATACAACCCGAACGATACTGTCTGGGGGACGCCATTGTTTTGGGGCCACGCCAC

GTCCGACGACCTGACCAATTGGGAGGACCAACCAATAGCTATCGCTCCGAAGAG

GAACGACTCCGGAGCATTCTCGGGTTCCATGGTGGTTGACTACAACAATACTTCC

GGCTTTTTCAACGATACCATTGACCCGAGACAACGCTGCGTGGCCATATGGACTT

ACAACACACCGGAGTCCGAGGAGCAGTACATCTCGTATAGCCTGGACGGTGGAT

ACACTTTTACAGAGTATCAGAAGAACCCTGTGCTTGCTGCAAATTCGACTCAGTT

CCGAGATCCGAAGGTCTTTTGGTACGAGCCCTCGCAGAAGTGGATCATGACAGC

GGCAAAGTCACAGGACTACAAGATCGAAATTTACTCGTCTGACGACCTTAAATCC

TGGAAGCTCGAATCCGCGTTCGCAAACGAGGGCTTTCTCGGCTACCAATACGAAT

GCCCAGGCCTGATAGAGGTCCCAACAGAGCAAGATCCCAGCAAGTCCTACTGGG

TGATGTTTATTTCCATTAATCCAGGAGCACCGGCAGGAGGTTCTTTTAATCAGTA

CTTCGTCGGAAGCTTTAACGGAACTCATTTCGAGGCATTTGATAACCAATCAAGA

GTAGTTGATTTTGGAAAGGACTACTATGCCCTGCAGACTTTCTTCAATACTGACC

CGACCTATGGGAGCGCTCTTGGCATTGCGTGGGCTTCTAACTGGGAGTATTCCGC

ATTCGTTCCTACAAACCCTTGGAGGTCCTCCATGTCGCTCGTGAGGAAATTCTCTC

TCAACACTGAGTACCAGGCCAACCCGGAAACCGAACTCATAAACCTGAAAGCCG

AACCGATCCTGAACATTAGCAACGCTGGCCCCTGGAGCCGGTTTGCAACCAACA

CCACGTTGACGAAAGCCAACAGCTACAACGTCGATCTTTCGAATAGCACCGGTA

CACTTGAATTTGAACTGGTGTATGCCGTCAATACCACCCAAACGATCTCGAAGTC

GGTGTTCGCGGACCTCTCCCTCTGGTTTAAAGGCCTGGAAGACCCCGAGGAGTAC

CTCAGAATGGGTTTCGAGGTTTCTGCGTCCTCCTTCTTCCTTGATCGCGGGAACAG

CAAAGTAAAATTTGTTAAGGAGAACCCATATTTTACCAACAGGATGAGCGTTAA

CAACCAACCATTCAAGAGCGAAAACGACCTGTCGTACTACAAAGTGTATGGTTT

GCTTGATCAAAATATCCTGGAACTCTACTTCAACGATGGTGATGTCGTGTCCACC

AACACATACTTCATGACAACCGGGAACGCACTGGGCTCCGTGAACATGACGACG

GGTGTGGATAACCTGTTCTACATCGACAAATTCCAGGTGAGGGAAGTCAAGTGA

GATCTGTCGATCGACAAGCTCGAGTTTCTCCATAATAATGTGTGAGTAGTTCCCA

GATAAGGGAATTAGGGTTCCTATAGGGTTTCGCTCATGTGTTGAGCATATAAGAA

ACCCTTAGTATGTATTTGTATTTGTAAAATACTTCTATCAATAAAATTTCTAATTC

CTAAAACCAAAATCCAGTACTAAAATCCAGATCCCCCGAATTAA

SEQ ID NO: 9

TGTTGAAGAATGAGCCGGCGAC SEQ ID NO: 10

CAGTGAGCTATTACGCACTC SEQ ID NO: 11

UTEX 329 Prototheca kruegani

TGTTGAAGAATGAGCCGGCGAGTTAAAAAGAGTGGCATGGTTAAAGAAAATACT

AAACCGAGTGATCTACCCATGATCAGGGTGAAGTGTTAGTAAAATAACATGGAG

GCCCGAACCGACTAATGTTGAAAAATTAGCGGATGAATTGTGGGTAGGGGCGAA

AAACCAATCGAACTCGGAGTTAGCTGGTTCTCCCCGAAATGCGTTTAGGCGCAGC

AGTAGCAGTACAAATAGAGGGGTAAAGCACTGTTTCTTTTGTGGGCTTCGAAAGT

TGTACCTCAAAGTGGCAAACTCTGAATACTCTATTTAGATATCTACTAGTGAGAC

CTTGGGGGATAAGCTCCTTGGTCAAAAGGGAAACAGCCCAGATCACCAGTTAAG

GCCCCAAAATGAAAATGATAGTGACTAAGGATGTGGGTATGTCAAAACCTCCAG

CAGGTTAGCTTAGAAGCAGCAATCCTTTCAAGAGTGCGTAATAGCTCACTG

SEQ ID NO: 12

UTEX 1440 Prototheca wickerhamii

TGTTGAAGAATGAGCCGGCGACTTAAAATAAATGGCAGGCTAAGAGATTTAATA

ACTCGAAACCTAAGCGAAAGCAAGTCTTAATAGGGCGTCAATTTAACAAAACTT

TAAATAAATTATAAAGTCATTTATTTTAGACCCGAACCTGAGTGATCTAACCATG

GTCAGGATGAAACTTGGGTGACACCAAGTGGAAGTCCGAACCGACCGATGTTGA

AAAATCGGCGGATGAACTGTGGTTAGTGGTGAAATACCAGTCGAACTCAGAGCT

AGCTGGTTCTCCCCGAAATGCGTTGAGGCGCAGCAATATATCTCGTCTATCTAGG

GGTAAAGCACTGTTTCGGTGCGGGCTATGAAAATGGTACCAAATCGTGGCAAAC

TCTGAATACTAGAAATGACGATATATTAGTGAGACTATGGGGGATAAGCTCCAT

AGTCGAGAGGGAAACAGCCCAGACCACCAGTTAAGGCCCCAAAATGATAATGAA

GTGGTAAAGGAGGTGAAAATGCAAATACAACCAGGAGGTTGGCTTAGAAGCAGC

CATCCTTTAAAGAGTGCGTAATAGCTCACTG

SEQ ID NO: 13

UTEX 1442 Prototheca stagnora

TGTTGAAGAATGAGCCGGCGAGTTAAAAAAAATGGCATGGTTAAAGATATTTCT

CTGAAGCCATAGCGAAAGCAAGTTTTACAAGCTATAGTCATTTTTTTTAGACCCG

AAACCGAGTGATCTACCCATGATCAGGGTGAAGTGTTGGTCAAATAACATGGAG

GCCCGAACCGACTAATGGTGAAAAATTAGCGGATGAATTGTGGGTAGGGGCGAA

AAACCAATCGAACTCGGAGTTAGCTGGTTCTCCCCGAAATGCGTTTAGGCGCAGC

AGTAGCAACACAAATAGAGGGGTAAAGCACTGTTTCTTTTGTGGGCTTCGAAAGT

TGTACCTCAAAGTGGCAAACTCTGAATACTCTATTTAGATATCTACTAGTGAGAC

CTTGGGGGATAAGCTCCTTGGTCAAAAGGGAAACAGCCCAGATCACCAGTTAAG

GCCCCAAAATGAAAATGATAGTGACTAAGGACGTGAGTATGTCAAAACCTCCAG

CAGGTTAGCTTAGAAGCAGCAATCCTTTCAAGAGTGCGTAATAGCTCACTG

SEQ ID NO: 14

UTEX 288 Prototheca moriformis

TGTTGAAGAATGAGCCGGCGAGTTAAAAAGAGTGGCATGGTTAAAGATAATTCT

AAACCGAGTGATCTACCCATGATCAGGGTGAAGTGTTGGTAAAATAACATGGAG GCCCGAACCGACTAATGGTGAAAAATTAGCGGATGAATTGTGGGTAGGGGCGAA

AAACCAATCGAACTCGGAGTTAGCTGGTTCTCCCCGAAATGCGTTTAGGCGCAGC

AGTAGCAACACAAATAGAGGGGTAAAGCACTGTTTCTTTTGTGGGCTTCGAAAGT

TGTACCTCAAAGTGGCAAACTCTGAATACTCTATTTAGATATCTACTAGTGAGAC

CTTGGGGGATAAGCTCCTTGGTCAAAAGGGAAACAGCCCAGATCACCAGTTAAG

GCCCCAAAATGAAAATGATAGTGACTAAGGATGTGGGTATGTTAAAACCTCCAG

CAGGTTAGCTTAGAAGCAGCAATCCTTTCAAGAGTGCGTAATAGCTCACTG

SEQ ID NO: 15

UTEX 1439, UTEX 1441, UTEX 1435, UTEX 1437 Prototheca moriformis

TGTTGAAGAATGAGCCGGCGACTTAAAATAAATGGCAGGCTAAGAGAATTAATA

ACTCGAAACCTAAGCGAAAGCAAGTCTTAATAGGGCGCTAATTTAACAAAACAT

TAAATAAAATCTAAAGTCATTTATTTTAGACCCGAACCTGAGTGATCTAACCATG

GTCAGGATGAAACTTGGGTGACACCAAGTGGAAGTCCGAACCGACCGATGTTGA

AAAATCGGCGGATGAACTGTGGTTAGTGGTGAAATACCAGTCGAACTCAGAGCT

AGCTGGTTCTCCCCGAAATGCGTTGAGGCGCAGCAATATATCTCGTCTATCTAGG

GGTAAAGCACTGTTTCGGTGCGGGCTATGAAAATGGTACCAAATCGTGGCAAAC

TCTGAATACTAGAAATGACGATATATTAGTGAGACTATGGGGGATAAGCTCCAT

AGTCGAGAGGGAAACAGCCCAGACCACCAGTTAAGGCCCCAAAATGATAATGAA

GTGGTAAAGGAGGTGAAAATGCAAATACAACCAGGAGGTTGGCTTAGAAGCAGC

CATCCTTTAAAGAGTGCGTAATAGCTCACTG

SEQ ID NO: 16

UTEX 1533 Prototheca wickerhamii

TGTTGAAGAATGAGCCGTCGACTTAAAATAAATGGCAGGCTAAGAGAATTAATA

ACTCGAAACCTAAGCGAAAGCAAGTCTTAATAGGGCGCTAATTTAACAAAACAT

TAAATAAAATCTAAAGTCATTTATTTTAGACCCGAACCTGAGTGATCTAACCATG

GTCAGGATGAAACTTGGGTGACACCAAGTGGAAGTCCGAACCGACCGATGTTGA

AAAATCGGCGGATGAACTGTGGTTAGTGGTGAAATACCAGTCGAACTCAGAGCT

AGCTGGTTCTCCCCGAAATGCGTTGAGGCGCAGCAATATATCTCGTCTATCTAGG

GGTAAAGCACTGTTTCGGTGCGGGCTATGAAAATGGTACCAAATCGTGGCAAAC

TCTGAATACTAGAAATGACGATATATTAGTGAGACTATGGGGGATAAGCTCCAT

AGTCGAGAGGGAAACAGCCCAGACCACCAGTTAAGGCCCCAAAATGATAATGAA

GTGGTAAAGGAGGTGAAAATGCAAATACAACCAGGAGGTTGGCTTAGAAGCAGC

CATCCTTTAAAGAGTGCGTAATAGCTCACTG

SEQ ID NO: 17

UTEX 1434 Prototheca moriformis

TGTTGAAGAATGAGCCGGCGAGTTAAAAAGAGTGGCGTGGTTAAAGAAAATTCT

CTGGAACCATAGCGAAAGCAAGTTTAACAAGCTTAAGTCACTTTTTTTAGACCCG

AAACCGAGTGATCTACCCATGATCAGGGTGAAGTGTTGGTAAAATAACATGGAG

GCCCGAACCGACTAATGGTGAAAAATTAGCGGATGAATTGTGGGTAGGGGCGAA

AAACCAATCGAACTCGGAGTTAGCTGGTTCTCCCCGAAATGCGTTTAGGCGCAGC

AGTAGCAACACAAATAGAGGGGTAAAGCACTGTTTCTTTTGTGGGCTCCGAAAG

TTGTACCTCAAAGTGGCAAACTCTGAATACTCTATTTAGATATCTACTAGTGAGA

CCTTGGGGGATAAGCTCCTTGGTCGAAAGGGAAACAGCCCAGATCACCAGTTAA GGCCCCAAAATGAAAATGATAGTGACTAAGGATGTGAGTATGTCAAAACCTCCA GCAGGTTAGCTTAGAAGCAGCAATCCTTTCAAGAGTGCGTAATAGCTCACTG

SEQ ID NO: 18

UTEX 1438 Prototheca zopfii

TGTTGAAGAATGAGCCGGCGAGTTAAAAAGAGTGGCATGGTTAAAGAAAATTCT

CTGGAGCCATAGCGAAAGCAAGTTTAACAAGCTTAAGTCACTTTTTTTAGACCCG

AAACCGAGTGATCTACCCATGATCAGGGTGAAGTGTTGGTAAAATAACATGGAG

GCCCGAACCGACTAATGGTGAAAAATTAGCGGATGAATTGTGGGTAGGGGCGAA

AAACCAATCGAACTCGGAGTTAGCTGGTTCTCCCCGAAATGCGTTTAGGCGCAGC

AGTAGCAACACAAATAGAGGGGTAAAGCACTGTTTCTTTCGTGGGCTTCGAAAG

TTGTACCTCAAAGTGGCAAACTCTGAATACTCTATTTAGATATCTACTAGTGAGA

CCTTGGGGGATAAGCTCCTTGGTCAAAAGGGAAACAGCCCAGATCACCAGTTAA

GGCCCCAAAATGAAAATGATAGTGACTAAGGATGTGAGTATGTCAAAACCTCCA

GCAGGTTAGCTTAGAAGCAGCAATCCTTTCAAGAGTGCGTAATAGCTCACTG

SEQ ID NO: 19

UTEX 1436 Prototheca moriformis

TGTTGAAGAATGAGCCGGCGACTTAGAAAAGGTGGCATGGTTAAGGAAATATTC CGAAGCCGTAGCAAAAGCGAGTCTGAATAGGGCGATAAAATATATTAATATTTA

GAAGCTTGGGTGATACCAAGTGAAGGTCCGAACCGACCGATGTTGAAAAATCGG

CGGATGAGTTGTGGTTAGCGGTGAAATACCAGTCGAACCCGGAGCTAGCTGGTT

CTCCCCGAAATGCGTTGAGGCGCAGCAGTACATCTAGTCTATCTAGGGGTAAAGC

ACTGTTTCGGTGCGGGCTGTGAGAACGGTACCAAATCGTGGCAAACTCTGAATAC

TAGAAATGACGATGTAGTAGTGAGACTGTGGGGGATAAGCTCCATTGTCAAGAG

GGAAACAGCCCAGACCACCAGCTAAGGCCCCAAAATGGTAATGTAGTGACAAAG

GAGGTGAAAATGCAAATACAACCAGGAGGTTGGCTTAGAAGCAGCCATCCTTTA

AAGAGTGCGTAATAGCTCACTG

SEQ ID NO: 20

Chicorium intybus invertase: Genbank Accession No. Y11124

MSNSSNASESLFPATSEQPYRTAFHFQPPQNWMNDPNGPMCYNGVYHLFYQYNPFG

PLWNLRMYWAHSVSHDLINWIHLDLAFAPTEPFDINGCLSGSATVLPGNKPIMLYTG

IDTENRQVQNLAVPKDLSDPYLREWVKHTGNPIISLPEEIQPDDFRDPTTTWLEEDG T

WRLLVGSQKDKTGIAFLYHSGDFVNWTKSDSPLHKVSGTGMWECVDFFPVWVDST

NGVDTSIINPSNRVKHVLKLGIQDHGKDCYLIGKYSADKENYVPEDELTLSTLRLDY

GMYYASKSFFDPVKNRRIMTAWVNESDSEADVIARGWSGVQSFPRSLWLDKNQKQ

LLQWPIEEIEMLHQNEVSFHNKKLDGGSSLEVLGITASQADVKISFKLANLEEAEEL D

PSWVDPQLICSENDASKKGKFGPFGLLALASSDLREQTAIFFRVFRKNGRYVVLMCS

DQSRSSMKNGIEKRTYGAFVDIDPQQDEISLRTLIDHSIVESFGGRGKTCITTRVYP TL

AIGEQARLFAFNHGTESVEISELSAWSMK AQM VEEP

SEQ ID NO: 21

Schizosaccharomyces pombe Invertase: Genbank Accession No. AB011433 MFLKYILASGICLVSLLSSTNAAPRHLYVKRYPVIYNASNITEVSNSTTVPPPPFVNTT

APNGTCLGNYNEYLPSGYYNATDRPKIHFTPSSGFMNDPNGLVYTGGVYHMFFQYS

PKTLTAGEVHWGHTVSKDLIHWENYPIAIYPDEHENGVLSLPFSGSAVVDVHNSSGL

FSNDTIPEERIVLIYTDHWTGVAERQAIAYTTDGGYTFKKYSGNPVLDINSLQFRDP

VIWDFDANRWVMIVAMSQNYGIAFYSSYDLIHWTELSVFSTSGYLGLQYECPGMAR

VPVEGTDEYKWVLFISINPGAPLGGSVVQYFVGDWNGTNFVPDDGQTRFVDLGKDF

YASALYHSSSANADVIGVGWASNWQYTNQAPTQVFRSAMTVARKFTLRDVPQNPM

TNLTSLIQTPLN VS LLRDETLFTAP VINS S S S LS GSPITLPSNT AFEFNVTLSIN YTEGCT

TGYCLGRIIIDSDDPYRLQSISVDVDFAASTLVINRAKAQMGWFNSLFTPSFANDIY IY

GNVTLYGIVDNGLLELYVNNGEKTYTNDFFFLQGATPGQISFAAFQGVSFNNVTVTP

LKTIWNC

SEQ ID NO: 22

Picha anomala beta-fructofuranosidase (invertase): Genbank Accession No. X80640

MIQLSPLLLLPLFSVFNSIADASTEYLRPQIHLTPDQGWMNDPNGMFYDRKDKLWHV

YFQHNPDKKSIWATPVTWGHSTSKDLLTWDYHGNALEPENDDEGIFSGSVVVDRNN

TSGFFNDSTDPEQRIVAIYTNNAQLQTQEIAYSLDKGYSFIKYDQNPVINVNSSQQR D

PKVLWHDESNQWIMVVAKTQEFKVQIYGSPDLKKWDLKSNFTSNGYLGFQYECPG

LFKLPIENPLNDTVTSKWVLLLAINPGSPLGGSINEYFIGDFDGTTFHPDDGATRFM DI

GKDFYAFQSFDNTEPEDGALGLAWASNWQYANTVPTENWRSSMSLVRNYTLKYVD

VNPENYGLTLIQKPVYDTKETRLNETLKTLETINEYEVNDLKLDKSSFVATDFNTER N

ATGVFEFDLKFTQTDLKMGYSNMTTQFGLYIHSQTVKGSQETLQLVFDTLSTTWYID

RTTQHSFQRNSPVFTERISTYVEKIDTTDQGNVYTLYGVVDRNILELYFNDGSIAMT N

TFFFREGKIPTSFEVVCDSEKSFITIDELSVRELARK

SEQ ID NO: 23

Debaryomyces occidentalis Invertase: Genbank Accession No. X17604

MVQVLSVLVIPLLTLFFGYVASSSIDLSVDTSEYNRPLIHFTPEKGWMNDPNGLFYD K

TAKLWHLYFQYNPNATAWGQPLYWGHATSNDLVHWDEHEIAIGPEHDNEGIFSGSI

VVDHNNTSGFFNSSIDPNQRIVAIYTNNIPDLQTQDIAFSLDGGYTFTKYENNPVID VS

SNQFRDPKVFWHERFKSMDHGCSEIARVKIQIFGSANLKNWVLNSNFSSGYYGNQY

GMSRLIEVPIENSD SKWVMFLAINPGSPLGGSINQYFVGDFDGFQFVPDDSQTRFVD

IGKDFYAFQTFSEVEHGVLGLAWASNWQYADQVPTNPWRSSTSLARNYTLRYVIQM

LKLTANID KS VLPD SINV VD KLKKKN VKLTNKKPIKTNFKGSTGLFDFNITFKVLNLN

VSPGKTHFDILINSQELNSSVDSIKIGFDSSQSLFYIDRHIPNVEFPRKQFFTDKLA AYL

EPLDYDQDLRVFSLYGIVDKNIIELYFNDGTVAMTNTFFMGEGKYPHDIQIVTDTEE P

LFELESVIIRELNK

SEQ ID NO: 24

Oryza sativa Invertase: Genbank Accession No. AF019113

MATSRLTPAYDLKNAAAAVYTPLPEQPHSAEVEIRDRKPFKIISAIILSSLLLLALI LVA

VNYQAPPSHSSGDNSQPAAVMPPSRGVSQGVSEKAFRGASGAGNGVSFAWSNLMLS

WQRTSYHFQPVKNWMNDPNGPLYYKGWYHLFYQYNPDSAVWGNITWGHAVSTD

LINWLHLPFAMVPDQWYDVNGVWTGSATILPDGRIVMLYTGDTDDYVQDQNLAFP

ANLSDPLLVDWVKYPNNPVIYPPPGIGVKDFRDPTTAGTAGMQNGQRLVTIGSKVG

KTGISLVYETTNFTTFKLLYGVLHAVPGTGMWECVDLYPVSTTGENGLDTSVNGLG

VKHVLKTSLDDDKHDYYALGTYDPV NKWTPDNPDLDVGIGLRLDYGKYYAARTF YDQNKQRRILWGWIGETDLEAVDLMKGWASLQAIPRTIVFDKKTGTNVLQRPEEEV

ESWSSGDPITQRRIFEPGSVVPIHVSGATQLDITASFEVDETLLETTSESHDAGYDC SN

SGGAGTRGSLGPFGLLVVADEKLSELTPVYLYVAKGGDGKAKAHLCAYQTRSSMAS

GVEKEVYGSAVPVLDGENYSARILIDHSIVESFAQAGRTCVRSRDYPTKDIYGAARC F

FFNNATEASVRASLKAWQMKSFIRPYPFIPDQKS

SEQ ID NO: 25

Allium cepa Invertase: Genbank Accession No. AJ006067

MSSDDLESPPSSYLPIPPSDEFHDQPPPLRSWLRLLSIPLALMFLLFLATFLSNLES PPSD

SGLVSDPVTFDVNPAVVRRGKDAGVSDKTSGVDSGFVLDPVAVDANSVVVHRGKD

AGVSDKTSGVDSGLLKDSPLGPYPWTNQMLSWQRTGFHFQPVKNWMNDPNGPLYY

KGWYHFFYQYNPEGAVWGNIAWGHAVSRDLVHWTHLPLAMVPDQWYDINGVWT

GSATILPDGQIVMLYTGATNESVQVQNLAVPADQSDTLLLRWKKSEANPILVPPPGI G

DKDFRDPTTAWYEPSDDTWRIVIGSKDSSHSGIAIVYST DFINYKLIPGILHAVERVG

MWECVDFYPVATADSSHANHGLDPSARPSPAVKHVLKASMDDDRHDYYAIGTYDP

AQNTWVPDDASVDVGIGLRYDWGKFYASKTFYDHAKKRRILWSWIGETDSETADIA

KGWASLQGVPRTVLLDVKTGSNLITWPVVEIESLRTRPRDFSGITVDAGSTFKLDVG

GAAQLDIEAEFKISSEELEAVKEADVSYNCSSSGGAAERGVLGPFGLLVLANQDLTE

QTATYFYVSRGMDGGLNTHFCQDEKRSSKASDIVKRIVGHSVPVLDGESFALRILVD

HSIVESFAQGGRASATSRVYPTEAIYNNARVFVFNNATGAKVTAQSLKVWHMSTAI

NEIYDPATSVM

SEQ ID NO: 26

Beta vulgaris subsp. vulgaris Invertase: Genbank Accession No. AJ278531

LFYQYNPNGVIWGPPVWGHSTSKDLVNWVPQPLTMEPEMAANINGSWSGSATILPG

NKPAILFTGLDPKYEQVQVLAYPKDTSDPNLKEWFLAPQNPVMFPTPQNQINATSFR

DPTTAWRLPDGVWRLLIGSKRGQRGLSLLFRSRDFVHWVQAKHPLYSDKLSGMWE

CPDFFPVYANGDQMGVDTSIIGSHVKHVLKNSLDITKHDIYTIGDYNIKKDAYTPDI G

YMNDSSLRYDYGKYYASKTFFDDAKKERILLGWANESSSVEDDIKKGWSGIHTIPRK

IWLDKLGKQLIQWPIANIEKLRQKPVNIYRKVLKGGSQIEVSGITAAQADVEISFKI KD

LKNVEKFDASWTSPQLLCSKKGASVKGGLGPFGLLTLASXGLEEYTAVFFRIFKAYD

NKFVVLMCSDQSRSSLNPTNDKTTYGTFVDVNPIREGLSLRVLIDHSVVESFGA GK

NVITARVYPTLAINE AHLYVFNRGTSNVEITGLTAWSMKKANIA

SEQ ID NO: 27

Bifidobacterium breve UCC2003 beta-fructofuranosidase (invertase): Genbank Accession No. AAT28190

MTDFTPETPVLTPIRDHAAELAKAEAGVAEMAAKRNNRWYPKYHIASNGGWINDPN

GLCFYKGRWHVFYQLHPYGTQWGPMHWGHVSSTDMLNWKREPIMFAPSLEQEKD

GVFSGSAVIDDNGDLRFYYTGHRWANGHDNTGGDWQVQMTALPDNDELTSATKQ

GMIIDCPTD VDHH YRDPKVWKTGDTW YMTFG VS SED KRGQM WLFS S KDM VRWE

YERVLFQHPDPDVFMLECPDFFPIKDKDGNEKWVIGFSAMGSKPSGFMNRNVNNAG

YMIGTWEPGGEFKPETEFRLWDCGHNYYAPQSFNVDGRQIVYGWMSPFVQPIPMED

DGWCGQLTLPREITLDDDGDVVTAPVAEMEGLREDTLDHGSITLDMDGEQVIADDA

EAVEIEMTIDLAASTADRAGLKIHATEDGAYTYVAYDDQIGRVVVDRQAMANGDH

GYRAAPLTDAELASGKLDLRVFVDRGSVEVYVNGGHQVLSSYSYASEGPRAIKLVA

EFGNL VESLKLHHMKSIGLE SEQ ID NO: 28

Saccharomyces cerevisiae Invertase: Genbank Accession No. NP_012104

MLLQAFLFLLAGFAAKISASMTNETSDRPLVHFTPNKGWMNDPNGLWYDEKDAKW

HLYFQYNPNDTVWGTPLFWGHATSDDLTNWEDQPIAIAPKRNDSGAFSGSMVVDY

NNTSGFFNDTIDPRQRCVAIWTYNTPESEEQYISYSLDGGYTFTEYQKNPVLAANST Q

FRDPKVFWYEPSQKWIMTAAKSQDYKIEIYSSDDLKSWKLESAFANEGFLGYQYECP

GLIEVPTEQDPSKSYWVMFISINPGAPAGGSFNQYFVGSFNGTHFEAFDNQSRVVDF G

KDYYALQTFFNTDPTYGSALGIAWASNWEYSAFVPTNPWRSSMSLVRKFSLNTEYQ

ANPETELINLKAEPILNISNAGPWSRFATNTTLTKANSYNVDLSNSTGTLEFELVYA V

NTTQTISKSVFADLSLWFKGLEDPEEYLRMGFEVSASSFFLDRGNSKVKFVKENPYF T

NRMSVNNQPFKSENDLSYYKVYGLLDQNILELYFNDGDVVSTNTYFMTTGNALGSV

NMTTGVDNLFYID FQVREVK

SEQ ID NO: 29

Zymomonas mobilis Invertase A: Genbank Accession No. AY171597

MESPSYKNLIKAEDAQKKAGKRLLSSEWYPGFHVTPLTGWMNDPNGLIFFKGEYHL

FYQYYPFAPVWGPMHWGHAKSRDLVHWETLPVALAPGDLFDRDGCFSGCAVDNN

GVLTLIYTGHIVLSNDSPDAIREVQCMATSIDGIHFQKEGIVLEKAPMPQVAHFRDP R

VWKENDHWFMVVGYRTDDEKHQGIGHVALYRSENLKDWIFVKTLLGDNSQLPLGK

RAFMWECPDFFSLGNRSVLMFSPQGLKASGYKNRNLFQNGYILGKWQAPQFTPETS

FQELDYGHDFYAAQRFEAKDGRQILIAWFDMWENQKPSQRDGWAGCMTLPRKLDL

IDNKIVMTPVREMEILRQSEKIESVVTLSDAEHPFTMDSPLQEIELIFDLEKSSAYQ AG

LALRCNGKGQETLLYIDRSQNRIILDRNRSGQNVKGIRSCPLPNTSKVRLHIFLDRS SIE

IFVGDDQTQGLYSISSRIFPDKDSLKGRLFAIEGYAVFDSFKRWTLQDANLAAFSSD A

C

SEQ ID NO: 30

Cinnamomum camphora FATB 1 (Genbank Q39473) amino acid sequence with Prototheca moriformis delta 12 fatty acid desaturase transit peptide.

MAIKTNRQPVEKPPFTIGTLRKAIPAHCFERSALRGRAPDWSMLFAVITTIFSAAEK Q

WTNLEWKPKPNPPQLLDDHFGPHGLVFRRTFAIRSYEVGPDRSTSIVAVMNHLQEAA

LNHAKSVGILGDGFGTTLEMSKRDLIWVVKRTHVAVERYPAWGDTVEVECWVGAS

GNNGRRHDFLVRDCKTGEILTRCTSLSVMMNTRTRRLSKIPEEVRGEIGPAFIDNVA V

KDEEIKKPQKLNDSTADYIQGGLTPRWNDLDINQHVNNIKYVDWILETVPDSIFESH H

ISSFTIEYRRECTMDSVLQSLTTVSGGSSEAGLVCEHLLQLEGGSEVLRA TEWRPKL

TDSFRGISVIPAESSV

SEQ ID NO: 31

Relevant codon optimized expression construct of Cinnamomum camphora FATB 1 cDNA with Prototheca moriformis delta 12 fatty acid desaturase transit peptide.

GGTACCCGCCTGCAACGCAAGGGCAGCCACAGCCGCTCCCACCCGCCGCTGAAC CGACACGTGCTTGGGCGCCTGCCGCCTGCCTGCCGCATGCTTGTGCTGGTGAGGC TGGGCAGTGCTGCCATGCTGATTGAGGCTTGGTTCATCGGGTGGAAGCTTATGTG TGTGCTGGGCTTGCATGCCGGGCAATGCGCATGGTGGCAAGAGGGCGGCAGCAC TTGCTGGAGCTGCCGCGGTGCCTCCAGGTGGTTCAATCGCGGCAGCCAGAGGGA TTTCAGATGATCGCGCGTACAGGTTGAGCAGCAGTGTCAGCAAAGGTAGCAGTTT

GCCAGAATGATCGGTTCAGCTGTTAATCAATGCCAGCAAGAGAAGGGGTCAAGT

GCAAACACGGGCATGCCACAGCACGGGCACCGGGGAGTGGAATGGCACCACCA

AGTGTGTGCGAGCCAGCATCGCCGCCTGGCTGTTTCAGCTACAACGGCAGGAGTC

ATCCAACGTAACCATGAGCTGATCAACACTGCAATCATCGGGCGGGCGTGATGC

AAGCATGCCTGGCGAAGACACATGGTGTGCGGATGCTGCCGGCTGCTGCCTGCT

GCGCACGCCGTTGAGTTGGCAGCAGGCTCAGCCATGCACTGGATGGCAGCTGGG

CTGCCACTGCAATGTGGTGGATAGGATGCAAGTGGAGCGAATACCAAACCCTCT

GGCTGCTTGCTGGGTTGCATGGCATCGCACCATCAGCAGGAGCGCATGCGAAGG

GACTGGCCCCATGCACGCCATGCCAAACCGGAGCGCACCGAGTGTCCACACTGT

CACCAGGCCCGCAAGCTTTGCAGAACCATGCTCATGGACGCATGTAGCGCTGAC

GTCCCTTGACGGCGCTCCTCTCGGGTGTGGGAAACGCAATGCAGCACAGGCAGC

AGAGGCGGCGGCAGCAGAGCGGCGGCAGCAGCGGCGGGGGCCACCCTTCTTGCG

GGGTCGCGCCCCAGCCAGCGGTGATGCGCTGATCCCAAACGAGTTCACATTCATT

TGCATGCCTGGAGAAGCGAGGCTGGGGCCTTTGGGCTGGTGCAGCCCGCAATGG

AATGCGGGACCGCCAGGCTAGCAGCAAAGGCGCCTCCCCTACTCCGCATCGATG

TTCCATAGTGCATTGGACTGCATTTGGGTGGGGCGGCCGGCTGTTTCTTTCGTGTT

GCAAAACGCGCCAGCTCAGCAACCTGTCCCGTGGGTCCCCCGTGCCGATGAAAT

CGTGTGCACGCCGATCAGCTGATTGCCCGGCTCGCGAAGTAGGCGCCCTCCTTTC

TGCTCGCCCTCTCTCCGTCCCGCCTCTAGAATATCAATGATCGAGCAGGACGGCC

TCCACGCCGGCTCCCCCGCCGCCTGGGTGGAGCGCCTGTTCGGCTACGACTGGGC

CCAGCAGACCATCGGCTGCTCCGACGCCGCCGTGTTCCGCCTGTCCGCCCAGGGC

CGCCCCGTGCTGTTCGTGAAGACCGACCTGTCCGGCGCCCTGAACGAGCTGCAGG

ACGAGGCCGCCCGCCTGTCCTGGCTGGCCACCACCGGCGTGCCCTGCGCCGCCGT

GCTGGACGTGGTGACCGAGGCCGGCCGCGACTGGCTGCTGCTGGGCGAGGTGCC

CGGCCAGGACCTGCTGTCCTCCCACCTGGCCCCCGCCGAGAAGGTGTCCATCATG

GCCGACGCCATGCGCCGCCTGCACACCCTGGACCCCGCCACCTGCCCCTTCGACC

ACCAGGCCAAGCACCGCATCGAGCGCGCCCGCACCCGCATGGAGGCCGGCCTGG

TGGACCAGGACGACCTGGACGAGGAGCACCAGGGCCTGGCCCCCGCCGAGCTGT

TCGCCCGCCTGAAGGCCCGCATGCCCGACGGCGAGGACCTGGTGGTGACCCACG

GCGACGCCTGCCTGCCCAACATCATGGTGGAGAACGGCCGCTTCTCCGGCTTCAT

CGACTGCGGCCGCCTGGGCGTGGCCGACCGCTACCAGGACATCGCCCTGGCCAC

CCGCGACATCGCCGAGGAGCTGGGCGGCGAGTGGGCCGACCGCTTCCTGGTGCT

GTACGGCATCGCCGCCCCCGACTCCCAGCGCATCGCCTTCTACCGCCTGCTGGAC

GAGTTCTTCTGACAATTGGCAGCAGCAGCTCGGATAGTATCGACACACTCTGGAC

GCTGGTCGTGTGATGGACTGTTGCCGCCACACTTGCTGCCTTGACCTGTGAATAT

CCCTGCCGCTTTTATCAAACAGCCTCAGTGTGTTTGATCTTGTGTGTACGCGCTTT

TGCGAGTTGCTAGCTGCTTGTGCTATTTGCGAATACCACCCCCAGCATCCCCTTCC

CTCGTTTCATATCGCTTGCATCCCAACCGCAACTTATCTACGCTGTCCTGCTATCC

CTCAGCGCTGCTCCTGCTCCTGCTCACTGCCCCTCGCACAGCCTTGGTTTGGGCTC

CGCCTGTATTCTCCTGGTACTGCAACCTGTAAACCAGCACTGCAATGCTGATGCA

CGGGAAGTAGTGGGATGGGAACACAAATGGAGGATCCCGCGTCTCGAACAGAGC

GCGCAGAGGAACGCTGAAGGTCTCGCCTCTGTCGCACCTCAGCGCGGCATACAC

CACAATAACCACCTGACGAATGCGCTTGGTTCTTCGTCCATTAGCGAAGCGTCCG

GTTCACACACGTGCCACGTTGGCGAGGTGGCAGGTGACAATGATCGGTGGAGCT

GATGGTCGAAACGTTCACAGCCTAGGGATATCGAATTCCTTTCTTGCGCTATGAC

ACTTCCAGCAAAAGGTAGGGCGGGCTGCGAGACGGCTTCCCGGCGCTGCATGCA

ACACCGATGATGCTTCGACCCCCCGAAGCTCCTTCGGGGCTGCATGGGCGCTCCG

ATGCCGCTCCAGGGCGAGCGCTGTTTAAATAGCCAGGCCCCCGATTGCAAAGAC

ATTATAGCGAGCTACCAAAGCCATATTCAAACACCTAGATCACTACCACTTCTAC ACAGGCCACTCGAGCTTGTGATCGCACTCCGCTAAGGGGGCGCCTCTTCCTCTTC

GTTTCAGTCACAACCCGCAAACACTAGTATGGCTATCAAGACGAACAGGCAGCC

TGTGGAGAAGCCTCCGTTCACGATCGGGACGCTGCGCAAGGCCATCCCCGCGCA

CTGTTTCGAGCGCTCGGCGCTTCGTGGGCGCGCCCCCGACTGGTCCATGCTGTTC

GCCGTGATCACCACCATCTTCTCCGCCGCCGAGAAGCAGTGGACCAACCTGGAGT

GGAAGCCCAAGCCCAACCCCCCCCAGCTGCTGGACGACCACTTCGGCCCCCACG

GCCTGGTGTTCCGCCGCACCTTCGCCATCCGCAGCTACGAGGTGGGCCCCGACCG

CTCCACCAGCATCGTGGCCGTGATGAACCACCTGCAGGAGGCCGCCCTGAACCA

CGCCAAGTCCGTGGGCATCCTGGGCGACGGCTTCGGCACCACCCTGGAGATGTCC

AAGCGCGACCTGATCTGGGTGGTGAAGCGCACCCACGTGGCCGTGGAGCGCTAC

CCCGCCTGGGGCGACACCGTGGAGGTGGAGTGCTGGGTGGGCGCCTCCGGCAAC

AACGGCCGCCGCCACGACTTCCTGGTGCGCGACTGCAAGACCGGCGAGATCCTG

ACCCGCTGCACCTCCCTGAGCGTGATGATGAACACCCGCACCCGCCGCCTGAGCA

AGATCCCCGAGGAGGTGCGCGGCGAGATCGGCCCCGCCTTCATCGACAACGTGG

CCGTGAAGGACGAGGAGATCAAGAAGCCCCAGAAGCTGAACGACTCCACCGCCG

ACTACATCCAGGGCGGCCTGACCCCCCGCTGGAACGACCTGGACATCAACCAGC

ACGTGAACAACATCAAGTACGTGGACTGGATCCTGGAGACCGTGCCCGACAGCA

TCTTCGAGAGCCACCACATCTCCTCCTTCACCATCGAGTACCGCCGCGAGTGCAC

CATGGACAGCGTGCTGCAGTCCCTGACCACCGTGAGCGGCGGCTCCTCCGAGGC

CGGCCTGGTGTGCGAGCACCTGCTGCAGCTGGAGGGCGGCAGCGAGGTGCTGCG

CGCCAAGACCGAGTGGCGCCCCAAGCTGACCGACTCCTTCCGCGGCATCAGCGT

GATCCCCGCCGAGTCCAGCGTGATGGACTACAAGGACCACGACGGCGACTACAA

GGACCACGACATCGACTACAAGGACGACGACGACAAGTGATGACTCGAGGCAGC

AGCAGCTCGGATAGTATCGACACACTCTGGACGCTGGTCGTGTGATGGACTGTTG

CCGCCACACTTGCTGCCTTGACCTGTGAATATCCCTGCCGCTTTTATCAAACAGCC

TCAGTGTGTTTGATCTTGTGTGTACGCGCTTTTGCGAGTTGCTAGCTGCTTGTGCT

ATTTGCGAATACCACCCCCAGCATCCCCTTCCCTCGTTTCATATCGCTTGCATCCC

AACCGCAACTTATCTACGCTGTCCTGCTATCCCTCAGCGCTGCTCCTGCTCCTGCT

CACTGCCCCTCGCACAGCCTTGGTTTGGGCTCCGCCTGTATTCTCCTGGTACTGCA

ACCTGTAAACCAGCACTGCAATGCTGATGCACGGGAAGTAGTGGGATGGGAACA

CAAATGGAAAGCTT

SEQ ID NO: 32

Cuphea hookeriana FATB2 (Genbank AAC49269) amino acid sequence with Prototheca moriformis delta 12 fatty acid desaturase transit peptide.

MAIKTNRQPVEKPPFTIGTLRKAIPAHCFERSALRGRAQLPDWSRLLTAITTVFVKS K

RPDMHDRKSKRPDMLVDSFGLESTVQDGLVFRQSFSIRSYEIGTDRTASIETLMNHL Q

ETSLNHCKSTGILLDGFGRTLEMCKRDLIWVVIKMQIKVNRYPAWGDTVEINTRFSR

LGKIGMGRDWLISDCNTGEILVRATSAYAMMNQKTRRLSKLPYEVHQEIVPLFVDSP

VIEDSDLKVHKFKVKTGDSIQKGLTPGWNDLDVNQHVSNVKYIGWILESMPTEVLET

QELCSLALEYRRECGRDSVLESVTAMDPSKVGVRSQYQHLLRLEDGTAIVNGATEW

RPKNAGANGAISTGKTSNGNSVS

SEQ ID NO: 33

Relevant codon optimized expression construct of Cuphea hookeriana FATB2 cDNA with Prototheca moriformis delta 12 fatty acid desaturase transit peptide.

GGTACCCGCCTGCAACGCAAGGGCAGCCACAGCCGCTCCCACCCGCCGCTGAAC CGACACGTGCTTGGGCGCCTGCCGCCTGCCTGCCGCATGCTTGTGCTGGTGAGGC TGGGCAGTGCTGCCATGCTGATTGAGGCTTGGTTCATCGGGTGGAAGCTTATGTG

TGTGCTGGGCTTGCATGCCGGGCAATGCGCATGGTGGCAAGAGGGCGGCAGCAC

TTGCTGGAGCTGCCGCGGTGCCTCCAGGTGGTTCAATCGCGGCAGCCAGAGGGA

TTTCAGATGATCGCGCGTACAGGTTGAGCAGCAGTGTCAGCAAAGGTAGCAGTTT

GCCAGAATGATCGGTTCAGCTGTTAATCAATGCCAGCAAGAGAAGGGGTCAAGT

GCAAACACGGGCATGCCACAGCACGGGCACCGGGGAGTGGAATGGCACCACCA

AGTGTGTGCGAGCCAGCATCGCCGCCTGGCTGTTTCAGCTACAACGGCAGGAGTC

ATCCAACGTAACCATGAGCTGATCAACACTGCAATCATCGGGCGGGCGTGATGC

AAGCATGCCTGGCGAAGACACATGGTGTGCGGATGCTGCCGGCTGCTGCCTGCT

GCGCACGCCGTTGAGTTGGCAGCAGGCTCAGCCATGCACTGGATGGCAGCTGGG

CTGCCACTGCAATGTGGTGGATAGGATGCAAGTGGAGCGAATACCAAACCCTCT

GGCTGCTTGCTGGGTTGCATGGCATCGCACCATCAGCAGGAGCGCATGCGAAGG

GACTGGCCCCATGCACGCCATGCCAAACCGGAGCGCACCGAGTGTCCACACTGT

CACCAGGCCCGCAAGCTTTGCAGAACCATGCTCATGGACGCATGTAGCGCTGAC

GTCCCTTGACGGCGCTCCTCTCGGGTGTGGGAAACGCAATGCAGCACAGGCAGC

AGAGGCGGCGGCAGCAGAGCGGCGGCAGCAGCGGCGGGGGCCACCCTTCTTGCG

GGGTCGCGCCCCAGCCAGCGGTGATGCGCTGATCCCAAACGAGTTCACATTCATT

TGCATGCCTGGAGAAGCGAGGCTGGGGCCTTTGGGCTGGTGCAGCCCGCAATGG

AATGCGGGACCGCCAGGCTAGCAGCAAAGGCGCCTCCCCTACTCCGCATCGATG

TTCCATAGTGCATTGGACTGCATTTGGGTGGGGCGGCCGGCTGTTTCTTTCGTGTT

GCAAAACGCGCCAGCTCAGCAACCTGTCCCGTGGGTCCCCCGTGCCGATGAAAT

CGTGTGCACGCCGATCAGCTGATTGCCCGGCTCGCGAAGTAGGCGCCCTCCTTTC

TGCTCGCCCTCTCTCCGTCCCGCCTCTAGAATATCAATGATCGAGCAGGACGGCC

TCCACGCCGGCTCCCCCGCCGCCTGGGTGGAGCGCCTGTTCGGCTACGACTGGGC

CCAGCAGACCATCGGCTGCTCCGACGCCGCCGTGTTCCGCCTGTCCGCCCAGGGC

CGCCCCGTGCTGTTCGTGAAGACCGACCTGTCCGGCGCCCTGAACGAGCTGCAGG

ACGAGGCCGCCCGCCTGTCCTGGCTGGCCACCACCGGCGTGCCCTGCGCCGCCGT

GCTGGACGTGGTGACCGAGGCCGGCCGCGACTGGCTGCTGCTGGGCGAGGTGCC

CGGCCAGGACCTGCTGTCCTCCCACCTGGCCCCCGCCGAGAAGGTGTCCATCATG

GCCGACGCCATGCGCCGCCTGCACACCCTGGACCCCGCCACCTGCCCCTTCGACC

ACCAGGCCAAGCACCGCATCGAGCGCGCCCGCACCCGCATGGAGGCCGGCCTGG

TGGACCAGGACGACCTGGACGAGGAGCACCAGGGCCTGGCCCCCGCCGAGCTGT

TCGCCCGCCTGAAGGCCCGCATGCCCGACGGCGAGGACCTGGTGGTGACCCACG

GCGACGCCTGCCTGCCCAACATCATGGTGGAGAACGGCCGCTTCTCCGGCTTCAT

CGACTGCGGCCGCCTGGGCGTGGCCGACCGCTACCAGGACATCGCCCTGGCCAC

CCGCGACATCGCCGAGGAGCTGGGCGGCGAGTGGGCCGACCGCTTCCTGGTGCT

GTACGGCATCGCCGCCCCCGACTCCCAGCGCATCGCCTTCTACCGCCTGCTGGAC

GAGTTCTTCTGACAATTGGCAGCAGCAGCTCGGATAGTATCGACACACTCTGGAC

GCTGGTCGTGTGATGGACTGTTGCCGCCACACTTGCTGCCTTGACCTGTGAATAT

CCCTGCCGCTTTTATCAAACAGCCTCAGTGTGTTTGATCTTGTGTGTACGCGCTTT

TGCGAGTTGCTAGCTGCTTGTGCTATTTGCGAATACCACCCCCAGCATCCCCTTCC

CTCGTTTCATATCGCTTGCATCCCAACCGCAACTTATCTACGCTGTCCTGCTATCC

CTCAGCGCTGCTCCTGCTCCTGCTCACTGCCCCTCGCACAGCCTTGGTTTGGGCTC

CGCCTGTATTCTCCTGGTACTGCAACCTGTAAACCAGCACTGCAATGCTGATGCA

CGGGAAGTAGTGGGATGGGAACACAAATGGAGGATCCCGCGTCTCGAACAGAGC

GCGCAGAGGAACGCTGAAGGTCTCGCCTCTGTCGCACCTCAGCGCGGCATACAC

CACAATAACCACCTGACGAATGCGCTTGGTTCTTCGTCCATTAGCGAAGCGTCCG

GTTCACACACGTGCCACGTTGGCGAGGTGGCAGGTGACAATGATCGGTGGAGCT

GATGGTCGAAACGTTCACAGCCTAGGGATATCGAATTCCTTTCTTGCGCTATGAC

ACTTCCAGCAAAAGGTAGGGCGGGCTGCGAGACGGCTTCCCGGCGCTGCATGCA ACACCGATGATGCTTCGACCCCCCGAAGCTCCTTCGGGGCTGCATGGGCGCTCCG

ATGCCGCTCCAGGGCGAGCGCTGTTTAAATAGCCAGGCCCCCGATTGCAAAGAC

ATTATAGCGAGCTACCAAAGCCATATTCAAACACCTAGATCACTACCACTTCTAC

ACAGGCCACTCGAGCTTGTGATCGCACTCCGCTAAGGGGGCGCCTCTTCCTCTTC

GTTTCAGTCACAACCCGCAAACACTAGTATGGCTATCAAGACGAACAGGCAGCC

TGTGGAGAAGCCTCCGTTCACGATCGGGACGCTGCGCAAGGCCATCCCCGCGCA

CTGTTTCGAGCGCTCGGCGCTTCGTGGGCGCGCCCAGCTGCCCGACTGGAGCCGC

CTGCTGACCGCCATCACCACCGTGTTCGTGAAGTCCAAGCGCCCCGACATGCACG

ACCGCAAGTCCAAGCGCCCCGACATGCTGGTGGACAGCTTCGGCCTGGAGTCCA

CCGTGCAGGACGGCCTGGTGTTCCGCCAGTCCTTCTCCATCCGCTCCTACGAGAT

CGGCACCGACCGCACCGCCAGCATCGAGACCCTGATGAACCACCTGCAGGAGAC

CTCCCTGAACCACTGCAAGAGCACCGGCATCCTGCTGGACGGCTTCGGCCGCACC

CTGGAGATGTGCAAGCGCGACCTGATCTGGGTGGTGATCAAGATGCAGATCAAG

GTGAACCGCTACCCCGCCTGGGGCGACACCGTGGAGATCAACACCCGCTTCAGC

CGCCTGGGCAAGATCGGCATGGGCCGCGACTGGCTGATCTCCGACTGCAACACC

GGCGAGATCCTGGTGCGCGCCACCAGCGCCTACGCCATGATGAACCAGAAGACC

CGCCGCCTGTCCAAGCTGCCCTACGAGGTGCACCAGGAGATCGTGCCCCTGTTCG

TGGACAGCCCCGTGATCGAGGACTCCGACCTGAAGGTGCACAAGTTCAAGGTGA

AGACCGGCGACAGCATCCAGAAGGGCCTGACCCCCGGCTGGAACGACCTGGACG

TGAACCAGCACGTGTCCAACGTGAAGTACATCGGCTGGATCCTGGAGAGCATGC

CCACCGAGGTGCTGGAGACCCAGGAGCTGTGCTCCCTGGCCCTGGAGTACCGCC

GCGAGTGCGGCCGCGACTCCGTGCTGGAGAGCGTGACCGCCATGGACCCCAGCA

AGGTGGGCGTGCGCTCCCAGTACCAGCACCTGCTGCGCCTGGAGGACGGCACCG

CCATCGTGAACGGCGCCACCGAGTGGCGCCCCAAGAACGCCGGCGCCAACGGCG

CCATCTCCACCGGCAAGACCAGCAACGGCAACTCCGTGTCCATGGACTACAAGG

ACCACGACGGCGACTACAAGGACCACGACATCGACTACAAGGACGACGACGAC

AAGTGACTCGAGGCAGCAGCAGCTCGGATAGTATCGACACACTCTGGACGCTGG

TCGTGTGATGGACTGTTGCCGCCACACTTGCTGCCTTGACCTGTGAATATCCCTGC

CGCTTTTATCAAACAGCCTCAGTGTGTTTGATCTTGTGTGTACGCGCTTTTGCGAG

TTGCTAGCTGCTTGTGCTATTTGCGAATACCACCCCCAGCATCCCCTTCCCTCGTT

TCATATCGCTTGCATCCCAACCGCAACTTATCTACGCTGTCCTGCTATCCCTCAGC

GCTGCTCCTGCTCCTGCTCACTGCCCCTCGCACAGCCTTGGTTTGGGCTCCGCCTG

TATTCTCCTGGTACTGCAACCTGTAAACCAGCACTGCAATGCTGATGCACGGGAA

GTAGTGGGATGGGAACACAAATGGAAAGCTT

SEQ ID NO: 34

Umbellularia calif ornica FATB 1 (Genbank Q41635) amino acid sequence with Prototheca moriformis delta 12 fatty acid desaturase transit peptide.

MAIKTNRQPVEKPPFTIGTLRKAIPAHCFERSALRGRAPDWSMLFAVITTIFSAAEK Q

WTNLEWKPKPKLPQLLDDHFGLHGLVFRRTFAIRSYEVGPDRSTSILAVMNHMQEA

TLNHAKSVGILGDGFGTTLEMSKRDLMWVVRRTHVAVERYPTWGDTVEVECWIGA

SGNNGMRRDFLVRDCKTGEILTRCTSLSVLMNTRTRRLSTIPDEVRGEIGPAFIDNV A

VKDDEIKKLQKLNDSTADYIQGGLTPRWNDLDVNQHVNNLKYVAWVFETVPDSIFE

SHHISSFTLEYRRECTRDSVLRSLTTVSGGSSEAGLVCDHLLQLEGGSEVLRARTEW R

PKLTDSFRGISVIPAEPRV

SEQ ID NO: 35

Relevant codon optimized expression construct of Umbellularia calif omica FATB1 cDNA with Prototheca moriformis delta 12 fatty acid desaturase transit peptide. GGTACCCGCCTGCAACGCAAGGGCAGCCACAGCCGCTCCCACCCGCCGCTGAAC

CGACACGTGCTTGGGCGCCTGCCGCCTGCCTGCCGCATGCTTGTGCTGGTGAGGC

TGGGCAGTGCTGCCATGCTGATTGAGGCTTGGTTCATCGGGTGGAAGCTTATGTG

TGTGCTGGGCTTGCATGCCGGGCAATGCGCATGGTGGCAAGAGGGCGGCAGCAC

TTGCTGGAGCTGCCGCGGTGCCTCCAGGTGGTTCAATCGCGGCAGCCAGAGGGA

TTTCAGATGATCGCGCGTACAGGTTGAGCAGCAGTGTCAGCAAAGGTAGCAGTTT

GCCAGAATGATCGGTTCAGCTGTTAATCAATGCCAGCAAGAGAAGGGGTCAAGT

GCAAACACGGGCATGCCACAGCACGGGCACCGGGGAGTGGAATGGCACCACCA

AGTGTGTGCGAGCCAGCATCGCCGCCTGGCTGTTTCAGCTACAACGGCAGGAGTC

ATCCAACGTAACCATGAGCTGATCAACACTGCAATCATCGGGCGGGCGTGATGC

AAGCATGCCTGGCGAAGACACATGGTGTGCGGATGCTGCCGGCTGCTGCCTGCT

GCGCACGCCGTTGAGTTGGCAGCAGGCTCAGCCATGCACTGGATGGCAGCTGGG

CTGCCACTGCAATGTGGTGGATAGGATGCAAGTGGAGCGAATACCAAACCCTCT

GGCTGCTTGCTGGGTTGCATGGCATCGCACCATCAGCAGGAGCGCATGCGAAGG

GACTGGCCCCATGCACGCCATGCCAAACCGGAGCGCACCGAGTGTCCACACTGT

CACCAGGCCCGCAAGCTTTGCAGAACCATGCTCATGGACGCATGTAGCGCTGAC

GTCCCTTGACGGCGCTCCTCTCGGGTGTGGGAAACGCAATGCAGCACAGGCAGC

AGAGGCGGCGGCAGCAGAGCGGCGGCAGCAGCGGCGGGGGCCACCCTTCTTGCG

GGGTCGCGCCCCAGCCAGCGGTGATGCGCTGATCCCAAACGAGTTCACATTCATT

TGCATGCCTGGAGAAGCGAGGCTGGGGCCTTTGGGCTGGTGCAGCCCGCAATGG

AATGCGGGACCGCCAGGCTAGCAGCAAAGGCGCCTCCCCTACTCCGCATCGATG

TTCCATAGTGCATTGGACTGCATTTGGGTGGGGCGGCCGGCTGTTTCTTTCGTGTT

GCAAAACGCGCCAGCTCAGCAACCTGTCCCGTGGGTCCCCCGTGCCGATGAAAT

CGTGTGCACGCCGATCAGCTGATTGCCCGGCTCGCGAAGTAGGCGCCCTCCTTTC

TGCTCGCCCTCTCTCCGTCCCGCCTCTAGAATATCAATGATCGAGCAGGACGGCC

TCCACGCCGGCTCCCCCGCCGCCTGGGTGGAGCGCCTGTTCGGCTACGACTGGGC

CCAGCAGACCATCGGCTGCTCCGACGCCGCCGTGTTCCGCCTGTCCGCCCAGGGC

CGCCCCGTGCTGTTCGTGAAGACCGACCTGTCCGGCGCCCTGAACGAGCTGCAGG

ACGAGGCCGCCCGCCTGTCCTGGCTGGCCACCACCGGCGTGCCCTGCGCCGCCGT

GCTGGACGTGGTGACCGAGGCCGGCCGCGACTGGCTGCTGCTGGGCGAGGTGCC

CGGCCAGGACCTGCTGTCCTCCCACCTGGCCCCCGCCGAGAAGGTGTCCATCATG

GCCGACGCCATGCGCCGCCTGCACACCCTGGACCCCGCCACCTGCCCCTTCGACC

ACCAGGCCAAGCACCGCATCGAGCGCGCCCGCACCCGCATGGAGGCCGGCCTGG

TGGACCAGGACGACCTGGACGAGGAGCACCAGGGCCTGGCCCCCGCCGAGCTGT

TCGCCCGCCTGAAGGCCCGCATGCCCGACGGCGAGGACCTGGTGGTGACCCACG

GCGACGCCTGCCTGCCCAACATCATGGTGGAGAACGGCCGCTTCTCCGGCTTCAT

CGACTGCGGCCGCCTGGGCGTGGCCGACCGCTACCAGGACATCGCCCTGGCCAC

CCGCGACATCGCCGAGGAGCTGGGCGGCGAGTGGGCCGACCGCTTCCTGGTGCT

GTACGGCATCGCCGCCCCCGACTCCCAGCGCATCGCCTTCTACCGCCTGCTGGAC

GAGTTCTTCTGACAATTGGCAGCAGCAGCTCGGATAGTATCGACACACTCTGGAC

GCTGGTCGTGTGATGGACTGTTGCCGCCACACTTGCTGCCTTGACCTGTGAATAT

CCCTGCCGCTTTTATCAAACAGCCTCAGTGTGTTTGATCTTGTGTGTACGCGCTTT

TGCGAGTTGCTAGCTGCTTGTGCTATTTGCGAATACCACCCCCAGCATCCCCTTCC

CTCGTTTCATATCGCTTGCATCCCAACCGCAACTTATCTACGCTGTCCTGCTATCC

CTCAGCGCTGCTCCTGCTCCTGCTCACTGCCCCTCGCACAGCCTTGGTTTGGGCTC

CGCCTGTATTCTCCTGGTACTGCAACCTGTAAACCAGCACTGCAATGCTGATGCA

CGGGAAGTAGTGGGATGGGAACACAAATGGAGGATCCCGCGTCTCGAACAGAGC

GCGCAGAGGAACGCTGAAGGTCTCGCCTCTGTCGCACCTCAGCGCGGCATACAC

CACAATAACCACCTGACGAATGCGCTTGGTTCTTCGTCCATTAGCGAAGCGTCCG GTTCACACACGTGCCACGTTGGCGAGGTGGCAGGTGACAATGATCGGTGGAGCT

GATGGTCGAAACGTTCACAGCCTAGGGATATCGAATTCCTTTCTTGCGCTATGAC

ACTTCCAGCAAAAGGTAGGGCGGGCTGCGAGACGGCTTCCCGGCGCTGCATGCA

ACACCGATGATGCTTCGACCCCCCGAAGCTCCTTCGGGGCTGCATGGGCGCTCCG

ATGCCGCTCCAGGGCGAGCGCTGTTTAAATAGCCAGGCCCCCGATTGCAAAGAC

ATTATAGCGAGCTACCAAAGCCATATTCAAACACCTAGATCACTACCACTTCTAC

ACAGGCCACTCGAGCTTGTGATCGCACTCCGCTAAGGGGGCGCCTCTTCCTCTTC

GTTTCAGTCACAACCCGCAAACACTAGTATGGCTATCAAGACGAACAGGCAGCC

TGTGGAGAAGCCTCCGTTCACGATCGGGACGCTGCGCAAGGCCATCCCCGCGCA

CTGTTTCGAGCGCTCGGCGCTTCGTGGGCGCGCCCCCGACTGGTCCATGCTGTTC

GCCGTGATCACCACCATCTTCAGCGCCGCCGAGAAGCAGTGGACCAACCTGGAG

TGGAAGCCCAAGCCCAAGCTGCCCCAGCTGCTGGACGACCACTTCGGCCTGCAC

GGCCTGGTGTTCCGCCGCACCTTCGCCATCCGCTCCTACGAGGTGGGCCCCGACC

GCAGCACCTCCATCCTGGCCGTGATGAACCACATGCAGGAGGCCACCCTGAACC

ACGCCAAGAGCGTGGGCATCCTGGGCGACGGCTTCGGCACCACCCTGGAGATGT

CCAAGCGCGACCTGATGTGGGTGGTGCGCCGCACCCACGTGGCCGTGGAGCGCT

ACCCCACCTGGGGCGACACCGTGGAGGTGGAGTGCTGGATCGGCGCCAGCGGCA

ACAACGGCATGCGCCGCGACTTCCTGGTGCGCGACTGCAAGACCGGCGAGATCC

TGACCCGCTGCACCTCCCTGAGCGTGCTGATGAACACCCGCACCCGCCGCCTGAG

CACCATCCCCGACGAGGTGCGCGGCGAGATCGGCCCCGCCTTCATCGACAACGT

GGCCGTGAAGGACGACGAGATCAAGAAGCTGCAGAAGCTGAACGACTCCACCGC

CGACTACATCCAGGGCGGCCTGACCCCCCGCTGGAACGACCTGGACGTGAACCA

GCACGTGAACAACCTGAAGTACGTGGCCTGGGTGTTCGAGACCGTGCCCGACAG

CATCTTCGAGTCCCACCACATCAGCTCCTTCACCCTGGAGTACCGCCGCGAGTGC

ACCCGCGACTCCGTGCTGCGCAGCCTGACCACCGTGAGCGGCGGCAGCTCCGAG

GCCGGCCTGGTGTGCGACCACCTGCTGCAGCTGGAGGGCGGCAGCGAGGTGCTG

CGCGCCCGCACCGAGTGGCGCCCCAAGCTGACCGACTCCTTCCGCGGCATCAGC

GTGATCCCCGCCGAGCCCCGCGTGATGGACTACAAGGACCACGACGGCGACTAC

AAGGACCACGACATCGACTACAAGGACGACGACGACAAGTGATGACTCGAGGC

AGCAGCAGCTCGGATAGTATCGACACACTCTGGACGCTGGTCGTGTGATGGACT

GTTGCCGCCACACTTGCTGCCTTGACCTGTGAATATCCCTGCCGCTTTTATCAAAC

AGCCTCAGTGTGTTTGATCTTGTGTGTACGCGCTTTTGCGAGTTGCTAGCTGCTTG

TGCTATTTGCGAATACCACCCCCAGCATCCCCTTCCCTCGTTTCATATCGCTTGCA

TCCCAACCGCAACTTATCTACGCTGTCCTGCTATCCCTCAGCGCTGCTCCTGCTCC

TGCTCACTGCCCCTCGCACAGCCTTGGTTTGGGCTCCGCCTGTATTCTCCTGGTAC

TGCAACCTGTAAACCAGCACTGCAATGCTGATGCACGGGAAGTAGTGGGATGGG

AACACAAATGGAAAGCTT

SEQ ID NO: 36

Cuphea palustris C8 preferring thioesterase (Genbank AAC49179) amino acid sequence with Prototheca moriformis delta 12 fatty acid desaturase transit peptide.

MAIKTNRQPVEKPPFTIGTLRKAIPAHCFERSALRGRAPANGSAVTLKSGSLNTQED T

LSSSPPPRAFFNQLPDWSMLLTAITTVFVAPEKRWTMFDRKSKRPNMLMDSFGLERV

VQDGLVFRQSFSIRSYEICADRTASIETVMNHVQETSLNQCKSIGLLDDGFGRSPEM C

KRDLIWVVTRMKIMVNRYPTWGDTIEVSTWLSQSGKIGMGRDWLISDCNTGEILVR

ATSVYAMMNQKTRRFSKLPHEVRQEFAPHFLDSPPAIEDNDGKLQKFDVKTGDSIRK

GLTPGWYDLDVNQHVSNVKYIGWILESMPTEVLETQELCSLTLEYRRECGRDSVLES

VTSMDPSKVGDRFQYRHLLRLEDGADIMKGRTEWRPKNAGTNGAISTGKT SEQ ID NO: 37

Relevant codon optimized expression construct of Cuphea palustris C8 preferring thioesterase cDNA with Prototheca moriformis delta 12 fatty acid desaturase transit peptide.

GGTACCCGCCTGCAACGCAAGGGCAGCCACAGCCGCTCCCACCCGCCGCTGAAC

CGACACGTGCTTGGGCGCCTGCCGCCTGCCTGCCGCATGCTTGTGCTGGTGAGGC

TGGGCAGTGCTGCCATGCTGATTGAGGCTTGGTTCATCGGGTGGAAGCTTATGTG

TGTGCTGGGCTTGCATGCCGGGCAATGCGCATGGTGGCAAGAGGGCGGCAGCAC

TTGCTGGAGCTGCCGCGGTGCCTCCAGGTGGTTCAATCGCGGCAGCCAGAGGGA

TTTCAGATGATCGCGCGTACAGGTTGAGCAGCAGTGTCAGCAAAGGTAGCAGTTT

GCCAGAATGATCGGTTCAGCTGTTAATCAATGCCAGCAAGAGAAGGGGTCAAGT

GCAAACACGGGCATGCCACAGCACGGGCACCGGGGAGTGGAATGGCACCACCA

AGTGTGTGCGAGCCAGCATCGCCGCCTGGCTGTTTCAGCTACAACGGCAGGAGTC

ATCCAACGTAACCATGAGCTGATCAACACTGCAATCATCGGGCGGGCGTGATGC

AAGCATGCCTGGCGAAGACACATGGTGTGCGGATGCTGCCGGCTGCTGCCTGCT

GCGCACGCCGTTGAGTTGGCAGCAGGCTCAGCCATGCACTGGATGGCAGCTGGG

CTGCCACTGCAATGTGGTGGATAGGATGCAAGTGGAGCGAATACCAAACCCTCT

GGCTGCTTGCTGGGTTGCATGGCATCGCACCATCAGCAGGAGCGCATGCGAAGG

GACTGGCCCCATGCACGCCATGCCAAACCGGAGCGCACCGAGTGTCCACACTGT

CACCAGGCCCGCAAGCTTTGCAGAACCATGCTCATGGACGCATGTAGCGCTGAC

GTCCCTTGACGGCGCTCCTCTCGGGTGTGGGAAACGCAATGCAGCACAGGCAGC

AGAGGCGGCGGCAGCAGAGCGGCGGCAGCAGCGGCGGGGGCCACCCTTCTTGCG

GGGTCGCGCCCCAGCCAGCGGTGATGCGCTGATCCCAAACGAGTTCACATTCATT

TGCATGCCTGGAGAAGCGAGGCTGGGGCCTTTGGGCTGGTGCAGCCCGCAATGG

AATGCGGGACCGCCAGGCTAGCAGCAAAGGCGCCTCCCCTACTCCGCATCGATG

TTCCATAGTGCATTGGACTGCATTTGGGTGGGGCGGCCGGCTGTTTCTTTCGTGTT

GCAAAACGCGCCAGCTCAGCAACCTGTCCCGTGGGTCCCCCGTGCCGATGAAAT

CGTGTGCACGCCGATCAGCTGATTGCCCGGCTCGCGAAGTAGGCGCCCTCCTTTC

TGCTCGCCCTCTCTCCGTCCCGCCTCTAGAATATCAATGATCGAGCAGGACGGCC

TCCACGCCGGCTCCCCCGCCGCCTGGGTGGAGCGCCTGTTCGGCTACGACTGGGC

CCAGCAGACCATCGGCTGCTCCGACGCCGCCGTGTTCCGCCTGTCCGCCCAGGGC

CGCCCCGTGCTGTTCGTGAAGACCGACCTGTCCGGCGCCCTGAACGAGCTGCAGG

ACGAGGCCGCCCGCCTGTCCTGGCTGGCCACCACCGGCGTGCCCTGCGCCGCCGT

GCTGGACGTGGTGACCGAGGCCGGCCGCGACTGGCTGCTGCTGGGCGAGGTGCC

CGGCCAGGACCTGCTGTCCTCCCACCTGGCCCCCGCCGAGAAGGTGTCCATCATG

GCCGACGCCATGCGCCGCCTGCACACCCTGGACCCCGCCACCTGCCCCTTCGACC

ACCAGGCCAAGCACCGCATCGAGCGCGCCCGCACCCGCATGGAGGCCGGCCTGG

TGGACCAGGACGACCTGGACGAGGAGCACCAGGGCCTGGCCCCCGCCGAGCTGT

TCGCCCGCCTGAAGGCCCGCATGCCCGACGGCGAGGACCTGGTGGTGACCCACG

GCGACGCCTGCCTGCCCAACATCATGGTGGAGAACGGCCGCTTCTCCGGCTTCAT

CGACTGCGGCCGCCTGGGCGTGGCCGACCGCTACCAGGACATCGCCCTGGCCAC

CCGCGACATCGCCGAGGAGCTGGGCGGCGAGTGGGCCGACCGCTTCCTGGTGCT

GTACGGCATCGCCGCCCCCGACTCCCAGCGCATCGCCTTCTACCGCCTGCTGGAC

GAGTTCTTCTGACAATTGGCAGCAGCAGCTCGGATAGTATCGACACACTCTGGAC

GCTGGTCGTGTGATGGACTGTTGCCGCCACACTTGCTGCCTTGACCTGTGAATAT

CCCTGCCGCTTTTATCAAACAGCCTCAGTGTGTTTGATCTTGTGTGTACGCGCTTT

TGCGAGTTGCTAGCTGCTTGTGCTATTTGCGAATACCACCCCCAGCATCCCCTTCC

CTCGTTTCATATCGCTTGCATCCCAACCGCAACTTATCTACGCTGTCCTGCTATCC

CTCAGCGCTGCTCCTGCTCCTGCTCACTGCCCCTCGCACAGCCTTGGTTTGGGCTC

CGCCTGTATTCTCCTGGTACTGCAACCTGTAAACCAGCACTGCAATGCTGATGCA CGGGAAGTAGTGGGATGGGAACACAAATGGAGGATCCCGCGTCTCGAACAGAGC

GCGCAGAGGAACGCTGAAGGTCTCGCCTCTGTCGCACCTCAGCGCGGCATACAC

CACAATAACCACCTGACGAATGCGCTTGGTTCTTCGTCCATTAGCGAAGCGTCCG

GTTCACACACGTGCCACGTTGGCGAGGTGGCAGGTGACAATGATCGGTGGAGCT

GATGGTCGAAACGTTCACAGCCTAGGGATATCGAATTCCTTTCTTGCGCTATGAC

ACTTCCAGCAAAAGGTAGGGCGGGCTGCGAGACGGCTTCCCGGCGCTGCATGCA

ACACCGATGATGCTTCGACCCCCCGAAGCTCCTTCGGGGCTGCATGGGCGCTCCG

ATGCCGCTCCAGGGCGAGCGCTGTTTAAATAGCCAGGCCCCCGATTGCAAAGAC

ATTATAGCGAGCTACCAAAGCCATATTCAAACACCTAGATCACTACCACTTCTAC

ACAGGCCACTCGAGCTTGTGATCGCACTCCGCTAAGGGGGCGCCTCTTCCTCTTC

GTTTCAGTCACAACCCGCAAACACTAGTATGGCTATCAAGACGAACAGGCAGCC

TGTGGAGAAGCCTCCGTTCACGATCGGGACGCTGCGCAAGGCCATCCCCGCGCA

CTGTTTCGAGCGCTCGGCGCTTCGTGGGCGCGCCCCCGCGAACGGCAGCGCGGTG

ACCCTGAAGTCGGGCTCCCTGAACACCCAGGAGGACACGCTGAGCTCGTCCCCC

CCCCCCCGCGCGTTCTTCAACCAGCTGCCCGACTGGAGCATGCTGCTGACCGCGA

TCACCACGGTCTTCGTGGCGCCCGAGAAGCGCTGGACCATGTTCGACCGCAAGTC

GAAGCGCCCCAACATGCTGATGGACTCCTTCGGCCTGGAGCGCGTGGTCCAGGA

CGGCCTGGTGTTCCGCCAGAGCTTCTCGATCCGCTCCTACGAGATCTGCGCGGAC

CGCACCGCGAGCATCGAGACGGTGATGAACCACGTCCAGGAGACCTCGCTGAAC

CAGTGCAAGTCCATCGGCCTGCTGGACGACGGCTTCGGCCGCAGCCCCGAGATG

TGCAAGCGCGACCTGATCTGGGTGGTCACCCGCATGAAGATCATGGTGAACCGC

TACCCCACGTGGGGCGACACCATCGAGGTCTCGACGTGGCTGTCCCAGAGCGGC

AAGATCGGCATGGGCCGCGACTGGCTGATCTCGGACTGCAACACCGGCGAGATC

CTGGTGCGCGCGACGTCCGTCTACGCGATGATGAACCAGAAGACCCGCCGCTTC

AGCAAGCTGCCCCACGAGGTGCGCCAGGAGTTCGCGCCCCACTTCCTGGACTCGC

CCCCCGCGATCGAGGACAACGACGGCAAGCTGCAGAAGTTCGACGTCAAGACGG

GCGACTCCATCCGCAAGGGCCTGACCCCCGGCTGGTACGACCTGGACGTGAACC

AGCACGTGAGCAACGTCAAGTACATCGGCTGGATCCTGGAGTCGATGCCCACCG

AGGTCCTGGAGACGCAGGAGCTGTGCTCCCTGACCCTGGAGTACCGCCGCGAGT

GCGGCCGCGACTCGGTGCTGGAGAGCGTCACCAGCATGGACCCCTCGAAGGTGG

GCGACCGCTTCCAGTACCGCCACCTGCTGCGCCTGGAGGACGGCGCGGACATCA

TGAAGGGCCGCACCGAGTGGCGCCCCAAGAACGCGGGCACGAACGGCGCGATCT

CCACCGGCAAGACGTGACTCGAGGCAGCAGCAGCTCGGATAGTATCGACACACT

CTGGACGCTGGTCGTGTGATGGACTGTTGCCGCCACACTTGCTGCCTTGACCTGT

GAATATCCCTGCCGCTTTTATCAAACAGCCTCAGTGTGTTTGATCTTGTGTGTACG

CGCTTTTGCGAGTTGCTAGCTGCTTGTGCTATTTGCGAATACCACCCCCAGCATCC

CCTTCCCTCGTTTCATATCGCTTGCATCCCAACCGCAACTTATCTACGCTGTCCTG

CTATCCCTCAGCGCTGCTCCTGCTCCTGCTCACTGCCCCTCGCACAGCCTTGGTTT

GGGCTCCGCCTGTATTCTCCTGGTACTGCAACCTGTAAACCAGCACTGCAATGCT

GATGCACGGGAAGTAGTGGGATGGGAACACAAATGGAAAGCTTGAGCTC

SEQ ID NO: 38

Cuphea lanceolata CIO preferring thioesterase (Genbank CAB60830) amino acid sequence with Prototheca moriformis delta 12 fatty acid desaturase transit peptide.

MAIKTNRQPVEKPPFTIGTLRKAIPAHCFERSALRGRAPANGSAVNLKSGSLNTQED T SSSPPPRAFLNQLPDWSMLLTAITTVFVAAEKQWTMLDRKSKRPDMLVDSVGLKSIV RDGLVSRQSFLIRSYEIGADRTASIETLMNHLQETSINHCKSLGLLNDGFGRTPGMCK NDLIWVLTKMQIMVNRYPTWGDTVEINTWFSQSGKIGMASDWLISDCNTGEILIRAT SVWAMMNQKTRRFSRLPYEVRQELTPHFVDSPHVIEDNDQKLH FDVKTGDSIRKG LTPRWNDLDVNQHVSNV YIGWILESMPIEVLETQELCSLTVEYRRECGMDSVLESV TAVDPSENGGRSQYKHLLRLEDGTDIVKSRTEWRPKNAGTNGAISTSTAKTSNGNSA SDDDDKLG

SEQ ID NO: 39

Relevant codon optimized coding region of Cuphea lanceolata CIO preferring thioesterase with Prototheca moriformis delta 12 fatty acid desaturase transit peptide.

ACTAGTATGGCTATCAAGACGAACAGGCAGCCTGTGGAGAAGCCTCCGTTCACG

ATCGGGACGCTGCGCAAGGCCATCCCCGCGCACTGTTTCGAGCGCTCGGCGCTTC

GTGGGCGCGCCCCCGCGAACGGCAGCGCGGTGAACCTGAAGTCGGGCTCCCTGA

ACACCCAGGAGGACACGAGCTCGTCCCCCCCCCCCCGCGCGTTCCTGAACCAGCT

GCCCGACTGGAGCATGCTGCTGACCGCGATCACCACCGTCTTCGTGGCGGCGGA

GAAGCAGTGGACGATGCTGGACCGCAAGTCGAAGCGCCCCGACATGCTGGTGGA

CTCCGTCGGCCTGAAGAGCATCGTGCGCGACGGCCTGGTCTCGCGCCAGTCCTTC

CTGATCCGCAGCTACGAGATCGGCGCGGACCGCACCGCGTCGATCGAGACCCTG

ATGAACCACCTGCAGGAGACGTCCATCAACCACTGCAAGAGCCTGGGCCTGCTG

AACGACGGCTTCGGCCGCACCCCCGGCATGTGCAAGAACGACCTGATCTGGGTG

CTGACCAAGATGCAGATCATGGTCAACCGCTACCCCACGTGGGGCGACACCGTC

GAGATCAACACGTGGTTCTCGCAGTCCGGCAAGATCGGCATGGCGAGCGACTGG

CTGATCTCGGACTGCAACACCGGCGAGATCCTGATCCGCGCGACCTCCGTGTGGG

CGATGATGAACCAGAAGACGCGCCGCTTCAGCCGCCTGCCCTACGAGGTCCGCC

AGGAGCTGACCCCCCACTTCGTGGACTCGCCCCACGTCATCGAGGACAACGACC

AGAAGCTGCACAAGTTCGACGTGAAGACCGGCGACTCCATCCGCAAGGGCCTGA

CGCCCCGCTGGAACGACCTGGACGTCAACCAGCACGTGTCGAACGTGAAGTACA

TCGGCTGGATCCTGGAGTCCATGCCCATCGAGGTCCTGGAGACCCAGGAGCTGTG

CTCGCTGACCGTGGAGTACCGCCGCGAGTGCGGCATGGACTCCGTGCTGGAGTC

GGTCACGGCGGTGGACCCCAGCGAGAACGGCGGCCGCAGCCAGTACAAGCACCT

GCTGCGCCTGGAGGACGGCACCGACATCGTCAAGTCGCGCACCGAGTGGCGCCC

CAAGAACGCGGGCACGAACGGCGCGATCTCCACCAGCACCGCGAAGACGTCGAA

CGGCAACTCCGCGAGCGATGACGATGACAAGCTGGGATGACTCGAG

SEQ ID NO: 40

Iris germanica C14 preferring thioesterase (Genbank AAG43858.1) amino acid sequence with Chlorella protothecoid.es stearoyl ACP desaturase chloroplast transit peptide.

MATASTFSAFNARCGDLRRSAGSGPRRPARPLPVRGRAAQAATRVNGSKVGLKTDT

NKLEDAPFIPSSAPRTFYNQLPDWSVLLAAITTIFLAAEKQWTLIDWKRGGPDMLSD A

FGLP IIENGLLYRQKFSIRSYEIGADQTASIETLMNHLQETALNHVKCAGLLGNGFG

STPEMSKMNLIWVVTKMQVLVEHYPSWGDVIEVDTWAAASGKNGMRRDWHVRD

WQTGQTIMRASSNWVMMNQNTRRLSKFPEEVRAEIEPYFMERAPVIDDDNRKLPKL

DDDTADHVRNGLTPRWSDLDVNQHVKNVKYIGWILESAPISILESHELASMTLEYRR

ECGRDSVLQSLTSVSNNCTDGSEELPIECQHLLRNEGGSEIVKGRTEWRPKKCGPFG A

GRP

SEQ ID NO: 41

Relevant codon optimized coding region of Iris germanica C14 preferring thioesterase with Chlorella protothecoides stearoyl ACP desaturase transit peptide. ACTAGTATGGCCACCGCATCCACTTTCTCGGCGTTCAATGCCCGCTGCGGCGACC

TGCGTCGCTCGGCGGGCTCCGGGCCCCGGCGCCCAGCGAGGCCCCTCCCCGTGCG

CGGGCGCGCCGCCCAGGCGGCCACCCGCGTGAACGGCAGCAAGGTGGGCCTGAA

GACCGACACCAACAAGCTGGAGGACGCGCCCTTCATCCCCTCGTCCGCCCCCCGC

ACCTTCTACAACCAGCTGCCCGACTGGAGCGTCCTGCTGGCGGCCATCACCACCA

TCTTCCTGGCGGCCGAGAAGCAGTGGACCCTGATCGACTGGAAGCGCGGCGGCC

CCGACATGCTGTCGGACGCGTTCGGCCTGCCCAAGATCATCGAGAACGGCCTGCT

GTACCGCCAGAAGTTCTCCATCCGCAGCTACGAGATCGGCGCCGACCAGACCGC

CTCGATCGAGACCCTGATGAACCACCTGCAGGAGACCGCGCTGAACCACGTCAA

GTGCGCCGGCCTGCTGGGCAACGGCTTCGGCTCCACCCCCGAGATGAGCAAGAT

GAACCTGATCTGGGTGGTCACCAAGATGCAGGTGCTGGTCGAGCACTACCCCTCG

TGGGGCGACGTGATCGAGGTGGACACCTGGGCGGCCGCGTCCGGCAAGAACGGC

ATGCGCCGCGACTGGCACGTCCGCGACTGGCAGACCGGCCAGACCATCATGCGC

GCCAGCTCGAACTGGGTGATGATGAACCAGAACACCCGCCGCCTGTCCAAGTTC

CCCGAGGAGGTCCGCGCCGAGATCGAGCCCTACTTCATGGAGCGCGCCCCCGTG

ATCGACGACGACAACCGCAAGCTGCCCAAGCTGGACGACGACACCGCGGACCAC

GTGCGCAACGGCCTGACCCCCCGCTGGAGCGACCTGGACGTGAACCAGCACGTC

AAGAACGTGAAGTACATCGGCTGGATCCTGGAGTCGGCCCCCATCTCCATCCTGG

AGAGCCACGAGCTGGCCTCGATGACCCTGGAGTACCGCCGCGAGTGCGGCCGCG

ACTCCGTCCTGCAGAGCCTGACCTCGGTGTCCAACAACTGCACCGACGGCAGCG

AGGAGCTGCCCATCGAGTGCCAGCACCTGCTGCGCAACGAGGGCGGCTCGGAGA

TCGTCAAGGGCCGCACCGAGTGGCGCCCCAAGAAGTGCGGCCCCTTCGGCGCCG

GCCGCCCCTGACTCGAG

SEQ ID NO: 42

Myristicafragrans fatty acyl thioesterase (Genbank AAB717291.1) amino acid sequence with Prototheca moriformis delta 12 fatty acid desaturase chloroplast transit peptide.

MAIKTNRQPVEKPPFTIGTLRKAIPAHCFERSALRGRAANAHTVPKINGNKAGLLTP M

ESTKDEDIVAAPTVAPKRTFINQLPDWSMLLAAITTIFLAAEKQWTNLDW PRRPDM

LVDFDPFSLGRFVQDGLIFRQNFSIRSYEIGADRTASIETLMNHLQETALNHVRCIG LL

DDGFGSTPEMTRRDLIWVVTRMQVLVDRYPSWGDVIEVDSWVTPSGKNGMKREWF

LRDCKTGEILTRATSVWVMMNKRTRRLSKIPEEVRVEIEPYFVEHGVLDEDSRKLPK

LNDNTANYIRRGLAPRWSDLDVNQHVNNVKYIGWILESVPSSLLESHELYGMTLEY

RKECGKDGLLQSLTAVASDYGGGSLEAGVECDHLLRLEDGSEIMRGKTEWRPKRAA

NTTYFGSVDDIPPANNA

SEQ ID NO: 43

Relevant codon optimized coding region of Mysistica fragrans fatty acyl thioesterase with Prototheca moriformis delta 12 fatty acid desaturase chloroplast transit peptide.

ACTAGTATGGCTATCAAGACGAACAGGCAGCCTGTGGAGAAGCCTCCGTTCACG

ATCGGGACGCTGCGCAAGGCCATCCCCGCGCACTGTTTCGAGCGCTCGGCGCTTC

GTGGGCGCGCCGCCAACGCCCACACCGTGCCCAAGATCAACGGCAACAAGGCCG

GCCTGCTGACCCCCATGGAGAGCACCAAGGACGAGGACATCGTCGCGGCCCCCA

CCGTGGCGCCCAAGCGCACCTTCATCAACCAGCTGCCCGACTGGTCGATGCTGCT

GGCCGCGATCACCACCATCTTCCTGGCGGCCGAGAAGCAGTGGACCAACCTGGA

CTGGAAGCCCCGCCGCCCCGACATGCTGGTCGACTTCGACCCCTTCTCCCTGGGC

CGCTTCGTGCAGGACGGCCTGATCTTCCGCCAGAACTTCAGCATCCGCTCGTACG

AGATCGGCGCGGACCGCACCGCCTCCATCGAGACCCTGATGAACCACCTGCAGG AGACCGCGCTGAACCACGTCCGCTGCATCGGCCTGCTGGACGACGGCTTCGGCA

GCACCCCCGAGATGACCCGCCGCGACCTGATCTGGGTGGTCACCCGCATGCAGG

TCCTGGTGGACCGCTACCCCTCGTGGGGCGACGTGATCGAGGTCGACTCCTGGGT

GACCCCCAGCGGCAAGAACGGCATGAAGCGCGAGTGGTTCCTGCGCGACTGCAA

GACCGGCGAGATCCTGACCCGCGCCACCTCGGTCTGGGTGATGATGAACAAGCG

CACCCGCCGCCTGTCCAAGATCCCCGAGGAGGTCCGCGTGGAGATCGAGCCCTA

CTTCGTCGAGCACGGCGTGCTGGACGAGGACTCGCGCAAGCTGCCCAAGCTGAA

CGACAACACCGCCAACTACATCCGCCGCGGCCTGGCGCCCCGCTGGTCCGACCTG

GACGTCAACCAGCACGTGAACAACGTCAAGTACATCGGCTGGATCCTGGAGAGC

GTGCCCAGCAGCCTGCTGGAGTCGCACGAGCTGTACGGCATGACCCTGGAGTAC

CGCAAGGAGTGCGGCAAGGACGGCCTGCTGCAGTCCCTGACCGCCGTCGCCAGC

GACTACGGCGGCGGCTCGCTGGAGGCCGGCGTGGAGTGCGACCACCTGCTGCGC

CTGGAGGACGGCTCCGAGATCATGCGCGGCAAGACCGAGTGGCGCCCCAAGCGC

GCCGCGAACACCACCTACTTCGGCAGCGTCGACGACATCCCCCCCGCCAACAAC

GCGTGACTCGAG

SEQ ID NO: 44

Cuphea palustris C14 preferring thioesterase (Genbank AAC49180) amino acid sequence with Chlorella protothecoid.es stearoyl ACP desaturase transit peptide.

MATASTFSAFNARCGDLRRSAGSGPRRPARPLPVRGRASMLLSAVTTVFGVAEKQW

PMLDRKSKRPDMLVEPLGVDRIVYDGVSFRQSFSIRSYEIGADRTASIETLMNMFQE T

SLNHCKIIGLLNDGFGRTPEMCKRDLIWVVTKMQIEVNRYPTWGDTIEVNTWVSASG

KHGMGRDWLISDCHTGEILIRATSVWAMMNQKTRRLSKIPYEVRQEIEPQFVDSAPV

IVDDRKFHKLDLKTGDSICNGLTPRWTDLDVNQHVNNVKYIGWILQSVPTEVFETQE

LCGLTLEYRRECGRDSVLESVTAMDPSKEGDRSLYQHLLRLEDGADIVKGRTEWRP

KNAGAKGAILTGKTSNGNSIS

SEQ ID NO: 45

Relevant codon optimized coding region of Cuphea palustris C14 preferring thioesterase with Chlorella protothecoides stearoyl ACP desaturase transit peptide.

ACTAGTATGGCCACCGCATCCACTTTCTCGGCGTTCAATGCCCGCTGCGGCGACC

TGCGTCGCTCGGCGGGCTCCGGGCCCCGGCGCCCAGCGAGGCCCCTCCCCGTGCG

CGGGCGCGCCAGCATGCTGCTGTCGGCGGTGACCACGGTCTTCGGCGTGGCCGA

GAAGCAGTGGCCCATGCTGGACCGCAAGTCCAAGCGCCCCGACATGCTGGTCGA

GCCCCTGGGCGTGGACCGCATCGTCTACGACGGCGTGAGCTTCCGCCAGTCGTTC

TCCATCCGCAGCTACGAGATCGGCGCCGACCGCACCGCCTCGATCGAGACGCTG

ATGAACATGTTCCAGGAGACCTCCCTGAACCACTGCAAGATCATCGGCCTGCTGA

ACGACGGCTTCGGCCGCACGCCCGAGATGTGCAAGCGCGACCTGATCTGGGTCG

TGACCAAGATGCAGATCGAGGTGAACCGCTACCCCACGTGGGGCGACACCATCG

AGGTCAACACGTGGGTGAGCGCCTCGGGCAAGCACGGCATGGGCCGCGACTGGC

TGATCTCCGACTGCCACACCGGCGAGATCCTGATCCGCGCGACGAGCGTCTGGGC

GATGATGAACCAGAAGACCCGCCGCCTGTCGAAGATCCCCTACGAGGTGCGCCA

GGAGATCGAGCCCCAGTTCGTCGACTCCGCCCCCGTGATCGTGGACGACCGCAA

GTTCCACAAGCTGGACCTGAAGACGGGCGACAGCATCTGCAACGGCCTGACCCC

CCGCTGGACGGACCTGGACGTGAACCAGCACGTCAACAACGTGAAGTACATCGG

CTGGATCCTGCAGTCGGTCCCCACCGAGGTGTTCGAGACGCAGGAGCTGTGCGG

CCTGACCCTGGAGTACCGCCGCGAGTGCGGCCGCGACTCCGTGCTGGAGAGCGT

CACGGCCATGGACCCCTCGAAGGAGGGCGACCGCTCCCTGTACCAGCACCTGCT GCGCCTGGAGGACGGCGCGGACATCGTGAAGGGCCGCACCGAGTGGCGCCCCAA GAACGCCGGCGCCAAGGGCGCCATCCTGACGGGCAAGACCAGCAACGGCAACTC GATCTCCTGACTCGAG

SEQ ID NO: 46

Ulmus americana broad specificity thioesterase (Genbank AAB71731) amino acid sequence with Chlorella protothecoides stearoyl ACP desaturase transit peptide.

MATASTFSAFNARCGDLRRSAGSGPRRPARPLPVRGRAQLPDWSMLLAAITTLFLAA

EKQWMMLDWKPKRPDMLVDPFGLGRFVQDGLVFRNNFSIRSYEIGADRTASIETLM

NHLQETALNHVKSVGLLEDGLGSTREMSLRNLIWVVTKMQVAVDRYPTWGDEVQV

SSWATAIGKNGMRREWIVTDFRTGETLLRATSVWVMMNKLTRRISKIPEEVWHEIGP

SFIDAPPLPTVEDDGRKLTRFDESSADFIRKGLTPRWSDLDINQHVNNVKYIGWLLE S

APPEIHESHEIASLTLEYRRECGRDSVLNSATKVSDSSQLGKSAVECNHLVRLQNGG E

IVKGRTVWRPKRPLYNDGAVVDVPAKTS

SEQ ID NO: 47

Relevant codon optimized coding region of Ulmus americana broad specificity thioesterase with Chlorella protothecoides stearoyl ACP desaturase transit peptide.

ACTAGTATGGCCACCGCATCCACTTTCTCGGCGTTCAATGCCCGCTGCGGCGACC

TGCGTCGCTCGGCGGGCTCCGGGCCCCGGCGCCCAGCGAGGCCCCTCCCCGTGCG

CGGGCGCGCCCAGCTGCCCGACTGGAGCATGCTGCTGGCCGCGATCACCACCCT

GTTCCTGGCGGCCGAGAAGCAGTGGATGATGCTGGACTGGAAGCCCAAGCGCCC

CGACATGCTGGTGGACCCCTTCGGCCTGGGCCGCTTCGTGCAGGACGGCCTGGTG

TTCCGCAACAACTTCAGCATCCGCAGCTACGAGATCGGCGCGGACCGCACCGCC

AGCATCGAGACCCTGATGAACCACCTGCAGGAGACCGCCCTGAACCACGTGAAG

AGCGTGGGCCTGCTGGAGGACGGCCTGGGCAGCACCCGCGAGATGAGCCTGCGC

AACCTGATCTGGGTGGTGACCAAGATGCAGGTGGCGGTGGACCGCTACCCCACC

TGGGGCGACGAGGTGCAGGTGAGCAGCTGGGCGACCGCCATCGGCAAGAACGG

CATGCGCCGCGAGTGGATCGTGACCGACTTCCGCACCGGCGAGACCCTGCTGCG

CGCCACCAGCGTGTGGGTGATGATGAACAAGCTGACCCGCCGCATCAGCAAGAT

CCCCGAGGAGGTGTGGCACGAGATCGGCCCCAGCTTCATCGACGCGCCCCCCCT

GCCCACCGTGGAGGACGACGGCCGCAAGCTGACCCGCTTCGACGAGAGCAGCGC

CGACTTCATCCGCAAGGGCCTGACCCCCCGCTGGAGCGACCTGGACATCAACCA

GCACGTGAACAACGTGAAGTACATCGGCTGGCTGCTGGAGAGCGCGCCCCCCGA

GATCCACGAGAGCCACGAGATCGCCAGCCTGACCCTGGAGTACCGCCGCGAGTG

CGGCCGCGACAGCGTGCTGAACAGCGCCACCAAGGTGAGCGACAGCAGCCAGCT

GGGCAAGAGCGCCGTGGAGTGCAACCACCTGGTGCGCCTGCAGAACGGCGGCGA

GATCGTGAAGGGCCGCACCGTGTGGCGCCCCAAGCGCCCCCTGTACAACGACGG

CGCCGTGGTGGACGTGCCCGCCAAGACCAGCTGACTCGAG

SEQ ID NO: 48

Codon optimized Prototheca moriformis (UTEX 1435) delta 12 fatty acid desaturase transit peptide cDNA sequence.

ACTAGTATGGCTATCAAGACGAACAGGCAGCCTGTGGAGAAGCCTCCGTTCACG

ATCGGGACGCTGCGCAAGGCCATCCCCGCGCACTGTTTCGAGCGCTCGGCGCTTC

GTGGGCGCGCC SEQ ID NO: 49

Codon optimized Chlorella protothecoides (UTEX 250) stearoyl ACP desaturase transit peptide cDNA sequence.

ACTAGTATGGCCACCGCATCCACTTTCTCGGCGTTCAATGCCCGCTGCGGCGACC

TGCGTCGCTCGGCGGGCTCCGGGCCCCGGCGCCCAGCGAGGCCCCTCCCCGTGCG

CGGGCGCGCC

SEQ ID NO: 50

Revelant homologous recombination expression construct of codon optimized coding region of Ulmus americana broad specificity thioesterase.

GCTCTTCGGCCGCCGCCACTCCTGCTCGAGCGCGCCCGACTCGCGCTCCGCCTGC

GCCCGCGCGTGCGCCGCCAGCGCCTTGGCCTTTTCGCCGCGCTCGTGCGCGTCGC

TGATGTCCATCACCAGGTCCATGAGGTCTGCCTTGCGCCGGCTGAGCCACTGC1T

CGTCCGGGCGGCCAAGAGGAGCATGAGGGAGGACTCCTGGTCCAGGGTCCTGAC

GTGGTCGCGGCTCTGGGAGCGGGCCAGCATCATCTGGCTCTGCCGCACCGAGGC

CGCCTCCAACTGGTCCTCCAGCAGCCGCAGTCGCCGCCGACCCTGGCAGAGGAA

GACAGGTGAGGGGGGTATGAATTGTACAGAACAACCACGAGCCTTGTCTAGGCA

GAATCCCTACCAGTCATGGCTTTACCTGGATGACGGCCTGCGAACAGCTGTCCAG

CGACCCTCGCTGCCGCCGCTTCTCCCGCACGCTTCTTTCCAGCACCGTGATGGCG

CGAGCCAGCGCCGCACGCTGGCGCTGCGCTTCGCCGATCTGAGGACAGTCGGGG

AACTCTGATCAGTCTAAACCCCCTTGCGCGTTAGTGTTGCCATCCTTTGCAGACC

GGTGAGAGCCGACTTGTTGTGCGCCACCCCCCACACCACCTCCTCCCAGACCAAT

CCCAGCCGCTGGGGGTTGGCGGATGCACGCTCAGGTACCCTTTCTTGCGCTATGA

CACTTCCAGCAAAAGGTAGGGCGGGCTGCGAGACGGCTTCCCGGCGCTGCATGC

AACACCGATGATGCTTCGACCCCCCGAAGCTCCTTCGGGGCTGCATGGGCGCTCC

GATGCCGCTCCAGGGCGAGCGCTGTTTAAATAGCCAGGCCCCCGATTGCAAAGA

CATTATAGCGAGCTACCAAAGCCATATTCAAACACCTAGATCACTACCACTTCTA

CACAGGCCACTCGAGCTTGTGATCGCACTCCGCTAAGGGGGCGCCTCTTCCTCTT

CGTTTCAGTCACAACCCGCAAACGGCGCGCCATGCTGCTGCAGGCCTTCCTGTTC

CTGCTGGCCGGCTTCGCCGCCAAGATCAGCGCCTCCATGACGAACGAGACGTCC

GACCGCCCCCTGGTGCACTTCACCCCCAACAAGGGCTGGATGAACGACCCCAAC

GGCCTGTGGTACGACGAGAAGGACGCCAAGTGGCACCTGTACTTCCAGTACAAC

CCGAACGACACCGTCTGGGGGACGCCCTTGTTCTGGGGCCACGCCACGTCCGAC

GACCTGACCAACTGGGAGGACCAGCCCATCGCCATCGCCCCGAAGCGCAACGAC

TCCGGCGCCTTCTCCGGCTCCATGGTGGTGGACTACAACAACACCTCCGGCTTCT

TCAACGACACCATCGACCCGCGCCAGCGCTGCGTGGCCATCTGGACCTACAACA

CCCCGGAGTCCGAGGAGCAGTACATCTCCTACAGCCTGGACGGCGGCTACACCTT

CACCGAGTACCAGAAGAACCCCGTGCTGGCCGCCAACTCCACCCAGTTCCGCGA

CCCGAAGGTCTTCTGGTACGAGCCCTCCCAGAAGTGGATCATGACCGCGGCCAA

GTCCCAGGACTACAAGATCGAGATCTACTCCTCCGACGACCTGAAGTCCTGGAA

GCTGGAGTCCGCGTTCGCCAACGAGGGCTTCCTCGGCTACCAGTACGAGTGCCCC

GGCCTGATCGAGGTCCCCACCGAGCAGGACCCCAGCAAGTCCTACTGGGTGATG

TTCATCTCCATCAACCCCGGCGCCCCGGCCGGCGGCTCCTTCAACCAGTACTTCG

TCGGCAGCTTCAACGGCACCCACTTCGAGGCCTTCGACAACCAGTCCCGCGTGGT

GGACTTCGGCAAGGACTACTACGCCCTGCAGACCTTCTTCAACACCGACCCGACC

TACGGGAGCGCCCTGGGCATCGCGTGGGCCTCCAACTGGGAGTACTCCGCCTTCG

TGCCCACCAACCCCTGGCGCTCCTCCATGTCCCTCGTGCGCAAGTTCTCCCTCAAC ACCGAGTACCAGGCCAACCCGGAGACGGAGCTGATCAACCTGAAGGCCGAGCCG

ATCCTGAACATCAGCAACGCCGGCCCCTGGAGCCGGTTCGCCACCAACACCACG

TTGACGAAGGCCAACAGCTACAACGTCGACCTGTCCAACAGCACCGGCACCCTG

GAGTTCGAGCTGGTGTACGCCGTCAACACCACCCAGACGATCTCCAAGTCCGTGT

TCGCGGACCTCTCCCTCTGGTTCAAGGGCCTGGAGGACCCCGAGGAGTACCTCCG

CATGGGCTTCGAGGTGTCCGCGTCCTCCTTCTTCCTGGACCGCGGGAACAGCAAG

GTGAAGTTCGTGAAGGAGAACCCCTACTTCACCAACCGCATGAGCGTGAACAAC

CAGCCCTTCAAGAGCGAGAACGACCTGTCCTACTACAAGGTGTACGGCTTGCTGG

ACCAGAACATCCTGGAGCTGTACTTCAACGACGGCGACGTCGTGTCCACCAACA

CCTACTTCATGACCACCGGGAACGCCCTGGGCTCCGTGAACATGACGACGGGGG

TGGACAACCTGTTCTACATCGACAAGTTCCAGGTGCGCGAGGTCAAGTGACAATT

GGCAGCAGCAGCTCGGATAGTATCGACACACTCTGGACGCTGGTCGTGTGATGG

ACTGTTGCCGCCACACTTGCTGCCTTGACCTGTGAATATCCCTGCCGCTTTTATCA

AACAGCCTCAGTGTGTTTGATCTTGTGTGTACGCGCTTTTGCGAGTTGCTAGCTGC

TTGTGCTATTTGCGAATACCACCCCCAGCATCCCCTTCCCTCGTTTCATATCGCTT

GCATCCCAACCGCAACTTATCTACGCTGTCCTGCTATCCCTCAGCGCTGCTCCTGC

TCCTGCTCACTGCCCCTCGCACAGCCTTGGTTTGGGCTCCGCCTGTATTCTCCTGG

TACTGCAACCTGTAAACCAGCACTGCAATGCTGATGCACGGGAAGTAGTGGGAT

GGGAACACAAATGGAGGATCCCGCGTCTCGAACAGAGCGCGCAGAGGAACGCT

GAAGGTCTCGCCTCTGTCGCACCTCAGCGCGGCATACACCACAATAACCACCTGA

CGAATGCGCTTGGTTCTTCGTCCATTAGCGAAGCGTCCGGTTCACACACGTGCCA

CGTTGGCGAGGTGGCAGGTGACAATGATCGGTGGAGCTGATGGTCGAAACGTTC

ACAGCCTAGGGATATCGAATTCCTTTCTTGCGCTATGACACTTCCAGCAAAAGGT

AGGGCGGGCTGCGAGACGGCTTCCCGGCGCTGCATGCAACACCGATGATGCTTC

GACCCCCCGAAGCTCCTTCGGGGCTGCATGGGCGCTCCGATGCCGCTCCAGGGCG

AGCGCTGTTTAAATAGCCAGGCCCCCGATTGCAAAGACATTATAGCGAGCTACC

AAAGCCATATTCAAACACCTAGATCACTACCACTTCTACACAGGCCACTCGAGCT

TGTGATCGCACTCCGCTAAGGGGGCGCCTCTTCCTCTTCGTTTCAGTCACAACCC

GCAAACACTAGTATGGCCACCGCATCCACTTTCTCGGCGTTCAATGCCCGCTGCG

GCGACCTGCGTCGCTCGGCGGGCTCCGGGCCCCGGCGCCCAGCGAGGCCCCTCC

CCGTGCGCGGGCGCGCCCAGCTGCCCGACTGGAGCATGCTGCTGGCCGCGATCA

CCACCCTGTTCCTGGCGGCCGAGAAGCAGTGGATGATGCTGGACTGGAAGCCCA

AGCGCCCCGACATGCTGGTGGACCCCTTCGGCCTGGGCCGCTTCGTGCAGGACGG

CCTGGTGTTCCGCAACAACTTCAGCATCCGCAGCTACGAGATCGGCGCGGACCGC

ACCGCCAGCATCGAGACCCTGATGAACCACCTGCAGGAGACCGCCCTGAACCAC

GTGAAGAGCGTGGGCCTGCTGGAGGACGGCCTGGGCAGCACCCGCGAGATGAGC

CTGCGCAACCTGATCTGGGTGGTGACCAAGATGCAGGTGGCGGTGGACCGCTAC

CCCACCTGGGGCGACGAGGTGCAGGTGAGCAGCTGGGCGACCGCCATCGGCAAG

AACGGCATGCGCCGCGAGTGGATCGTGACCGACTTCCGCACCGGCGAGACCCTG

CTGCGCGCCACCAGCGTGTGGGTGATGATGAACAAGCTGACCCGCCGCATCAGC

AAGATCCCCGAGGAGGTGTGGCACGAGATCGGCCCCAGCTTCATCGACGCGCCC

CCCCTGCCCACCGTGGAGGACGACGGCCGCAAGCTGACCCGCTTCGACGAGAGC

AGCGCCGACTTCATCCGCAAGGGCCTGACCCCCCGCTGGAGCGACCTGGACATC

AACCAGCACGTGAACAACGTGAAGTACATCGGCTGGCTGCTGGAGAGCGCGCCC

CCCGAGATCCACGAGAGCCACGAGATCGCCAGCCTGACCCTGGAGTACCGCCGC

GAGTGCGGCCGCGACAGCGTGCTGAACAGCGCCACCAAGGTGAGCGACAGCAGC

CAGCTGGGCAAGAGCGCCGTGGAGTGCAACCACCTGGTGCGCCTGCAGAACGGC

GGCGAGATCGTGAAGGGCCGCACCGTGTGGCGCCCCAAGCGCCCCCTGTACAAC

GACGGCGCCGTGGTGGACGTGCCCGCCAAGACCAGCGATGACGATGACAAGCTG

GGATGACTCGAGTTAATTAACTCGAGGCAGCAGCAGCTCGGATAGTATCGACAC ACTCTGGACGCTGGTCGTGTGATGGACTGTTGCCGCCACACTTGCTGCCTTGACC

TGTGAATATCCCTGCCGCTTTTATCAAACAGCCTCAGTGTGTTTGATCTTGTGTGT

ACGCGCTTTTGCGAGTTGCTAGCTGCTTGTGCTATTTGCGAATACCACCCCCAGC

ATCCCCTTCCCTCGTTTCATATCGCTTGCATCCCAACCGCAACTTATCTACGCTGT

CCTGCTATCCCTCAGCGCTGCTCCTGCTCCTGCTCACTGCCCCTCGCACAGCCTTG

GTTTGGGCTCCGCCTGTATTCTCCTGGTACTGCAACCTGTAAACCAGCACTGCAA

TGCTGATGCACGGGAAGTAGTGGGATGGGAACACAAATGGAAAGCTGTAGAGCT

CCTTGTTTTCCAGAAGGAGTTGCTCCTTGAGCCTTTCATTCTCAGCCTCGATAACC

TCCAAAGCCGCTCTAATTGTGGAGGGGGTTCGAATTTAAAAGCTTGGAATGTTGG

TTCGTGCGTCTGGAACAAGCCCAGACTTGTTGCTCACTGGGAAAAGGACCATCAG

CTCCAAAAAACTTGCCGCTCAAACCGCGTACCTCTGCTTTCGCGCAATCTGCCCT

GTTGAAATCGCCACCACATTCATATTGTGACGCTTGAGCAGTCTGTAATTGCCTC

AGAATGTGGAATCATCTGCCCCCTGTGCGAGCCCATGCCAGGCATGTCGCGGGC

GAGGACACCCGCCACTCGTACAGCAGACCATTATGCTACCTCACAATAGTTCATA

ACAGTGACCATATTTCTCGAAGCTCCCCAACGAGCACCTCCATGCTCTGAGTGGC

CACCCCCCGGCCCTGGTGCTTGCGGAGGGCAGGTCAACCGGCATGGGGCTACCG

AAATCCCCGACCGGATCCCACCACCCCCGCGATGGGAAGAATCTCTCCCCGGGA

TGTGGGCCCACCACCAGCACAACCTGCTGGCCCAGGCGAGCGTCAAACCATACC

ACACAAATATCCTTGGCATCGGCCCTGAATTCCTTCTGCCGCTCTGCTACCCGGT

GCTTCTGTCCGAAGCAGGGGTTGCTAGGGATCGCTCCGAGTCCGCAAACCCTTGT

CGCGTGGCGGGGCTTGTTCGAGCTTGTTCGAGCTTGAAGAGCCTCTAGAGTCGAC

CTGCAGGCATGCAAGCTTGGCGTAATCATGGTCATAGCTGTTTCCTGTGTGAAAT

TGTTATCCGCTCACAATTCCACACAACATACGAGCCGGAAGCATAAAGTGTAAA

GCCTGGGGTGCCTAATGAGTGAGCTAACTCACATTAATTGCGTTGCGCTCACTGC

CCGCTTTCCAGTCGGGAAACCTGTCGTGCCAGCTGCATTAATGAATCGGCCAACG

CGCGGGGAGAGGCGGTTTGCGTATTGGGCGCTCTTCC

SEQ ID NO: 51

Revelant homologous recombination expression construct of codon optimized coding region of Cinnamomum camphor a C14 preferring thioesterase.

GAATTCGCCCTCCCGTGATCACACAGGTGCCTTGCGAGCGTGATCACACTATTTT

GGGGGTCCTACAGTACTGAAATGGTGAGAAGTCGTACTGAAATCAAGGATGAAC

AATGAAAATGGTGCTGTGGTGGCTTCTCAAAGGTCAAGAATCAGTCGCTCGCGTC

AGGAAATCGCGGCGTCAACCAGCGTGGGCGCGGTCAGTGGCCCCGCACTGGTCA

CCATAGCCTCTCCTGCCACAGTAGCGATCCCCTGGGCGTTCACTCTCAGCAGCGG

CTGTACTGCCTCCCAGATTTTCTTCTTCTGGACCTGCGGGCGTGAGAGGATGAGC

AGGGTGGGCCAAGGGCTCAATCCTGAACGGCCCTCATTCGGTTTCCAATCCCACA

ACACATACCCACAGCAGGTCAGACCACGCATTCCACCATGCGCACCAATAACGT

GTCCTTACCTGATTGGGTGTGGCAGGCTCCGTGGACAGGAGTGCCTCGTCCCCCG

CCCAGACCCGCTCCCCCGTCACGGCGGCGTCCGGGACCCGCAGCGGCTCCACCG

CGGTGTGATCCGCGTTGGCGGCGCAGAGCAGCATCCCAGCCGATTTGACCCCGC

GCATGCTCCGAGGCTTGAGGTTGGCCAGCACCACCACCCGCCGGCCGACAAGGT

CCTCCAGGGTCACGTGCCGGACCAGGCCACTCACGATGGTGCGAGGGCCCCCCT

CCTCGCCGAGGTCGATCTGCTCGACGTACAGACTGCGACATGCGTGGCGAGTGGT

CATCAGAAGGAAGCAGGTGTGCAGAAGGGGCACGTGGTTGGTATTGAGAGTAGC

CAAAGCTTTGTGCCAATCAGAAAGTCAACGCAGCTGCCTGCCTGGCTCGCGTACA

ATTCCTTTCTTGCGCTATGACACTTCCAGCAAAAGGTAGGGCGGGCTGCGAGACG

GCTTCCCGGCGCTGCATGCAACACCGATGATGCTTCGACCCCCCGAAGCTCCTTC

GGGGCTGCATGGGCGCTCCGATGCCGCTCCAGGGCGAGCGCTGTTTAAATAGCC AGGCCCCCGATTGCAAAGACATTATAGCGAGCTACCAAAGCATATTCAAACACC

TAGATCACTACCACTTCTACACAGGCCACTCGAGCTTGTGATCGCACTCCGCTAA

GGGGGCGCCTCTTCCCTTCGTTTCAGTCACAACCCGCAAACGGCGCGCCATGCTG

CTGCAGGCCTTCCTGTTCCTGCTGGCCGGCTTCGCCGCCAAGATCAGCGCCTCCA

TGACGAACGAGACGTCCGACCGCCCCCTGGTGCACTTCACCCCCAACAAGGGCT

GGATGAACGACCCCAACGGCCTGTGGTACGACGAGAAGGACGCCAAGTGGCACC

TGTACTTCCAGTACAACCCGAACGACACCGTCTGGGGGACGCCCTTGTTCTGGGG

CCACGCCACGTCCGACGACCTGACCAACTGGGAGGACCAGCCCATCGCCATCGC

CCCGAAGCGCAACGACTCCGGCGCCTTCTCCGGCTCCATGGTGGTGGACTACAAC

AACACCTCCGGCTTCTTCAACGACACCATCGACCCGCGCCAGCGCTGCGTGGCCA

TCTGGACCTACAACACCCCGGAGTCCGAGGAGCAGTACATCTCCTACAGCCTGG

ACGGCGGCTACACCTTCACCGAGTACCAGAAGAACCCCGTGCTGGCCGCCAACT

CCACCCAGTTCCGCGACCCGAAGGTCTTCTGGTACGAGCCCTCCCAGAAGTGGAT

CATGACCGCGGCCAAGTCCCAGGACTACAAGATCGAGATCTACTCCTCCGACGA

CCTGAAGTCCTGGAAGCTGGAGTCCGCGTTCGCCAACGAGGGCTTCCTCGGCTAC

CAGTACGAGTGCCCCGGCCTGATCGAGGTCCCCACCGAGCAGGACCCCAGCAAG

TCCTACTGGGTGATGTTCATCTCCATCAACCCCGGCGCCCCGGCCGGCGGCTCCT

TCAACCAGTACTTCGTCGGCAGCTTCAACGGCACCCACTTCGAGGCCTTCGACAA

CCAGTCCCGCGTGGTGGACTTCGGCAAGGACTACTACGCCCTGCAGACCTTCTTC

AACACCGACCCGACCTACGGGAGCGCCCTGGGCATCGCGTGGGCCTCCAACTGG

GAGTACTCCGCCTTCGTGCCCACCAACCCCTGGCGCTCCTCCATGTCCCTCGTGC

GCAAGTTCTCCCTCAACACCGAGTACCAGGCCAACCCGGAGACGGAGCTGATCA

ACCTGAAGGCCGAGCCGATCCTGAACATCAGCAACGCCGGCCCCTGGAGCCGGT

TCGCCACCAACACCACGTTGACGAAGGCCAACAGCTACAACGTCGACCTGTCCA

ACAGCACCGGCACCCTGGAGTTCGAGCTGGTGTACGCCGTCAACACCACCCAGA

CGATCTCCAAGTCCGTGTTCGCGGACCTCTCCCTCTGGTTCAAGGGCCTGGAGGA

CCCCGAGGAGTACCTCCGCATGGGCTTCGAGGTGTCCGCGTCCTCCTTCTTCCTG

GACCGCGGGAACAGCAAGGTGAAGTTCGTGAAGGAGAACCCCTACTTCACCAAC

CGCATGAGCGTGAACAACCAGCCCTTCAAGAGCGAGAACGACCTGTCCTACTAC

AAGGTGTACGGCTTGCTGGACCAGAACATCCTGGAGCTGTACTTCAACGACGGC

GACGTCGTGTCCACCAACACCTACTTCATGACCACCGGGAACGCCCTGGGCTCCG

TGAACATGACGACGGGGGTGGACAACCTGTTCTACATCGACAAGTTCCAGGTGC

GCGAGGTCAAGTGATTAATTAACTCGAGGCAGCAGCAGCTCGGATAGTATCGAC

ACACTCTGGACGCTGGTCGTGTGATGGACTGTTGCCGCCACACTTGCTGCCTTGA

CCTGTGAATATCCCTGCCGCTTTTATCAAACAGCCTCAGTGTGTTTGATCTTGTGT

GTACGCGCTTTTGCGAGTTGCTAGCTGCTTGTGCTATTTGCGAATACCACCCCCA

GCATCCCCTCCCTCGTTTCATATCGCTTGCATCCCAACCGCAACTTATCTACGCTG

TCCTGCTATCCCTCAGCGCTGCTCCTGCTCCTGCTCACTGCCCCTCGCACAGCCTT

GGTTTGGGCTCCGCCTGTATTCTCCTGGTACTGCAACCTGTAAACCAGCACTGCA

ATGCTGATGCACGGGAAGTAGTGGGATGGGAACACAAATGGAAAGCTTGAGCTC

CTTTCTTGCGCTATGACACTTCCAGCAAAAGGTAGGGCGGGCTGCGAGACGGCTT

CCCGGCGCTGCATGCAACACCGATGATGCTTCGACCCCCCGAAGCTCCTTCGGGG

CTGCATGGGCGCTCCGATGCCGCTCCAGGGCGAGCGCTGTTTAAATAGCCAGGCC

CCCGATTGCAAAGACATTATAGCGAGCTACCAAAGCCATATTCAAACACCTAGA

TCACTACCACTTCTACACAGGCCACTCGAGCTTGTGATCGCACTCCGCTAAGGGG

GCGCCTCTTCCTCTTCGTTTCAGTCACAACCCGCAAACACTAGTATGACGTTCGG

GGTCGCCCTCCCGGCCATGGGCCGCGGTGTCTCCCTTCCCCGGCCCAGGGTCGCG

TCCATGCTGTTCGCCGTGATCACCACCATCTTCTCCGCCGCCGAGAAGCAGTGGA CCAACCTGGAGTGGAAGCCCAAGCCCAACCCCCCCCAGCTGCTGGACGACCACT TCGGCCCCCACGGCCTGGTGTTCCGCCGCACCTTCGCCATCCGCAGCTACGAGGT

GGGCCCCGACCGCTCCACCAGCATCGTGGCCGTGATGAACCACCTGCAGGAGGC

CGCCCTGAACCACGCCAAGTCCGTGGGCATCCTGGGCGACGGCTTCGGCACCAC

CCTGGAGATGTCCAAGCGCGACCTGATCTGGGTGGTGAAGCGCACCCACGTGGC

CGTGGAGCGCTACCCCGCCTGGGGCGACACCGTGGAGGTGGAGTGCTGGGTGGG

CGCCTCCGGCAACAACGGCCGCCGCCACGACTTCCTGGTGCGCGACTGCAAGAC

CGGCGAGATCCTGACCCGCTGCACCTCCCTGAGCGTGATGATGAACACCCGCACC

CGCCGCCTGAGCAAGATCCCCGAGGAGGTGCGCGGCGAGATCGGCCCCGCCTTC

ATCGACAACGTGGCCGTGAAGGACGAGGAGATCAAGAAGCCCCAGAAGCTGAA

CGACTCCACCGCCGACTACATCCAGGGCGGCCTGACCCCCCGCTGGAACGACCT

GGACATCAACCAGCACGTGAACAACATCAAGTACGTGGACTGGATCCTGGAGAC

CGTGCCCGACAGCATCTTCGAGAGCCACCACATCTCCTCCTTCACCATCGAGTAC

CGCCGCGAGTGCACCATGGACAGCGTGCTGCAGTCCCTGACCACCGTGAGCGGC

GGCTCCTCCGAGGCCGGCCTGGTGTGCGAGCACCTGCTGCAGCTGGAGGGCGGC

AGCGAGGTGCTGCGCGCCAAGACCGAGTGGCGCCCCAAGCTGACCGACTCCTTC

CGCGGCATCAGCGTGATCCCCGCCGAGTCCAGCGTGATGGACTACAAGGACCAC

GACGGCGACTACAAGGACCACGACATCGACTACAAGGACGACGACGACAAGTG

ACTCGAGGCAGCAGCAGCTCGGATAGTATCGACACACTCTGGACGCTGGTCGTG

TGATGGACTGTTGCCGCCACACTTGCTGCCTTGACCTGTGAATATCCCTGCCGCTT

TTATCAAACAGCCTCAGTGTGTTTGATCTTGTGTGTACGCGCTTTTGCGAGTTGCT

AGCTCTTGTGCTATTTGCGAATACCACCCCCAGCATCCCCTTCCCTCGTTTCATAT

CGCTTGCATCCCAACCGCAACTTATCTACGCTGTCCTGCTATCCCTCAGCGCTGCT

CCTGCTCCTGCTCACTGCCCCTCGCACAGCCTTGGTTTGGGCTCCGCCTGTATTCT

CCTGGTACTGCAACCTGTAAACCAGCACTGCAATGCTGATGCACGGGAAGTAGT

GGGATGGGAACACAAATGGAAAGCTGGTACCCGTACCCATCAGCATCCGGGTGA

ATCTTGGCCTCCAAGATATGGCCAATCCTCACATCCAGCTTGGCAAAATCGACTA

GACTGTCTGCAAGTGGGAATGTGGAGCACAAGGTTGCTTGTAGCGATCGACAGA

CTGGTGGGGTACATTGACAGGTGGGCAGCGCCGCATCCATCGTGCCTGACGCGA

GCGCCGCCGGTTGCTCGCCCGTGCCTGCCGTCAAAGAGCGGCAGAGAAATCGGG

AACCGAAAACGTCACATTGCCTGATGTTGTTACATGCTGGACTAGACTTTCTTGG

CGTGGGTCTGCTCCTCGCCAGGTGCGCGACGCCTCGGGGCTGGGTGCGAGGGAG

CCGTGCGGCCACGCATTTGACAAGACCCAAAGCTCGCATCTCAGACGGTCAACC

GTTCGTATTATACATTCAACATATGGTACATACGCAAAAAGCATGCCAACGATGA

CATAGGCGAATTC

SEQ ID NO: 52

Relevant expression construct for codon optimized coding region of Cuphea hookeriana CIO preferring thioesterase with Chlorella protothecoides stearoyl ACP desaturase transit peptide.

GGTACCCGCCTGCAACGCAAGGGCAGCCACAGCCGCTCCCACCCGCCGCTGAAC

CGACACGTGCTTGGGCGCCTGCCGCCTGCCTGCCGCATGCTTGTGCTGGTGAGGC

TGGGCAGTGCTGCCATGCTGATTGAGGCTTGGTTCATCGGGTGGAAGCTTATGTG

TGTGCTGGGCTTGCATGCCGGGCAATGCGCATGGTGGCAAGAGGGCGGCAGCAC

TTGCTGGAGCTGCCGCGGTGCCTCCAGGTGGTTCAATCGCGGCAGCCAGAGGGA

TTTCAGATGATCGCGCGTACAGGTTGAGCAGCAGTGTCAGCAAAGGTAGCAGTTT

GCCAGAATGATCGGTTCAGCTGTTAATCAATGCCAGCAAGAGAAGGGGTCAAGT

GCAAACACGGGCATGCCACAGCACGGGCACCGGGGAGTGGAATGGCACCACCA

AGTGTGTGCGAGCCAGCATCGCCGCCTGGCTGTTTCAGCTACAACGGCAGGAGTC

ATCCAACGTAACCATGAGCTGATCAACACTGCAATCATCGGGCGGGCGTGATGC

AAGCATGCCTGGCGAAGACACATGGTGTGCGGATGCTGCCGGCTGCTGCCTGCT GCGCACGCCGTTGAGTTGGCAGCAGGCTCAGCCATGCACTGGATGGCAGCTGGG

CTGCCACTGCAATGTGGTGGATAGGATGCAAGTGGAGCGAATACCAAACCCTCT

GGCTGCTTGCTGGGTTGCATGGCATCGCACCATCAGCAGGAGCGCATGCGAAGG

GACTGGCCCCATGCACGCCATGCCAAACCGGAGCGCACCGAGTGTCCACACTGT

CACCAGGCCCGCAAGCTTTGCAGAACCATGCTCATGGACGCATGTAGCGCTGAC

GTCCCTTGACGGCGCTCCTCTCGGGTGTGGGAAACGCAATGCAGCACAGGCAGC

AGAGGCGGCGGCAGCAGAGCGGCGGCAGCAGCGGCGGGGGCCACCCTTCTTGCG

GGGTCGCGCCCCAGCCAGCGGTGATGCGCTGATCCCAAACGAGTTCACATTCATT

TGCATGCCTGGAGAAGCGAGGCTGGGGCCTTTGGGCTGGTGCAGCCCGCAATGG

AATGCGGGACCGCCAGGCTAGCAGCAAAGGCGCCTCCCCTACTCCGCATCGATG

TTCCATAGTGCATTGGACTGCATTTGGGTGGGGCGGCCGGCTGTTTCTTTCGTGTT

GCAAAACGCGCCAGCTCAGCAACCTGTCCCGTGGGTCCCCCGTGCCGATGAAAT

CGTGTGCACGCCGATCAGCTGATTGCCCGGCTCGCGAAGTAGGCGCCCTCCTTTC

TGCTCGCCCTCTCTCCGTCCCGCCTCTAGAATATCAATGATCGAGCAGGACGGCC

TCCACGCCGGCTCCCCCGCCGCCTGGGTGGAGCGCCTGTTCGGCTACGACTGGGC

CCAGCAGACCATCGGCTGCTCCGACGCCGCCGTGTTCCGCCTGTCCGCCCAGGGC

CGCCCCGTGCTGTTCGTGAAGACCGACCTGTCCGGCGCCCTGAACGAGCTGCAGG

ACGAGGCCGCCCGCCTGTCCTGGCTGGCCACCACCGGCGTGCCCTGCGCCGCCGT

GCTGGACGTGGTGACCGAGGCCGGCCGCGACTGGCTGCTGCTGGGCGAGGTGCC

CGGCCAGGACCTGCTGTCCTCCCACCTGGCCCCCGCCGAGAAGGTGTCCATCATG

GCCGACGCCATGCGCCGCCTGCACACCCTGGACCCCGCCACCTGCCCCTTCGACC

ACCAGGCCAAGCACCGCATCGAGCGCGCCCGCACCCGCATGGAGGCCGGCCTGG

TGGACCAGGACGACCTGGACGAGGAGCACCAGGGCCTGGCCCCCGCCGAGCTGT

TCGCCCGCCTGAAGGCCCGCATGCCCGACGGCGAGGACCTGGTGGTGACCCACG

GCGACGCCTGCCTGCCCAACATCATGGTGGAGAACGGCCGCTTCTCCGGCTTCAT

CGACTGCGGCCGCCTGGGCGTGGCCGACCGCTACCAGGACATCGCCCTGGCCAC

CCGCGACATCGCCGAGGAGCTGGGCGGCGAGTGGGCCGACCGCTTCCTGGTGCT

GTACGGCATCGCCGCCCCCGACTCCCAGCGCATCGCCTTCTACCGCCTGCTGGAC

GAGTTCTTCTGACAATTGGCAGCAGCAGCTCGGATAGTATCGACACACTCTGGAC

GCTGGTCGTGTGATGGACTGTTGCCGCCACACTTGCTGCCTTGACCTGTGAATAT

CCCTGCCGCTTTTATCAAACAGCCTCAGTGTGTTTGATCTTGTGTGTACGCGCTTT

TGCGAGTTGCTAGCTGCTTGTGCTATTTGCGAATACCACCCCCAGCATCCCCTTCC

CTCGTTTCATATCGCTTGCATCCCAACCGCAACTTATCTACGCTGTCCTGCTATCC

CTCAGCGCTGCTCCTGCTCCTGCTCACTGCCCCTCGCACAGCCTTGGTTTGGGCTC

CGCCTGTATTCTCCTGGTACTGCAACCTGTAAACCAGCACTGCAATGCTGATGCA

CGGGAAGTAGTGGGATGGGAACACAAATGGAGGATCCCGCGTCTCGAACAGAGC

GCGCAGAGGAACGCTGAAGGTCTCGCCTCTGTCGCACCTCAGCGCGGCATACAC

CACAATAACCACCTGACGAATGCGCTTGGTTCTTCGTCCATTAGCGAAGCGTCCG

GTTCACACACGTGCCACGTTGGCGAGGTGGCAGGTGACAATGATCGGTGGAGCT

GATGGTCGAAACGTTCACAGCCTAGGGATATCGAATTCCTTTCTTGCGCTATGAC

ACTTCCAGCAAAAGGTAGGGCGGGCTGCGAGACGGCTTCCCGGCGCTGCATGCA

ACACCGATGATGCTTCGACCCCCCGAAGCTCCTTCGGGGCTGCATGGGCGCTCCG

ATGCCGCTCCAGGGCGAGCGCTGTTTAAATAGCCAGGCCCCCGATTGCAAAGAC

ATTATAGCGAGCTACCAAAGCCATATTCAAACACCTAGATCACTACCACTTCTAC

ACAGGCCACTCGAGCTTGTGATCGCACTCCGCTAAGGGGGCGCCTCTTCCTCTTC

GTTTCAGTCACAACCCGCAAACACTAGTATGGCCACCGCATCCACTTTCTCGGCG

TTCAATGCCCGCTGCGGCGACCTGCGTCGCTCGGCGGGCTCCGGGCCCCGGCGCC

CAGCGAGGCCCCTCCCCGTGCGCGGGCGCGCCCAGCTGCCCGACTGGAGCCGCC

TGCTGACCGCCATCACCACCGTGTTCGTGAAGTCCAAGCGCCCCGACATGCACGA

CCGCAAGTCCAAGCGCCCCGACATGCTGGTGGACAGCTTCGGCCTGGAGTCCAC CGTGCAGGACGGCCTGGTGTTCCGCCAGTCCTTCTCCATCCGCTCCTACGAGATC

GGCACCGACCGCACCGCCAGCATCGAGACCCTGATGAACCACCTGCAGGAGACC

TCCCTGAACCACTGCAAGAGCACCGGCATCCTGCTGGACGGCTTCGGCCGCACCC

TGGAGATGTGCAAGCGCGACCTGATCTGGGTGGTGATCAAGATGCAGATCAAGG

TGAACCGCTACCCCGCCTGGGGCGACACCGTGGAGATCAACACCCGCTTCAGCC

GCCTGGGCAAGATCGGCATGGGCCGCGACTGGCTGATCTCCGACTGCAACACCG

GCGAGATCCTGGTGCGCGCCACCAGCGCCTACGCCATGATGAACCAGAAGACCC

GCCGCCTGTCCAAGCTGCCCTACGAGGTGCACCAGGAGATCGTGCCCCTGTTCGT

GGACAGCCCCGTGATCGAGGACTCCGACCTGAAGGTGCACAAGTTCAAGGTGAA

GACCGGCGACAGCATCCAGAAGGGCCTGACCCCCGGCTGGAACGACCTGGACGT

GAACCAGCACGTGTCCAACGTGAAGTACATCGGCTGGATCCTGGAGAGCATGCC

CACCGAGGTGCTGGAGACCCAGGAGCTGTGCTCCCTGGCCCTGGAGTACCGCCG

CGAGTGCGGCCGCGACTCCGTGCTGGAGAGCGTGACCGCCATGGACCCCAGCAA

GGTGGGCGTGCGCTCCCAGTACCAGCACCTGCTGCGCCTGGAGGACGGCACCGC

CATCGTGAACGGCGCCACCGAGTGGCGCCCCAAGAACGCCGGCGCCAACGGCGC

CATCTCCACCGGCAAGACCAGCAACGGCAACTCCGTGTCCATGGACTACAAGGA

CCACGACGGCGACTACAAGGACCACGACATCGACTACAAGGACGACGACGACA

AGTGACTCGAGGCAGCAGCAGCTCGGATAGTATCGACACACTCTGGACGCTGGT

CGTGTGATGGACTGTTGCCGCCACACTTGCTGCCTTGACCTGTGAATATCCCTGC

CGCTTTTATCAAACAGCCTCAGTGTGTTTGATCTTGTGTGTACGCGCTTTTGCGAG

TTGCTAGCTGCTTGTGCTATTTGCGAATACCACCCCCAGCATCCCCTTCCCTCGTT

TCATATCGCTTGCATCCCAACCGCAACTTATCTACGCTGTCCTGCTATCCCTCAGC

GCTGCTCCTGCTCCTGCTCACTGCCCCTCGCACAGCCTTGGTTTGGGCTCCGCCTG

TATTCTCCTGGTACTGCAACCTGTAAACCAGCACTGCAATGCTGATGCACGGGAA

GTAGTGGGATGGGAACACAAATGGAAAGCTTGAGCTC

SEQ ID NO: 53

Relevant expression construct for codon optimized coding region of Umbellularia californica C12 preferring thioesterase with Chlorella protothecoid.es stearoyl ACP desaturase transit peptide.

GGTACCCGCCTGCAACGCAAGGGCAGCCACAGCCGCTCCCACCCGCCGCTGAAC

CGACACGTGCTTGGGCGCCTGCCGCCTGCCTGCCGCATGCTTGTGCTGGTGAGGC

TGGGCAGTGCTGCCATGCTGATTGAGGCTTGGTTCATCGGGTGGAAGCTTATGTG

TGTGCTGGGCTTGCATGCCGGGCAATGCGCATGGTGGCAAGAGGGCGGCAGCAC

TTGCTGGAGCTGCCGCGGTGCCTCCAGGTGGTTCAATCGCGGCAGCCAGAGGGA

TTTCAGATGATCGCGCGTACAGGTTGAGCAGCAGTGTCAGCAAAGGTAGCAGTTT

GCCAGAATGATCGGTTCAGCTGTTAATCAATGCCAGCAAGAGAAGGGGTCAAGT

GCAAACACGGGCATGCCACAGCACGGGCACCGGGGAGTGGAATGGCACCACCA

AGTGTGTGCGAGCCAGCATCGCCGCCTGGCTGTTTCAGCTACAACGGCAGGAGTC

ATCCAACGTAACCATGAGCTGATCAACACTGCAATCATCGGGCGGGCGTGATGC

AAGCATGCCTGGCGAAGACACATGGTGTGCGGATGCTGCCGGCTGCTGCCTGCT

GCGCACGCCGTTGAGTTGGCAGCAGGCTCAGCCATGCACTGGATGGCAGCTGGG

CTGCCACTGCAATGTGGTGGATAGGATGCAAGTGGAGCGAATACCAAACCCTCT

GGCTGCTTGCTGGGTTGCATGGCATCGCACCATCAGCAGGAGCGCATGCGAAGG

GACTGGCCCCATGCACGCCATGCCAAACCGGAGCGCACCGAGTGTCCACACTGT

CACCAGGCCCGCAAGCTTTGCAGAACCATGCTCATGGACGCATGTAGCGCTGAC

GTCCCTTGACGGCGCTCCTCTCGGGTGTGGGAAACGCAATGCAGCACAGGCAGC

AGAGGCGGCGGCAGCAGAGCGGCGGCAGCAGCGGCGGGGGCCACCCTTCTTGCG

GGGTCGCGCCCCAGCCAGCGGTGATGCGCTGATCCCAAACGAGTTCACATTCATT TGCATGCCTGGAGAAGCGAGGCTGGGGCCTTTGGGCTGGTGCAGCCCGCAATGG

AATGCGGGACCGCCAGGCTAGCAGCAAAGGCGCCTCCCCTACTCCGCATCGATG

TTCCATAGTGCATTGGACTGCATTTGGGTGGGGCGGCCGGCTGTTTCTTTCGTGTT

GCAAAACGCGCCAGCTCAGCAACCTGTCCCGTGGGTCCCCCGTGCCGATGAAAT

CGTGTGCACGCCGATCAGCTGATTGCCCGGCTCGCGAAGTAGGCGCCCTCCTTTC

TGCTCGCCCTCTCTCCGTCCCGCCTCTAGAATATCAATGATCGAGCAGGACGGCC

TCCACGCCGGCTCCCCCGCCGCCTGGGTGGAGCGCCTGTTCGGCTACGACTGGGC

CCAGCAGACCATCGGCTGCTCCGACGCCGCCGTGTTCCGCCTGTCCGCCCAGGGC

CGCCCCGTGCTGTTCGTGAAGACCGACCTGTCCGGCGCCCTGAACGAGCTGCAGG

ACGAGGCCGCCCGCCTGTCCTGGCTGGCCACCACCGGCGTGCCCTGCGCCGCCGT

GCTGGACGTGGTGACCGAGGCCGGCCGCGACTGGCTGCTGCTGGGCGAGGTGCC

CGGCCAGGACCTGCTGTCCTCCCACCTGGCCCCCGCCGAGAAGGTGTCCATCATG

GCCGACGCCATGCGCCGCCTGCACACCCTGGACCCCGCCACCTGCCCCTTCGACC

ACCAGGCCAAGCACCGCATCGAGCGCGCCCGCACCCGCATGGAGGCCGGCCTGG

TGGACCAGGACGACCTGGACGAGGAGCACCAGGGCCTGGCCCCCGCCGAGCTGT

TCGCCCGCCTGAAGGCCCGCATGCCCGACGGCGAGGACCTGGTGGTGACCCACG

GCGACGCCTGCCTGCCCAACATCATGGTGGAGAACGGCCGCTTCTCCGGCTTCAT

CGACTGCGGCCGCCTGGGCGTGGCCGACCGCTACCAGGACATCGCCCTGGCCAC

CCGCGACATCGCCGAGGAGCTGGGCGGCGAGTGGGCCGACCGCTTCCTGGTGCT

GTACGGCATCGCCGCCCCCGACTCCCAGCGCATCGCCTTCTACCGCCTGCTGGAC

GAGTTCTTCTGACAATTGGCAGCAGCAGCTCGGATAGTATCGACACACTCTGGAC

GCTGGTCGTGTGATGGACTGTTGCCGCCACACTTGCTGCCTTGACCTGTGAATAT

CCCTGCCGCTTTTATCAAACAGCCTCAGTGTGTTTGATCTTGTGTGTACGCGCTTT

TGCGAGTTGCTAGCTGCTTGTGCTATTTGCGAATACCACCCCCAGCATCCCCTTCC

CTCGTTTCATATCGCTTGCATCCCAACCGCAACTTATCTACGCTGTCCTGCTATCC

CTCAGCGCTGCTCCTGCTCCTGCTCACTGCCCCTCGCACAGCCTTGGTTTGGGCTC

CGCCTGTATTCTCCTGGTACTGCAACCTGTAAACCAGCACTGCAATGCTGATGCA

CGGGAAGTAGTGGGATGGGAACACAAATGGAGGATCCCGCGTCTCGAACAGAGC

GCGCAGAGGAACGCTGAAGGTCTCGCCTCTGTCGCACCTCAGCGCGGCATACAC

CACAATAACCACCTGACGAATGCGCTTGGTTCTTCGTCCATTAGCGAAGCGTCCG

GTTCACACACGTGCCACGTTGGCGAGGTGGCAGGTGACAATGATCGGTGGAGCT

GATGGTCGAAACGTTCACAGCCTAGGGATATCGAATTCCTTTCTTGCGCTATGAC

ACTTCCAGCAAAAGGTAGGGCGGGCTGCGAGACGGCTTCCCGGCGCTGCATGCA

ACACCGATGATGCTTCGACCCCCCGAAGCTCCTTCGGGGCTGCATGGGCGCTCCG

ATGCCGCTCCAGGGCGAGCGCTGTTTAAATAGCCAGGCCCCCGATTGCAAAGAC

ATTATAGCGAGCTACCAAAGCCATATTCAAACACCTAGATCACTACCACTTCTAC

ACAGGCCACTCGAGCTTGTGATCGCACTCCGCTAAGGGGGCGCCTCTTCCTCTTC

GTTTCAGTCACAACCCGCAAACACTAGTATGGCCACCGCATCCACTTTCTCGGCG

TTCAATGCCCGCTGCGGCGACCTGCGTCGCTCGGCGGGCTCCGGGCCCCGGCGCC

CAGCGAGGCCCCTCCCCGTGCGCGGGCGCGCCCCCGACTGGTCCATGCTGTTCGC

CGTGATCACCACCATCTTCAGCGCCGCCGAGAAGCAGTGGACCAACCTGGAGTG

GAAGCCCAAGCCCAAGCTGCCCCAGCTGCTGGACGACCACTTCGGCCTGCACGG

CCTGGTGTTCCGCCGCACCTTCGCCATCCGCTCCTACGAGGTGGGCCCCGACCGC

AGCACCTCCATCCTGGCCGTGATGAACCACATGCAGGAGGCCACCCTGAACCAC

GCCAAGAGCGTGGGCATCCTGGGCGACGGCTTCGGCACCACCCTGGAGATGTCC

AAGCGCGACCTGATGTGGGTGGTGCGCCGCACCCACGTGGCCGTGGAGCGCTAC

CCCACCTGGGGCGACACCGTGGAGGTGGAGTGCTGGATCGGCGCCAGCGGCAAC

AACGGCATGCGCCGCGACTTCCTGGTGCGCGACTGCAAGACCGGCGAGATCCTG

ACCCGCTGCACCTCCCTGAGCGTGCTGATGAACACCCGCACCCGCCGCCTGAGCA

CCATCCCCGACGAGGTGCGCGGCGAGATCGGCCCCGCCTTCATCGACAACGTGG CCGTGAAGGACGACGAGATCAAGAAGCTGCAGAAGCTGAACGACTCCACCGCCG

ACTACATCCAGGGCGGCCTGACCCCCCGCTGGAACGACCTGGACGTGAACCAGC

ACGTGAACAACCTGAAGTACGTGGCCTGGGTGTTCGAGACCGTGCCCGACAGCA

TCTTCGAGTCCCACCACATCAGCTCCTTCACCCTGGAGTACCGCCGCGAGTGCAC

CCGCGACTCCGTGCTGCGCAGCCTGACCACCGTGAGCGGCGGCAGCTCCGAGGC

CGGCCTGGTGTGCGACCACCTGCTGCAGCTGGAGGGCGGCAGCGAGGTGCTGCG

CGCCCGCACCGAGTGGCGCCCCAAGCTGACCGACTCCTTCCGCGGCATCAGCGTG

ATCCCCGCCGAGCCCCGCGTGATGGACTACAAGGACCACGACGGCGACTACAAG

GACCACGACATCGACTACAAGGACGACGACGACAAGTGACTCGAGGCAGCAGC

AGCTCGGATAGTATCGACACACTCTGGACGCTGGTCGTGTGATGGACTGTTGCCG

CCACACTTGCTGCCTTGACCTGTGAATATCCCTGCCGCTTTTATCAAACAGCCTCA

GTGTGTTTGATCTTGTGTGTACGCGCTTTTGCGAGTTGCTAGCTGCTTGTGCTATT

TGCGAATACCACCCCCAGCATCCCCTTCCCTCGTTTCATATCGCTTGCATCCCAAC

CGCAACTTATCTACGCTGTCCTGCTATCCCTCAGCGCTGCTCCTGCTCCTGCTCAC

TGCCCCTCGCACAGCCTTGGTTTGGGCTCCGCCTGTATTCTCCTGGTACTGCAACC

TGTAAACCAGCACTGCAATGCTGATGCACGGGAAGTAGTGGGATGGGAACACAA

ATGGAAAGCTTGAGCTC

SEQ ID NO: 54

Relevant expression construct for codon optimized coding region of Ulmus americana broad specificity thioesterase with Chlorella protothecoides stearoyl ACP desaturase transit peptide.

GGTACCCGCCTGCAACGCAAGGGCAGCCACAGCCGCTCCCACCCGCCGCTGAAC

CGACACGTGCTTGGGCGCCTGCCGCCTGCCTGCCGCATGCTTGTGCTGGTGAGGC

TGGGCAGTGCTGCCATGCTGATTGAGGCTTGGTTCATCGGGTGGAAGCTTATGTG

TGTGCTGGGCTTGCATGCCGGGCAATGCGCATGGTGGCAAGAGGGCGGCAGCAC

TTGCTGGAGCTGCCGCGGTGCCTCCAGGTGGTTCAATCGCGGCAGCCAGAGGGA

TTTCAGATGATCGCGCGTACAGGTTGAGCAGCAGTGTCAGCAAAGGTAGCAGTTT

GCCAGAATGATCGGTTCAGCTGTTAATCAATGCCAGCAAGAGAAGGGGTCAAGT

GCAAACACGGGCATGCCACAGCACGGGCACCGGGGAGTGGAATGGCACCACCA

AGTGTGTGCGAGCCAGCATCGCCGCCTGGCTGTTTCAGCTACAACGGCAGGAGTC

ATCCAACGTAACCATGAGCTGATCAACACTGCAATCATCGGGCGGGCGTGATGC

AAGCATGCCTGGCGAAGACACATGGTGTGCGGATGCTGCCGGCTGCTGCCTGCT

GCGCACGCCGTTGAGTTGGCAGCAGGCTCAGCCATGCACTGGATGGCAGCTGGG

CTGCCACTGCAATGTGGTGGATAGGATGCAAGTGGAGCGAATACCAAACCCTCT

GGCTGCTTGCTGGGTTGCATGGCATCGCACCATCAGCAGGAGCGCATGCGAAGG

GACTGGCCCCATGCACGCCATGCCAAACCGGAGCGCACCGAGTGTCCACACTGT

CACCAGGCCCGCAAGCTTTGCAGAACCATGCTCATGGACGCATGTAGCGCTGAC

GTCCCTTGACGGCGCTCCTCTCGGGTGTGGGAAACGCAATGCAGCACAGGCAGC

AGAGGCGGCGGCAGCAGAGCGGCGGCAGCAGCGGCGGGGGCCACCCTTCTTGCG

GGGTCGCGCCCCAGCCAGCGGTGATGCGCTGATCCCAAACGAGTTCACATTCATT

TGCATGCCTGGAGAAGCGAGGCTGGGGCCTTTGGGCTGGTGCAGCCCGCAATGG

AATGCGGGACCGCCAGGCTAGCAGCAAAGGCGCCTCCCCTACTCCGCATCGATG

TTCCATAGTGCATTGGACTGCATTTGGGTGGGGCGGCCGGCTGTTTCTTTCGTGTT

GCAAAACGCGCCAGCTCAGCAACCTGTCCCGTGGGTCCCCCGTGCCGATGAAAT

CGTGTGCACGCCGATCAGCTGATTGCCCGGCTCGCGAAGTAGGCGCCCTCCTTTC

TGCTCGCCCTCTCTCCGTCCCGCCTCTAGAATATCAATGATCGAGCAGGACGGCC

TCCACGCCGGCTCCCCCGCCGCCTGGGTGGAGCGCCTGTTCGGCTACGACTGGGC

CCAGCAGACCATCGGCTGCTCCGACGCCGCCGTGTTCCGCCTGTCCGCCCAGGGC CGCCCCGTGCTGTTCGTGAAGACCGACCTGTCCGGCGCCCTGAACGAGCTGCAGG

ACGAGGCCGCCCGCCTGTCCTGGCTGGCCACCACCGGCGTGCCCTGCGCCGCCGT

GCTGGACGTGGTGACCGAGGCCGGCCGCGACTGGCTGCTGCTGGGCGAGGTGCC

CGGCCAGGACCTGCTGTCCTCCCACCTGGCCCCCGCCGAGAAGGTGTCCATCATG

GCCGACGCCATGCGCCGCCTGCACACCCTGGACCCCGCCACCTGCCCCTTCGACC

ACCAGGCCAAGCACCGCATCGAGCGCGCCCGCACCCGCATGGAGGCCGGCCTGG

TGGACCAGGACGACCTGGACGAGGAGCACCAGGGCCTGGCCCCCGCCGAGCTGT

TCGCCCGCCTGAAGGCCCGCATGCCCGACGGCGAGGACCTGGTGGTGACCCACG

GCGACGCCTGCCTGCCCAACATCATGGTGGAGAACGGCCGCTTCTCCGGCTTCAT

CGACTGCGGCCGCCTGGGCGTGGCCGACCGCTACCAGGACATCGCCCTGGCCAC

CCGCGACATCGCCGAGGAGCTGGGCGGCGAGTGGGCCGACCGCTTCCTGGTGCT

GTACGGCATCGCCGCCCCCGACTCCCAGCGCATCGCCTTCTACCGCCTGCTGGAC

GAGTTCTTCTGACAATTGGCAGCAGCAGCTCGGATAGTATCGACACACTCTGGAC

GCTGGTCGTGTGATGGACTGTTGCCGCCACACTTGCTGCCTTGACCTGTGAATAT

CCCTGCCGCTTTTATCAAACAGCCTCAGTGTGTTTGATCTTGTGTGTACGCGCTTT

TGCGAGTTGCTAGCTGCTTGTGCTATTTGCGAATACCACCCCCAGCATCCCCTTCC

CTCGTTTCATATCGCTTGCATCCCAACCGCAACTTATCTACGCTGTCCTGCTATCC

CTCAGCGCTGCTCCTGCTCCTGCTCACTGCCCCTCGCACAGCCTTGGTTTGGGCTC

CGCCTGTATTCTCCTGGTACTGCAACCTGTAAACCAGCACTGCAATGCTGATGCA

CGGGAAGTAGTGGGATGGGAACACAAATGGAGGATCCCGCGTCTCGAACAGAGC

GCGCAGAGGAACGCTGAAGGTCTCGCCTCTGTCGCACCTCAGCGCGGCATACAC

CACAATAACCACCTGACGAATGCGCTTGGTTCTTCGTCCATTAGCGAAGCGTCCG

GTTCACACACGTGCCACGTTGGCGAGGTGGCAGGTGACAATGATCGGTGGAGCT

GATGGTCGAAACGTTCACAGCCTAGGGATATCGAATTCCTTTCTTGCGCTATGAC

ACTTCCAGCAAAAGGTAGGGCGGGCTGCGAGACGGCTTCCCGGCGCTGCATGCA

ACACCGATGATGCTTCGACCCCCCGAAGCTCCTTCGGGGCTGCATGGGCGCTCCG

ATGCCGCTCCAGGGCGAGCGCTGTTTAAATAGCCAGGCCCCCGATTGCAAAGAC

ATTATAGCGAGCTACCAAAGCCATATTCAAACACCTAGATCACTACCACTTCTAC

ACAGGCCACTCGAGCTTGTGATCGCACTCCGCTAAGGGGGCGCCTCTTCCTCTTC

GTTTCAGTCACAACCCGCAAACACTAGTATGGCCACCGCATCCACTTTCTCGGCG

TTCAATGCCCGCTGCGGCGACCTGCGTCGCTCGGCGGGCTCCGGGCCCCGGCGCC

CAGCGAGGCCCCTCCCCGTGCGCGGGCGCGCCCAGCTGCCCGACTGGAGCATGC

TGCTGGCCGCGATCACCACCCTGTTCCTGGCGGCCGAGAAGCAGTGGATGATGCT

GGACTGGAAGCCCAAGCGCCCCGACATGCTGGTGGACCCCTTCGGCCTGGGCCG

CTTCGTGCAGGACGGCCTGGTGTTCCGCAACAACTTCAGCATCCGCAGCTACGAG

ATCGGCGCGGACCGCACCGCCAGCATCGAGACCCTGATGAACCACCTGCAGGAG

ACCGCCCTGAACCACGTGAAGAGCGTGGGCCTGCTGGAGGACGGCCTGGGCAGC

ACCCGCGAGATGAGCCTGCGCAACCTGATCTGGGTGGTGACCAAGATGCAGGTG

GCGGTGGACCGCTACCCCACCTGGGGCGACGAGGTGCAGGTGAGCAGCTGGGCG

ACCGCCATCGGCAAGAACGGCATGCGCCGCGAGTGGATCGTGACCGACTTCCGC

ACCGGCGAGACCCTGCTGCGCGCCACCAGCGTGTGGGTGATGATGAACAAGCTG

ACCCGCCGCATCAGCAAGATCCCCGAGGAGGTGTGGCACGAGATCGGCCCCAGC

TTCATCGACGCGCCCCCCCTGCCCACCGTGGAGGACGACGGCCGCAAGCTGACC

CGCTTCGACGAGAGCAGCGCCGACTTCATCCGCAAGGGCCTGACCCCCCGCTGG

AGCGACCTGGACATCAACCAGCACGTGAACAACGTGAAGTACATCGGCTGGCTG

CTGGAGAGCGCGCCCCCCGAGATCCACGAGAGCCACGAGATCGCCAGCCTGACC

CTGGAGTACCGCCGCGAGTGCGGCCGCGACAGCGTGCTGAACAGCGCCACCAAG

GTGAGCGACAGCAGCCAGCTGGGCAAGAGCGCCGTGGAGTGCAACCACCTGGTG

CGCCTGCAGAACGGCGGCGAGATCGTGAAGGGCCGCACCGTGTGGCGCCCCAAG

CGCCCCCTGTACAACGACGGCGCCGTGGTGGACGTGCCCGCCAAGACCAGCGAT GACGATGACAAGCTGGGATGACTCGAGGCAGCAGCAGCTCGGATAGTATCGACA CACTCTGGACGCTGGTCGTGTGATGGACTGTTGCCGCCACACTTGCTGCCTTGAC

TACGCGCTTTTGCGAGTTGCTAGCTGCTTGTGCTATTTGCGAATACCACCCCCAGC

ATCCCCTTCCCTCGTTTCATATCGCTTGCATCCCAACCGCAACTTATCTACGCTGT

CCTGCTATCCCTCAGCGCTGCTCCTGCTCCTGCTCACTGCCCCTCGCACAGCCTTG

GTTTGGGCTCCGCCTGTATTCTCCTGGTACTGCAACCTGTAAACCAGCACTGCAA

TGCTGATGCACGGGAAGTAGTGGGATGGGAACACAAATGGAAAGCTTGAGCTC

SEQ ID NO: 55

Prototheca moriformis FatB/A promoter/5 'UTR

CCTGTCGATCGAAGAGAAGGAGACATGTGTACATTATTGGTGTGAGGGCGCTGA

ATCGGCCATTTTTTAAAATGATCACGCTCATGCCAATAGACGCGGCACATAACGA

CGTTCAAACCCCCGCCAAAGCCGCGGACAACCCCATCCCTCCACACCCCCCACAC

AAAGAACCCGCCACCGCTTACCTTGCCCACGAGGTAGGCCTTTCGTTGCGCAAAA

CCGGCCTCGGTGATGAATGCATGCCCGTTCCTGACGAGCGCTGCCCGGGCCAACA

CGCTCTTTTGCTGCGTCTCCTCAGGCTTGGGGGCCTCCTTGGGCTTGGGTGCCGCC

ATGATCTGCGCGCATCAGAGAAACGTTGCTGGTAAAAAGGAGCGCCCGGCTGCG

CAATATATATATAGGCATGCCAACACAGCCCAACCTCACTCGGGAGCCCGTCCCA

CCACCCCCAAGTCGCGTGCCTTGACGGCATACTGCTGCAGAAGCTTCATGAGAAT

GATGCCGAACAAGAGGGGCACGAGGACCCAATCCCGGACATCCTTGTCGATAAT

GATCTCGTGAGTCCCCATCGTCCGCCCGACGCTCCGGGGAGCCCGCCGATGCTCA

AGACGAGAGGGCCCTCGACCAGGAGGGGCTGGCCCGGGCGGGCACTGGCGTCG

AAGGTGCGCCCGTCGTTCGCCTGCAGTCCTATGCCACAAAACAAGTCTTCTGACG

GGGTGCGTTTGCTCCCGTGCGGGCAGGCAACAGAGGTATTCACCCTGGTCATGGG

GAGATCGGCGATCGAGCTGGGATAAGAGATACTTCTGGCAAGCAATGACAACTT

GTCAGGACCGGACCGTGCCATATATTTCTCACCTAGCGCCGCAAAACCTAACAAT

TTGGGAGTCACTGTGCCACTGAGTTCGACTGGTAGCTGAATGGAGTCGCTGCTCC

ACTAAACGAATTGTCAGCACCGCCAGCCGGCCGAGGACCCGAGTCATAGCGAGG

GTAGTAGCGCGCC

SEQ ID NO: 56

Prototheca moriformis NRAMP metal transporter promoter/5 'UTR

ACTAATTGCAATCGTGCAGTAATCATCGATATGGTCACAAGTAGATCCCCTACTG

ACACCCTCTCGTACATGTAGGCAATGTCATCGGCGCCGTCCTGCTGACCGATGCC

GACGTAGCAGAGCAGACCCGGGCCGATCTGGGATACGAGCCGGCCCTCCACCTG

CGCTCGAGGTGGAATCAAGTAAATAACCAATACACTTTTCGACACCACACAGAG

TTGCACGGACGGTGGCGTACCTCTACGCTCGCGCTCTTCACGCGCTGGACGACCG

CACGCATGAGCCCGGGTGGCTTGGTCTGGGCTGCAAAAATGCACAACAAACAAG

TATCAGACGCTCATGGATGCACACGCGCTCCCAAGCACGCTCAGACTAAATATTA

CAGTAGCTCGTATCTGATAAGATATCGAGACATACCGCTCAACTCACCCGCAAAC

TGCGCCCCGCCAGGTGATGCGCACAGGGCCCCACCATGCGATCCATCGCATCGCT

CCTCGAGGGCGCTATCACGTGGCCGGAGAGCGTTCACAGCGTACGCCACTGTATC

TGGGCGGTATGCGGTCCGTCAACATGGAGACAGATACCCGCACCACCACCTTGC

AAGCTCTTCCATATTGGAAGTAGAAAATTGTAATTGTATCATCGCACGAGGGGCC

AACTTGCCGTCGGCGAGCTGGGCGACGAACACCACCTGGACGTTGTCGAGACTC

GCTCGTGCCGTGCGCCGGGCCGCTGGGTATCCAGACCGTCGCC SEQ ID NO: 57

Prototheca moriformis FLAP Flagellar-associated protein promoter/5 'UTR

CAACGACAACCAGCAGGCAACTCGGTCAGCGACCCAACACGCGAGTCAAATTGT

TGCGTGTTCTTGCCTTGTCTATTTACTGTGATAGCAAGACTGTCGGTCAGTCAATA

CCGCGGTGCGCACGTCGGGGTGCCAAGCCTAGCAGAGCACGGGACGGCTGGTGC

TGTGCGCCAGCTCAGCTCGCTTCGCGACCAATTGTAGGACCGGCAAAGTCACCAA

AACATGCCAGCGGTGCGATTCAATTGGTCATGAGCTCTACAAAATTGTTTTGTGC

GTCGCGCAGGTATCCAACGGCGCGGCAGAGAAAGTTTGACAGCTCTCGATTTCAT

CTCGGAAAAATGGGGAGAATTTATGACACACAAGTGCGCAGGCGGCCCAGGCGG

CCAGCATATTCTGGCGTGACCTGGGCCGCCCACAAAATGCTTGGATGCACTCTAA

AATAATTATATTTGCCATGAACAAGGGAAGAGTTACCGCACCCAGCCCTAGACTT

GGGCGCCCGAGCAAGGTTACGTCAAGCCACCTTCGCCCATCGCCCAACTCCGTAT

TCCCCGACAGCCGCACGTGGCCCTCGCCGGAATGAACCCTGAATCGGCATCACG

CCACGCGTTCGCCAATCGTTCCGCTCTCTGGCTTCATCGGCCTGCGCCTTCACGTC

GTGGTCACGACAGTGCATTCATACTTCCATTTGCACCTCGGCACACACTTTTACG

CATCGCCTACCCTTGCTGCGGCAGTCTAGGGTCACTTTGCAGCCATGGGACAGTG

CTACACCACCGTCGGTGCGCAAAGCTATTTCAAGTGAACCGTGGGCGGAAAAAA

GGAATGTACACTGTCTCAACCGACTCCTACAATTGTTTACCATGCAGATCAGAGC

TCGACGGCCATCATCGAGCAGGTGTGGGGCCTTGGTGGCGCGGCGCGGGGCCCC

AGGGCGTCGCAGGCATTGATGGCACTCTGAGACTTTCGCACGCGCATGAGGGAC

CCCATCAAGAGAAGAGTGTGTCTTTATGTCCCCATTCATGATGATGTATCTTGTG

ATTGTCGCAGTTTGGCAAGTTTAACCGGATCGCCGCTCCAGGTGTGGCGTGGCGG

ATTTTTCTAGGGGTGCTTGAGCAGTCG

SEQ ID NO: 58

Prototheca moriformis SulfRed Sulfite reductase promoter/5 'UTR

GGCCCAGGGCCCTGCGGATGGCCCACACCAGATCTAGCCTCTCTTATGCCATGCC

CGCCTCGCTGCCCGTCGTATCCCCCCGCCGATCCGCGCGTAGGGGACCGCGGCCT

GACCCACGCCACGAAAGAGCTTTGCTCCTCAATTTCTCGCCAACAGAACCGTATC

AAACGCTCAACGCCTATCCCGAACAATCCGTATTCACACCAAATCGAGTATACCG

GACTGGTTTGCCTAGTCTTGAAGGAAATGATCCCGTCCATGCTCGGAAGGGGGA

GCGGGCGGAGGATCCTACTCATCTCTGAAATGGGATTGGTCCGAAGATGGGTTG

GGCAAGCACGTGCCAAACCCCAGCGAGTTGCTGACGAGCAGGCTCATCCAATCC

CCCGGCGAATCCTCCCTCACGCCCCGCATGCATACAAGTCCCTCCCACACGCCCC

CTCCCATCCATTTTCGCCTGGTCCGAACGCGAGCGGCGTCGAGGCGGACCACTTG

CTCCGCAGCGCCGTCTGGGTCTCCACCCCACAGCGGCTTTGCTGCCAGAGGCACC

CCCCTTGCCCCACCTCCTCTTGCAGCC

SEQ ID NO: 59

Prototheca moriformis SugT Sugar tranporter promoter/5"UTR

CCAGGCAGGCGGTAGGGTTGCCGATTGCTTGAGCGAATTGGAAGATATAATTTTT

TGTGGTGTCCCTGGACGCTGTTTGTGGCGCTCCTTTTTGGAGAAGATTGCGTGGG

GGAGCTTTCCATGTACCACGCTTCCTTCTGAAAGGATTCTGGCCGAGTCCTGATG

AGCCCAAAGAAAACACCTGCCTTTCAGTGCTGGCACTCTGAAAACGTCAACAGA

TGATTATACATGTCACAAAAGGCAGCCGATTAGGAACGGGAGCTCTGGCCGTTC

GTTTGGCTGCCTGGGCTGATTGAAGTGATCCACCCTGTTCGAATGAAGGCGGTCG

AGTCGAATTATCGACCGGAGCTGTCGGGAAGGCGTCCGGGGCAGAGTGAGGTGC TGCGGCCTGGTTGTCGTTCAAAAAGACCCCGGTAGCCCAACAATCACGAACGAA AGGAATATAATTGCTTGCATACTATACATTCAGTTTCTATGTGGCGGGTAGACAA GTCTCATGGGCTTCTAAAGGCTGTCCCTTGAAGGCTACTTATAAAAACTTGCTGC GCCATGGCACGGATCGCGCTTGCGCAGGCTGCAACCCTGCGCGCAAGGTCAAAT ACACAGCAAAAGATACTAACAGAATTTCTAAAAACATTTAAATATTTGTTTCGAC

CCACGCTGGCAGTCAAGCCAGTCCGATGTGCATTGCGTGGCAGCATCGAGGAGC

ATACCCGATGCATCGCGGTGCGCAGCGCGCCACGCGTCCCAGACCCGCCCAAAA ACCCAGCAGCGGCGAAAGCAAATCTTCACTTGCCCGAAACCCCGAGCAGCGGCA TTCACACGTGGGCGAAAACCCCACTTGCCCTAACAGGCGTATGTCTGCTGTCACG ATGCCTGACAACGGTATTATAGATATACACTGATTAATGTTTGAGTGTGTGCGAG TCGCGAATCAGGAATGAATTGCTAGTAGGCACTCCGACCGGGCGGGGGCCGAGG GACCA

SEQ ID NO: 60

Prototheca moriformis Amt03 -Ammonium transporter promoter/5 'UTR

GGCCGACAGGACGCGCGTCAAAGGTGCTGGGCGTGTATGCCCTGGTCGGCAGGT

CGTTGCTGTTGCTGCGCTCGTGGTTCCGCAACCCTGATTTTGGCGTCTTATTCTGG

CGTGGCAAGCGCTGACGCCCGCGAGCCGGGCCGGCGGCGATGCGGTGTCTCACG

GCTGCCGAGCTCCAAGGGAGGCAAGAGCGCCCGGATCAGCTGAAGGGCTTTACA

CGCAAGGTACAGCCGCTCCTGCAAGGCTGCGTGGTGGACTTGAACCTGTAGGTCC

TCTGCTGAAGTTCCTCCACTACCTCACCAGGCCCAGCAGACCAAAGCACAGGCTT

TTCAGGTCCGTGTCATCCACTCTAAAACACTCGACTACGACCTACTGATGGCCCT

AGATTCTTCATCAACAATGCCTGAGACACTTGCTCAGAATTGAAACTCCCTGAAG

GGACCACCAGAGGCCCTGAGTTGTTCCTTCCCCCCGTGGCGAGCTGCCAGCCAGG

CTGTACCTGTGATCGAGGCTGGCGGGAAAATAGGCTTCGTGTGCTCAGGTCATGG

GAGGTGCAGGACAGCTCATGAAACGCCAACAATCGCACAATTCATGTCAAGCTA

ATCAGCTATTTCCTCTTCACGAGCTGTAATTGTCCCAAAATTCTGGTCTACCGGGG

GTGATCCTTCGTGTACGGGCCCTTCCCTCAACCCTAGGTATGCGCGCATGCGGTC

GCCGCGCAACTCGCGCGAGGGCCGAGGGTTTGGGACGGGCCGTCCCGAAATGCA

GTTGCACCCGGATGCGCGGCGCCTTTCTTGCGATAATTTATGCAATGGACTGCTC

TGCAAATTTCTGGGTCTGTCGCCAACCCTAGGATCAGCGGCGTAGGATTTCGTAA

TCATTCGTCCTGATGGGGAGCTACCGACTACCCTAATATCAGCCCGGCTGCCTGA

CGCCAGCGTCCACTTTTGCGTACACATTCCATTCGTGCCCAAGACATTTCATTGTG

GTGCGAAGCGTCCCCAGTTACGCTCACCTGTTTCCCGACCTCCTTACTGTTCTGTC

GACAGAGCGGGCCCACAGGCCGGTCGCAGCC

SEQ ID NO: 61

Prototheca moriformis Amt02- Ammonium transporter promoter/5 'UTR

TCACCAGCGGACAAAGCACCGGTGTATCAGGTCCGTGTCATCCACTCTAAAGAG

CTCGACTACGACCTACTGATGGCCCTAGATTCTTCATCAAAAACGCCTGAGACAC

TTGCCCAGGATTGAAACTCCCTGAAGGGACCACCAGGGGCCCTGAGTTGTTCCTT

CCCCCCGTGGCGAGCTGCCAGCCAGGCTGTACCTGTGATCGGGGCTGGCGGGAA

AACAGGCTTCGTGTGCTCAGGTTATGGGAGGTGCAGGACAGCTCATTAAACGCC

AACAATCGCACAATTCATGGCAAGCTAATCAGTTATTTCCCATTAACGAGCTATA

ATTGTCCCAAAATTCTGGTCTACCGGGGGTGATCCTTCGTGTACGGGCCCTTCCCT

CAACCCTAGGTATGCGCACATGCGGTCGCCGCGCAACGCGCGCGAGGGCCGAGG GTTTGGGACGGGCCGTCCCGAAATGCAGTTGCACCCGGATGCGTGGCACCTTTTT

TGCGATAATTTATGCAATGGACTGCTCTGCAAAATTCTGGCTCTGTCGCCAACCC

TAGGATCAGCGGTGTAGGATTTCGTAATCATTCGTCCTGATGGGGAGCTACCGAC

TGCCCTAGTATCAGCCCGACTGCCTGACGCCAGCGTCCACTTTTGTGCACACATT

CCATTCGTGCCCAAGACATTTCATTGTGGTGCGAAGCGTCCCCAGTTACGCTCAC

CTGATCCCCAACCTCCTTATTGTTCTGTCGACAGAGTGGGCCCAGAGGCCGGTCG

CAGCC

SEQ ID NO: 62

Protoheca moriformis Aat-OlAmino Acid Transporter promoter/5 'UTR

CGAAGGGGTCTGCATCGATTCGCGCGGTCTGGAGGCCAGCGTGACTGCTCGCGA

AAATGCTCTGCCGTGTCGGGCTCTGGCTGGGGCGGCCAGAGATCTCACCGTGCCA

CACGCAACTGCCGCACTCTGTGCCCGCCACCTGGCGCGCACATGCGACCTCTTCC

CCGTCATACCCTCTCCTCATGTGATCTTTCCACACGAGTGACGCAGGTGCGCGGA

GTGGAGGGAATCAGGACGTTTTCAAGGTACCTGCTCGAGCCGTACCAACAGCTG

CCGCCCGGCAAGGAAGAGATCGAGGCAGAGATTGCCCGGCTGGAGGCCCGGATA

ACGGAGCTCAAGAGCAAGCTGTCCGAGTGAGACCGCCCAGGTGCACGTGTCGAC

TCGCTATGACATGTACTCGACACAACATGAGGAATTCATCGAATTTGTAGGAAGC

GGGCATTGGTACGGGAGTGGGAAAGCGAAAAAACCTCCCTCCGGCAGTGCCATC

TGCCGGAGTCGAACGTTGATAGGGTTCTCGTGACAGGGTGTGACCTCTCAGCCTT

GCATCAATTAAACGCTATAGACATTATCAGTAACCGTGAATCCCGCATTGGATGC

CACCCGCGCGACCATTGGGGACCTGCATTACAGATCTAGGTGAGATGACAGCGA

GGCAACTTCGGCCCGCGGCCCAGCTTGCGGCGCACCAATATTGGTCACGGGAAG

CCACACACCGACCATAAATGAATACTTGTAAGCTATGTCAACCGATCAATGGCGT

CGAAAGTGTGCCACGAGGATCCATCTGGCGGGGCGGCGTGGCGCACAAGCGCAG

TCGCAATTTCTCGGACCCATCTGACCTAGGCCCAGCGCCGCGGGAGAAATCCCCG

GCGGGTCCTCCACGCAGTAACCCTAATGAGTATCGAGCGCCGACCATTTACACCA

TCGCCCCCGAAATCCTTCCGACATTATTATTATCTTTTAGATCTTGGAACAGACTC

TGCCAACC

SEQ ID NO: 63

Prototheca moriformis Aat02- Amino acid transporter promoter/5 'UTR

AGAGAGCGGAGGTGGGGTTGTGAGGTGGGGTTGCTGACCAGGAGCTCGCGTCGC

CGAGCGCGACTCGCACACGGTCCAGTTACCCCCCCCTCCGCCCAAACGCAAGCCT

CCCATCTTGATGCCTTTCCGGCCACCTATACTATTTCTTAGTTCGCTGTAACATCC

AGACCGTCCTGAATAATAACAATGCCCTGTGTCAAGTGCATTCCTAAAAAAATTC

TGTCCCAACCAACAATCCCACCTGAAATACCACCAGCCCTGCCCAGTACACTCTT

CCAATACCATCTCCCTACCTCCACGCGCAAGCGACCCCCATGCGCGACCAGGCTC

GAAAGTGATTTATGACTTGAGACGAGCGAGTGGCGGCGCGGTCGACTGCCTTTTC

ATCACGTGCCGTACGTCGGCGACCGCTAGGGCTTTGCACGGCAACGCACGGCTTC

GCCAACCCGACCAGCCAGGACCTCGACTACTCTACCGCGAATTCGCCTCAAGAA

GTCGCCAAATGTGCCATACACCATTCCTTACAGCACTGTTCAAACTTGATGCCAA

TTTTGACATTCGGGTTGCTCGTTGGCTGCGCCCACATCGGCCGTGAGTGCAGCAG

GCGGGATCGGACACGGAGGACGCGGCGTCACGCCCCGAACGCAGCCCGTAACTC

TACATCAACACGACGTGTTGCGTAATCCCGCCCGGCTGCGCATCGTGCCAACCCA

TTCGCGATGGATGGTCGGAAAATGGTGTGCCAACTGCCCTGAGGGAGGCTCTCG

CGAAACGGGCACGTCCCTGAAACCGAAACTGTGGCCTTGTCGTCGGCCACGCAA

GCACGTGGACCCTAAACACCAAGAAAATCAGTAAACAAGGTTGACATCCTCTAC GGGCGAATTGTTTGCCCAACCCTTCATCGCACACTGCCATTATAATGCATCTAGC

TCGGCGACAAGTTTAGAAAAGGCAGGCTGCATTGTTCCATTTCGCCGTGGCGGCG

TGGGTGCCCATTTTACGAGGTTTGGGCTCCCGGGCAGCGACCGAGCCAGGTCGA

GTCCCTCTCGCCCGTCGACAATGTTGCGAACCCCACAAGCGGCTAACAACAACTT

GATGGTACCTGTACACTGCCAATTCCTTCTTCCCCGGCCGAGGTTTACACGTGAT

GGCCATGGCTTCGCATTCAGGCCGACTTCCCATTCCGACTTTCCAGAGGGTCCGC

GGACGCTGGGGGTTGGCTGCCTGAGGCCCACCCTTTGTTCCCCGCGTCCCGACAA

ACACAATTGCGTTACATAAGGGGGAGCCGCCCCCGTTCAGAGTGCAGAAATCTTT

CACTATATTTTCCAGTCGTCAGCGAAATCAAGT

SEQ ID NO: 64

Prototheca moriformis Aat03- Amino acid transporter promoter/5 'UTR

GATGGTGGGGTGTCTGCCTTGGGCTGGGTGATGGAGGCTGGTGGTGCGCGGGTTT

CCTGATGCATTCTATCTACGCAGTGTCATGGTGTCCATTCCACACACCAGTACAC

CCTTACACTAAGGATCCATCCCTCCTTCCCTCTTCAGGACTACATGGACCCCACG

AGCTACCGACCGGGCTTTCTCAAAAACGTCAAGGTCATGTTTGACATGCGGGACG

TGGTGGACGACGTGCAAGGTGCGTCCGGAGTGCGCGCAAATGAGCAAGTCGGGC

AATGTGTCGGGGTGGGCACCGGGGCTGGAGATCCGCGATCCCCGAGAAAACGCC

GTACCACCCCCCGCGCTATTCCCTCGATTGCGCGCAGATGTGGTGACCGACACGG

GGGACAACCTGGCGGACATGGGGCGCCGGACCTGGAAGCACGCCAAGTCGCACA

CGGGGAGGCTCGTGCAGTCCCCCCCATCGTACCTCAAGGGTCTCTTTGGTCGCGA

TCCAAAGTACGCTGGTGGCATGGCATGCCCGAAATGAACATCATGTGTGATCTCC

GATTGCCAATGGCCACCTCCACGGACCACCTTGCAGGCGGAAGCGCAATCCAGG

GCCCGAGCCTGACGAGGACGGAGACTCCTCGTCCAGCGCGGGGTCCCCGACCCG

ACGCAGCAGCCGACCCCTGCTAACCCGGCAACGATCGGACCAGCAACCTTGCTG

TAGTTCCGATCCGTGATGACGGGCATTGCCGCCGCTCGATCCGCTTTGATGACTG

TCTATTATTTGCGCGGAGCCCCCTCGGAACCCTACCCCGCTCTTGCAAGCCCCTTG

CATCGGAGATCCTCGTGCGCCCGCCATGACCCCACTGGATTGCCCAACATCCTTC

TTTATCGTGTAAAATGTGATTCCTCGGCTGCAATCGACTGGCCTTCGCTTCTGGCC

CCAAGAGGGCTCGAACGTGCGGCAGCGAGGGCGCTGACACACCCAAGCCCTAGG

GCTTTCAACGTCGGCTGCCAGGCCGGATAGGGGGATCGCCTCCTTTCCACCACCC

ACCTACGAGGGATTCGAGTCGGCTTCCAGCTCAGCTATTCGGCCGCGCCCCCGGC

CCTGCAGACGTCCTCCAGTTTCCGAACAGGTCGCTCTCAGAACACCTGCCGCGGC

TGCGATACGGCAGGCTCTCAAAGCGTCGAC

SEQ ID NO: 65

Prototheca moriformis Aat04- Amino acid transporter promoter/5 'UTR

CGCGTGGAGCGGTGCGTGCGGATGCCGCGCGCCTGCCAAGGCCTTTTGTATGCCT

GGCCTGGGAAGTTTCCTGACTGAAGCATCTTCAAGATGCTCTCTCACGACCAGCG

ACACCAACACCGTCACTTTTTGCCCCTCCTGCCGCAGGTGCCACTTTCTACTTTGA

CGTCTTCTCCAGGCGGTACATTGCGGGACTGAGCGCCAATTCGGCCAAGAACAG

CGCTGTCGACTTGAGGAGGCAGGGGTCCGTCGACTCTGCCGAGTGACACGCCTTC

GACCCGACTGTACTACGGCCTGCTGAAGAGTGGGTCTCGCCGGCCGGCGTGACC

GGCCCTGTGCCCACAATCGACCATCTATTCGCTCCTTGTCATCTGGCGCCGTCAAT

TGCCCGCGACTTGACGGCAACTGGCTCGATCGAGTCGTATTGAAAAAGCACGTTT

TGTCCTACAGGGCCGCGGTCCGTTACCAACGTGGTTCTCGTTAGGTTTTCGTCGG

GCGGTGGTGCGCGAACTGTCCGATGCCATCCCGGCAAACCCCAGCAAGGTCGCC

AGTCTGGTTCTGACGCAATAGAGTGCGTTTTGGGCCAGTCTAAAAATTCGTCTGG CATGACGTGGCTCCACATCGTACCCGGAGCCTGCCTTGGTAATGTGAGGCACCGG

TGCCAACTCCATTATGGCAGGCATCGAGCGCGCAGGTGAGTACATGACCTTCCGT

GAATTGGGAAGGCGAGCTTGTGTAACGCCTGCGATCGTGCCAGTGAGGCATCGT

AAACTCAAAATATTTTGTAGAAAGTGTCTGATGCCTGGTGAGGCTGCGTAGGGCA

AGGGCAAGCCCTTGGCAGATGGGTAATGGGTCCGGACCTCACAACAGCAACCCC

GCGTCCCCCTTAGGGCCCCTGAGGCTCGATGGCAGGGCCAGCGAGCCCGCGGCC

AAAGGGCGCCATCCCACGGTCGCCCAACGACTCCACGGGTCCTATACCTCATCTT

GAATGGCACTAAAAACTATAGAATATCGGGCACTGGTGGGCGTCTGGGGTACAG

CTGGCCGAGCGCAGTGGCAAACCCTAGGTCCCGCCTCAAGGGCGATTCCCGGGT

CAATGACACGCAAGCAAGATCACATGGCGCGGTCCGCCTCGCGGCTCCACACCC

AGGCCCTAGTTTCGCAACCCATAAATATCGCCCCGATACCATCATAAGCCAGCAA

ATAATTTTTTATCAGAGTTCCAAACCTCCTCAGCTGTGGGAAACCAGCCCACTCT

GAACG

SEQ ID NO: 66

Prototheca moriformis Aat05- Amino acid transporter promoter/5 'UTR

CCGAGCAGTTCATGGCCAAGTACAAGGACTAGAGACCGGAGGTCGGTAGGCTGA

ATGGAGCTGGCGTCGTCGTGCGCGACGTGCACGCGATGCGATACTACGACCCCA

CAAACGCATGCCTCCCATCTTGATGCCTTTCCGGCCATTTATACTATTTCTCATTT

CGCTGTAACATCTTGAATAATAGAATTGCCCTGTGTCAAGTGGATTCCAAGAAAT

ATTCTGTCCCAACAAAACAACCCAACCTGAAAACAACCTCAAATACCACCAGCC

CGGCACCCATGCGCGACCAGGCTCGAAAGGATTTCACGACTCAGGACGAGCGAG

TGGCGGCGCGACCGCCTGCCTGTTCGTCACGTGCCGTACGTCGGCGACCGCTAGA

GCTTTGCCTGGCAACCCCCGGCTTCGTCAACCCGGCCAGCCAGGATCTCGACCAC

TCTACCGCGAAATCGCCTCAAGAAGTCGCCAAAAGTGCCGTACACCATGCTTCGC

AGCGCTGTTCAAACTTGATGCCAATCTTGACAATCAGGTTGCTCGTTGGCTGCGT

CCACATCGGCCGTGATTGCAGCAGGCGGGGATCGGACACGGAGGACGCGGCGTC

ACGCCGCGAACGCAGCCCGTAACTCTACATCAACGCGATATGTTGCGTAATCCCG

CCCGGCTGCGCATTGTGACAACCCATTCGCGATGGATGGTCGGAAAATGGTGTGC

CAACTGCCCTGAGGGACTCTCTCGCGAAACGGGCACGTCCCTGTATCCGAAACTG

TGGCATGGCCTTGTCGACCACGCAAGCACGTGGACCCTAACACCACGAAAATAA

GTAAAAAAGGTTGACATCCTCTACGAGCGAATTGTTTGCTCGACCCTTCATCGCA

CACTGTCATTATAATGCATCTAGCTCGGCGACAAGTTTAAAAAAGGCAGGCTGCA

TTATTCCATTTTGCCGTGGCGGCATGGGTGCCCATTTTATGAGGTTTGGGCTCTTG

GGCAGCGACCGAGCCAGGTTGAGTCCCTCTCGCCCGTCGACAACGTTCCAAAGC

CCATAAGTGGCTAATAAACAACTTGATGGTACCTGTACACTGCCAGTTCCTTCTT

CCCCGGCCGAGGTTTACACGTGATGGCCATGGCTTCGCGTTTCAGGCTGACTTCC

CATTCCGACTTTCCAGAGGGTCCGCGGACGCCGGGGGTTGGCTGCGTGAGGCCC

ACCCCTTGTTCCCCGCGTCCCGACAAACACAATTGCGTTACATAAGGGGGAAGCC

GCCCCCCGTTCAGAGTGCAAACATCTTTCATTATATTTTTCAGTCGTCAGCGAAAT

CAAGTATGTCGCTGACAGGCATGAAGGCC

SEQ ID NO: 67

Relevant portions of the reporter construct for testing putative promoter/5 'UTR of Prototheca moriformis AatOl.

GCTCTTCGGCCGCCGCCACTCCTGCTCGAGCGCGCCCGACTCGCGCTCCGCCTGC GCCCGCGCGTGCGCCGCCAGCGCCTTGGCCTTTTCGCCGCGCTCGTGCGCGTCGC TGATGTCCATCACCAGGTCCATGAGGTCTGCCTTGCGCCGGCTGAGCCACTGCTT

CGTCCGGGCGGCCAAGAGGAGCATGAGGGAGGACTCCTGGTCCAGGGTCCTGAC

GTGGTCGCGGCTCTGGGAGCGGGCCAGCATCATCTGGCTCTGCCGCACCGAGGC

CGCCTCCAACTGGTCCTCCAGCAGCCGCAGTCGCCGCCGACCCTGGCAGAGGAA

GACAGGTGAGGGGGGTATGAATTGTACAGAACAACCACGAGCCTTGTCTAGGCA

GAATCCCTACCAGTCATGGCTTTACCTGGATGACGGCCTGCGAACAGCTGTCCAG

CGACCCTCGCTGCCGCCGCTTCTCCCGCACGCTTCTTTCCAGCACCGTGATGGCG

CGAGCCAGCGCCGCACGCTGGCGCTGCGCTTCGCCGATCTGAGGACAGTCGGGG

AACTCTGATCAGTCTAAACCCCCTTGCGCGTTAGTGTTGCCATCCTTTGCAGACC

GGTGAGAGCCGACTTGTTGTGCGCCACCCCCCACACCACCTCCTCCCAGACCAAT

CCCAGCCGCTGGGGGTTGGCGGATGCACGCTCAGGTACCCTTTCTTGCGCTATGA

CACTTCCAGCAAAAGGTAGGGCGGGCTGCGAGACGGCTTCCCGGCGCTGCATGC

AACACCGATGATGCTTCGACCCCCCGAAGCTCCTTCGGGGCTGCATGGGCGCTCC

GATGCCGCTCCAGGGCGAGCGCTGTTTAAATAGCCAGGCCCCCGATTGCAAAGA

CATTATAGCGAGCTACCAAAGCCATATTCAAACACCTAGATCACTACCACTTCTA

CACAGGCCACTCGAGCTTGTGATCGCACTCCGCTAAGGGGGCGCCTCTTCCTCTT

CGTTTCAGTCACAACCCGCAAACGGCGCGCCATGCTGCTGCAGGCCTTCCTGTTC

CTGCTGGCCGGCTTCGCCGCCAAGATCAGCGCCTCCATGACGAACGAGACGTCC

GACCGCCCCCTGGTGCACTTCACCCCCAACAAGGGCTGGATGAACGACCCCAAC

GGCCTGTGGTACGACGAGAAGGACGCCAAGTGGCACCTGTACTTCCAGTACAAC

CCGAACGACACCGTCTGGGGGACGCCCTTGTTCTGGGGCCACGCCACGTCCGAC

GACCTGACCAACTGGGAGGACCAGCCCATCGCCATCGCCCCGAAGCGCAACGAC

TCCGGCGCCTTCTCCGGCTCCATGGTGGTGGACTACAACAACACCTCCGGCTTCT

TCAACGACACCATCGACCCGCGCCAGCGCTGCGTGGCCATCTGGACCTACAACA

CCCCGGAGTCCGAGGAGCAGTACATCTCCTACAGCCTGGACGGCGGCTACACCTT

CACCGAGTACCAGAAGAACCCCGTGCTGGCCGCCAACTCCACCCAGTTCCGCGA

CCCGAAGGTCTTCTGGTACGAGCCCTCCCAGAAGTGGATCATGACCGCGGCCAA

GTCCCAGGACTACAAGATCGAGATCTACTCCTCCGACGACCTGAAGTCCTGGAA

GCTGGAGTCCGCGTTCGCCAACGAGGGCTTCCTCGGCTACCAGTACGAGTGCCCC

GGCCTGATCGAGGTCCCCACCGAGCAGGACCCCAGCAAGTCCTACTGGGTGATG

TTCATCTCCATCAACCCCGGCGCCCCGGCCGGCGGCTCCTTCAACCAGTACTTCG

TCGGCAGCTTCAACGGCACCCACTTCGAGGCCTTCGACAACCAGTCCCGCGTGGT

GGACTTCGGCAAGGACTACTACGCCCTGCAGACCTTCTTCAACACCGACCCGACC

TACGGGAGCGCCCTGGGCATCGCGTGGGCCTCCAACTGGGAGTACTCCGCCTTCG

TGCCCACCAACCCCTGGCGCTCCTCCATGTCCCTCGTGCGCAAGTTCTCCCTCAAC

ACCGAGTACCAGGCCAACCCGGAGACGGAGCTGATCAACCTGAAGGCCGAGCCG

ATCCTGAACATCAGCAACGCCGGCCCCTGGAGCCGGTTCGCCACCAACACCACG

TTGACGAAGGCCAACAGCTACAACGTCGACCTGTCCAACAGCACCGGCACCCTG

GAGTTCGAGCTGGTGTACGCCGTCAACACCACCCAGACGATCTCCAAGTCCGTGT

TCGCGGACCTCTCCCTCTGGTTCAAGGGCCTGGAGGACCCCGAGGAGTACCTCCG

CATGGGCTTCGAGGTGTCCGCGTCCTCCTTCTTCCTGGACCGCGGGAACAGCAAG

GTGAAGTTCGTGAAGGAGAACCCCTACTTCACCAACCGCATGAGCGTGAACAAC

CAGCCCTTCAAGAGCGAGAACGACCTGTCCTACTACAAGGTGTACGGCTTGCTGG

ACCAGAACATCCTGGAGCTGTACTTCAACGACGGCGACGTCGTGTCCACCAACA

CCTACTTCATGACCACCGGGAACGCCCTGGGCTCCGTGAACATGACGACGGGGG

TGGACAACCTGTTCTACATCGACAAGTTCCAGGTGCGCGAGGTCAAGTGACAATT

GGCAGCAGCAGCTCGGATAGTATCGACACACTCTGGACGCTGGTCGTGTGATGG

AACAGCCTCAGTGTGTTTGATCTTGTGTGTACGCGCTTTTGCGAGTTGCTAGCTGC TTGTGCTATTTGCGAATACCACCCCCAGCATCCCCTTCCCTCGTTTCATATCGCTT

GCATCCCAACCGCAACTTATCTACGCTGTCCTGCTATCCCTCAGCGCTGCTCCTGC

TCCTGCTCACTGCCCCTCGCACAGCCTTGGTTTGGGCTCCGCCTGTATTCTCCTGG

TACTGCAACCTGTAAACCAGCACTGCAATGCTGATGCACGGGAAGTAGTGGGAT

GGGAACACAAATGGAGGATCCCGCGTCTCGAACAGAGCGCGCAGAGGAACGCT

GAAGGTCTCGCCTCTGTCGCACCTCAGCGCGGCATACACCACAATAACCACCTGA

CGAATGCGCTTGGTTCTTCGTCCATTAGCGAAGCGTCCGGTTCACACACGTGCCA

CGTTGGCGAGGTGGCAGGTGACAATGATCGGTGGAGCTGATGGTCGAAACGTTC

ACAGCCTAGGGATATCGAATTCATCGAATTTGTAGGAAGCGGGCATTGGTACGG

GAGTGGGAAAGCGAAAAAACCTCCCTCCGGCAGTGCCATCTGCCGGAGTCGAAC

GTTGATAGGGTTCTCGTGACAGGGTGTGACCTCTCAGCCTTGCATCAATTAAACG

CTATAGACATTATCAGTAACCGTGAATCCCGCATTGGATGCCACCCGCGCGACCA

TTGGGGACCTGCATTACAGATCTAGGTGAGATGACAGCGAGGCAACTTCGGCCC

GCGGCCCAGCTTGCGGCGCACCAATATTGGTCACGGGAAGCCACACACCGACCA

TAAATGAATACTTGTAAGCTATGTCAACCGATCAATGGCGTCGAAAGTGTGCCAC

GAGGATCCATCTGGCGGGGCGGCGTGGCGCACAAGCGCAGTCGCAATTTCTCGG

ACCCATCTGACCTAGGCCCAGCGCCGCGGGAGAAATCCCCGGCGGGTCCTCCAC

GCAGTAACCCTAATGAGTATCGAGCGCCGACCATTTACACCATCGCCCCCCGAAA

TCCTTCCGACATTATTATTATCTTTTAGATCTTGGAACAGACTCTGCCAACCACTA

GTATGGCCACCGCATCCACTTTCTCGGCGTTCAATGCCCGCTGCGGCGACCTGCG

TCGCTCGGCGGGCTCCGGGCCCCGGCGCCCAGCGAGGCCCCTCCCCGTGCGCGG

GCGCGCCCCCGACTGGTCCATGCTGTTCGCCGTGATCACCACCATCTTCTCCGCC

GCCGAGAAGCAGTGGACCAACCTGGAGTGGAAGCCCAAGCCCAACCCCCCCCAG

CTGCTGGACGACCACTTCGGCCCCCACGGCCTGGTGTTCCGCCGCACCTTCGCCA

TCCGCAGCTACGAGGTGGGCCCCGACCGCTCCACCAGCATCGTGGCCGTGATGA

ACCACCTGCAGGAGGCCGCCCTGAACCACGCCAAGTCCGTGGGCATCCTGGGCG

ACGGCTTCGGCACCACCCTGGAGATGTCCAAGCGCGACCTGATCTGGGTGGTGA

AGCGCACCCACGTGGCCGTGGAGCGCTACCCCGCCTGGGGCGACACCGTGGAGG

TGGAGTGCTGGGTGGGCGCCTCCGGCAACAACGGCCGCCGCCACGACTTCCTGG

TGCGCGACTGCAAGACCGGCGAGATCCTGACCCGCTGCACCTCCCTGAGCGTGAT

GATGAACACCCGCACCCGCCGCCTGAGCAAGATCCCCGAGGAGGTGCGCGGCGA

GATCGGCCCCGCCTTCATCGACAACGTGGCCGTGAAGGACGAGGAGATCAAGAA

GCCCCAGAAGCTGAACGACTCCACCGCCGACTACATCCAGGGCGGCCTGACCCC

CCGCTGGAACGACCTGGACATCAACCAGCACGTGAACAACATCAAGTACGTGGA

CTGGATCCTGGAGACCGTGCCCGACAGCATCTTCGAGAGCCACCACATCTCCTCC

TTCACCATCGAGTACCGCCGCGAGTGCACCATGGACAGCGTGCTGCAGTCCCTGA

CCACCGTGAGCGGCGGCTCCTCCGAGGCCGGCCTGGTGTGCGAGCACCTGCTGC

AGCTGGAGGGCGGCAGCGAGGTGCTGCGCGCCAAGACCGAGTGGCGCCCCAAGC

TGACCGACTCCTTCCGCGGCATCAGCGTGATCCCCGCCGAGTCCAGCGTGATGGA

CTACAAGGACCACGACGGCGACTACAAGGACCACGACATCGACTACAAGGACGA

CGACGACAAGTGACTCGAGTTAATTAACTCGAGGCAGCAGCAGCTCGGATAGTA

TCGACACACTCTGGACGCTGGTCGTGTGATGGACTGTTGCCGCCACACTTGCTGC

CTTGACCTGTGAATATCCCTGCCGCTTTTATCAAACAGCCTCAGTGTGTTTGATCT

TGTGTGTACGCGCTTTTGCGAGTTGCTAGCTGCTTGTGCTATTTGCGAATACCACC

CCCAGCATCCCCTTCCCTCGTTTCATATCGCTTGCATCCCAACCGCAACTTATCTA

CGCTGTCCTGCTATCCCTCAGCGCTGCTCCTGCTCCTGCTCACTGCCCCTCGCACA

GCCTTGGTTTGGGCTCCGCCTGTATTCTCCTGGTACTGCAACCTGTAAACCAGCA

CTGCAATGCTGATGCACGGGAAGTAGTGGGATGGGAACACAAATGGAAAGCTGT

AGAGCTCCTTGTTTTCCAGAAGGAGTTGCTCCTTGAGCCTTTCATTCTCAGCCTCG

ATAACCTCCAAAGCCGCTCTAATTGTGGAGGGGGTTCGAATTTAAAAGCTTGGAA TGTTGGTTCGTGCGTCTGGAACAAGCCCAGACTTGTTGCTCACTGGGAAAAGGAC

CATCAGCTCCAAAAAACTTGCCGCTCAAACCGCGTACCTCTGCTTTCGCGCAATC

TGCCCTGTTGAAATCGCCACCACATTCATATTGTGACGCTTGAGCAGTCTGTAAT

TGCCTCAGAATGTGGAATCATCTGCCCCCTGTGCGAGCCCATGCCAGGCATGTCG

CGGGCGAGGACACCCGCCACTCGTACAGCAGACCATTATGCTACCTCACAATAG

TTCATAACAGTGACCATATTTCTCGAAGCTCCCCAACGAGCACCTCCATGCTCTG

AGTGGCCACCCCCCGGCCCTGGTGCTTGCGGAGGGCAGGTCAACCGGCATGGGG

CTACCGAAATCCCCGACCGGATCCCACCACCCCCGCGATGGGAAGAATCTCTCCC

CGGGATGTGGGCCCACCACCAGCACAACCTGCTGGCCCAGGCGAGCGTCAAACC

ATACCACACAAATATCCTTGGCATCGGCCCTGAATTCCTTCTGCCGCTCTGCTACC

CGGTGCTTCTGTCCGAAGCAGGGGTTGCTAGGGATCGCTCCGAGTCCGCAAACCC

TTGTCGCGTGGCGGGGCTTGTTCGAGCTTGTTCGAGCTTGAAGAGCCTCTAGAGT

CGACCTGCAGGCATGCAAGCTTGGCGTAATCATGGTCATAGCTGTTTCCTGTGTG

AAATTGTTATCCGCTCACAATTCCACACAACATACGAGCCGGAAGCATAAAGTGT

AAAGCCTGGGGTGCCTAATGAGTGAGCTAACTCACATTAATTGCGTTGCGCTCAC

TGCCCGCTTTCCAGTCGGGAAACCTGTCGTGCCAGCTGCATTAATGAATCGGCCA

ACGCGCGGGGAGAGGCGGTTTGCGTATTGGGCGCTCTTCC

SEQ ID NO: 68

C. camphora thioesterase forward primer TACCCCGCCTGGGGCGACAC SEQ ID NO: 69

C. camphora thioesterase reverse primer CTTGCTCAGGCGGCGGGTGC

SEQ ID NO: 70

cdl89 forward primer

CCGGATCTCGGCCAGGGCTA

SEQ ID NO: 71

cdl89 reverse primer

TCGATGTCGTGCACCGTCGC SEQ ID NO: 72

5' donor DNA sequence of Prototheca moriformis delta 12 FAD knockout homologous recombination targeting construct

GCTCTTCGGGTTTGCTCACCCGCGAGGTCGACGCCCAGCATGGCTATCAAGACGA

ACAGGCAGCCTGTGGAGAAGCCTCCGTTCACGATCGGGACGCTGCGCAAGGCCA

TCCCCGCGCACTGTTTCGAGCGCTCGGCGCTTCGTAGCAGCATGTACCTGGCCTT

TGACATCGCGGTCATGTCCCTGCTCTACGTCGCGTCGACGTACATCGACCCTGCG

CCGGTGCCTACGTGGGTCAAGTATGGCGTCATGTGGCCGCTCTACTGGTTCTTCC

AGGTGTGTGTGAGGGTTGTGGTTGCCCGTATCGAGGTCCTGGTGGCGCGCATGGG

GGAGAAGGCGCCTGTCCCGCTGACCCCCCCGGCTACCCTCCCGGCACCTTCCAGG

GCGCCTTCGGCACGGGTGTCTGGGTGTGCGCGCACGAGTGCGGCCACCAGGCCTT TTCCTCCAGCCAGGCCATCAACGACGGCGTGGGCCTGGTGTTCCACAGCCTGCTG

CTGGTGCCCTACTACTCCTGGAAGCACTCGCACCGCCGCCACCACTCCAACACGG

GGTGCCTGGACAAGGACGAGGTGTTTGTGCCGCCGCACCGCGCAGTGGCGCACG

AGGGCCTGGAGTGGGAGGAGTGGCTGCCCATCCGCATGGGCAAGGTGCTGGTCA

CCCTGACCCTGGGCTGGCCGCTGTACCTCATGTTCAACGTCGCCTCGCGGCCGTA

CCCGCGCTTCGCCAACCACTTTGACCCGTGGTCGCCCATCTTCAGCAAGCGCGAG

GTACCCTTTCTTGCGCTATGACACTTCCAGCAAAAGGTAGGGCGGGCTGCGAGAC

GGCTTCCCGGCGCTGCATGCAACACCGATGATGCTTCGACCCCCCGAAGCTCCTT

CGGGGCTGCATGGGCGCTCCGATGCCGCTCCAGGGCGAGCGCTGTTTAAATAGC

CAGGCCCCCGATTGCAAAGACATTATAGCGAGCTACCAAAGCCATATTCAAACA

CCTAGATCACTACCACTTCTACACAGGCCACTCGAGCTTGTGATCGCACTCCGCT

AAGGGGGCGCCTCTTCCTCTTCGTTTCAGTCACAACCCGCAAACGGCGCGCC

SEQ ID NO: 73

3' donor DNA sequence of Prototheca moriformis delta 12 FAD knockout homologous recombination targeting construct

CAATTGGCAGCAGCAGCTCGGATAGTATCGACACACTCTGGACGCTGGTCGTGTG

ATGGACTGTTGCCGCCACACTTGCTGCCTTGACCTGTGAATATCCCTGCCGCTTTT

ATCAAACAGCCTCAGTGTGTTTGATCTTGTGTGTACGCGCTTTTGCGAGTTGCTAG

CTGCTTGTGCTATTTGCGAATACCACCCCCAGCATCCCCTTCCCTCGTTTCATATC

GCTTGCATCCCAACCGCAACTTATCTACGCTGTCCTGCTATCCCTCAGCGCTGCTC

CTGCTCCTGCTCACTGCCCCTCGCACAGCCTTGGTTTGGGCTCCGCCTGTATTCTC

CTGGTACTGCAACCTGTAAACCAGCACTGCAATGCTGATGCACGGGAAGTAGTG

GGATGGGAACACAAATGGAGCATCGAGGTGGTCATCTCCGACCTCGCGTTGGTG

GCGGTGCTCAGCGGGCTCAGCGTGCTGGGCCGCACCATGGGCTGGGCCTGGCTG

GTCAAGACCTACGTGGTGCCCTACATGATCGTGAACATGTGGCTGGTGCTCATCA

CGCTGCTCCAGCACACGCACCCGGCCCTGCCGCACTACTTCGAGAAGGACTGGG

ACTGGCTACGCGGCGCCATGGCCACCGTCGACCGCTCCATGGGCCCGCCCTTCAT

GGACAGCATCCTGCACCACATCTCCGACACCCACGTGCTGCACCACCTCTTCAGC

ACCATCCCGCACTACCACGCCGAGGAGGCCTCCGCCGCCATCCGGCCCATCCTGG

GCAAGTACTACCAATCCGACAGCCGCTGGGTCGGCCGCGCCCTGTGGGAGGACT

GGCGCGACTGCCGCTACGTCGTCCCCGACGCGCCCGAGGACGACTCCGCGCTCTG

GTTCCACAAGTGAGCGCGCCTGCGCGAGGACGCAGAACAACGCTGCCGCCGTGT

CAATTCTGTTTGTGGAAGACACGGTGTACCCCCACCCACCCACCTGCACCTCTAT

TATTGGTATTATTGACGCGGGAGTGGGCGTTGTACCCTACAACGTAGCTTCTCTA

GTTTTCAGCTGGCTCCCACCATTGTAAAGAGCCTCTAGAGTCGACCTGCAGGCAT

GCAAGCTTGGCGTAATCATGGTCATAGCTGTTTCCTGTGTGAAATTGTTATCCGCT

CACAATTCCACACAACATACGAGCCGGAAGCATAAAGTGTAAAGCCTGGGGTGC

CTAATGAGTGAGCTAACTCACATTAATTGCGTTGCGCTCACTGCCCGCTTTCCAG

TCGGGAAACCTGTCGTGCCAGCTGCATTAATGAATCGGCCAACGCGCGGGGAGA

GGCGGTTTGCGTATTGGGCGCTCTTCC

SEQ ID NO: 74

Prototheca moriformis delta 12 FAD knockout homologous recombination targeting construct

GCTCTTCGGGTTTGCTCACCCGCGAGGTCGACGCCCAGCATGGCTATCAAGACGA ACAGGCAGCCTGTGGAGAAGCCTCCGTTCACGATCGGGACGCTGCGCAAGGCCA TCCCCGCGCACTGTTTCGAGCGCTCGGCGCTTCGTAGCAGCATGTACCTGGCCTT

TGACATCGCGGTCATGTCCCTGCTCTACGTCGCGTCGACGTACATCGACCCTGCG

CCGGTGCCTACGTGGGTCAAGTATGGCGTCATGTGGCCGCTCTACTGGTTCTTCC

AGGTGTGTGTGAGGGTTGTGGTTGCCCGTATCGAGGTCCTGGTGGCGCGCATGGG

GGAGAAGGCGCCTGTCCCGCTGACCCCCCCGGCTACCCTCCCGGCACCTTCCAGG

GCGCCTTCGGCACGGGTGTCTGGGTGTGCGCGCACGAGTGCGGCCACCAGGCCTT

TTCCTCCAGCCAGGCCATCAACGACGGCGTGGGCCTGGTGTTCCACAGCCTGCTG

CTGGTGCCCTACTACTCCTGGAAGCACTCGCACCGCCGCCACCACTCCAACACGG

GGTGCCTGGACAAGGACGAGGTGTTTGTGCCGCCGCACCGCGCAGTGGCGCACG

AGGGCCTGGAGTGGGAGGAGTGGCTGCCCATCCGCATGGGCAAGGTGCTGGTCA

CCCTGACCCTGGGCTGGCCGCTGTACCTCATGTTCAACGTCGCCTCGCGGCCGTA

CCCGCGCTTCGCCAACCACTTTGACCCGTGGTCGCCCATCTTCAGCAAGCGCGAG

GTACCCTTTCTTGCGCTATGACACTTCCAGCAAAAGGTAGGGCGGGCTGCGAGAC

GGCTTCCCGGCGCTGCATGCAACACCGATGATGCTTCGACCCCCCGAAGCTCCTT

CGGGGCTGCATGGGCGCTCCGATGCCGCTCCAGGGCGAGCGCTGTTTAAATAGC

CAGGCCCCCGATTGCAAAGACATTATAGCGAGCTACCAAAGCCATATTCAAACA

CCTAGATCACTACCACTTCTACACAGGCCACTCGAGCTTGTGATCGCACTCCGCT

AAGGGGGCGCCTCTTCCTCTTCGTTTCAGTCACAACCCGCAAACGGCGCGCCATG

CTGCTGCAGGCCTTCCTGTTCCTGCTGGCCGGCTTCGCCGCCAAGATCAGCGCCT

CCATGACGAACGAGACGTCCGACCGCCCCCTGGTGCACTTCACCCCCAACAAGG

GCTGGATGAACGACCCCAACGGCCTGTGGTACGACGAGAAGGACGCCAAGTGGC

ACCTGTACTTCCAGTACAACCCGAACGACACCGTCTGGGGGACGCCCTTGTTCTG

GGGCCACGCCACGTCCGACGACCTGACCAACTGGGAGGACCAGCCCATCGCCAT

CGCCCCGAAGCGCAACGACTCCGGCGCCTTCTCCGGCTCCATGGTGGTGGACTAC

AACAACACCTCCGGCTTCTTCAACGACACCATCGACCCGCGCCAGCGCTGCGTGG

CCATCTGGACCTACAACACCCCGGAGTCCGAGGAGCAGTACATCTCCTACAGCCT

GGACGGCGGCTACACCTTCACCGAGTACCAGAAGAACCCCGTGCTGGCCGCCAA

CTCCACCCAGTTCCGCGACCCGAAGGTCTTCTGGTACGAGCCCTCCCAGAAGTGG

ATCATGACCGCGGCCAAGTCCCAGGACTACAAGATCGAGATCTACTCCTCCGAC

GACCTGAAGTCCTGGAAGCTGGAGTCCGCGTTCGCCAACGAGGGCTTCCTCGGCT

ACCAGTACGAGTGCCCCGGCCTGATCGAGGTCCCCACCGAGCAGGACCCCAGCA

AGTCCTACTGGGTGATGTTCATCTCCATCAACCCCGGCGCCCCGGCCGGCGGCTC

CTTCAACCAGTACTTCGTCGGCAGCTTCAACGGCACCCACTTCGAGGCCTTCGAC

AACCAGTCCCGCGTGGTGGACTTCGGCAAGGACTACTACGCCCTGCAGACCTTCT

TCAACACCGACCCGACCTACGGGAGCGCCCTGGGCATCGCGTGGGCCTCCAACT

GGGAGTACTCCGCCTTCGTGCCCACCAACCCCTGGCGCTCCTCCATGTCCCTCGT

GCGCAAGTTCTCCCTCAACACCGAGTACCAGGCCAACCCGGAGACGGAGCTGAT

CAACCTGAAGGCCGAGCCGATCCTGAACATCAGCAACGCCGGCCCCTGGAGCCG

GTTCGCCACCAACACCACGTTGACGAAGGCCAACAGCTACAACGTCGACCTGTC

CAACAGCACCGGCACCCTGGAGTTCGAGCTGGTGTACGCCGTCAACACCACCCA

GACGATCTCCAAGTCCGTGTTCGCGGACCTCTCCCTCTGGTTCAAGGGCCTGGAG

GACCCCGAGGAGTACCTCCGCATGGGCTTCGAGGTGTCCGCGTCCTCCTTCTTCC

TGGACCGCGGGAACAGCAAGGTGAAGTTCGTGAAGGAGAACCCCTACTTCACCA

ACCGCATGAGCGTGAACAACCAGCCCTTCAAGAGCGAGAACGACCTGTCCTACT

ACAAGGTGTACGGCTTGCTGGACCAGAACATCCTGGAGCTGTACTTCAACGACG

GCGACGTCGTGTCCACCAACACCTACTTCATGACCACCGGGAACGCCCTGGGCTC

CGTGAACATGACGACGGGGGTGGACAACCTGTTCTACATCGACAAGTTCCAGGT

GCGCGAGGTCAAGTGACAATTGGCAGCAGCAGCTCGGATAGTATCGACACACTC

TGGACGCTGGTCGTGTGATGGACTGTTGCCGCCACACTTGCTGCCTTGACCTGTG

AATATCCCTGCCGCTTTTATCAAACAGCCTCAGTGTGTTTGATCTTGTGTGTACGC GCTTTTGCGAGTTGCTAGCTGCTTGTGCTATTTGCGAATACCACCCCCAGCATCCC

CTTCCCTCGTTTCATATCGCTTGCATCCCAACCGCAACTTATCTACGCTGTCCTGC

TATCCCTCAGCGCTGCTCCTGCTCCTGCTCACTGCCCCTCGCACAGCCTTGGTTTG

GGCTCCGCCTGTATTCTCCTGGTACTGCAACCTGTAAACCAGCACTGCAATGCTG

ATGCACGGGAAGTAGTGGGATGGGAACACAAATGGAGCATCGAGGTGGTCATCT

CCGACCTCGCGTTGGTGGCGGTGCTCAGCGGGCTCAGCGTGCTGGGCCGCACCAT

GGGCTGGGCCTGGCTGGTCAAGACCTACGTGGTGCCCTACATGATCGTGAACATG

TGGCTGGTGCTCATCACGCTGCTCCAGCACACGCACCCGGCCCTGCCGCACTACT

TCGAGAAGGACTGGGACTGGCTACGCGGCGCCATGGCCACCGTCGACCGCTCCA

TGGGCCCGCCCTTCATGGACAGCATCCTGCACCACATCTCCGACACCCACGTGCT

GCACCACCTCTTCAGCACCATCCCGCACTACCACGCCGAGGAGGCCTCCGCCGCC

ATCCGGCCCATCCTGGGCAAGTACTACCAATCCGACAGCCGCTGGGTCGGCCGC

GCCCTGTGGGAGGACTGGCGCGACTGCCGCTACGTCGTCCCCGACGCGCCCGAG

GACGACTCCGCGCTCTGGTTCCACAAGTGAGCGCGCCTGCGCGAGGACGCAGAA

CAACGCTGCCGCCGTGTCTTTTGCACGCGCGACTCCGGCGCTTCGCTGGTGGCAC

CCCCATAAAGAAACCCTCAATTCTGTTTGTGGAAGACACGGTGTACCCCCACCCA

CCCACCTGCACCTCTATTATTGGTATTATTGACGCGGGAGTGGGCGTTGTACCCT

AGTCGACCTGCAGGCATGCAAGCTTGGCGTAATCATGGTCATAGCTGTTTCCTGT

GTGAAATTGTTATCCGCTCACAATTCCACACAACATACGAGCCGGAAGCATAAA

GTGTAAAGCCTGGGGTGCCTAATGAGTGAGCTAACTCACATTAATTGCGTTGCGC

TCACTGCCCGCTTTCCAGTCGGGAAACCTGTCGTGCCAGCTGCATTAATGAATCG

GCCAACGCGCGGGGAGAGGCGGTTTGCGTATTGGGCGCTCTTCC

SEQ ID NO: 75

5' donor DNA sequence of Prototheca moriformis SAD2A knockout homologous recombination targeting construct

GCTCTTCCGCCTGGAGCTGGTGCAGAGCATGGGTCAGTTTGCGGAGGAGAGGGT

GCTCCCCGTGCTGCACCCCGTGGACAAGCTGTGGCAGCCGCAGGACTTCCTGCCC

GACCCCGAGTCGCCCGACTTCGAGGACCAGGTGGCGGAGCTGCGCGCGCGCGCC

AAGGACCTGCCCGACGAGTACTTTGTGGTGCTGGTGGGCGACATGATCACGGAG

GAGGCGCTGCCGACCTACATGGCCATGCTCAACACCTTGGACGGTGTGCGCGAC

GACACGGGCGCGGCTGACCACCCGTGGGCGCGCTGGACGCGGCAGTGGGTGGCC

GAGGAGAACCGGCACGGCGACCTGCTGAACAAGTACTGTTGGCTGACGGGGCGC

GTCAACATGCGGGCCGTGGAGGTGACCATCAACAACCTGATCAAGAGCGGCATG

AACCCGCAGACGGACAACAACCCTTACTTGGGCTTCGTCTACACCTCCTTCCAGG

AGCGCGCCACCAAGTAGGTACC

SEQ ID NO: 76

3' donor DNA sequence of Prototheca moriformis SAD2A knockout homologous recombination targeting construct

CAATTGGCAGCAGCAGCTCGGATAGTATCGACACACTCTGGACGCTGGTCGTGTG

ATGGACTGTTGCCGCCACACTTGCTGCCTTGACCTGTGAATATCCCTGCCGCTTTT

ATCAAACAGCCTCAGTGTGTTTGATCTTGTGTGTACGCGCTTTTGCGAGTTGCTAG

CTGCTTGTGCTATTTGCGAATACCACCCCCAGCATCCCCTTCCCTCGTTTCATATC

GCTTGCATCCCAACCGCAACTTATCTACGCTGTCCTGCTATCCCTCAGCGCTGCTC

CTGCTCCTGCTCACTGCCCCTCGCACAGCCTTGGTTTGGGCTCCGCCTGTATTCTC

CTGGTACTGCAACCTGTAAACCAGCACTGCAATGCTGATGCACGGGAAGTAGTG GGATGGGAACACAAATGGAAGGATCGTAGAGCTCCAGCCACGGCAACACCGCGC

GCCTGGCGGCCGAGCACGGCGACAAGGGCCTGAGCAAGATCTGCGGGCTGATCG

CCAGCGACGAGGGCCGGCACGAGATCGCCTACACGCGCATCGTGGACGAGTTCT

TCCGCCTCGACCCCGAGGGCGCCGTCGCCGCCTACGCCAACATGATGCGCAAGC

AGATCACCATGCCCGCGCACCTCATGGACGACATGGGCCACGGCGAGGCCAACC

CGGGCCGCAACCTCTTCGCCGACTTCTCCGCCGTCGCCGAGAAGATCGACGTCTA

CGACGCCGAGGACTACTGCCGCATCCTGGAGCACCTCAACGCGCGCTGGAAGGT

GGACGAGCGCCAGGTCAGCGGCCAGGCCGCCGCGGACCAGGAGTACGTTCTGGG

CCTGCCCCAGCGCTTCCGGAAACTCGCCGAGAAGACCGCCGCCAAGCGCAAGCG

CGTCGCGCGCAGGCCCGTCGCCTTCTCCTGGAGAGAAGAGCCTCTAGAGTCGACC

TGCAGGCATGCAAGCTTGGCGTAATCATGGTCATAGCTGTTTCCTGTGTGAAATT

GTTATCCGCTCACAATTCCACACAACATACGAGCCGGAAGCATAAAGTGTAAAG

CCTGGGGTGCCTAATGAGTGAGCTAACTCACATTAATTGCGTTGCGCTCACTGCC

CGCTTTCCAGTCGGGAAACCTGTCGTGCCAGCTGCATTAATGAATCGGCCAACGC

GCGGGGAGAGGCGGTTTGCGTATTGGGCGCTCTTCC

SEQ ID NO: 77

Prototheca moriformis SAD2A knockout homologous recombination targeting construct

GCTCTTCCGCCTGGAGCTGGTGCAGAGCATGGGTCAGTTTGCGGAGGAGAGGGT

GCTCCCCGTGCTGCACCCCGTGGACAAGCTGTGGCAGCCGCAGGACTTCCTGCCC

GACCCCGAGTCGCCCGACTTCGAGGACCAGGTGGCGGAGCTGCGCGCGCGCGCC

AAGGACCTGCCCGACGAGTACTTTGTGGTGCTGGTGGGCGACATGATCACGGAG

GAGGCGCTGCCGACCTACATGGCCATGCTCAACACCTTGGACGGTGTGCGCGAC

GACACGGGCGCGGCTGACCACCCGTGGGCGCGCTGGACGCGGCAGTGGGTGGCC

GAGGAGAACCGGCACGGCGACCTGCTGAACAAGTACTGTTGGCTGACGGGGCGC

GTCAACATGCGGGCCGTGGAGGTGACCATCAACAACCTGATCAAGAGCGGCATG

AACCCGCAGACGGACAACAACCCTTACTTGGGCTTCGTCTACACCTCCTTCCAGG

AGCGCGCCACCAAGTAGGTACCCTTTCTTGCGCTATGACACTTCCAGCAAAAGGT

AGGGCGGGCTGCGAGACGGCTTCCCGGCGCTGCATGCAACACCGATGATGCTTC

GACCCCCCGAAGCTCCTTCGGGGCTGCATGGGCGCTCCGATGCCGCTCCAGGGCG

AGCGCTGTTTAAATAGCCAGGCCCCCGATTGCAAAGACATTATAGCGAGCTACC

AAAGCCATATTCAAACACCTAGATCACTACCACTTCTACACAGGCCACTCGAGCT

TGTGATCGCACTCCGCTAAGGGGGCGCCTCTTCCTCTTCGTTTCAGTCACAACCC

GCAAACGGCGCGCCATGCTGCTGCAGGCCTTCCTGTTCCTGCTGGCCGGCTTCGC

CGCCAAGATCAGCGCCTCCATGACGAACGAGACGTCCGACCGCCCCCTGGTGCA

CTTCACCCCCAACAAGGGCTGGATGAACGACCCCAACGGCCTGTGGTACGACGA

GAAGGACGCCAAGTGGCACCTGTACTTCCAGTACAACCCGAACGACACCGTCTG

GGGGACGCCCTTGTTCTGGGGCCACGCCACGTCCGACGACCTGACCAACTGGGA

GGACCAGCCCATCGCCATCGCCCCGAAGCGCAACGACTCCGGCGCCTTCTCCGGC

TCCATGGTGGTGGACTACAACAACACCTCCGGCTTCTTCAACGACACCATCGACC

CGCGCCAGCGCTGCGTGGCCATCTGGACCTACAACACCCCGGAGTCCGAGGAGC

AGTACATCTCCTACAGCCTGGACGGCGGCTACACCTTCACCGAGTACCAGAAGA

ACCCCGTGCTGGCCGCCAACTCCACCCAGTTCCGCGACCCGAAGGTCTTCTGGTA

CGAGCCCTCCCAGAAGTGGATCATGACCGCGGCCAAGTCCCAGGACTACAAGAT

CGAGATCTACTCCTCCGACGACCTGAAGTCCTGGAAGCTGGAGTCCGCGTTCGCC

AACGAGGGCTTCCTCGGCTACCAGTACGAGTGCCCCGGCCTGATCGAGGTCCCCA

CCGAGCAGGACCCCAGCAAGTCCTACTGGGTGATGTTCATCTCCATCAACCCCGG

CGCCCCGGCCGGCGGCTCCTTCAACCAGTACTTCGTCGGCAGCTTCAACGGCACC

CACTTCGAGGCCTTCGACAACCAGTCCCGCGTGGTGGACTTCGGCAAGGACTACT ACGCCCTGCAGACCTTCTTCAACACCGACCCGACCTACGGGAGCGCCCTGGGCAT

CGCGTGGGCCTCCAACTGGGAGTACTCCGCCTTCGTGCCCACCAACCCCTGGCGC

TCCTCCATGTCCCTCGTGCGCAAGTTCTCCCTCAACACCGAGTACCAGGCCAACC

CGGAGACGGAGCTGATCAACCTGAAGGCCGAGCCGATCCTGAACATCAGCAACG

CCGGCCCCTGGAGCCGGTTCGCCACCAACACCACGTTGACGAAGGCCAACAGCT

ACAACGTCGACCTGTCCAACAGCACCGGCACCCTGGAGTTCGAGCTGGTGTACG

CCGTCAACACCACCCAGACGATCTCCAAGTCCGTGTTCGCGGACCTCTCCCTCTG

GTTCAAGGGCCTGGAGGACCCCGAGGAGTACCTCCGCATGGGCTTCGAGGTGTC

CGCGTCCTCCTTCTTCCTGGACCGCGGGAACAGCAAGGTGAAGTTCGTGAAGGA

GAACCCCTACTTCACCAACCGCATGAGCGTGAACAACCAGCCCTTCAAGAGCGA

GAACGACCTGTCCTACTACAAGGTGTACGGCTTGCTGGACCAGAACATCCTGGA

GCTGTACTTCAACGACGGCGACGTCGTGTCCACCAACACCTACTTCATGACCACC

GGGAACGCCCTGGGCTCCGTGAACATGACGACGGGGGTGGACAACCTGTTCTAC

ATCGACAAGTTCCAGGTGCGCGAGGTCAAGTGACAATTGGCAGCAGCAGCTCGG

ATAGTATCGACACACTCTGGACGCTGGTCGTGTGATGGACTGTTGCCGCCACACT

TGCTGCCTTGACCTGTGAATATCCCTGCCGCTTTTATCAAACAGCCTCAGTGTGTT

TGATCTTGTGTGTACGCGCTTTTGCGAGTTGCTAGCTGCTTGTGCTATTTGCGAAT

ACCACCCCCAGCATCCCCTTCCCTCGTTTCATATCGCTTGCATCCCAACCGCAACT

TATCTACGCTGTCCTGCTATCCCTCAGCGCTGCTCCTGCTCCTGCTCACTGCCCCT

CGCACAGCCTTGGTTTGGGCTCCGCCTGTATTCTCCTGGTACTGCAACCTGTAAA

CCAGCACTGCAATGCTGATGCACGGGAAGTAGTGGGATGGGAACACAAATGGAA

GGATCGTAGAGCTCCAGCCACGGCAACACCGCGCGCCTGGCGGCCGAGCACGGC

GACAAGGGCCTGAGCAAGATCTGCGGGCTGATCGCCAGCGACGAGGGCCGGCAC

GAGATCGCCTACACGCGCATCGTGGACGAGTTCTTCCGCCTCGACCCCGAGGGCG

CCGTCGCCGCCTACGCCAACATGATGCGCAAGCAGATCACCATGCCCGCGCACCT

CATGGACGACATGGGCCACGGCGAGGCCAACCCGGGCCGCAACCTCTTCGCCGA

CTTCTCCGCCGTCGCCGAGAAGATCGACGTCTACGACGCCGAGGACTACTGCCGC

ATCCTGGAGCACCTCAACGCGCGCTGGAAGGTGGACGAGCGCCAGGTCAGCGGC

CAGGCCGCCGCGGACCAGGAGTACGTTCTGGGCCTGCCCCAGCGCTTCCGGAAA

CTCGCCGAGAAGACCGCCGCCAAGCGCAAGCGCGTCGCGCGCAGGCCCGTCGCC

TTCTCCTGGAGAGAAGAGCCTCTAGAGTCGACCTGCAGGCATGCAAGCTTGGCGT

AATCATGGTCATAGCTGTTTCCTGTGTGAAATTGTTATCCGCTCACAATTCCACAC

AACATACGAGCCGGAAGCATAAAGTGTAAAGCCTGGGGTGCCTAATGAGTGAGC

TAACTCACATTAATTGCGTTGCGCTCACTGCCCGCTTTCCAGTCGGGAAACCTGT

CGTGCCAGCTGCATTAATGAATCGGCCAACGCGCGGGGAGAGGCGGTTTGCGTA

TTGGGCGCTCTTCC

SEQ ID NO: 78

5' donor DNA sequence of Prototheca moriformis SAD2B knockout homologous recombination targeting construct

GCTCTTCCCGCCTGGAGCTGGTGCAGAGCATGGGGCAGTTTGCGGAGGAGAGGG

TGCTCCCCGTGCTGCACCCCGTGGACAAGCTGTGGCAGCCGCAGGACTTCCTGCC

CGACCCCGAGTCGCCCGACTTCGAGGACCAGGTGGCGGAGCTGCGCGCGCGCGC

CAAGGACCTGCCCGACGAGTACTTTGTGGTGCTGGTGGGCGACATGATCACGGA

GGAGGCGCTGCCGACCTACATGGCCATGCTCAACACCTTGGACGGTGTGCGCGA

CGACACGGGCGCGGCTGACCACCCGTGGGCGCGCTGGACGCGGCAGTGGGTGGC

CGAGGAGAACCGGCACGGCGACCTGCTGAACAAGTACTGTTGGCTGACGGGGCG

CGTCAACATGCGGGCCGTGGAGGTGACCATCAACAACCTGATCAAGAGCGGCAT GAACCCGCAGACGGACAACAACCCTTACTTGGGCTTCGTCTACACCTCCTTCCAG GAGCGCGCCACCAAGTAGGTACC

SEQ ID NO: 79

3' donor DNA sequence of Prototheca moriformis SAD2B knockout homologous recombination targeting construct

CAGCCACGGCAACACCGCGCGCCTTGCGGCCGAGCACGGCGACAAGAACCTGAG

CAAGATCTGCGGGCTGATCGCCAGCGACGAGGGCCGGCACGAGATCGCCTACAC

GCGCATCGTGGACGAGTTCTTCCGCCTCGACCCCGAGGGCGCCGTCGCCGCCTAC

GCCAACATGATGCGCAAGCAGATCACCATGCCCGCGCACCTCATGGACGACATG

GGCCACGGCGAGGCCAACCCGGGCCGCAACCTCTTCGCCGACTTCTCCGCGGTCG

CCGAGAAGATCGACGTCTACGACGCCGAGGACTACTGCCGCATCCTGGAGCACC

TCAACGCGCGCTGGAAGGTGGACGAGCGCCAGGTCAGCGGCCAGGCCGCCGCGG

ACCAGGAGTACGTCCTGGGCCTGCCCCAGCGCTTCCGGAAACTCGCCGAGAAGA

CCGCCGCCAAGCGCAAGCGCGTCGCGCGCAGGCCCGTCGCCTTCTCCTGGAGAA

GAGCCTCTAGAGTCGACCTGCAGGCATGCAAGCTTGGCGTAATCATGGTCATAGC

TGTTTCCTGTGTGAAATTGTTATCCGCTCACAATTCCACACAACATACGAGCCGG

AAGCATAAAGTGTAAAGCCTGGGGTGCCTAATGAGTGAGCTAACTCACATTAAT

TGCGTTGCGCTCACTGCCCGCTTTCCAGTCGGGAAACCTGTCGTGCCAGCTGCAT

TAATGAATCGGCCAACGCGCGGGGAGAGGCGGTTTGCGTATTGGGCGCTCTTCC

SEQ ID NO: 80

Prototheca moriformis SAD2B knockout homologous recombination targeting construct

GCTCTTCCCGCCTGGAGCTGGTGCAGAGCATGGGGCAGTTTGCGGAGGAGAGGG

TGCTCCCCGTGCTGCACCCCGTGGACAAGCTGTGGCAGCCGCAGGACTTCCTGCC

CGACCCCGAGTCGCCCGACTTCGAGGACCAGGTGGCGGAGCTGCGCGCGCGCGC

CAAGGACCTGCCCGACGAGTACTTTGTGGTGCTGGTGGGCGACATGATCACGGA

GGAGGCGCTGCCGACCTACATGGCCATGCTCAACACCTTGGACGGTGTGCGCGA

CGACACGGGCGCGGCTGACCACCCGTGGGCGCGCTGGACGCGGCAGTGGGTGGC

CGAGGAGAACCGGCACGGCGACCTGCTGAACAAGTACTGTTGGCTGACGGGGCG

CGTCAACATGCGGGCCGTGGAGGTGACCATCAACAACCTGATCAAGAGCGGCAT

GAACCCGCAGACGGACAACAACCCTTACTTGGGCTTCGTCTACACCTCCTTCCAG

GAGCGCGCCACCAAGTAGGTACCCTTTCTTGCGCTATGACACTTCCAGCAAAAGG

TAGGGCGGGCTGCGAGACGGCTTCCCGGCGCTGCATGCAACACCGATGATGCTT

CGACCCCCCGAAGCTCCTTCGGGGCTGCATGGGCGCTCCGATGCCGCTCCAGGGC

GAGCGCTGTTTAAATAGCCAGGCCCCCGATTGCAAAGACATTATAGCGAGCTAC

CAAAGCCATATTCAAACACCTAGATCACTACCACTTCTACACAGGCCACTCGAGC

TTGTGATCGCACTCCGCTAAGGGGGCGCCTCTTCCTCTTCGTTTCAGTCACAACCC

GCAAACGGCGCGCCATGCTGCTGCAGGCCTTCCTGTTCCTGCTGGCCGGCTTCGC

CGCCAAGATCAGCGCCTCCATGACGAACGAGACGTCCGACCGCCCCCTGGTGCA

CTTCACCCCCAACAAGGGCTGGATGAACGACCCCAACGGCCTGTGGTACGACGA

GAAGGACGCCAAGTGGCACCTGTACTTCCAGTACAACCCGAACGACACCGTCTG

GGGGACGCCCTTGTTCTGGGGCCACGCCACGTCCGACGACCTGACCAACTGGGA

GGACCAGCCCATCGCCATCGCCCCGAAGCGCAACGACTCCGGCGCCTTCTCCGGC

TCCATGGTGGTGGACTACAACAACACCTCCGGCTTCTTCAACGACACCATCGACC

CGCGCCAGCGCTGCGTGGCCATCTGGACCTACAACACCCCGGAGTCCGAGGAGC

AGTACATCTCCTACAGCCTGGACGGCGGCTACACCTTCACCGAGTACCAGAAGA

ACCCCGTGCTGGCCGCCAACTCCACCCAGTTCCGCGACCCGAAGGTCTTCTGGTA CGAGCCCTCCCAGAAGTGGATCATGACCGCGGCCAAGTCCCAGGACTACAAGAT

CGAGATCTACTCCTCCGACGACCTGAAGTCCTGGAAGCTGGAGTCCGCGTTCGCC

AACGAGGGCTTCCTCGGCTACCAGTACGAGTGCCCCGGCCTGATCGAGGTCCCCA

CCGAGCAGGACCCCAGCAAGTCCTACTGGGTGATGTTCATCTCCATCAACCCCGG

CGCCCCGGCCGGCGGCTCCTTCAACCAGTACTTCGTCGGCAGCTTCAACGGCACC

CACTTCGAGGCCTTCGACAACCAGTCCCGCGTGGTGGACTTCGGCAAGGACTACT

ACGCCCTGCAGACCTTCTTCAACACCGACCCGACCTACGGGAGCGCCCTGGGCAT

CGCGTGGGCCTCCAACTGGGAGTACTCCGCCTTCGTGCCCACCAACCCCTGGCGC

TCCTCCATGTCCCTCGTGCGCAAGTTCTCCCTCAACACCGAGTACCAGGCCAACC

CGGAGACGGAGCTGATCAACCTGAAGGCCGAGCCGATCCTGAACATCAGCAACG

CCGGCCCCTGGAGCCGGTTCGCCACCAACACCACGTTGACGAAGGCCAACAGCT

ACAACGTCGACCTGTCCAACAGCACCGGCACCCTGGAGTTCGAGCTGGTGTACG

CCGTCAACACCACCCAGACGATCTCCAAGTCCGTGTTCGCGGACCTCTCCCTCTG

GTTCAAGGGCCTGGAGGACCCCGAGGAGTACCTCCGCATGGGCTTCGAGGTGTC

CGCGTCCTCCTTCTTCCTGGACCGCGGGAACAGCAAGGTGAAGTTCGTGAAGGA

GAACCCCTACTTCACCAACCGCATGAGCGTGAACAACCAGCCCTTCAAGAGCGA

GAACGACCTGTCCTACTACAAGGTGTACGGCTTGCTGGACCAGAACATCCTGGA

GCTGTACTTCAACGACGGCGACGTCGTGTCCACCAACACCTACTTCATGACCACC

GGGAACGCCCTGGGCTCCGTGAACATGACGACGGGGGTGGACAACCTGTTCTAC

ATCGACAAGTTCCAGGTGCGCGAGGTCAAGTGACAATTGGCAGCAGCAGCTCGG

ATAGTATCGACACACTCTGGACGCTGGTCGTGTGATGGACTGTTGCCGCCACACT

TGCTGCCTTGACCTGTGAATATCCCTGCCGCTTTTATCAAACAGCCTCAGTGTGTT

TGATCTTGTGTGTACGCGCTTTTGCGAGTTGCTAGCTGCTTGTGCTATTTGCGAAT

ACCACCCCCAGCATCCCCTTCCCTCGTTTCATATCGCTTGCATCCCAACCGCAACT

TATCTACGCTGTCCTGCTATCCCTCAGCGCTGCTCCTGCTCCTGCTCACTGCCCCT

CGCACAGCCTTGGTTTGGGCTCCGCCTGTATTCTCCTGGTACTGCAACCTGTAAA

CCAGCACTGCAATGCTGATGCACGGGAAGTAGTGGGATGGGAACACAAATGGAC

AGCCACGGCAACACCGCGCGCCTTGCGGCCGAGCACGGCGACAAGAACCTGAGC

AAGATCTGCGGGCTGATCGCCAGCGACGAGGGCCGGCACGAGATCGCCTACACG

CGCATCGTGGACGAGTTCTTCCGCCTCGACCCCGAGGGCGCCGTCGCCGCCTACG

CCAACATGATGCGCAAGCAGATCACCATGCCCGCGCACCTCATGGACGACATGG

GCCACGGCGAGGCCAACCCGGGCCGCAACCTCTTCGCCGACTTCTCCGCGGTCGC

CGAGAAGATCGACGTCTACGACGCCGAGGACTACTGCCGCATCCTGGAGCACCT

CAACGCGCGCTGGAAGGTGGACGAGCGCCAGGTCAGCGGCCAGGCCGCCGCGG

ACCAGGAGTACGTCCTGGGCCTGCCCCAGCGCTTCCGGAAACTCGCCGAGAAGA

CCGCCGCCAAGCGCAAGCGCGTCGCGCGCAGGCCCGTCGCCTTCTCCTGGAGAA

GAGCCTCTAGAGTCGACCTGCAGGCATGCAAGCTTGGCGTAATCATGGTCATAGC

TGTTTCCTGTGTGAAATTGTTATCCGCTCACAATTCCACACAACATACGAGCCGG

AAGCATAAAGTGTAAAGCCTGGGGTGCCTAATGAGTGAGCTAACTCACATTAAT

TGCGTTGCGCTCACTGCCCGCTTTCCAGTCGGGAAACCTGTCGTGCCAGCTGCAT

TAATGAATCGGCCAACGCGCGGGGAGAGGCGGTTTGCGTATTGGGCGCTCTTCC

SEQ ID NO: 81

P.moriformis Aat02 promoter/5 'UTR .C.protothecoides stearoyl ACP desaturase

YTS .Cinnamomum camphora C14 thioesterase::C.vi /gara nitrate reductase 3"UTR

AGAGAGCGGAGGTGGGGTTGTGAGGTGGGGTTGCTGACCAGGAGCTCGCGTCGC CGAGCGCGACTCGCACACGGTCCAGTTACCCCCCCCTCCGCCCAAACGCAAGCCT CCCATCTTGATGCCTTTCCGGCCACCTATACTATTTCTTAGTTCGCTGTAACATCC AGACCGTCCTGAATAATAACAATGCCCTGTGTCAAGTGCATTCCTAAAAAAATTC TGTCCCAACCAACAATCCCACCTGAAATACCACCAGCCCTGCCCAGTACACTCTT

CCAATACCATCTCCCTACCTCCACGCGCAAGCGACCCCCATGCGCGACCAGGCTC

GAAAGTGATTTATGACTTGAGACGAGCGAGTGGCGGCGCGGTCGACTGCCTTTTC

ATCACGTGCCGTACGTCGGCGACCGCTAGGGCTTTGCACGGCAACGCACGGCTTC

GCCAACCCGACCAGCCAGGACCTCGACTACTCTACCGCGAATTCGCCTCAAGAA

GTCGCCAAATGTGCCATACACCATTCCTTACAGCACTGTTCAAACTTGATGCCAA

TTTTGACATTCGGGTTGCTCGTTGGCTGCGCCCACATCGGCCGTGAGTGCAGCAG

GCGGGATCGGACACGGAGGACGCGGCGTCACGCCCCGAACGCAGCCCGTAACTC

TACATCAACACGACGTGTTGCGTAATCCCGCCCGGCTGCGCATCGTGCCAACCCA

TTCGCGATGGATGGTCGGAAAATGGTGTGCCAACTGCCCTGAGGGAGGCTCTCG

CGAAACGGGCACGTCCCTGAAACCGAAACTGTGGCCTTGTCGTCGGCCACGCAA

GCACGTGGACCCTAAACACCAAGAAAATCAGTAAACAAGGTTGACATCCTCTAC

GGGCGAATTGTTTGCCCAACCCTTCATCGCACACTGCCATTATAATGCATCTAGC

TCGGCGACAAGTTTAGAAAAGGCAGGCTGCATTGTTCCATTTCGCCGTGGCGGCG

TGGGTGCCCATTTTACGAGGTTTGGGCTCCCGGGCAGCGACCGAGCCAGGTCGA

GTCCCTCTCGCCCGTCGACAATGTTGCGAACCCCACAAGCGGCTAACAACAACTT

GATGGTACCTGTACACTGCCAATTCCTTCTTCCCCGGCCGAGGTTTACACGTGAT

GGCCATGGCTTCGCATTCAGGCCGACTTCCCATTCCGACTTTCCAGAGGGTCCGC

GGACGCTGGGGGTTGGCTGCCTGAGGCCCACCCTTTGTTCCCCGCGTCCCGACAA

ACACAATTGCGTTACATAAGGGGGAGCCGCCCCCGTTCAGAGTGCAGAAATCTTT

CACTATATTTTCCAGTCGTCAGCGAAATCAAGTACTAGTATGGCCACCGCATCCA

CTTTCTCGGCGTTCAATGCCCGCTGCGGCGACCTGCGTCGCTCGGCGGGCTCCGG

GCCCCGGCGCCCAGCGAGGCCCCTCCCCGTGCGCGGGCGCGCCCCCGACTGGTC

CATGCTGTTCGCCGTGATCACCACCATCTTCTCCGCCGCCGAGAAGCAGTGGACC

AACCTGGAGTGGAAGCCCAAGCCCAACCCCCCCCAGCTGCTGGACGACCACTTC

GGCCCCCACGGCCTGGTGTTCCGCCGCACCTTCGCCATCCGCAGCTACGAGGTGG

GCCCCGACCGCTCCACCAGCATCGTGGCCGTGATGAACCACCTGCAGGAGGCCG

CCCTGAACCACGCCAAGTCCGTGGGCATCCTGGGCGACGGCTTCGGCACCACCCT

GGAGATGTCCAAGCGCGACCTGATCTGGGTGGTGAAGCGCACCCACGTGGCCGT

GGAGCGCTACCCCGCCTGGGGCGACACCGTGGAGGTGGAGTGCTGGGTGGGCGC

CTCCGGCAACAACGGCCGCCGCCACGACTTCCTGGTGCGCGACTGCAAGACCGG

CGAGATCCTGACCCGCTGCACCTCCCTGAGCGTGATGATGAACACCCGCACCCGC

CGCCTGAGCAAGATCCCCGAGGAGGTGCGCGGCGAGATCGGCCCCGCCTTCATC

GACAACGTGGCCGTGAAGGACGAGGAGATCAAGAAGCCCCAGAAGCTGAACGA

CTCCACCGCCGACTACATCCAGGGCGGCCTGACCCCCCGCTGGAACGACCTGGA

CATCAACCAGCACGTGAACAACATCAAGTACGTGGACTGGATCCTGGAGACCGT

GCCCGACAGCATCTTCGAGAGCCACCACATCTCCTCCTTCACCATCGAGTACCGC

CGCGAGTGCACCATGGACAGCGTGCTGCAGTCCCTGACCACCGTGAGCGGCGGC

TCCTCCGAGGCCGGCCTGGTGTGCGAGCACCTGCTGCAGCTGGAGGGCGGCAGC

GAGGTGCTGCGCGCCAAGACCGAGTGGCGCCCCAAGCTGACCGACTCCTTCCGC

GGCATCAGCGTGATCCCCGCCGAGTCCAGCGTGATGGACTACAAGGACCACGAC

GGCGACTACAAGGACCACGACATCGACTACAAGGACGACGACGACAAGTGACTC

GAGTTAATTAACTCGAGGCAGCAGCAGCTCGGATAGTATCGACACACTCTGGAC

GCTGGTCGTGTGATGGACTGTTGCCGCCACACTTGCTGCCTTGACCTGTGAATAT

CCCTGCCGCTTTTATCAAACAGCCTCAGTGTGTTTGATCTTGTGTGTACGCGCTTT

TGCGAGTTGCTAGCTGCTTGTGCTATTTGCGAATACCACCCCCAGCATCCCCTTCC

CTCGTTTCATATCGCTTGCATCCCAACCGCAACTTATCTACGCTGTCCTGCTATCC

CTCAGCGCTGCTCCTGCTCCTGCTCACTGCCCCTCGCACAGCCTTGGTTTGGGCTC

CGCCTGTATTCTCCTGGTACTGCAACCTGTAAACCAGCACTGCAATGCTGATGCA

CGGGAAGTAGTGGGATGGGAACACAAATGGAAAGCTT SEQ ID NO: 82

5' 6S rRNA genomic donor sequence

GCTCTTCGCCGCCGCCACTCCTGCTCGAGCGCGCCCGCGCGTGCGCCGCCAGCGC

AGGTCTGCCTTGCGCCGGCTGAGCCACTGCTTCGTCCGGGCGGCCAAGAGGAGC

ATGAGGGAGGACTCCTGGTCCAGGGTCCTGACGTGGTCGCGGCTCTGGGAGCGG

GCCAGCATCATCTGGCTCTGCCGCACCGAGGCCGCCTCCAACTGGTCCTCCAGCA

GCCGCAGTCGCCGCCGACCCTGGCAGAGGAAGACAGGTGAGGGGGGTATGAATT

GTACAGAACAACCACGAGCCTTGTCTAGGCAGAATCCCTACCAGTCATGGCTTTA

CCTGGATGACGGCCTGCGAACAGCTGTCCAGCGACCCTCGCTGCCGCCGCTTCTC

CCGCACGCTTCTTTCCAGCACCGTGATGGCGCGAGCCAGCGCCGCACGCTGGCGC

TGCGCTTCGCCGATCTGAGGACAGTCGGGGAACTCTGATCAGTCTAAACCCCCTT

GCGCGTTAGTGTTGCCATCCTTTGCAGACCGGTGAGAGCCGACTTGTTGTGCGCC

ACCCCCCACACCACCTCCTCCCAGACCAATTCTGTCACCTTTTTGGCGAAGGCAT

CGGCCTCGGCCTGCAGAGAGGACAGCAGTGCCCAGCCGCTGGGGGTTGGCGGAT

GCACGCTCAGGTACC

SEQ ID NO: 83

Relevant expression construct for Cinnamomum camphora thioesterase

( tub: :neo: :nitred: :βηΛ: :C.camphora TE: :nitred)

CTTTCTTGCGCTATGACACTTCCAGCAAAAGGTAGGGCGGGCTGCGAGACGGCTT

CCCGGCGCTGCATGCAACACCGATGATGCTTCGACCCCCCGAAGCTCCTTCGGGG

CTGCATGGGCGCTCCGATGCCGCTCCAGGGCGAGCGCTGTTTAAATAGCCAGGCC

CCCGATTGCAAAGACATTATAGCGAGCTACCAAAGCCATATTCAAACACCTAGA

TCACTACCACTTCTACACAGGCCACTCGAGCTTGTGATCGCACTCCGCTAAGGGG

GCGCCTCTTCCTCTTCGTTTCAGTCACAACCCGCAAACTCTAGAATATCAATGATC

GAGCAGGACGGCCTCCACGCCGGCTCCCCCGCCGCCTGGGTGGAGCGCCTGTTC

GGCTACGACTGGGCCCAGCAGACCATCGGCTGCTCCGACGCCGCCGTGTTCCGCC

TGTCCGCCCAGGGCCGCCCCGTGCTGTTCGTGAAGACCGACCTGTCCGGCGCCCT

GAACGAGCTGCAGGACGAGGCCGCCCGCCTGTCCTGGCTGGCCACCACCGGCGT

GCCCTGCGCCGCCGTGCTGGACGTGGTGACCGAGGCCGGCCGCGACTGGCTGCT

GCTGGGCGAGGTGCCCGGCCAGGACCTGCTGTCCTCCCACCTGGCCCCCGCCGAG

AAGGTGTCCATCATGGCCGACGCCATGCGCCGCCTGCACACCCTGGACCCCGCCA

CCTGCCCCTTCGACCACCAGGCCAAGCACCGCATCGAGCGCGCCCGCACCCGCAT

GGAGGCCGGCCTGGTGGACCAGGACGACCTGGACGAGGAGCACCAGGGCCTGG

CCCCCGCCGAGCTGTTCGCCCGCCTGAAGGCCCGCATGCCCGACGGCGAGGACC

TGGTGGTGACCCACGGCGACGCCTGCCTGCCCAACATCATGGTGGAGAACGGCC

GCTTCTCCGGCTTCATCGACTGCGGCCGCCTGGGCGTGGCCGACCGCTACCAGGA

CATCGCCCTGGCCACCCGCGACATCGCCGAGGAGCTGGGCGGCGAGTGGGCCGA

CCGCTTCCTGGTGCTGTACGGCATCGCCGCCCCCGACTCCCAGCGCATCGCCTTC

TACCGCCTGCTGGACGAGTTCTTCTGACAATTGGCAGCAGCAGCTCGGATAGTAT

CGACACACTCTGGACGCTGGTCGTGTGATGGACTGTTGCCGCCACACTTGCTGCC

TTGACCTGTGAATATCCCTGCCGCTTTTATCAAACAGCCTCAGTGTGTTTGATCTT

GTGTGTACGCGCTTTTGCGAGTTGCTAGCTGCTTGTGCTATTTGCGAATACCACCC

CCAGCATCCCCTTCCCTCGTTTCATATCGCTTGCATCCCAACCGCAACTTATCTAC

GCTGTCCTGCTATCCCTCAGCGCTGCTCCTGCTCCTGCTCACTGCCCCTCGCACAG

CCTTGGTTTGGGCTCCGCCTGTATTCTCCTGGTACTGCAACCTGTAAACCAGCACT GCAATGCTGATGCACGGGAAGTAGTGGGATGGGAACACAAATGGAGGATCCCGC

GTCTCGAACAGAGCGCGCAGAGGAACGCTGAAGGTCTCGCCTCTGTCGCACCTC

AGCGCGGCATACACCACAATAACCACCTGACGAATGCGCTTGGTTCTTCGTCCAT

TAGCGAAGCGTCCGGTTCACACACGTGCCACGTTGGCGAGGTGGCAGGTGACAA

TGATCGGTGGAGCTGATGGTCGAAACGTTCACAGCCTAGGGATATCGAATTCCTT

TCTTGCGCTATGACACTTCCAGCAAAAGGTAGGGCGGGCTGCGAGACGGCTTCCC

GGCGCTGCATGCAACACCGATGATGCTTCGACCCCCCGAAGCTCCTTCGGGGCTG

CATGGGCGCTCCGATGCCGCTCCAGGGCGAGCGCTGTTTAAATAGCCAGGCCCCC

GATTGCAAAGACATTATAGCGAGCTACCAAAGCCATATTCAAACACCTAGATCA

CTACCACTTCTACACAGGCCACTCGAGCTTGTGATCGCACTCCGCTAAGGGGGCG

CCTCTTCCTCTTCGTTTCAGTCACAACCCGCAAACACTAGTATGGCCACCGCATCC

ACTTTCTCGGCGTTCAATGCCCGCTGCGGCGACCTGCGTCGCTCGGCGGGCTCCG

GGCCCCGGCGCCCAGCGAGGCCCCTCCCCGTGCGCGGGCGCGCCCCCGACTGGT

CCATGCTGTTCGCCGTGATCACCACCATCTTCTCCGCCGCCGAGAAGCAGTGGAC

CAACCTGGAGTGGAAGCCCAAGCCCAACCCCCCCCAGCTGCTGGACGACCACTT

CGGCCCCCACGGCCTGGTGTTCCGCCGCACCTTCGCCATCCGCAGCTACGAGGTG

GGCCCCGACCGCTCCACCAGCATCGTGGCCGTGATGAACCACCTGCAGGAGGCC

GCCCTGAACCACGCCAAGTCCGTGGGCATCCTGGGCGACGGCTTCGGCACCACC

CTGGAGATGTCCAAGCGCGACCTGATCTGGGTGGTGAAGCGCACCCACGTGGCC

GTGGAGCGCTACCCCGCCTGGGGCGACACCGTGGAGGTGGAGTGCTGGGTGGGC

GCCTCCGGCAACAACGGCCGCCGCCACGACTTCCTGGTGCGCGACTGCAAGACC

GGCGAGATCCTGACCCGCTGCACCTCCCTGAGCGTGATGATGAACACCCGCACCC

GCCGCCTGAGCAAGATCCCCGAGGAGGTGCGCGGCGAGATCGGCCCCGCCTTCA

TCGACAACGTGGCCGTGAAGGACGAGGAGATCAAGAAGCCCCAGAAGCTGAAC

GACTCCACCGCCGACTACATCCAGGGCGGCCTGACCCCCCGCTGGAACGACCTG

GACATCAACCAGCACGTGAACAACATCAAGTACGTGGACTGGATCCTGGAGACC

GTGCCCGACAGCATCTTCGAGAGCCACCACATCTCCTCCTTCACCATCGAGTACC

GCCGCGAGTGCACCATGGACAGCGTGCTGCAGTCCCTGACCACCGTGAGCGGCG

GCTCCTCCGAGGCCGGCCTGGTGTGCGAGCACCTGCTGCAGCTGGAGGGCGGCA

GCGAGGTGCTGCGCGCCAAGACCGAGTGGCGCCCCAAGCTGACCGACTCCTTCC

GCGGCATCAGCGTGATCCCCGCCGAGTCCAGCGTGATGGACTACAAGGACCACG

ACGGCGACTACAAGGACCACGACATCGACTACAAGGACGACGACGACAAGTGA

CTCGAGGCAGCAGCAGCTCGGATAGTATCGACACACTCTGGACGCTGGTCGTGT

GATGGACTGTTGCCGCCACACTTGCTGCCTTGACCTGTGAATATCCCTGCCGCTTT

TATCAAACAGCCTCAGTGTGTTTGATCTTGTGTGTACGCGCTTTTGCGAGTTGCTA

GCTGCTTGTGCTATTTGCGAATACCACCCCCAGCATCCCCTTCCCTCGTTTCATAT

CGCTTGCATCCCAACCGCAACTTATCTACGCTGTCCTGCTATCCCTCAGCGCTGCT

CCTGCTCCTGCTCACTGCCCCTCGCACAGCCTTGGTTTGGGCTCCGCCTGTATTCT

CCTGGTACTGCAACCTGTAAACCAGCACTGCAATGCTGATGCACGGGAAGTAGT

GGGATGGGAACACAAATGGAAAGCTT

SEQ ID NO: 84

3'6S rRNA genomic donor sequence

TAACCTCCAAAGCCGCTCTAATTGTGGAGGGGGTTCGAATTTAAAAGCTTGGAAT

GTTGGTTCGTGCGTCTGGAACAAGCCCAGACTTGTTGCTCACTGGGAAAAGGACC

ATCAGCTCCAAAAAACTTGCCGCTCAAACCGCGTACCTCTGCTTTCGCGCAATCT

GCCCTGTTGAAATCGCCACCACATTCATATTGTGACGCTTGAGCAGTCTGTAATT

GCCTCAGAATGTGGAATCATCTGCCCCCTGTGCGAGCCCATGCCAGGCATGTCGC GGGCGAGGACACCCGCCACTCGTACAGCAGACCATTATGCTACCTCACAATAGTT

CATAACAGTGACCATATTTCTCGAAGCTCCCCAACGAGCACCTCCATGCTCTGAG

TGGCCACCCCCCGGCCCTGGTGCTTGCGGAGGGCAGGTCAACCGGCATGGGGCT

ACCGAAATCCCCGACCGGATCCCACCACCCCCGCGATGGGAAGAATCTCTCCCC

GGGATGTGGGCCCACCACCAGCACAACCTGCTGGCCCAGGCGAGCGTCAAACCA

TACCACACAAATATCCTTGGCATCGGCCCTGAATTCCTTCTGCCGCTCTGCTACCC

GGTGCTTCTGTCCGAAGCAGGGGTTGCTAGGGATCGCTCCGAGTCCGCAAACCCT

TGTCGCGTGGCGGGGCTTGTTCGAGCTTGAAGAGC

SEQ ID NO: 85

Amt03 forward primer

5 ' -GGAGGAATTCGGCCGACAGGACGCGCGTCA-3 '

SEQ ID NO: 86

Amt03 reverse primer

5 ' -GGAGACTAGTGGCTGCGACCGGCCTGTG-3 '

SEQ ID NO: 87

Amt02 forward primer

5 ' -GGAGGAATTCTCACCAGCGGACAAAGCACCG-3 '

SEQ ID NO: 88

Amt02 reverse primer

5 ' -GGAGACTAGTGGCTGCGACCGGCCTCTGG-3 '

SEQ ID NO: 89

amt03 promoter/UTR sequence

GGCCGACAGGACGCGCGTCAAAGGTGCTGGTCGTGTATGCCCTGGCCGGCAGGT

CGTTGCTGCTGCTGGTTAGTGATTCCGCAACCCTGATTTTGGCGTCTTATTTTGGC

GTGGCAAACGCTGGCGCCCGCGAGCCGGGCCGGCGGCGATGCGGTGCCCCACGG

CTGCCGGAATCCAAGGGAGGCAAGAGCGCCCGGGTCAGTTGAAGGGCTTTACGC

GCAAGGTACAGCCGCTCCTGCAAGGCTGCGTGGTGGAATTGGACGTGCAGGTCC

TGCTGAAGTTCCTCCACCGCCTCACCAGCGGACAAAGCACCGGTGTATCAGGTCC

GTGTCATCCACTCTAAAGAGCTCGACTACGACCTACTGATGGCCCTAGATTCTTC

ATCAAAAACGCCTGAGACACTTGCCCAGGATTGAAACTCCCTGAAGGGACCACC

AGGGGCCCTGAGTTGTTCCTTCCCCCCGTGGCGAGCTGCCAGCCAGGCTGTACCT

GTGATCGAGGCTGGCGGGAAAATAGGCTTCGTGTGCTCAGGTCATGGGAGGTGC

AGGACAGCTCATGAAACGCCAACAATCGCACAATTCATGTCAAGCTAATCAGCT

ATTTCCTCTTCACGAGCTGTAATTGTCCCAAAATTCTGGTCTACCGGGGGTGATCC

TTCGTGTACGGGCCCTTCCCTCAACCCTAGGTATGCGCGCATGCGGTCGCCGCGC

AACTCGCGCGAGGGCCGAGGGTTTGGGACGGGCCGTCCCGAAATGCAGTTGCAC

CCGGATGCGTGGCACCTTTTTTGCGATAATTTATGCAATGGACTGCTCTGCAAAA

TTCTGGCTCTGTCGCCAACCCTAGGATCAGCGGCGTAGGATTTCGTAATCATTCG

TCCTGATGGGGAGCTACCGACTACCCTAATATCAGCCCGACTGCCTGACGCCAGC

GTCCACTTTTGTGCACACATTCCATTCGTGCCCAAGACATTTCATTGTGGTGCGAA GCGTCCCCAGTTACGCTCACCTGTTTCCCGACCTCCTTACTGTTCTGTCGACAGAG CGGGCCCACAGGCCGGTCGCAGCC

SEQ ID NO: 90

relevant sequence of the amt promoter/UTR::C. camphora thioesterase expression construct

GCTCTTCGGCCGCCGCCACTCCTGCTCGAGCGCGCCCGACTCGCGCTCCGCCTGC

GCCCGCGCGTGCGCCGCCAGCGCCTTGGCCTTTTCGCCGCGCTCGTGCGCGTCGC

TGATGTCCATCACCAGGTCCATGAGGTCTGCCTTGCGCCGGCTGAGCCACTGCTT

CGTCCGGGCGGCCAAGAGGAGCATGAGGGAGGACTCCTGGTCCAGGGTCCTGAC

GTGGTCGCGGCTCTGGGAGCGGGCCAGCATCATCTGGCTCTGCCGCACCGAGGC

CGCCTCCAACTGGTCCTCCAGCAGCCGCAGTCGCCGCCGACCCTGGCAGAGGAA

GACAGGTGAGGGGGGTATGAATTGTACAGAACAACCACGAGCCTTGTCTAGGCA

GAATCCCTACCAGTCATGGCTTTACCTGGATGACGGCCTGCGAACAGCTGTCCAG

CGACCCTCGCTGCCGCCGCTTCTCCCGCACGCTTCTTTCCAGCACCGTGATGGCG

CGAGCCAGCGCCGCACGCTGGCGCTGCGCTTCGCCGATCTGAGGACAGTCGGGG

AACTCTGATCAGTCTAAACCCCCTTGCGCGTTAGTGTTGCCATCCTTTGCAGACC

GGTGAGAGCCGACTTGTTGTGCGCCACCCCCCACACCACCTCCTCCCAGACCAAT

CCCAGCCGCTGGGGGTTGGCGGATGCACGCTCAGGTACCCTTTCTTGCGCTATGA

CACTTCCAGCAAAAGGTAGGGCGGGCTGCGAGACGGCTTCCCGGCGCTGCATGC

AACACCGATGATGCTTCGACCCCCCGAAGCTCCTTCGGGGCTGCATGGGCGCTCC

GATGCCGCTCCAGGGCGAGCGCTGTTTAAATAGCCAGGCCCCCGATTGCAAAGA

CATTATAGCGAGCTACCAAAGCCATATTCAAACACCTAGATCACTACCACTTCTA

CACAGGCCACTCGAGCTTGTGATCGCACTCCGCTAAGGGGGCGCCTCTTCCTCTT

CGTTTCAGTCACAACCCGCAAACGGCGCGCCATGCTGCTGCAGGCCTTCCTGTTC

CTGCTGGCCGGCTTCGCCGCCAAGATCAGCGCCTCCATGACGAACGAGACGTCC

GACCGCCCCCTGGTGCACTTCACCCCCAACAAGGGCTGGATGAACGACCCCAAC

GGCCTGTGGTACGACGAGAAGGACGCCAAGTGGCACCTGTACTTCCAGTACAAC

CCGAACGACACCGTCTGGGGGACGCCCTTGTTCTGGGGCCACGCCACGTCCGAC

GACCTGACCAACTGGGAGGACCAGCCCATCGCCATCGCCCCGAAGCGCAACGAC

TCCGGCGCCTTCTCCGGCTCCATGGTGGTGGACTACAACAACACCTCCGGCTTCT

TCAACGACACCATCGACCCGCGCCAGCGCTGCGTGGCCATCTGGACCTACAACA

CCCCGGAGTCCGAGGAGCAGTACATCTCCTACAGCCTGGACGGCGGCTACACCTT

CACCGAGTACCAGAAGAACCCCGTGCTGGCCGCCAACTCCACCCAGTTCCGCGA

CCCGAAGGTCTTCTGGTACGAGCCCTCCCAGAAGTGGATCATGACCGCGGCCAA

GTCCCAGGACTACAAGATCGAGATCTACTCCTCCGACGACCTGAAGTCCTGGAA

GCTGGAGTCCGCGTTCGCCAACGAGGGCTTCCTCGGCTACCAGTACGAGTGCCCC

GGCCTGATCGAGGTCCCCACCGAGCAGGACCCCAGCAAGTCCTACTGGGTGATG

TTCATCTCCATCAACCCCGGCGCCCCGGCCGGCGGCTCCTTCAACCAGTACTTCG

TCGGCAGCTTCAACGGCACCCACTTCGAGGCCTTCGACAACCAGTCCCGCGTGGT

GGACTTCGGCAAGGACTACTACGCCCTGCAGACCTTCTTCAACACCGACCCGACC

TACGGGAGCGCCCTGGGCATCGCGTGGGCCTCCAACTGGGAGTACTCCGCCTTCG

TGCCCACCAACCCCTGGCGCTCCTCCATGTCCCTCGTGCGCAAGTTCTCCCTCAAC

ACCGAGTACCAGGCCAACCCGGAGACGGAGCTGATCAACCTGAAGGCCGAGCCG

ATCCTGAACATCAGCAACGCCGGCCCCTGGAGCCGGTTCGCCACCAACACCACG

TTGACGAAGGCCAACAGCTACAACGTCGACCTGTCCAACAGCACCGGCACCCTG

GAGTTCGAGCTGGTGTACGCCGTCAACACCACCCAGACGATCTCCAAGTCCGTGT

TCGCGGACCTCTCCCTCTGGTTCAAGGGCCTGGAGGACCCCGAGGAGTACCTCCG

CATGGGCTTCGAGGTGTCCGCGTCCTCCTTCTTCCTGGACCGCGGGAACAGCAAG GTGAAGTTCGTGAAGGAGAACCCCTACTTCACCAACCGCATGAGCGTGAACAAC

CAGCCCTTCAAGAGCGAGAACGACCTGTCCTACTACAAGGTGTACGGCTTGCTGG

ACCAGAACATCCTGGAGCTGTACTTCAACGACGGCGACGTCGTGTCCACCAACA

CCTACTTCATGACCACCGGGAACGCCCTGGGCTCCGTGAACATGACGACGGGGG

TGGACAACCTGTTCTACATCGACAAGTTCCAGGTGCGCGAGGTCAAGTGACAATT

GGCAGCAGCAGCTCGGATAGTATCGACACACTCTGGACGCTGGTCGTGTGATGG

ACTGTTGCCGCCACACTTGCTGCCTTGACCTGTGAATATCCCTGCCGCTTTTATCA

AACAGCCTCAGTGTGTTTGATCTTGTGTGTACGCGCTTTTGCGAGTTGCTAGCTGC

TTGTGCTATTTGCGAATACCACCCCCAGCATCCCCTTCCCTCGTTTCATATCGCTT

GCATCCCAACCGCAACTTATCTACGCTGTCCTGCTATCCCTCAGCGCTGCTCCTGC

TCCTGCTCACTGCCCCTCGCACAGCCTTGGTTTGGGCTCCGCCTGTATTCTCCTGG

TACTGCAACCTGTAAACCAGCACTGCAATGCTGATGCACGGGAAGTAGTGGGAT

GGGAACACAAATGGAGGATCCCGCGTCTCGAACAGAGCGCGCAGAGGAACGCT

GAAGGTCTCGCCTCTGTCGCACCTCAGCGCGGCATACACCACAATAACCACCTGA

CGAATGCGCTTGGTTCTTCGTCCATTAGCGAAGCGTCCGGTTCACACACGTGCCA

CGTTGGCGAGGTGGCAGGTGACAATGATCGGTGGAGCTGATGGTCGAAACGTTC

ACAGCCTAGGGATATCGAATTCGGCCGACAGGACGCGCGTCAAAGGTGCTGGTC

GTGTATGCCCTGGCCGGCAGGTCGTTGCTGCTGCTGGTTAGTGATTCCGCAACCC

TGATTTTGGCGTCTTATTTTGGCGTGGCAAACGCTGGCGCCCGCGAGCCGGGCCG

GCGGCGATGCGGTGCCCCACGGCTGCCGGAATCCAAGGGAGGCAAGAGCGCCCG

GGTCAGTTGAAGGGCTTTACGCGCAAGGTACAGCCGCTCCTGCAAGGCTGCGTG

GTGGAATTGGACGTGCAGGTCCTGCTGAAGTTCCTCCACCGCCTCACCAGCGGAC

AAAGCACCGGTGTATCAGGTCCGTGTCATCCACTCTAAAGAGCTCGACTACGACC

TACTGATGGCCCTAGATTCTTCATCAAAAACGCCTGAGACACTTGCCCAGGATTG

AAACTCCCTGAAGGGACCACCAGGGGCCCTGAGTTGTTCCTTCCCCCCGTGGCGA

GCTGCCAGCCAGGCTGTACCTGTGATCGAGGCTGGCGGGAAAATAGGCTTCGTG

TGCTCAGGTCATGGGAGGTGCAGGACAGCTCATGAAACGCCAACAATCGCACAA

TTCATGTCAAGCTAATCAGCTATTTCCTCTTCACGAGCTGTAATTGTCCCAAAATT

CTGGTCTACCGGGGGTGATCCTTCGTGTACGGGCCCTTCCCTCAACCCTAGGTAT

GCGCGCATGCGGTCGCCGCGCAACTCGCGCGAGGGCCGAGGGTTTGGGACGGGC

CGTCCCGAAATGCAGTTGCACCCGGATGCGTGGCACCTTTTTTGCGATAATTTAT

GCAATGGACTGCTCTGCAAAATTCTGGCTCTGTCGCCAACCCTAGGATCAGCGGC

GTAGGATTTCGTAATCATTCGTCCTGATGGGGAGCTACCGACTACCCTAATATCA

GCCCGACTGCCTGACGCCAGCGTCCACTTTTGTGCACACATTCCATTCGTGCCCA

AGACATTTCATTGTGGTGCGAAGCGTCCCCAGTTACGCTCACCTGTTTCCCGACC

TCCTTACTGTTCTGTCGACAGAGCGGGCCCACAGGCCGGTCGCAGCCACTAGTAT

GGCCACCGCATCCACTTTCTCGGCGTTCAATGCCCGCTGCGGCGACCTGCGTCGC

TCGGCGGGCTCCGGGCCCCGGCGCCCAGCGAGGCCCCTCCCCGTGCGCGGGCGC

GCCCCCGACTGGTCCATGCTGTTCGCCGTGATCACCACCATCTTCTCCGCCGCCG

AGAAGCAGTGGACCAACCTGGAGTGGAAGCCCAAGCCCAACCCCCCCCAGCTGC

TGGACGACCACTTCGGCCCCCACGGCCTGGTGTTCCGCCGCACCTTCGCCATCCG

CAGCTACGAGGTGGGCCCCGACCGCTCCACCAGCATCGTGGCCGTGATGAACCA

CCTGCAGGAGGCCGCCCTGAACCACGCCAAGTCCGTGGGCATCCTGGGCGACGG

CTTCGGCACCACCCTGGAGATGTCCAAGCGCGACCTGATCTGGGTGGTGAAGCG

CACCCACGTGGCCGTGGAGCGCTACCCCGCCTGGGGCGACACCGTGGAGGTGGA

GTGCTGGGTGGGCGCCTCCGGCAACAACGGCCGCCGCCACGACTTCCTGGTGCG

CGACTGCAAGACCGGCGAGATCCTGACCCGCTGCACCTCCCTGAGCGTGATGAT

GAACACCCGCACCCGCCGCCTGAGCAAGATCCCCGAGGAGGTGCGCGGCGAGAT

CGGCCCCGCCTTCATCGACAACGTGGCCGTGAAGGACGAGGAGATCAAGAAGCC

CCAGAAGCTGAACGACTCCACCGCCGACTACATCCAGGGCGGCCTGACCCCCCG CTGGAACGACCTGGACATCAACCAGCACGTGAACAACATCAAGTACGTGGACTG

GATCCTGGAGACCGTGCCCGACAGCATCTTCGAGAGCCACCACATCTCCTCCTTC

ACCATCGAGTACCGCCGCGAGTGCACCATGGACAGCGTGCTGCAGTCCCTGACC

ACCGTGAGCGGCGGCTCCTCCGAGGCCGGCCTGGTGTGCGAGCACCTGCTGCAG

CTGGAGGGCGGCAGCGAGGTGCTGCGCGCCAAGACCGAGTGGCGCCCCAAGCTG

ACCGACTCCTTCCGCGGCATCAGCGTGATCCCCGCCGAGTCCAGCGTGATGGACT

ACAAGGACCACGACGGCGACTACAAGGACCACGACATCGACTACAAGGACGAC

GACGACAAGTGACTCGAGTTAATTAACTCGAGGCAGCAGCAGCTCGGATAGTAT

CGACACACTCTGGACGCTGGTCGTGTGATGGACTGTTGCCGCCACACTTGCTGCC

TTGACCTGTGAATATCCCTGCCGCTTTTATCAAACAGCCTCAGTGTGTTTGATCTT

GTGTGTACGCGCTTTTGCGAGTTGCTAGCTGCTTGTGCTATTTGCGAATACCACCC

CCAGCATCCCCTTCCCTCGTTTCATATCGCTTGCATCCCAACCGCAACTTATCTAC

GCTGTCCTGCTATCCCTCAGCGCTGCTCCTGCTCCTGCTCACTGCCCCTCGCACAG

CCTTGGTTTGGGCTCCGCCTGTATTCTCCTGGTACTGCAACCTGTAAACCAGCACT

GCAATGCTGATGCACGGGAAGTAGTGGGATGGGAACACAAATGGAAAGCTGTAG

AACCTCCAAAGCCGCTCTAATTGTGGAGGGGGTTCGAATTTAAAAGCTTGGAATG

TTGGTTCGTGCGTCTGGAACAAGCCCAGACTTGTTGCTCACTGGGAAAAGGACCA

TCAGCTCCAAAAAACTTGCCGCTCAAACCGCGTACCTCTGCTTTCGCGCAATCTG

CCCTGTTGAAATCGCCACCACATTCATATTGTGACGCTTGAGCAGTCTGTAATTG

CCTCAGAATGTGGAATCATCTGCCCCCTGTGCGAGCCCATGCCAGGCATGTCGCG

GGCGAGGACACCCGCCACTCGTACAGCAGACCATTATGCTACCTCACAATAGTTC

ATAACAGTGACCATATTTCTCGAAGCTCCCCAACGAGCACCTCCATGCTCTGAGT

GGCCACCCCCCGGCCCTGGTGCTTGCGGAGGGCAGGTCAACCGGCATGGGGCTA

CCGAAATCCCCGACCGGATCCCACCACCCCCGCGATGGGAAGAATCTCTCCCCG

GGATGTGGGCCCACCACCAGCACAACCTGCTGGCCCAGGCGAGCGTCAAACCAT

ACCACACAAATATCCTTGGCATCGGCCCTGAATTCCTTCTGCCGCTCTGCTACCC

GGTGCTTCTGTCCGAAGCAGGGGTTGCTAGGGATCGCTCCGAGTCCGCAAACCCT

TGTCGCGTGGCGGGGCTTGTTCGAGCTTGTTCGAGCTTGAAGAGCCTCTAGAGTC

GACCTGCAGGCATGCAAGCTTGGCGTAATCATGGTCATAGCTGTTTCCTGTGTGA

AATTGTTATCCGCTCACAATTCCACACAACATACGAGCCGGAAGCATAAAGTGTA

AAGCCTGGGGTGCCTAATGAGTGAGCTAACTCACATTAATTGCGTTGCGCTCACT

GCCCGCTTTCCAGTCGGGAAACCTGTCGTGCCAGCTGCATTAATGAATCGGCCAA

CGCGCGGGGAGAGGCGGTTTGCGTATTGGGCGCTCTTCC

SEQ ID NO: 91

Codon-optimized C. camphora thioesterase sequence

ATGGCCACCGCATCCACTTTCTCGGCGTTCAATGCCCGCTGCGGCGACCTGCGTC

GCTCGGCGGGCTCCGGGCCCCGGCGCCCAGCGAGGCCCCTCCCCGTGCGCGGGC

GCGCCCCCGACTGGTCCATGCTGTTCGCCGTGATCACCACCATCTTCTCCGCCGC

CGAGAAGCAGTGGACCAACCTGGAGTGGAAGCCCAAGCCCAACCCCCCCCAGCT

GCTGGACGACCACTTCGGCCCCCACGGCCTGGTGTTCCGCCGCACCTTCGCCATC

CGCAGCTACGAGGTGGGCCCCGACCGCTCCACCAGCATCGTGGCCGTGATGAAC

CACCTGCAGGAGGCCGCCCTGAACCACGCCAAGTCCGTGGGCATCCTGGGCGAC

GGCTTCGGCACCACCCTGGAGATGTCCAAGCGCGACCTGATCTGGGTGGTGAAG

CGCACCCACGTGGCCGTGGAGCGCTACCCCGCCTGGGGCGACACCGTGGAGGTG

GAGTGCTGGGTGGGCGCCTCCGGCAACAACGGCCGCCGCCACGACTTCCTGGTG

CGCGACTGCAAGACCGGCGAGATCCTGACCCGCTGCACCTCCCTGAGCGTGATG

ATGAACACCCGCACCCGCCGCCTGAGCAAGATCCCCGAGGAGGTGCGCGGCGAG ATCGGCCCCGCCTTCATCGACAACGTGGCCGTGAAGGACGAGGAGATCAAGAAG

CCCCAGAAGCTGAACGACTCCACCGCCGACTACATCCAGGGCGGCCTGACCCCC

CGCTGGAACGACCTGGACATCAACCAGCACGTGAACAACATCAAGTACGTGGAC

TGGATCCTGGAGACCGTGCCCGACAGCATCTTCGAGAGCCACCACATCTCCTCCT

TCACCATCGAGTACCGCCGCGAGTGCACCATGGACAGCGTGCTGCAGTCCCTGAC

CACCGTGAGCGGCGGCTCCTCCGAGGCCGGCCTGGTGTGCGAGCACCTGCTGCA

GCTGGAGGGCGGCAGCGAGGTGCTGCGCGCCAAGACCGAGTGGCGCCCCAAGCT

GACCGACTCCTTCCGCGGCATCAGCGTGATCCCCGCCGAGTCCAGCGTGATGGAC

TACAAGGACCACGACGGCGACTACAAGGACCACGACATCGACTACAAGGACGAC

GACGACAAGTGA

SEQ ID NO: 92

Codon-optimized U. californica thioesterase sequence

GGCGCGCCCCCGACTGGTCCATGCTGTTCGCCGTGATCACCACCATCTTCAGCGC

CGCCGAGAAGCAGTGGACCAACCTGGAGTGGAAGCCCAAGCCCAAGCTGCCCCA

GCTGCTGGACGACCACTTCGGCCTGCACGGCCTGGTGTTCCGCCGCACCTTCGCC

ATCCGCTCCTACGAGGTGGGCCCCGACCGCAGCACCTCCATCCTGGCCGTGATGA

ACCACATGCAGGAGGCCACCCTGAACCACGCCAAGAGCGTGGGCATCCTGGGCG

ACGGCTTCGGCACCACCCTGGAGATGTCCAAGCGCGACCTGATGTGGGTGGTGC

GCCGCACCCACGTGGCCGTGGAGCGCTACCCCACCTGGGGCGACACCGTGGAGG

TGGAGTGCTGGATCGGCGCCAGCGGCAACAACGGCATGCGCCGCGACTTCCTGG

TGCGCGACTGCAAGACCGGCGAGATCCTGACCCGCTGCACCTCCCTGAGCGTGCT

GATGAACACCCGCACCCGCCGCCTGAGCACCATCCCCGACGAGGTGCGCGGCGA

GATCGGCCCCGCCTTCATCGACAACGTGGCCGTGAAGGACGACGAGATCAAGAA

GCTGCAGAAGCTGAACGACTCCACCGCCGACTACATCCAGGGCGGCCTGACCCC

CCGCTGGAACGACCTGGACGTGAACCAGCACGTGAACAACCTGAAGTACGTGGC

CTGGGTGTTCGAGACCGTGCCCGACAGCATCTTCGAGTCCCACCACATCAGCTCC

TTCACCCTGGAGTACCGCCGCGAGTGCACCCGCGACTCCGTGCTGCGCAGCCTGA

CCACCGTGAGCGGCGGCAGCTCCGAGGCCGGCCTGGTGTGCGACCACCTGCTGC

AGCTGGAGGGCGGCAGCGAGGTGCTGCGCGCCCGCACCGAGTGGCGCCCCAAGC

TGACCGACTCCTTCCGCGGCATCAGCGTGATCCCCGCCGAGCCCCGCGTGATGGA

CTACAAGGACCACGACGGCGACTACAAGGACCACGACATCGACTACAAGGACGA

CGACGACAAGTGACTCGAG

SEQ ID NO: 93

Codon-optimized U. americana thioesterase sequence

GGCGCGCCCAGCTGCCCGACTGGAGCATGCTGCTGGCCGCGATCACCACCCTGTT

CCTGGCGGCCGAGAAGCAGTGGATGATGCTGGACTGGAAGCCCAAGCGCCCCGA

CATGCTGGTGGACCCCTTCGGCCTGGGCCGCTTCGTGCAGGACGGCCTGGTGTTC

CGCAACAACTTCAGCATCCGCAGCTACGAGATCGGCGCGGACCGCACCGCCAGC

ATCGAGACCCTGATGAACCACCTGCAGGAGACCGCCCTGAACCACGTGAAGAGC

GTGGGCCTGCTGGAGGACGGCCTGGGCAGCACCCGCGAGATGAGCCTGCGCAAC

CTGATCTGGGTGGTGACCAAGATGCAGGTGGCGGTGGACCGCTACCCCACCTGG

GGCGACGAGGTGCAGGTGAGCAGCTGGGCGACCGCCATCGGCAAGAACGGCAT

GCGCCGCGAGTGGATCGTGACCGACTTCCGCACCGGCGAGACCCTGCTGCGCGC

CACCAGCGTGTGGGTGATGATGAACAAGCTGACCCGCCGCATCAGCAAGATCCC

CGAGGAGGTGTGGCACGAGATCGGCCCCAGCTTCATCGACGCGCCCCCCCTGCC

CACCGTGGAGGACGACGGCCGCAAGCTGACCCGCTTCGACGAGAGCAGCGCCGA CTTCATCCGCAAGGGCCTGACCCCCCGCTGGAGCGACCTGGACATCAACCAGCA

CGTGAACAACGTGAAGTACATCGGCTGGCTGCTGGAGAGCGCGCCCCCCGAGAT

CCACGAGAGCCACGAGATCGCCAGCCTGACCCTGGAGTACCGCCGCGAGTGCGG

CCGCGACAGCGTGCTGAACAGCGCCACCAAGGTGAGCGACAGCAGCCAGCTGGG

CAAGAGCGCCGTGGAGTGCAACCACCTGGTGCGCCTGCAGAACGGCGGCGAGAT

CGTGAAGGGCCGCACCGTGTGGCGCCCCAAGCGCCCCCTGTACAACGACGGCGC

CGTGGTGGACGTGCCCGCCAAGACCAGCGATGACGATGACAAGCTGGGATGACT

CGAG

SEQ ID NO: 94

Codon-optimized C. hookeriana thioesterase sequence

ACTAGTATGGCTATCAAGACGAACAGGCAGCCTGTGGAGAAGCCTCCGTTCACG

ATCGGGACGCTGCGCAAGGCCATCCCCGCGCACTGTTTCGAGCGCTCGGCGCTTC

GTGGGCGCGCCCAGCTGCCCGACTGGAGCCGCCTGCTGACCGCCATCACCACCGT

GTTCGTGAAGTCCAAGCGCCCCGACATGCACGACCGCAAGTCCAAGCGCCCCGA

CATGCTGGTGGACAGCTTCGGCCTGGAGTCCACCGTGCAGGACGGCCTGGTGTTC

CGCCAGTCCTTCTCCATCCGCTCCTACGAGATCGGCACCGACCGCACCGCCAGCA

TCGAGACCCTGATGAACCACCTGCAGGAGACCTCCCTGAACCACTGCAAGAGCA

CCGGCATCCTGCTGGACGGCTTCGGCCGCACCCTGGAGATGTGCAAGCGCGACCT

GATCTGGGTGGTGATTAAGATGCAGATCAAGGTGAACCGCTACCCCGCCTGGGG

CGACACCGTGGAGATCAACACCCGCTTCAGCCGCCTGGGCAAGATCGGCATGGG

CCGCGACTGGCTGATCTCCGACTGCAACACCGGCGAGATCCTGGTGCGCGCCACC

AGCGCCTACGCCATGATGAACCAGAAGACCCGCCGCCTGTCCAAGCTGCCCTAC

GAGGTGCACCAGGAGATCGTGCCCCTGTTCGTGGACAGCCCCGTGATCGAGGAC

TCCGACCTGAAGGTGCACAAGTTCAAGGTGAAGACCGGCGACAGCATCCAGAAG

GGCCTGACCCCCGGCTGGAACGACCTGGACGTGAACCAGCACGTGTCCAACGTG

AAGTACATCGGCTGGATCCTGGAGAGCATGCCCACCGAGGTGCTGGAGACCCAG

GAGCTGTGCTCCCTGGCCCTGGAGTACCGCCGCGAGTGCGGCCGCGACTCCGTGC

TGGAGAGCGTGACCGCCATGGACCCCAGCAAGGTGGGCGTGCGCTCCCAGTACC

AGCACCTGCTGCGCCTGGAGGACGGCACCGCCATCGTGAACGGCGCCACCGAGT

GGCGCCCCAAGAACGCCGGCGCCAACGGCGCCATCTCCACCGGCAAGACCAGCA

ACGGCAACTCCGTGTCCATGGACTACAAGGACCACGACGGCGACTACAAGGACC

ACGACATCGACTACAAGGACGACGACGACAAGTGACTCGAG

SEQ ID NO: 95

U. californica thioesterase forward primer CTGGGCGACGGCTTCGGCAC SEQ ID NO: 96

U. californica thiesterase reverse primer AAGTCGCGGCGCATGCCGTT SEQ ID NO: 97

U. americana thioesterase forward primer

CCCAGCTGCTCACCTGCACC SEQ ID NO: 98

U. americana thioesterase reverse primer CACCCAAGGCCAACGGCAGCGCCGTG SEQ ID NO: 99

C. hookeriana thioesterase forward primer TACCCCGCCTGGGGCGACAC SEQ ID NO: 100

C. hookeriana thioesterase reverse primer AGCTTGGACAGGCGGCGGGT

SEQ ID NO: 101

FAD primer 1

5 ' -TC ACTTCATGCCGGCGGTCC-3 '

SEQ ID NO: 102

FAD primer 2

5'- GCGCTCCTGCTTGGCTCGAA-3 ' SEQ ID NO: 103

pSZ1124 (FAD2B) 5' genomic targeting sequence

GCTCTTCGAGACGTGGTCTGAATCCTCCAGGCGGGTTTCCCCGAGAAAGAAAGG

GTGCCGATTTCAAAGCAGAGCCATGTGCCGGGCCCTGTGGCCTGTGTTGGCGCCT

ATGTAGTCACCCCCCCTCACCCAATTGTCGCCAGTTTGCGCAATCCATAAACTCA

AAACTGCAGCTTCTGAGCTGCGCTGTTCAAGAACACCTCTGGGGTTTGCTCACCC

GCGAGGTCGACGCCCAGCATGGCTATCAAGACGAACAGGCAGCCTGTGGAGAAG

CCTCCGTTCACGATCGGGACGCTGCGCAAGGCCATCCCCGCGCACTGTTTCGAGC

GCTCGGCGCTTCGTAGCAGCATGTACCTGGCCTTTGACATCGCGGTCATGTCCCT

GCTCTACGTCGCGTCGACGTACATCGACCCTGCGCCGGTGCCTACGTGGGTCAAG

TATGGCGTCATGTGGCCGCTCTACTGGTTCTTCCAGGTGTGTGTGAGGGTTGTGG

TTGCCCGTATCGAGGTCCTGGTGGCGCGCATGGGGGAGAAGGCGCCTGTCCCGCT

GACCCCCCCGGCTACCCTCCCGGCACCTTCCAGGGCGCCTTCGGCACGGGTGTCT

CGACGGCGTGGGCCTGGTGTTCCACAGCCTGCTGCTGGTGCCCTACTACTCCTGG AAGCACTCGCACCGGGTACC

SEQ ID NO: 104

pSZ1124 (FAD2B) 3' genomic targeting sequence

CCGCCACCACTCCAACACGGGGTGCCTGGACAAGGACGAGGTGTTTGTGCCGCC

GCACCGCGCAGTGGCGCACGAGGGCCTGGAGTGGGAGGAGTGGCTGCCCATCCG

CATGGGCAAGGTGCTGGTCACCCTGACCCTGGGCTGGCCGCTGTACCTCATGTTC AACGTCGCCTCGCGGCCGTACCCGCGCTTCGCCAACCACTTTGACCCGTGGTCGC

CCATCTTCAGCAAGCGCGAGCGCATCGAGGTGGTCATCTCCGACCTGGCGCTGGT

GGCGGTGCTCAGCGGGCTCAGCGTGCTGGGCCGCACCATGGGCTGGGCCTGGCT

GGTCAAGACCTACGTGGTGCCCTACCTGATCGTGAACATGTGGCTCGTGCTCATC

ACGCTGCTCCAGCACACGCACCCGGCGCTGCCGCACTACTTCGAGAAGGACTGG

GACTGGCTGCGCGGCGCCATGGCCACCGTGGACCGCTCCATGGGCCCGCCCTTCA

TGGACAACATCCTGCACCACATCTCCGACACCCACGTGCTGCACCACCTCTTCAG

CACCATCCCGCACTACCACGCCGAGGAGGCCTCCGCCGCCATCAGGCCCATCCTG

GGCAAGTACTACCAGTCCGACAGCCGCTGGGTCGGCCGCGCCCTGTGGGAGGAC

TGGCGCGACTGCCGCTACGTCGTCCCGGACGCGCCCGAGGACGACTCCGCGCTCT

GGTTCCACAAGTGAGTGAGTGAGAAGAGC

SEQ ID NO: 105

S. cerevisiae suc2 cassette

CTTTCTTGCGCTATGACACTTCCAGCAAAAGGTAGGGCGGGCTGCGAGACGGCTT

CCCGGCGCTGCATGCAACACCGATGATGCTTCGACCCCCCGAAGCTCCTTCGGGG

CTGCATGGGCGCTCCGATGCCGCTCCAGGGCGAGCGCTGTTTAAATAGCCAGGCC

CCCGATTGCAAAGACATTATAGCGAGCTACCAAAGCCATATTCAAACACCTAGA

TCACTACCACTTCTACACAGGCCACTCGAGCTTGTGATCGCACTCCGCTAAGGGG

GCGCCTCTTCCTCTTCGTTTCAGTCACAACCCGCAAACGGCGCGCCATGCTGCTG

CAGGCCTTCCTGTTCCTGCTGGCCGGCTTCGCCGCCAAGATCAGCGCCTCCATGA

CGAACGAGACGTCCGACCGCCCCCTGGTGCACTTCACCCCCAACAAGGGCTGGA

TGAACGACCCCAACGGCCTGTGGTACGACGAGAAGGACGCCAAGTGGCACCTGT

ACTTCCAGTACAACCCGAACGACACCGTCTGGGGGACGCCCTTGTTCTGGGGCCA

CGCCACGTCCGACGACCTGACCAACTGGGAGGACCAGCCCATCGCCATCGCCCC

GAAGCGCAACGACTCCGGCGCCTTCTCCGGCTCCATGGTGGTGGACTACAACAA

CACCTCCGGCTTCTTCAACGACACCATCGACCCGCGCCAGCGCTGCGTGGCCATC

TGGACCTACAACACCCCGGAGTCCGAGGAGCAGTACATCTCCTACAGCCTGGAC

GGCGGCTACACCTTCACCGAGTACCAGAAGAACCCCGTGCTGGCCGCCAACTCC

ACCCAGTTCCGCGACCCGAAGGTCTTCTGGTACGAGCCCTCCCAGAAGTGGATCA

TGACCGCGGCCAAGTCCCAGGACTACAAGATCGAGATCTACTCCTCCGACGACCT

GAAGTCCTGGAAGCTGGAGTCCGCGTTCGCCAACGAGGGCTTCCTCGGCTACCA

GTACGAGTGCCCCGGCCTGATCGAGGTCCCCACCGAGCAGGACCCCAGCAAGTC

CTACTGGGTGATGTTCATCTCCATCAACCCCGGCGCCCCGGCCGGCGGCTCCTTC

AACCAGTACTTCGTCGGCAGCTTCAACGGCACCCACTTCGAGGCCTTCGACAACC

AGTCCCGCGTGGTGGACTTCGGCAAGGACTACTACGCCCTGCAGACCTTCTTCAA

CACCGACCCGACCTACGGGAGCGCCCTGGGCATCGCGTGGGCCTCCAACTGGGA

GTACTCCGCCTTCGTGCCCACCAACCCCTGGCGCTCCTCCATGTCCCTCGTGCGCA

AGTTCTCCCTCAACACCGAGTACCAGGCCAACCCGGAGACGGAGCTGATCAACC

TGAAGGCCGAGCCGATCCTGAACATCAGCAACGCCGGCCCCTGGAGCCGGTTCG

CCACCAACACCACGTTGACGAAGGCCAACAGCTACAACGTCGACCTGTCCAACA

GCACCGGCACCCTGGAGTTCGAGCTGGTGTACGCCGTCAACACCACCCAGACGA

TCTCCAAGTCCGTGTTCGCGGACCTCTCCCTCTGGTTCAAGGGCCTGGAGGACCC

CGAGGAGTACCTCCGCATGGGCTTCGAGGTGTCCGCGTCCTCCTTCTTCCTGGAC

CGCGGGAACAGCAAGGTGAAGTTCGTGAAGGAGAACCCCTACTTCACCAACCGC

ATGAGCGTGAACAACCAGCCCTTCAAGAGCGAGAACGACCTGTCCTACTACAAG

GTGTACGGCTTGCTGGACCAGAACATCCTGGAGCTGTACTTCAACGACGGCGAC

GTCGTGTCCACCAACACCTACTTCATGACCACCGGGAACGCCCTGGGCTCCGTGA

ACATGACGACGGGGGTGGACAACCTGTTCTACATCGACAAGTTCCAGGTGCGCG AGGTCAAGTGACAATTGGCAGCAGCAGCTCGGATAGTATCGACACACTCTGGAC

GCTGGTCGTGTGATGGACTGTTGCCGCCACACTTGCTGCCTTGACCTGTGAATAT

CCCTGCCGCTTTTATCAAACAGCCTCAGTGTGTTTGATCTTGTGTGTACGCGCTTT

TGCGAGTTGCTAGCTGCTTGTGCTATTTGCGAATACCACCCCCAGCATCCCCTTCC

CTCGTTTCATATCGCTTGCATCCCAACCGCAACTTATCTACGCTGTCCTGCTATCC

CTCAGCGCTGCTCCTGCTCCTGCTCACTGCCCCTCGCACAGCCTTGGTTTGGGCTC

CGCCTGTATTCTCCTGGTACTGCAACCTGTAAACCAGCACTGCAATGCTGATGCA

CGGGAAGTAGTGGGATGGGAACACAAATGGA

SEQ ID NO: 106

pSZ1125 (FAD2C) 5' genomic targeting sequence

GCTCTTCGAGGGGCTGGTCTGAATCCTTCAGGCGGGTGTTACCCGAGAAAGAAA

GGGTGCCGATTTCAAAGCAGACCCATGTGCCGGGCCCTGTGGCCTGTGTTGGCGC

CTATGTAGTCACCCCCCCTCACCCAATTGTCGCCAGTTTGCGCACTCCATAAACTC

AAAACAGCAGCTTCTGAGCTGCGCTGTTCAAGAACACCTCTGGGGTTTGCTCACC

CGCGAGGTCGACGCCCAGCATGGCTATCAAGACGAACAGGCAGCCTGTGGAGAA

GCCTCCGTTCACGATCGGGACGCTGCGCAAGGCCATCCCCGCGCACTGTTTCGAG

CGCTCGGCGCTTCGTAGCAGCATGTACCTGGCCTTTGACATCGCGGTCATGTCCC

TGCTCTACGTCGCGTCGACGTACATCGACCCTGCACCGGTGCCTACGTGGGTCAA

TTGCCCGTATTGAGGTCCTGGTGGCGCGCATGGAGGAGAAGGCGCCTGTCCCGCT GACCCCCCCGGCTACCCTCCCGGCACCTTCCAGGGCGCCTTCGGCACGGGTGTCT

CGACGGCGTGGGCCTGGTGTTCCACAGCCTGCTGCTGGTGCCCTACTACTCCTGG AAGCACTCGCACCGGGTACC

SEQ ID NO: 107

pSZ1125 (FAD2C) 3' genomic targeting sequence

CCGCCACCACTCCAACACGGGGTGCCTGGACAAGGACGAGGTGTTTGTGCCGCC

GCACCGCGCAGTGGCGCACGAGGGCCTGGAGTGGGAGGAGTGGCTGCCCATCCG

CATGGGCAAGGTGCTGGTCACCCTGACCCTGGGCTGGCCGCTGTACCTCATGTTC

AACGTCGCCTCGCGGCCGTACCCGCGCTTCGCCAACCACTTTGACCCGTGGTCGC

CCATCTTCAGCAAGCGCGAGCGCATCGAGGTGGTCATCTCCGACCTGGCGCTGGT

GGCGGTGCTCAGCGGGCTCAGCGTGCTGGGCCGCACCATGGGCTGGGCCTGGCT

GGTCAAGACCTACGTGGTGCCCTACCTGATCGTGAACATGTGGCTCGTGCTCATC

ACGCTGCTCCAGCACACGCACCCGGCGCTGCCGCACTACTTCGAGAAGGACTGG

GACTGGCTGCGCGGCGCCATGGCCACCGTGGACCGCTCCATGGGCCCGCCCTTCA

TGGACAACATCCTGCACCACATCTCCGACACCCACGTGCTGCACCACCTCTTCAG

CACCATCCCGCACTACCACGCCGAGGAGGCCTCCGCCGCCATCAGGCCCATCCTG

GGCAAGTACTACCAGTCCGACAGCCGCTGGGTCGGCCGCGCCCTGTGGGAGGAC

TGGCGCGACTGCCGCTACGTCGTCCCGGACGCGCCCGAGGACGACTCCGCGCTCT

GGTTCCACAAGTGAGTGAGTGAGAAGAGC

SEQ ID NO: 108

S. carlbergensis MEL1 amino acid sequence

MFAFYFLTACISLKGVFGVSPSYNGLGLTPQMGWDNWNTFACDVSEQLLLDTADRI SDLGLKDMGYKYIILDDCWSSGRDSDGFLVADEQKFPNGMGHVADHLHNNSFLFG MYSSAGEYTCAGYPGSLGREEEDAQFFANNRVDYLKYDNCYNKGQFGTPEISYHRY

KAMSDALNKTGRPIFYSLCNWGQDLTFYWGSGIANSWRMSGDVTAEFTRPDSRCPC

DGDEYDCKYAGFHCSIMNILNKAAPMGQNAGVGGWNDLDNLEVGVGNLTDDEEK

AHFSMWAMVKSPLIIGANVNNLKASSYSIYSQASVIAINQDSNGIPATRVWRYYVSD

TDEYGQGEIQMWSGPLDNGDQVVALLNGGSVSRPMNTTLEEIFFDSNLGSKKLTST

WDIYDLWANRVDNSTASAILGRNKTATGILYNATEQSYKDGLSKNDTRLFGQKIGSL

SPNAILNTTVPAHGIAFYRLRPSS

SEQ ID NO: 109

S. carlbergensis MEL1 native signal peptide amino acid sequence

MFAFYFLTACISLKGVFG

SEQ ID NO: 110

Relevant sequence of S. carlbergensis MEL1 expression cassette

GCGGCCGCGTGGACGAGGGCTACAACCCCGCCTACGGCGCGCGCCCGCTGCGCC GCGCCATCATGCGCCTGCTGGAGGACGCGCTGGCCGAGCGCATGCTCGCCGGCG ACGTCAAGGAGGGCGACTCGGTCATCATGGACGTCGATGGCGATGGCGCCATCA GCGTCCTCAACGGAGACCGCACCCACACCACCACCATCGACTCCTCCCCGGCGG

CGAGGACTGGGCCGGGGGCCTTGGGGCGCTGCTGGAGCGTGGTGAGAGCGCGGC

GGACGTGCCTTTTCTTCTTCCGTGCGCGCGCTCTTGGCCATTGATCCCCGATTCGC

GCCCGCATCCCCCCACTGCCCCCATCATCTTGCCTGTTGTCGTGGCACTGACATA

AACCCCCTGCGCTGCGCTGCTCCGCTACTATTGATATAGGTCTCACGCGCCAATC

TTTTTTGCTCCGGGTAACCGTCTGGACGCCAGAATTCCTTTCTTGCGCTATGACAC

TTCCAGCAAAAGGTAGGGCGGGCTGCGAGACGGCTTCCCGGCGCTGCATGCAAC

ACCGATGATGCTTCGACCCCCCGAAGCTCCTTCGGGGCTGCATGGGCGCTCCGAT

GCCGCTCCAGGGCGAGCGCTGTTTAAATAGCCAGGCCCCCGATTGCAAAGACAT

TATAGCGAGCTACCAAAGCCATATTCAAACACCTAGATCACTACCACTTCTACAC

AGGCCACTCGAGCTTGTGATCGCACTCCGCTAAGGGGGCGCCTCTTCCTCTTCGT

TTCAGTCACAACCCGCAAACACTAGTATGTTCGCGTTCTACTTCCTGACGGCCTG

CATCTCCCTGAAGGGCGTGTTCGGCGTCTCCCCCTCCTACAACGGCCTGGGCCTG

ACGCCCCAGATGGGCTGGGACAACTGGAACACGTTCGCCTGCGACGTCTCCGAG

CAGCTGCTGCTGGACACGGCCGACCGCATCTCCGACCTGGGCCTGAAGGACATG

GGCTACAAGTACATCATCCTGGACGACTGCTGGTCCTCCGGCCGCGACTCCGACG

GCTTCCTGGTCGCCGACGAGCAGAAGTTCCCCAACGGCATGGGCCACGTCGCCG

ACCACCTGCACAACAACTCCTTCCTGTTCGGCATGTACTCCTCCGCGGGCGAGTA

CACGTGCGCCGGCTACCCCGGCTCCCTGGGCCGCGAGGAGGAGGACGCCCAGTT

CTTCGCGAACAACCGCGTGGACTACCTGAAGTACGACAACTGCTACAACAAGGG

CCAGTTCGGCACGCCCGAGATCTCCTACCACCGCTACAAGGCCATGTCCGACGCC

CTGAACAAGACGGGCCGCCCCATCTTCTACTCCCTGTGCAACTGGGGCCAGGACC

TGACCTTCTACTGGGGCTCCGGCATCGCGAACTCCTGGCGCATGTCCGGCGACGT

CACGGCGGAGTTCACGCGCCCCGACTCCCGCTGCCCCTGCGACGGCGACGAGTA

CGACTGCAAGTACGCCGGCTTCCACTGCTCCATCATGAACATCCTGAACAAGGCC

GCCCCCATGGGCCAGAACGCGGGCGTCGGCGGCTGGAACGACCTGGACAACCTG

GAGGTCGGCGTCGGCAACCTGACGGACGACGAGGAGAAGGCGCACTTCTCCATG

TGGGCCATGGTGAAGTCCCCCCTGATCATCGGCGCGAACGTGAACAACCTGAAG

GCCTCCTCCTACTCCATCTACTCCCAGGCGTCCGTCATCGCCATCAACCAGGACT

CCAACGGCATCCCCGCCACGCGCGTCTGGCGCTACTACGTGTCCGACACGGACG AGTACGGCCAGGGCGAGATCCAGATGTGGTCCGGCCCCCTGGACAACGGCGACC

AGGTCGTGGCGCTGCTGAACGGCGGCTCCGTGTCCCGCCCCATGAACACGACCCT

GGAGGAGATCTTCTTCGACTCCAACCTGGGCTCCAAGAAGCTGACCTCCACCTGG

GACATCTACGACCTGTGGGCGAACCGCGTCGACAACTCCACGGCGTCCGCCATCC

TGGGCCGCAACAAGACCGCCACCGGCATCCTGTACAACGCCACCGAGCAGTCCT

ACAAGGACGGCCTGTCCAAGAACGACACCCGCCTGTTCGGCCAGAAGATCGGCT

CCCTGTCCCCCAACGCGATCCTGAACACGACCGTCCCCGCCCACGGCATCGCGTT

CTACCGCCTGCGCCCCTCCTCCTGACAATTGAAGCAGCAGCAGCTCGGATAGTAT

CGACACACTCTGGACGCTGGTCGTGTGATGGACTGTTGCCGCCACACTTGCTGCC

TTGACCTGTGAATATCCCTGCCGCTTTTATCAAACAGCCTCAGTGTGTTTGATCTT

GTGTGTACGCGCTTTTGCGAGTTGCTAGCTGCTTGTGCTATTTGCGAATACCACCC

CCAGCATCCCCTTCCCTCGTTTCATATCGCTTGCATCCCAACCGCAACTTATCTAC

GCTGTCCTGCTATCCCTCAGCGCTGCTCCTGCTCCTGCTCACTGCCCCTCGCACAG

CCTTGGTTTGGGCTCCGCCTGTATTCTCCTGGTACTGCAACCTGTAAACCAGCACT

GCAATGCTGATGCACGGGAAGTAGTGGGATGGGAACACAAATGGAAAGCTTGAG

CTCAGAATAGTATCGGGTGATGCGAAGTCAGAACCAGGCAGGGCCTGTCGCCTG

AGGTGGCAACGATGGGAAGCAATCAATCTGGGTACAGTCGTCCGCACGATCCCG

TGATCTCCCCCACCGACACCTATCCCCGCCCATCCCGGCCCACCCTTTCAGTCCCC

TCAGCATGCATTGTGCACCGCGACAAAGCATGTCTGCTCGTGCACTGGTTCAGGC

CACGGCGCACCGAGTCCTCGCCCTTCGCAGAGTGATCACCCTCCCCGGAACCAGC

CACGCTCGCTGCTGCGGGCCGATCAGCCGCGCGCACTCCCTGCAACTAGGGACA

ACTCAGGCAACCACGCGCCTCACAAGCATGGCCGCCGTGGCATCCAACCCGCTC

GTGACGGTGGGTGCGCAAGTGCCAGGGGCCTCGTCGTCACGGCGTGCATCCTCG

AGGGATGCGATCCGGCAACTATATGTCGTTTATCTCCCCACCAATCACAGGATGA

GCCCCTGTCTAGA

SEQ ID NO: 111

Codon-optimized S. carbergensis MEL1 sequence

ATGTTCGCGTTCTACTTCCTGACGGCCTGCATCTCCCTGAAGGGCGTGTTCGGCGT

CTCCCCCTCCTACAACGGCCTGGGCCTGACGCCCCAGATGGGCTGGGACAACTGG

AACACGTTCGCCTGCGACGTCTCCGAGCAGCTGCTGCTGGACACGGCCGACCGC

ATCTCCGACCTGGGCCTGAAGGACATGGGCTACAAGTACATCATCCTGGACGACT

GCTGGTCCTCCGGCCGCGACTCCGACGGCTTCCTGGTCGCCGACGAGCAGAAGTT

CCCCAACGGCATGGGCCACGTCGCCGACCACCTGCACAACAACTCCTTCCTGTTC

GGCATGTACTCCTCCGCGGGCGAGTACACGTGCGCCGGCTACCCCGGCTCCCTGG

GCCGCGAGGAGGAGGACGCCCAGTTCTTCGCGAACAACCGCGTGGACTACCTGA

AGTACGACAACTGCTACAACAAGGGCCAGTTCGGCACGCCCGAGATCTCCTACC

ACCGCTACAAGGCCATGTCCGACGCCCTGAACAAGACGGGCCGCCCCATCTTCTA

CTCCCTGTGCAACTGGGGCCAGGACCTGACCTTCTACTGGGGCTCCGGCATCGCG

AACTCCTGGCGCATGTCCGGCGACGTCACGGCGGAGTTCACGCGCCCCGACTCCC

GCTGCCCCTGCGACGGCGACGAGTACGACTGCAAGTACGCCGGCTTCCACTGCTC

CATCATGAACATCCTGAACAAGGCCGCCCCCATGGGCCAGAACGCGGGCGTCGG

CGGCTGGAACGACCTGGACAACCTGGAGGTCGGCGTCGGCAACCTGACGGACGA

CGAGGAGAAGGCGCACTTCTCCATGTGGGCCATGGTGAAGTCCCCCCTGATCATC

GGCGCGAACGTGAACAACCTGAAGGCCTCCTCCTACTCCATCTACTCCCAGGCGT

CCGTCATCGCCATCAACCAGGACTCCAACGGCATCCCCGCCACGCGCGTCTGGCG

CTACTACGTGTCCGACACGGACGAGTACGGCCAGGGCGAGATCCAGATGTGGTC

CGGCCCCCTGGACAACGGCGACCAGGTCGTGGCGCTGCTGAACGGCGGCTCCGT

GTCCCGCCCCATGAACACGACCCTGGAGGAGATCTTCTTCGACTCCAACCTGGGC TCCAAGAAGCTGACCTCCACCTGGGACATCTACGACCTGTGGGCGAACCGCGTC

GACAACTCCACGGCGTCCGCCATCCTGGGCCGCAACAAGACCGCCACCGGCATC

CTGTACAACGCCACCGAGCAGTCCTACAAGGACGGCCTGTCCAAGAACGACACC

CGCCTGTTCGGCCAGAAGATCGGCTCCCTGTCCCCCAACGCGATCCTGAACACGA

CCGTCCCCGCCCACGGCATCGCGTTCTACCGCCTGCGCCCCTCCTCCTGA

SEQ ID NO: 112

5' Clp homologous recombination targeting sequence

GCGGCCGCGTGGACGAGGGCTACAACCCCGCCTACGGCGCGCGCCCGCTGCGCC GCGCCATCATGCGCCTGCTGGAGGACGCGCTGGCCGAGCGCATGCTCGCCGGCG ACGTCAAGGAGGGCGACTCGGTCATCATGGACGTCGATGGCGATGGCGCCATCA GCGTCCTCAACGGAGACCGCACCCACACCACCACCATCGACTCCTCCCCGGCGG

CGAGGACTGGGCCGGGGGCCTTGGGGCGCTGCTGGAGCGTGGTGAGAGCGCGGC

GGACGTGCCTTTTCTTCTTCCGTGCGCGCGCTCTTGGCCATTGATCCCCGATTCGC

GCCCGCATCCCCCCACTGCCCCCATCATCTTGCCTGTTGTCGTGGCACTGACATA

AACCCCCTGCGCTGCGCTGCTCCGCTACTATTGATATAGGTCTCACGCGCCAATC

TTTTTTGCTCCGGGTAACCGTCTGGACGCCAGAATTC

SEQ ID NO: 113

3' Clp homologous recombination targeting sequence

GAGCTCAGAATAGTATCGGGTGATGCGAAGTCAGAACCAGGCAGGGCCTGTCGC

CTGAGGTGGCAACGATGGGAAGCAATCAATCTGGGTACAGTCGTCCGCACGATC

CCGTGATCTCCCCCACCGACACCTATCCCCGCCCATCCCGGCCCACCCTTTCAGTC

CCCTCAGCATGCATTGTGCACCGCGACAAAGCATGTCTGCTCGTGCACTGGTTCA

GGCCACGGCGCACCGAGTCCTCGCCCTTCGCAGAGTGATCACCCTCCCCGGAACC

AGCCACGCTCGCTGCTGCGGGCCGATCAGCCGCGCGCACTCCCTGCAACTAGGG

ACAACTCAGGCAACCACGCGCCTCACAAGCATGGCCGCCGTGGCATCCAACCCG

CTCGTGACGGTGGGTGCGCAAGTGCCAGGGGCCTCGTCGTCACGGCGTGCATCCT

CGAGGGATGCGATCCGGCAACTATATGTCGTTTATCTCCCCACCAATCACAGGAT

GAGCCCCTGTCTAGA

SEQ ID NO: 114

Chlamydomonas reinhardtii TUB 2 promoter/5' UTR

CTTTCTTGCGCTATGACACTTCCAGCAAAAGGTAGGGCGGGCTGCGAGACGGCTT

CCCGGCGCTGCATGCAACACCGATGATGCTTCGACCCCCCGAAGCTCCTTCGGGG

CTGCATGGGCGCTCCGATGCCGCTCCAGGGCGAGCGCTGTTTAAATAGCCAGGCC

CCCGATTGCAAAGACATTATAGCGAGCTACCAAAGCCATATTCAAACACCTAGA

TCACTACCACTTCTACACAGGCCACTCGAGCTTGTGATCGCACTCCGCTAAGGGG

GCGCCTCTTCCTCTTCGTTTCAGTCACAACCCGCAAAC

SEQ ID NO: 115

Chlorella vulgaris nitrate reductase 3 'UTR

GCAGCAGCAGCTCGGATAGTATCGACACACTCTGGACGCTGGTCGTGTGATGGA CTGTTGCCGCCACACTTGCTGCCTTGACCTGTGAATATCCCTGCCGCTTTTATCAA ACAGCCTCAGTGTGTTTGATCTTGTGTGTACGCGCTTTTGCGAGTTGCTAGCTGCT TGTGCTATTTGCGAATACCACCCCCAGCATCCCCTTCCCTCGTTTCATATCGCTTG CATCCCAACCGCAACTTATCTACGCTGTCCTGCTATCCCTCAGCGCTGCTCCTGCT CCTGCTCACTGCCCCTCGCACAGCCTTGGTTTGGGCTCCGCCTGTATTCTCCTGGT ACTGCAACCTGTAAACCAGCACTGCAATGCTGATGCACGGGAAGTAGTGGGATG GGAACACAAATGGAAAGCTT

SEQ ID NO: 116

A. niger AlgC amino acid sequence

MIGSSHAVVALGLFTLYGHSAAAPAIGASNSQTIVTNGTSFALNGDNVSYRFHVNSS

TGDLISDHFGGVVSGTIPSPVEPAVNGWVGMPGRIRREFPDQGRGDFRIPAVRIRES A

GYTVSDLQYVSHEVIEGKYALPGLPATFGDAQDATTLVVHLYDNYSSVAADLSYSIF

PKYDAIVRSVNVTNQGPGNITIEALASISIDFPYEDLDMVSLRGDWAREANVQRSKV

QYGVQGFGSSTGYSSHLHNPFLAIVDPATTESQGEAWGFNLVYTGSFSAQVEKGSQG

FTRALLGFNPDQLSWNLGPGETLTSPECVAVYSDKGLGSVSRKFHRLYRNHLMKSK

FATSDRPVLLNSWEGVYFDYNQSSIETLAEESAALGVHLFVMDDGWFGDKYPRVSD

NAGLGDWMPNPARFPDGLTPVVQDITNLTVNGTESTKLRFGIWVEPEMVNPNSTLY

HEHPEWALHAGPYPRTERRNQLVLNLALPAVQDFIIDFMTNLLQDTGISYVKWDNN

RGIHETPSPSTDHQYMLGLYRVFDTLTTRFPDVLWEGCASGGGRFDAGMLQYVPQI

WTSDNTDAIDRITIQFGTSLAYPPSAMGAHLSAVPNAQTGRTVPFTFRAHVAMMGGS

FGLELDPATVEGDEIVPELLALAEKVNPIILNGDLYRLRLPQDSQWPAALFVSQDGA Q

AVLFYFQVQPNVNHAVPWVRLQGLDPKADYTVDGDQTYSGATLMNLGLQYSFDTE

YGSKVVFLERQ

SEQ ID NO: 117

A. niger AlgC native signal peptide amino acid sequence

MIGSSHAVVALGLFTLYGHSAAAPAIGA

SEQ ID NO: 118

Codon-optimized A. niger AlgC coding sequence

ATGATCGGCTCCTCCCACGCGGTCGTCGCCCTGGGCCTGTTCACCCTGTACGGCC

ACTCCGCCGCCGCGCCCGCCATCGGCGCCTCCAACTCCCAGACCATCGTCACGAA

CGGCACCTCCTTCGCCCTGAACGGCGACAACGTGTCCTACCGCTTCCACGTGAAC

TCCTCCACGGGCGACCTGATCTCCGACCACTTCGGCGGCGTGGTGTCCGGCACCA

TCCCCTCCCCCGTGGAGCCCGCGGTCAACGGCTGGGTGGGCATGCCCGGCCGCAT

CCGCCGCGAGTTCCCCGACCAGGGCCGCGGCGACTTCCGCATCCCCGCGGTGCGC

ATCCGCGAGTCCGCCGGCTACACCGTCTCCGACCTGCAGTACGTGTCCCACGAGG

TGATCGAGGGCAAGTACGCGCTGCCCGGCCTGCCCGCCACGTTCGGCGACGCCC

AGGACGCCACCACCCTGGTGGTGCACCTGTACGACAACTACTCCTCCGTCGCGGC

CGACCTGTCCTACTCCATCTTCCCCAAGTACGACGCGATCGTCCGCTCCGTGAAC

GTGACCAACCAGGGCCCCGGCAACATCACCATCGAGGCGCTGGCCTCCATCTCC

ATCGACTTCCCCTACGAGGACCTGGACATGGTGTCCCTGCGCGGCGACTGGGCCC

GCGAGGCGAACGTGCAGCGCTCCAAGGTCCAGTACGGCGTGCAGGGCTTCGGCT

CCTCCACCGGCTACTCCTCCCACCTGCACAACCCCTTCCTGGCGATCGTCGACCC

CGCGACCACCGAGTCCCAGGGCGAGGCCTGGGGCTTCAACCTGGTCTACACCGG

CTCCTTCTCCGCCCAGGTCGAGAAGGGCTCCCAGGGCTTCACGCGCGCCCTGCTG

GGCTTCAACCCCGACCAGCTGTCCTGGAACCTGGGCCCCGGCGAGACGCTGACG TCCCCCGAGTGCGTCGCCGTCTACTCCGACAAGGGCCTGGGCTCCGTCTCCCGCA

AGTTCCACCGCCTGTACCGCAACCACCTGATGAAGTCCAAGTTCGCCACGTCCGA

CCGCCCCGTGCTGCTGAACTCCTGGGAGGGCGTCTACTTCGACTACAACCAGTCC

TCCATCGAGACGCTGGCGGAGGAGTCCGCCGCCCTGGGCGTGCACCTGTTCGTCA

TGGACGACGGCTGGTTCGGCGACAAGTACCCCCGCGTGTCCGACAACGCCGGCC

TGGGCGACTGGATGCCCAACCCCGCCCGCTTCCCCGACGGCCTGACGCCCGTCGT

GCAGGACATCACCAACCTGACCGTCAACGGCACCGAGTCCACCAAGCTGCGCTT

CGGCATCTGGGTGGAGCCCGAGATGGTGAACCCCAACTCCACCCTGTACCACGA

GCACCCCGAGTGGGCGCTGCACGCGGGCCCCTACCCCCGCACCGAGCGCCGCAA

CCAGCTGGTCCTGAACCTGGCCCTGCCCGCGGTCCAGGACTTCATCATCGACTTC

ATGACCAACCTGCTGCAGGACACCGGCATCTCCTACGTCAAGTGGGACAACAAC

CGCGGCATCCACGAGACGCCCTCCCCCTCCACGGACCACCAGTACATGCTGGGCC

TGTACCGCGTGTTCGACACGCTGACCACGCGCTTCCCCGACGTCCTGTGGGAGGG

CTGCGCGTCCGGCGGCGGCCGGTTCGACGCCGGCATGCTGCAGTACGTCCCCCAG

ATCTGGACGTCCGACAACACGGACGCGATCGACCGCATCACCATCCAGTTCGGC

ACCTCCCTGGCCTACCCCCCCTCCGCCATGGGCGCCCACCTGTCCGCGGTGCCCA

ACGCCCAGACCGGCCGCACGGTGCCCTTCACCTTCCGCGCCCACGTCGCGATGAT

GGGCGGCTCCTTCGGCCTGGAGCTGGACCCCGCCACCGTGGAGGGCGACGAGAT

CGTGCCCGAGCTGCTGGCGCTGGCCGAGAAGGTGAACCCCATCATCCTGAACGG

CGACCTGTACCGCCTGCGCCTGCCCCAGGACTCCCAGTGGCCCGCGGCCCTGTTC

GTGTCCCAGGACGGCGCCCAGGCCGTCCTGTTCTACTTCCAGGTGCAGCCCAACG

TCAACCACGCCGTCCCCTGGGTCCGCCTGCAGGGCCTGGACCCCAAGGCCGACTA

CACGGTGGACGGCGACCAGACGTACTCCGGCGCGACCCTGATGAACCTGGGCCT

GCAGTACTCCTTCGACACCGAGTACGGCTCCAAGGTGGTGTTCCTGGAGCGCCAG

TAA

SEQ ID NO: 119

Relevant sequence of A. niger AlgC expression cassette

GCGGCCGCGTGGACGAGGGCTACAACCCCGCCTACGGCGCGCGCCCGCTGCGCC GCGCCATCATGCGCCTGCTGGAGGACGCGCTGGCCGAGCGCATGCTCGCCGGCG ACGTCAAGGAGGGCGACTCGGTCATCATGGACGTCGATGGCGATGGCGCCATCA GCGTCCTCAACGGAGACCGCACCCACACCACCACCATCGACTCCTCCCCGGCGG

CGAGGACTGGGCCGGGGGCCTTGGGGCGCTGCTGGAGCGTGGTGAGAGCGCGGC

GGACGTGCCTTTTCTTCTTCCGTGCGCGCGCTCTTGGCCATTGATCCCCGATTCGC

GCCCGCATCCCCCCACTGCCCCCATCATCTTGCCTGTTGTCGTGGCACTGACATA

AACCCCCTGCGCTGCGCTGCTCCGCTACTATTGATATAGGTCTCACGCGCCAATC

TTTTTTGCTCCGGGTAACCGTCTGGACGCCAGAATTCCTTTCTTGCGCTATGACAC

TTCCAGCAAAAGGTAGGGCGGGCTGCGAGACGGCTTCCCGGCGCTGCATGCAAC

ACCGATGATGCTTCGACCCCCCGAAGCTCCTTCGGGGCTGCATGGGCGCTCCGAT

GCCGCTCCAGGGCGAGCGCTGTTTAAATAGCCAGGCCCCCGATTGCAAAGACAT

TATAGCGAGCTACCAAAGCCATATTCAAACACCTAGATCACTACCACTTCTACAC

AGGCCACTCGAGCTTGTGATCGCACTCCGCTAAGGGGGCGCCTCTTCCTCTTCGT

TTCAGTCACAACCCGCAAACACTAGTATGATCGGCTCCTCCCACGCGGTCGTCGC

CCTGGGCCTGTTCACCCTGTACGGCCACTCCGCCGCCGCGCCCGCCATCGGCGCC

TCCAACTCCCAGACCATCGTCACGAACGGCACCTCCTTCGCCCTGAACGGCGACA

ACGTGTCCTACCGCTTCCACGTGAACTCCTCCACGGGCGACCTGATCTCCGACCA

CTTCGGCGGCGTGGTGTCCGGCACCATCCCCTCCCCCGTGGAGCCCGCGGTCAAC

GGCTGGGTGGGCATGCCCGGCCGCATCCGCCGCGAGTTCCCCGACCAGGGCCGC GGCGACTTCCGCATCCCCGCGGTGCGCATCCGCGAGTCCGCCGGCTACACCGTCT

CCGACCTGCAGTACGTGTCCCACGAGGTGATCGAGGGCAAGTACGCGCTGCCCG

GCCTGCCCGCCACGTTCGGCGACGCCCAGGACGCCACCACCCTGGTGGTGCACCT

GTACGACAACTACTCCTCCGTCGCGGCCGACCTGTCCTACTCCATCTTCCCCAAG

TACGACGCGATCGTCCGCTCCGTGAACGTGACCAACCAGGGCCCCGGCAACATC

ACCATCGAGGCGCTGGCCTCCATCTCCATCGACTTCCCCTACGAGGACCTGGACA

TGGTGTCCCTGCGCGGCGACTGGGCCCGCGAGGCGAACGTGCAGCGCTCCAAGG

TCCAGTACGGCGTGCAGGGCTTCGGCTCCTCCACCGGCTACTCCTCCCACCTGCA

CAACCCCTTCCTGGCGATCGTCGACCCCGCGACCACCGAGTCCCAGGGCGAGGC

CTGGGGCTTCAACCTGGTCTACACCGGCTCCTTCTCCGCCCAGGTCGAGAAGGGC

TCCCAGGGCTTCACGCGCGCCCTGCTGGGCTTCAACCCCGACCAGCTGTCCTGGA

ACCTGGGCCCCGGCGAGACGCTGACGTCCCCCGAGTGCGTCGCCGTCTACTCCGA

CAAGGGCCTGGGCTCCGTCTCCCGCAAGTTCCACCGCCTGTACCGCAACCACCTG

ATGAAGTCCAAGTTCGCCACGTCCGACCGCCCCGTGCTGCTGAACTCCTGGGAGG

GCGTCTACTTCGACTACAACCAGTCCTCCATCGAGACGCTGGCGGAGGAGTCCGC

CGCCCTGGGCGTGCACCTGTTCGTCATGGACGACGGCTGGTTCGGCGACAAGTAC

CCCCGCGTGTCCGACAACGCCGGCCTGGGCGACTGGATGCCCAACCCCGCCCGCT

TCCCCGACGGCCTGACGCCCGTCGTGCAGGACATCACCAACCTGACCGTCAACG

GCACCGAGTCCACCAAGCTGCGCTTCGGCATCTGGGTGGAGCCCGAGATGGTGA

ACCCCAACTCCACCCTGTACCACGAGCACCCCGAGTGGGCGCTGCACGCGGGCC

CCTACCCCCGCACCGAGCGCCGCAACCAGCTGGTCCTGAACCTGGCCCTGCCCGC

GGTCCAGGACTTCATCATCGACTTCATGACCAACCTGCTGCAGGACACCGGCATC

TCCTACGTCAAGTGGGACAACAACCGCGGCATCCACGAGACGCCCTCCCCCTCCA

CGGACCACCAGTACATGCTGGGCCTGTACCGCGTGTTCGACACGCTGACCACGCG

CTTCCCCGACGTCCTGTGGGAGGGCTGCGCGTCCGGCGGCGGCCGGTTCGACGCC

GGCATGCTGCAGTACGTCCCCCAGATCTGGACGTCCGACAACACGGACGCGATC

GACCGCATCACCATCCAGTTCGGCACCTCCCTGGCCTACCCCCCCTCCGCCATGG

GCGCCCACCTGTCCGCGGTGCCCAACGCCCAGACCGGCCGCACGGTGCCCTTCAC

CTTCCGCGCCCACGTCGCGATGATGGGCGGCTCCTTCGGCCTGGAGCTGGACCCC

GCCACCGTGGAGGGCGACGAGATCGTGCCCGAGCTGCTGGCGCTGGCCGAGAAG

GTGAACCCCATCATCCTGAACGGCGACCTGTACCGCCTGCGCCTGCCCCAGGACT

CCCAGTGGCCCGCGGCCCTGTTCGTGTCCCAGGACGGCGCCCAGGCCGTCCTGTT

CTACTTCCAGGTGCAGCCCAACGTCAACCACGCCGTCCCCTGGGTCCGCCTGCAG

GGCCTGGACCCCAAGGCCGACTACACGGTGGACGGCGACCAGACGTACTCCGGC

GCGACCCTGATGAACCTGGGCCTGCAGTACTCCTTCGACACCGAGTACGGCTCCA

AGGTGGTGTTCCTGGAGCGCCAGTAACAATTGAAGCAGCAGCAGCTCGGATAGT

ATCGACACACTCTGGACGCTGGTCGTGTGATGGACTGTTGCCGCCACACTTGCTG

CCTTGACCTGTGAATATCCCTGCCGCTTTTATCAAACAGCCTCAGTGTGTTTGATC

TTGTGTGTACGCGCTTTTGCGAGTTGCTAGCTGCTTGTGCTATTTGCGAATACCAC

CCCCAGCATCCCCTTCCCTCGTTTCATATCGCTTGCATCCCAACCGCAACTTATCT

ACGCTGTCCTGCTATCCCTCAGCGCTGCTCCTGCTCCTGCTCACTGCCCCTCGCAC

AGCCTTGGTTTGGGCTCCGCCTGTATTCTCCTGGTACTGCAACCTGTAAACCAGC

ACTGCAATGCTGATGCACGGGAAGTAGTGGGATGGGAACACAAATGGAAAGCTT

GAGCTCAGAATAGTATCGGGTGATGCGAAGTCAGAACCAGGCAGGGCCTGTCGC

CTGAGGTGGCAACGATGGGAAGCAATCAATCTGGGTACAGTCGTCCGCACGATC

CCGTGATCTCCCCCACCGACACCTATCCCCGCCCATCCCGGCCCACCCTTTCAGTC

CCCTCAGCATGCATTGTGCACCGCGACAAAGCATGTCTGCTCGTGCACTGGTTCA

GGCCACGGCGCACCGAGTCCTCGCCCTTCGCAGAGTGATCACCCTCCCCGGAACC

AGCCACGCTCGCTGCTGCGGGCCGATCAGCCGCGCGCACTCCCTGCAACTAGGG

ACAACTCAGGCAACCACGCGCCTCACAAGCATGGCCGCCGTGGCATCCAACCCG CTCGTGACGGTGGGTGCGCAAGTGCCAGGGGCCTCGTCGTCACGGCGTGCATCCT CGAGGGATGCGATCCGGCAACTATATGTCGTTTATCTCCCCACCAATCACAGGAT GAGCCCCTGTCTAGA

SEQ ID NO: 120

C. tetragonobola a-galactosidase amino acid sequence

MATHYSIIGGMIIVVLLMIIGSEGGRLLEKKNRTSAEAEHYNVRRYLAENGLGQTPP M

GWNSWNHFGCDINENVVRETADAMVSTGLAALGYQYINLDDCWAELNRDSEGNM

VPNAAAFPSGIKALADYVHSKGLKLGVYSDAGNQTCSKRMPGSLGHEEQDAKTFAS

WGVDYLKYDNCENLGISVKERYPPMGKALLSSGRPIFFSMCEWGWEDPQIWAKSIG

NSWRTTGDIEDNWNSMTSIADSNDKWASYAGPGGWNDPDMLEVGNGGMTTEEYR

SHFSIWALAKAPLLVGCDIRAMDDTTHELISNAEVIAVNQDKLGVQGKKVKSTNDLE

VWAGPLSDNKVAVILWNRSSSRATVTASWSDIGLQQGTTVDARDLWEHSTQSLVSG

EISAEIDSHACKMYVLTPRS

SEQ ID NO: 121

Codon-optimized C. tetragonobola a-galactosidase sequence

ATGGCCACCCACTACTCCATCATCGGCGGCATGATCATCGTCGTCCTGCTGATGA

TCATCGGCTCCGAGGGCGGCCGGCTGCTGGAGAAGAAGAACCGCACCTCCGCCG

AGGCGGAGCACTACAACGTGCGCCGCTACCTGGCCGAGAACGGCCTGGGCCAGA

CCCCCCCCATGGGCTGGAACTCCTGGAACCACTTCGGCTGCGACATCAACGAGA

ACGTCGTCCGCGAGACGGCGGACGCCATGGTGTCCACCGGCCTGGCCGCCCTGG

GCTACCAGTACATCAACCTGGACGACTGCTGGGCGGAGCTGAACCGCGACTCCG

AGGGCAACATGGTGCCCAACGCCGCCGCGTTCCCCTCCGGCATCAAGGCGCTGG

CGGACTACGTCCACTCCAAGGGCCTGAAGCTGGGCGTCTACTCCGACGCGGGCA

ACCAGACCTGCTCCAAGCGCATGCCCGGCTCCCTGGGCCACGAGGAGCAGGACG

CGAAGACGTTCGCCTCCTGGGGCGTCGACTACCTGAAGTACGACAACTGCGAGA

ACCTGGGCATCTCCGTGAAGGAGCGCTACCCCCCCATGGGCAAGGCGCTGCTGTC

CTCCGGCCGCCCCATCTTCTTCTCCATGTGCGAGTGGGGCTGGGAGGACCCCCAG

ATCTGGGCCAAGTCCATCGGCAACTCCTGGCGCACCACCGGCGACATCGAGGAC

AACTGGAACTCCATGACCTCCATCGCCGACTCCAACGACAAGTGGGCCTCCTACG

CCGGCCCCGGCGGCTGGAACGACCCCGACATGCTGGAGGTCGGCAACGGCGGCA

TGACGACGGAGGAGTACCGCTCCCACTTCTCCATCTGGGCCCTGGCCAAGGCGCC

CCTGCTGGTGGGCTGCGACATCCGCGCGATGGACGACACGACGCACGAGCTGAT

CTCCAACGCCGAGGTGATCGCGGTGAACCAGGACAAGCTGGGCGTGCAGGGCAA

GAAGGTCAAGTCCACGAACGACCTGGAGGTCTGGGCCGGCCCCCTGTCCGACAA

CAAGGTGGCGGTGATCCTGTGGAACCGCTCCTCCTCCCGCGCCACCGTCACCGCG

TCCTGGTCCGACATCGGCCTGCAGCAGGGCACCACCGTCGACGCGCGCGACCTGT

GGGAGCACTCCACGCAGTCCCTGGTGTCCGGCGAGATCTCCGCCGAGATCGACTC

CCACGCCTGCAAGATGTACGTCCTGACGCCCCGCTCCTAA

SEQ ID NO: 122

Relevant sequence of C. tetragonobola α-galactosidase expression cassette

GCGGCCGCGTGGACGAGGGCTACAACCCCGCCTACGGCGCGCGCCCGCTGCGCC GCGCCATCATGCGCCTGCTGGAGGACGCGCTGGCCGAGCGCATGCTCGCCGGCG ACGTCAAGGAGGGCGACTCGGTCATCATGGACGTCGATGGCGATGGCGCCATCA GCGTCCTCAACGGAGACCGCACCCACACCACCACCATCGACTCCTCCCCGGCGG CGAGGACTGGGCCGGGGGCCTTGGGGCGCTGCTGGAGCGTGGTGAGAGCGCGGC

GGACGTGCCTTTTCTTCTTCCGTGCGCGCGCTCTTGGCCATTGATCCCCGATTCGC

GCCCGCATCCCCCCACTGCCCCCATCATCTTGCCTGTTGTCGTGGCACTGACATA

AACCCCCTGCGCTGCGCTGCTCCGCTACTATTGATATAGGTCTCACGCGCCAATC

TTTTTTGCTCCGGGTAACCGTCTGGACGCCAGAATTCCTTTCTTGCGCTATGACAC

TTCCAGCAAAAGGTAGGGCGGGCTGCGAGACGGCTTCCCGGCGCTGCATGCAAC

ACCGATGATGCTTCGACCCCCCGAAGCTCCTTCGGGGCTGCATGGGCGCTCCGAT

GCCGCTCCAGGGCGAGCGCTGTTTAAATAGCCAGGCCCCCGATTGCAAAGACAT

TATAGCGAGCTACCAAAGCCATATTCAAACACCTAGATCACTACCACTTCTACAC

AGGCCACTCGAGCTTGTGATCGCACTCCGCTAAGGGGGCGCCTCTTCCTCTTCGT

TTCAGTCACAACCCGCAAACACTAGTATGGCCACCCACTACTCCATCATCGGCGG

CATGATCATCGTCGTCCTGCTGATGATCATCGGCTCCGAGGGCGGCCGGCTGCTG

GAGAAGAAGAACCGCACCTCCGCCGAGGCGGAGCACTACAACGTGCGCCGCTAC

CTGGCCGAGAACGGCCTGGGCCAGACCCCCCCCATGGGCTGGAACTCCTGGAAC

CACTTCGGCTGCGACATCAACGAGAACGTCGTCCGCGAGACGGCGGACGCCATG

GTGTCCACCGGCCTGGCCGCCCTGGGCTACCAGTACATCAACCTGGACGACTGCT

GGGCGGAGCTGAACCGCGACTCCGAGGGCAACATGGTGCCCAACGCCGCCGCGT

TCCCCTCCGGCATCAAGGCGCTGGCGGACTACGTCCACTCCAAGGGCCTGAAGCT

GGGCGTCTACTCCGACGCGGGCAACCAGACCTGCTCCAAGCGCATGCCCGGCTC

CCTGGGCCACGAGGAGCAGGACGCGAAGACGTTCGCCTCCTGGGGCGTCGACTA

CCTGAAGTACGACAACTGCGAGAACCTGGGCATCTCCGTGAAGGAGCGCTACCC

CCCCATGGGCAAGGCGCTGCTGTCCTCCGGCCGCCCCATCTTCTTCTCCATGTGC

GAGTGGGGCTGGGAGGACCCCCAGATCTGGGCCAAGTCCATCGGCAACTCCTGG

CGCACCACCGGCGACATCGAGGACAACTGGAACTCCATGACCTCCATCGCCGAC

TCCAACGACAAGTGGGCCTCCTACGCCGGCCCCGGCGGCTGGAACGACCCCGAC

ATGCTGGAGGTCGGCAACGGCGGCATGACGACGGAGGAGTACCGCTCCCACTTC

TCCATCTGGGCCCTGGCCAAGGCGCCCCTGCTGGTGGGCTGCGACATCCGCGCGA

TGGACGACACGACGCACGAGCTGATCTCCAACGCCGAGGTGATCGCGGTGAACC

AGGACAAGCTGGGCGTGCAGGGCAAGAAGGTCAAGTCCACGAACGACCTGGAG

GTCTGGGCCGGCCCCCTGTCCGACAACAAGGTGGCGGTGATCCTGTGGAACCGCT

CCTCCTCCCGCGCCACCGTCACCGCGTCCTGGTCCGACATCGGCCTGCAGCAGGG

CACCACCGTCGACGCGCGCGACCTGTGGGAGCACTCCACGCAGTCCCTGGTGTCC

GGCGAGATCTCCGCCGAGATCGACTCCCACGCCTGCAAGATGTACGTCCTGACGC

CCCGCTCCTAACAATTGAAGCAGCAGCAGCTCGGATAGTATCGACACACTCTGG

ACGCTGGTCGTGTGATGGACTGTTGCCGCCACACTTGCTGCCTTGACCTGTGAAT

ATCCCTGCCGCTTTTATCAAACAGCCTCAGTGTGTTTGATCTTGTGTGTACGCGCT

TTTGCGAGTTGCTAGCTGCTTGTGCTATTTGCGAATACCACCCCCAGCATCCCCTT

CCCTCGTTTCATATCGCTTGCATCCCAACCGCAACTTATCTACGCTGTCCTGCTAT

CCCTCAGCGCTGCTCCTGCTCCTGCTCACTGCCCCTCGCACAGCCTTGGTTTGGGC

TCCGCCTGTATTCTCCTGGTACTGCAACCTGTAAACCAGCACTGCAATGCTGATG

CACGGGAAGTAGTGGGATGGGAACACAAATGGAAAGCTTGAGCTCAGAATAGTA

TCGGGTGATGCGAAGTCAGAACCAGGCAGGGCCTGTCGCCTGAGGTGGCAACGA

TGGGAAGCAATCAATCTGGGTACAGTCGTCCGCACGATCCCGTGATCTCCCCCAC

CGACACCTATCCCCGCCCATCCCGGCCCACCCTTTCAGTCCCCTCAGCATGCATT

GTGCACCGCGACAAAGCATGTCTGCTCGTGCACTGGTTCAGGCCACGGCGCACC

GAGTCCTCGCCCTTCGCAGAGTGATCACCCTCCCCGGAACCAGCCACGCTCGCTG

CTGCGGGCCGATCAGCCGCGCGCACTCCCTGCAACTAGGGACAACTCAGGCAAC

CACGCGCCTCACAAGCATGGCCGCCGTGGCATCCAACCCGCTCGTGACGGTGGG

TGCGCAAGTGCCAGGGGCCTCGTCGTCACGGCGTGCATCCTCGAGGGATGCGAT CCGGCAACTATATGTCGTTTATCTCCCCACCAATCACAGGATGAGCCCCTGTCTA GA

SEQ ID NO: 123

5' primer C. vulgaris 3'UTR:downstream Clp sequence 5 ' - ACTGC AATGCTGATGC ACGGGA-3 ' SEQ ID NO: 124

3' primer C. vulgaris 3'UTR:downstream Clp sequence 5 ' -TCCAGGTCCTTTTCGCACT-3 ' SEQ ID NO: 125

Coccomyxa C-169 THIC amino acid sequence

MTTNLAKLPLGGLSSRSSIAGAPLRVNSHSRERHLGAKTTAIAAPERLDYLDNAEEA

RLQQTDAFAELKALSSRQSVNRPQKGELSFRQSPTFQDCFPGSEKCYREVEHDGKTL

KVPFRRVHLQDDNGHFDLYDTSGPQGVNPREGLPKIRSSWVEPREARGDKVQTQQY

YAKQGIITEEMAFCAARERMDPEFIRSEVARGRAIIPANKRHLELEPTVVGRNFLVK V

NANIGNSAVSSSIEEEVEKLQWSTIWGADTAMDLSTGNNIHETREWVMRNSPVPVGT

VPIYQCLEKAGGIVENITWELFRETLIEQAEQGVDYFTIHAGVLLRYIPLTANRVTG IV

SRGGSIHAKLCLLDHTENFAYMHWDEILDICAQYDITLSIGDGLRPGCIADANDAAQ F

AELKTQGELTRRAWAKDVQVMNEGPGHVPLHKIPENMEKQLDWCSEAPFYTLGPL

ATDIAPAYDHITSAIGAATIGALGTALLCYVTPKEHLGLPDRDDVKAGVIAYKIAAH A

ADLAKGHPYAQEWDNALSKARFEFRWYDQFHLSLDPVTARLFHDATLPQEPAKTA

HFCSMCGPKFCSMQITQDLREYAKNHQMEEDEAIQTGMAEMSEQFKSIGAEVYLDE

AVLEHA

SEQ ID NO: 126

Coccomyxa C-169 THIC native transit peptide amino acid sequence MTTNLAKLPLGGLSSRSSIAGAPLRVNSHSRERHLGAKTT SEQ ID NO: 127

Relevant sequence of Coccomyxa C-169 THIC expression cassette

GCTCTTCGCCGCCGCCACTCCTGCTCGAGCGCGCCCGCGCGTGCGCCGCCAGCGC

AGGTCTGCCTTGCGCCGGCTGAGCCACTGCTTCGTCCGGGCGGCCAAGAGGAGC

ATGAGGGAGGACTCCTGGTCCAGGGTCCTGACGTGGTCGCGGCTCTGGGAGCGG

GCCAGCATCATCTGGCTCTGCCGCACCGAGGCCGCCTCCAACTGGTCCTCCAGCA

GCCGCAGTCGCCGCCGACCCTGGCAGAGGAAGACAGGTGAGGGGGGTATGAATT

GTACAGAACAACCACGAGCCTTGTCTAGGCAGAATCCCTACCAGTCATGGCTTTA

CCTGGATGACGGCCTGCGAACAGCTGTCCAGCGACCCTCGCTGCCGCCGCTTCTC

CCGCACGCTTCTTTCCAGCACCGTGATGGCGCGAGCCAGCGCCGCACGCTGGCGC

TGCGCTTCGCCGATCTGAGGACAGTCGGGGAACTCTGATCAGTCTAAACCCCCTT

GCGCGTTAGTGTTGCCATCCTTTGCAGACCGGTGAGAGCCGACTTGTTGTGCGCC

ACCCCCCACACCACCTCCTCCCAGACCAATTCTGTCACCTTTTTGGCGAAGGCAT

CGGCCTCGGCCTGCAGAGAGGACAGCAGTGCCCAGCCGCTGGGGGTTGGCGGAT GCACGCTCAGGTACCCTTTCTTGCGCTATGACACTTCCAGCAAAAGGTAGGGCGG

GCTGCGAGACGGCTTCCCGGCGCTGCATGCAACACCGATGATGCTTCGACCCCCC

GAAGCTCCTTCGGGGCTGCATGGGCGCTCCGATGCCGCTCCAGGGCGAGCGCTGT

TTAAATAGCCAGGCCCCCGATTGCAAAGACATTATAGCGAGCTACCAAAGCCAT

ATTCAAACACCTAGATCACTACCACTTCTACACAGGCCACTCGAGCTTGTGATCG

CACTCCGCTAAGGGGGCGCCTCTTCCTCTTCGTTTCAGTCACAACCCGCAAACGG

CGCGCCATGCTGCTGCAGGCCTTCCTGTTCCTGCTGGCCGGCTTCGCCGCCAAGA

TCAGCGCCTCCATGACGAACGAGACGTCCGACCGCCCCCTGGTGCACTTCACCCC

CAACAAGGGCTGGATGAACGACCCCAACGGCCTGTGGTACGACGAGAAGGACGC

CAAGTGGCACCTGTACTTCCAGTACAACCCGAACGACACCGTCTGGGGGACGCC

CTTGTTCTGGGGCCACGCCACGTCCGACGACCTGACCAACTGGGAGGACCAGCC

CATCGCCATCGCCCCGAAGCGCAACGACTCCGGCGCCTTCTCCGGCTCCATGGTG

GTGGACTACAACAACACCTCCGGCTTCTTCAACGACACCATCGACCCGCGCCAGC

GCTGCGTGGCCATCTGGACCTACAACACCCCGGAGTCCGAGGAGCAGTACATCT

CCTACAGCCTGGACGGCGGCTACACCTTCACCGAGTACCAGAAGAACCCCGTGC

TGGCCGCCAACTCCACCCAGTTCCGCGACCCGAAGGTCTTCTGGTACGAGCCCTC

CCAGAAGTGGATCATGACCGCGGCCAAGTCCCAGGACTACAAGATCGAGATCTA

CTCCTCCGACGACCTGAAGTCCTGGAAGCTGGAGTCCGCGTTCGCCAACGAGGG

CTTCCTCGGCTACCAGTACGAGTGCCCCGGCCTGATCGAGGTCCCCACCGAGCAG

GACCCCAGCAAGTCCTACTGGGTGATGTTCATCTCCATCAACCCCGGCGCCCCGG

CCGGCGGCTCCTTCAACCAGTACTTCGTCGGCAGCTTCAACGGCACCCACTTCGA

GGCCTTCGACAACCAGTCCCGCGTGGTGGACTTCGGCAAGGACTACTACGCCCTG

CAGACCTTCTTCAACACCGACCCGACCTACGGGAGCGCCCTGGGCATCGCGTGG

GCCTCCAACTGGGAGTACTCCGCCTTCGTGCCCACCAACCCCTGGCGCTCCTCCA

TGTCCCTCGTGCGCAAGTTCTCCCTCAACACCGAGTACCAGGCCAACCCGGAGAC

GGAGCTGATCAACCTGAAGGCCGAGCCGATCCTGAACATCAGCAACGCCGGCCC

CTGGAGCCGGTTCGCCACCAACACCACGTTGACGAAGGCCAACAGCTACAACGT

CGACCTGTCCAACAGCACCGGCACCCTGGAGTTCGAGCTGGTGTACGCCGTCAAC

ACCACCCAGACGATCTCCAAGTCCGTGTTCGCGGACCTCTCCCTCTGGTTCAAGG

GCCTGGAGGACCCCGAGGAGTACCTCCGCATGGGCTTCGAGGTGTCCGCGTCCTC

CTTCTTCCTGGACCGCGGGAACAGCAAGGTGAAGTTCGTGAAGGAGAACCCCTA

CTTCACCAACCGCATGAGCGTGAACAACCAGCCCTTCAAGAGCGAGAACGACCT

GTCCTACTACAAGGTGTACGGCTTGCTGGACCAGAACATCCTGGAGCTGTACTTC

AACGACGGCGACGTCGTGTCCACCAACACCTACTTCATGACCACCGGGAACGCC

CTGGGCTCCGTGAACATGACGACGGGGGTGGACAACCTGTTCTACATCGACAAG

TTCCAGGTGCGCGAGGTCAAGTGACAATTGGCAGCAGCAGCTCGGATAGTATCG

ACACACTCTGGACGCTGGTCGTGTGATGGACTGTTGCCGCCACACTTGCTGCCTT

GACCTGTGAATATCCCTGCCGCTTTTATCAAACAGCCTCAGTGTGTTTGATCTTGT

GTGTACGCGCTTTTGCGAGTTGCTAGCTGCTTGTGCTATTTGCGAATACCACCCCC

AGCATCCCCTTCCCTCGTTTCATATCGCTTGCATCCCAACCGCAACTTATCTACGC

TGTCCTGCTATCCCTCAGCGCTGCTCCTGCTCCTGCTCACTGCCCCTCGCACAGCC

TTGGTTTGGGCTCCGCCTGTATTCTCCTGGTACTGCAACCTGTAAACCAGCACTGC

AATGCTGATGCACGGGAAGTAGTGGGATGGGAACACAAATGGAGGATCCCGCGT

CTCGAACAGAGCGCGCAGAGGAACGCTGAAGGTCTCGCCTCTGTCGCACCTCAG

CGCGGCATACACCACAATAACCACCTGACGAATGCGCTTGGTTCTTCGTCCATTA

GCGAAGCGTCCGGTTCACACACGTGCCACGTTGGCGAGGTGGCAGGTGACAATG

ATCGGTGGAGCTGATGGTCGAAACGTTCACAGCCTAGGGATATCGAATTCCTTTC

TTGCGCTATGACACTTCCAGCAAAAGGTAGGGCGGGCTGCGAGACGGCTTCCCG

GCGCTGCATGCAACACCGATGATGCTTCGACCCCCCGAAGCTCCTTCGGGGCTGC

ATGGGCGCTCCGATGCCGCTCCAGGGCGAGCGCTGTTTAAATAGCCAGGCCCCC GATTGCAAAGACATTATAGCGAGCTACCAAAGCCATATTCAAACACCTAGATCA

CTACCACTTCTACACAGGCCACTCGAGCTTGTGATCGCACTCCGCTAAGGGGGCG

CCTCTTCCTCTTCGTTTCAGTCACAACCCGCAAACACTAGTATGACCACCAACCT

GGCTAAGCTGCCTCTCGGCGGCCTCTCTTCTCGGTCGTCCATCGCCGGGGCTCCTC

TGCGCGTGAACAGCCACAGCCGCGAGCGCCACCTGGGCGCCAAGACCACCGCGA

TCGCCGCTCCCGAGCGGCTGGACTACCTCGACAACGCGGAGGAGGCGCGGCTGC

AGCAGACGGACGCCTTCGCCGAGCTGAAGGCGCTGTCGAGCCGCCAGTCCGTGA

ACCGGCCCCAAAAGGGCGAGCTGTCGTTCCGGCAGTCGCCGACCTTTCAGGACT

GCTTCCCTGGCTCGGAGAAGTGCTACCGCGAGGTGGAGCACGATGGCAAGACGC

TCAAGGTGCCTTTTCGCCGGGTGCACCTGCAGGACGACAACGGCCATTTCGACCT

GTACGACACCTCTGGCCCCCAGGGCGTGAACCCCCGCGAGGGTCTGCCCAAGAT

CCGCTCCTCCTGGGTGGAGCCGCGCGAGGCGCGGGGCGACAAGGTGCAAACGCA

GCAGTACTACGCCAAGCAAGGCATCATCACCGAGGAGATGGCCTTCTGCGCTGC

GCGCGAGCGCATGGACCCCGAGTTCATCCGGTCTGAGGTGGCCCGGGGTCGGGC

CATCATCCCTGCGAACAAGCGCCATCTGGAGCTGGAGCCGACCGTCGTGGGCCG

CAACTTTCTGGTCAAGGTGAACGCCAACATCGGCAACTCTGCGGTCAGCTCGAGC

ATCGAGGAGGAGGTGGAGAAGCTGCAGTGGAGCACCATCTGGGGTGCCGACACG

GCCATGGACCTGAGCACCGGCAACAACATCCACGAGACGCGCGAGTGGGTGATG

CGCAACAGCCCGGTCCCTGTGGGTACGGTCCCTATCTACCAGTGCCTGGAGAAGG

CGGGCGGCATCGTGGAGAACATCACCTGGGAGCTGTTCCGGGAGACGCTGATCG

AGCAGGCCGAGCAGGGCGTCGACTACTTTACCATCCACGCGGGGGTCCTGCTCC

GCTACATCCCCCTGACGGCGAACCGCGTCACCGGCATCGTCAGCCGCGGCGGCTC

TATCCATGCCAAGCTCTGCCTGCTGGACCACACCGAGAACTTCGCCTACATGCAC

TGGGACGAGATCCTGGACATCTGCGCCCAGTACGACATCACCCTGTCGATCGGCG

ACGGCCTGCGCCCCGGCTGCATCGCGGACGCCAACGACGCTGCCCAGTTTGCTGA

GCTGAAGACCCAGGGCGAGCTGACCCGCCGCGCCTGGGCGAAGGACGTGCAGGT

GATGAACGAGGGTCCCGGCCATGTGCCGCTGCACAAGATCCCCGAGAACATGGA

GAAGCAGCTGGACTGGTGCAGCGAGGCGCCCTTCTACACGCTCGGCCCCCTGGC

GACGGACATCGCGCCTGCGTACGACCACATCACCTCCGCGATCGGCGCCGCCAC

GATCGGCGCGCTGGGGACCGCGCTGCTGTGTTACGTGACCCCGAAGGAGCACCT

GGGCCTCCCCGATCGCGACGACGTGAAGGCCGGGGTGATCGCCTACAAGATCGC

GGCGCACGCGGCTGACCTGGCGAAGGGTCACCCCTACGCTCAGGAGTGGGATAA

CGCCCTCTCTAAGGCGCGCTTCGAGTTCCGGTGGTACGACCAGTTCCACCTGTCG

CTGGACCCCGTCACCGCCCGCCTGTTCCATGACGCGACCCTGCCGCAGGAGCCCG

CCAAGACCGCCCACTTCTGCTCCATGTGCGGCCCCAAGTTCTGCTCCATGCAGAT

CACCCAGGACCTCCGCGAGTACGCCAAGAACCATCAAATGGAGGAGGACGAGGC

GATCCAAACCGGCATGGCCGAGATGTCCGAGCAGTTCAAGAGCATCGGCGCGGA

GGTCTACCTCGACGAGGCGGTGCTGGAGCACGCGTAAGCAGCAGCAGCTCGGAT

AGTATCGACACACTCTGGACGCTGGTCGTGTGATGGACTGTTGCCGCCACACTTG

CTGCCTTGACCTGTGAATATCCCTGCCGCTTTTATCAAACAGCCTCAGTGTGTTTG

ATCTTGTGTGTACGCGCTTTTGCGAGTTGCTAGCTGCTTGTGCTATTTGCGAATAC

CACCCCCAGCATCCCCTTCCCTCGTTTCATATCGCTTGCATCCCAACCGCAACTTA

TCTACGCTGTCCTGCTATCCCTCAGCGCTGCTCCTGCTCCTGCTCACTGCCCCTCG

CACAGCCTTGGTTTGGGCTCCGCCTGTATTCTCCTGGTACTGCAACCTGTAAACC

AGCACTGCAATGCTGATGCACGGGAAGTAGTGGGATGGGAACACAAATGGAAA

GCTTGAGCTCTTGTTTTCCAGAAGGAGTTGCTCCTTGAGCCTTTCATTCTCAGCCT

CGATAACCTCCAAAGCCGCTCTAATTGTGGAGGGGGTTCGAATTTAAAAGCTTGG

AATGTTGGTTCGTGCGTCTGGAACAAGCCCAGACTTGTTGCTCACTGGGAAAAGG

ACCATCAGCTCCAAAAAACTTGCCGCTCAAACCGCGTACCTCTGCTTTCGCGCAA

TCTGCCCTGTTGAAATCGCCACCACATTCATATTGTGACGCTTGAGCAGTCTGTA ATTGCCTCAGAATGTGGAATCATCTGCCCCCTGTGCGAGCCCATGCCAGGCATGT

CGCGGGCGAGGACACCCGCCACTCGTACAGCAGACCATTATGCTACCTCACAAT

AGTTCATAACAGTGACCATATTTCTCGAAGCTCCCCAACGAGCACCTCCATGCTC

TGAGTGGCCACCCCCCGGCCCTGGTGCTTGCGGAGGGCAGGTCAACCGGCATGG

GGCTACCGAAATCCCCGACCGGATCCCACCACCCCCGCGATGGGAAGAATCTCT

CCCCGGGATGTGGGCCCACCACCAGCACAACCTGCTGGCCCAGGCGAGCGTCAA

ACCATACCACACAAATATCCTTGGCATCGGCCCTGAATTCCTTCTGCCGCTCTGCT

ACCCGGTGCTTCTGTCCGAAGCAGGGGTTGCTAGGGATCGCTCCGAGTCCGCAAA

CCCTTGTCGCGTGGCGGGGCTTGTTCGAGCTGAAGAGC

SEQ ID NO: 128

Codon-optimized Coccomyxa C-169 THIC sequence

ATGACCACCAACCTGGCTAAGCTGCCTCTCGGCGGCCTCTCTTCTCGGTCGTCCA

TCGCCGGGGCTCCTCTGCGCGTGAACAGCCACAGCCGCGAGCGCCACCTGGGCG

CCAAGACCACCGCGATCGCCGCTCCCGAGCGGCTGGACTACCTCGACAACGCGG

AGGAGGCGCGGCTGCAGCAGACGGACGCCTTCGCCGAGCTGAAGGCGCTGTCGA

GCCGCCAGTCCGTGAACCGGCCCCAAAAGGGCGAGCTGTCGTTCCGGCAGTCGC

CGACCTTTCAGGACTGCTTCCCTGGCTCGGAGAAGTGCTACCGCGAGGTGGAGCA

CGATGGCAAGACGCTCAAGGTGCCTTTTCGCCGGGTGCACCTGCAGGACGACAA

CGGCCATTTCGACCTGTACGACACCTCTGGCCCCCAGGGCGTGAACCCCCGCGAG

GGTCTGCCCAAGATCCGCTCCTCCTGGGTGGAGCCGCGCGAGGCGCGGGGCGAC

AAGGTGCAAACGCAGCAGTACTACGCCAAGCAAGGCATCATCACCGAGGAGATG

GCCTTCTGCGCTGCGCGCGAGCGCATGGACCCCGAGTTCATCCGGTCTGAGGTGG

CCCGGGGTCGGGCCATCATCCCTGCGAACAAGCGCCATCTGGAGCTGGAGCCGA

CCGTCGTGGGCCGCAACTTTCTGGTCAAGGTGAACGCCAACATCGGCAACTCTGC

GGTCAGCTCGAGCATCGAGGAGGAGGTGGAGAAGCTGCAGTGGAGCACCATCTG

GGGTGCCGACACGGCCATGGACCTGAGCACCGGCAACAACATCCACGAGACGCG

CGAGTGGGTGATGCGCAACAGCCCGGTCCCTGTGGGTACGGTCCCTATCTACCAG

TGCCTGGAGAAGGCGGGCGGCATCGTGGAGAACATCACCTGGGAGCTGTTCCGG

GAGACGCTGATCGAGCAGGCCGAGCAGGGCGTCGACTACTTTACCATCCACGCG

GGGGTCCTGCTCCGCTACATCCCCCTGACGGCGAACCGCGTCACCGGCATCGTCA

GCCGCGGCGGCTCTATCCATGCCAAGCTCTGCCTGCTGGACCACACCGAGAACTT

CGCCTACATGCACTGGGACGAGATCCTGGACATCTGCGCCCAGTACGACATCACC

CTGTCGATCGGCGACGGCCTGCGCCCCGGCTGCATCGCGGACGCCAACGACGCT

GCCCAGTTTGCTGAGCTGAAGACCCAGGGCGAGCTGACCCGCCGCGCCTGGGCG

AAGGACGTGCAGGTGATGAACGAGGGTCCCGGCCATGTGCCGCTGCACAAGATC

CCCGAGAACATGGAGAAGCAGCTGGACTGGTGCAGCGAGGCGCCCTTCTACACG

CTCGGCCCCCTGGCGACGGACATCGCGCCTGCGTACGACCACATCACCTCCGCGA

TCGGCGCCGCCACGATCGGCGCGCTGGGGACCGCGCTGCTGTGTTACGTGACCCC

GAAGGAGCACCTGGGCCTCCCCGATCGCGACGACGTGAAGGCCGGGGTGATCGC

CTACAAGATCGCGGCGCACGCGGCTGACCTGGCGAAGGGTCACCCCTACGCTCA

GGAGTGGGATAACGCCCTCTCTAAGGCGCGCTTCGAGTTCCGGTGGTACGACCA

GTTCCACCTGTCGCTGGACCCCGTCACCGCCCGCCTGTTCCATGACGCGACCCTG

CCGCAGGAGCCCGCCAAGACCGCCCACTTCTGCTCCATGTGCGGCCCCAAGTTCT

GCTCCATGCAGATCACCCAGGACCTCCGCGAGTACGCCAAGAACCATCAAATGG

AGGAGGACGAGGCGATCCAAACCGGCATGGCCGAGATGTCCGAGCAGTTCAAGA

GCATCGGCGCGGAGGTCTACCTCGACGAGGCGGTGCTGGAGCACGCGTAA

SEQ ID NO: 129 Codon-optimized S. cerevisiae suc2 sequence

GGCGCGCCATGCTGCTGCAGGCCTTCCTGTTCCTGCTGGCCGGCTTCGCCGCCAA

GATCAGCGCCTCCATGACGAACGAGACGTCCGACCGCCCCCTGGTGCACTTCACC

CCCAACAAGGGCTGGATGAACGACCCCAACGGCCTGTGGTACGACGAGAAGGAC

GCCAAGTGGCACCTGTACTTCCAGTACAACCCGAACGACACCGTCTGGGGGACG

CCCTTGTTCTGGGGCCACGCCACGTCCGACGACCTGACCAACTGGGAGGACCAG

CCCATCGCCATCGCCCCGAAGCGCAACGACTCCGGCGCCTTCTCCGGCTCCATGG

TGGTGGACTACAACAACACCTCCGGCTTCTTCAACGACACCATCGACCCGCGCCA

GCGCTGCGTGGCCATCTGGACCTACAACACCCCGGAGTCCGAGGAGCAGTACAT

CTCCTACAGCCTGGACGGCGGCTACACCTTCACCGAGTACCAGAAGAACCCCGT

GCTGGCCGCCAACTCCACCCAGTTCCGCGACCCGAAGGTCTTCTGGTACGAGCCC

TCCCAGAAGTGGATCATGACCGCGGCCAAGTCCCAGGACTACAAGATCGAGATC

TACTCCTCCGACGACCTGAAGTCCTGGAAGCTGGAGTCCGCGTTCGCCAACGAGG

GCTTCCTCGGCTACCAGTACGAGTGCCCCGGCCTGATCGAGGTCCCCACCGAGCA

GGACCCCAGCAAGTCCTACTGGGTGATGTTCATCTCCATCAACCCCGGCGCCCCG

GCCGGCGGCTCCTTCAACCAGTACTTCGTCGGCAGCTTCAACGGCACCCACTTCG

AGGCCTTCGACAACCAGTCCCGCGTGGTGGACTTCGGCAAGGACTACTACGCCCT

GCAGACCTTCTTCAACACCGACCCGACCTACGGGAGCGCCCTGGGCATCGCGTG

GGCCTCCAACTGGGAGTACTCCGCCTTCGTGCCCACCAACCCCTGGCGCTCCTCC

ATGTCCCTCGTGCGCAAGTTCTCCCTCAACACCGAGTACCAGGCCAACCCGGAGA

CGGAGCTGATCAACCTGAAGGCCGAGCCGATCCTGAACATCAGCAACGCCGGCC

CCTGGAGCCGGTTCGCCACCAACACCACGTTGACGAAGGCCAACAGCTACAACG

TCGACCTGTCCAACAGCACCGGCACCCTGGAGTTCGAGCTGGTGTACGCCGTCAA

CACCACCCAGACGATCTCCAAGTCCGTGTTCGCGGACCTCTCCCTCTGGTTCAAG

GGCCTGGAGGACCCCGAGGAGTACCTCCGCATGGGCTTCGAGGTGTCCGCGTCCT

CCTTCTTCCTGGACCGCGGGAACAGCAAGGTGAAGTTCGTGAAGGAGAACCCCT

ACTTCACCAACCGCATGAGCGTGAACAACCAGCCCTTCAAGAGCGAGAACGACC

TGTCCTACTACAAGGTGTACGGCTTGCTGGACCAGAACATCCTGGAGCTGTACTT

CAACGACGGCGACGTCGTGTCCACCAACACCTACTTCATGACCACCGGGAACGC

CCTGGGCTCCGTGAACATGACGACGGGGGTGGACAACCTGTTCTACATCGACAA

GTTCCAGGTGCGCGAGGTCAAGTGACAATTG

SEQ ID NO: 130

Relevant sequence of Coccomyxa C-169 THIC expression construct with C. protothecoides transit peptide

GCGGCCGCGTGGACGAGGGCTACAACCCCGCCTACGGCGCGCGCCCGCTGCGCC GCGCCATCATGCGCCTGCTGGAGGACGCGCTGGCCGAGCGCATGCTCGCCGGCG ACGTCAAGGAGGGCGACTCGGTCATCATGGACGTCGATGGCGATGGCGCCATCA GCGTCCTCAACGGAGACCGCACCCACACCACCACCATCGACTCCTCCCCGGCGG

CGAGGACTGGGCCGGGGGCCTTGGGGCGCTGCTGGAGCGTGGTGAGAGCGCGGC

GGACGTGCCTTTTCTTCTTCCGTGCGCGCGCTCTTGGCCATTGATCCCCGATTCGC

GCCCGCATCCCCCCACTGCCCCCATCATCTTGCCTGTTGTCGTGGCACTGACATA

AACCCCCTGCGCTGCGCTGCTCCGCTACTATTGATATAGGTCTCACGCGCCAATC

TTTTTTGCTCCGGGTAACCGTCTGGACGCCAGAATTCCTTTCTTGCGCTATGACAC

TTCCAGCAAAAGGTAGGGCGGGCTGCGAGACGGCTTCCCGGCGCTGCATGCAAC

ACCGATGATGCTTCGACCCCCCGAAGCTCCTTCGGGGCTGCATGGGCGCTCCGAT

GCCGCTCCAGGGCGAGCGCTGTTTAAATAGCCAGGCCCCCGATTGCAAAGACAT TATAGCGAGCTACCAAAGCCATATTCAAACACCTAGATCACTACCACTTCTACAC

AGGCCACTCGAGCTTGTGATCGCACTCCGCTAAGGGGGCGCCTCTTCCTCTTCGT

TTCAGTCACAACCCGCAAACACTAGTATGGCCACCGCATCCACTTTCTCGGCGTT

CAATGCCCGCTGCGGCGACCTGCGTCGCTCGGCGGGCTCCGGGCCCCGGCGCCC

AGCGAGGCCCCTCCCCGTGCGCGGGCGCGCCGCGATCGCCGCTCCCGAGCGGCT

GGACTACCTCGACAACGCGGAGGAGGCGCGGCTGCAGCAGACGGACGCCTTCGC

CGAGCTGAAGGCGCTGTCGAGCCGCCAGTCCGTGAACCGGCCCCAAAAGGGCGA

GCTGTCGTTCCGGCAGTCGCCGACCTTTCAGGACTGCTTCCCTGGCTCGGAGAAG

GTGCACCTGCAGGACGACAACGGCCATTTCGACCTGTACGACACCTCTGGCCCCC

AGGGCGTGAACCCCCGCGAGGGTCTGCCCAAGATCCGCTCCTCCTGGGTGGAGC

CGCGCGAGGCGCGGGGCGACAAGGTGCAAACGCAGCAGTACTACGCCAAGCAA

GGCATCATCACCGAGGAGATGGCCTTCTGCGCTGCGCGCGAGCGCATGGACCCC

GAGTTCATCCGGTCTGAGGTGGCCCGGGGTCGGGCCATCATCCCTGCGAACAAG

CGCCATCTGGAGCTGGAGCCGACCGTCGTGGGCCGCAACTTTCTGGTCAAGGTGA

ACGCCAACATCGGCAACTCTGCGGTCAGCTCGAGCATCGAGGAGGAGGTGGAGA

AGCTGCAGTGGAGCACCATCTGGGGTGCCGACACGGCCATGGACCTGAGCACCG

GCAACAACATCCACGAGACGCGCGAGTGGGTGATGCGCAACAGCCCGGTCCCTG

TGGGTACGGTCCCTATCTACCAGTGCCTGGAGAAGGCGGGCGGCATCGTGGAGA

ACATCACCTGGGAGCTGTTCCGGGAGACGCTGATCGAGCAGGCCGAGCAGGGCG

TCGACTACTTTACCATCCACGCGGGGGTCCTGCTCCGCTACATCCCCCTGACGGC

GAACCGCGTCACCGGCATCGTCAGCCGCGGCGGCTCTATCCATGCCAAGCTCTGC

CTGCTGGACCACACCGAGAACTTCGCCTACATGCACTGGGACGAGATCCTGGAC

ATCTGCGCCCAGTACGACATCACCCTGTCGATCGGCGACGGCCTGCGCCCCGGCT

GCATCGCGGACGCCAACGACGCTGCCCAGTTTGCTGAGCTGAAGACCCAGGGCG

AGCTGACCCGCCGCGCCTGGGCGAAGGACGTGCAGGTGATGAACGAGGGTCCCG

GCCATGTGCCGCTGCACAAGATCCCCGAGAACATGGAGAAGCAGCTGGACTGGT

GCAGCGAGGCGCCCTTCTACACGCTCGGCCCCCTGGCGACGGACATCGCGCCTGC

GTACGACCACATCACCTCCGCGATCGGCGCCGCCACGATCGGCGCGCTGGGGAC

CGCGCTGCTGTGTTACGTGACCCCGAAGGAGCACCTGGGCCTCCCCGATCGCGAC

GACGTGAAGGCCGGGGTGATCGCCTACAAGATCGCGGCGCACGCGGCTGACCTG

GCGAAGGGTCACCCCTACGCTCAGGAGTGGGATAACGCCCTCTCTAAGGCGCGC

TTCGAGTTCCGGTGGTACGACCAGTTCCACCTGTCGCTGGACCCCGTCACCGCCC

GCCTGTTCCATGACGCGACCCTGCCGCAGGAGCCCGCCAAGACCGCCCACTTCTG

CTCCATGTGCGGCCCCAAGTTCTGCTCCATGCAGATCACCCAGGACCTCCGCGAG

TACGCCAAGAACCATCAAATGGAGGAGGACGAGGCGATCCAAACCGGCATGGCC

GAGATGTCCGAGCAGTTCAAGAGCATCGGCGCGGAGGTCTACCTCGACGAGGCG

GTGCTGGAGCACGCGTAACAATTGAAGCAGCAGCAGCTCGGATAGTATCGACAC

ACTCTGGACGCTGGTCGTGTGATGGACTGTTGCCGCCACACTTGCTGCCTTGACC

TGTGAATATCCCTGCCGCTTTTATCAAACAGCCTCAGTGTGTTTGATCTTGTGTGT

ACGCGCTTTTGCGAGTTGCTAGCTGCTTGTGCTATTTGCGAATACCACCCCCAGC

ATCCCCTTCCCTCGTTTCATATCGCTTGCATCCCAACCGCAACTTATCTACGCTGT

CCTGCTATCCCTCAGCGCTGCTCCTGCTCCTGCTCACTGCCCCTCGCACAGCCTTG

GTTTGGGCTCCGCCTGTATTCTCCTGGTACTGCAACCTGTAAACCAGCACTGCAA

TGCTGATGCACGGGAAGTAGTGGGATGGGAACACAAATGGAAAGCTTGAGCTCA

GAATAGTATCGGGTGATGCGAAGTCAGAACCAGGCAGGGCCTGTCGCCTGAGGT

GGCAACGATGGGAAGCAATCAATCTGGGTACAGTCGTCCGCACGATCCCGTGAT

CTCCCCCACCGACACCTATCCCCGCCCATCCCGGCCCACCCTTTCAGTCCCCTCAG

CATGCATTGTGCACCGCGACAAAGCATGTCTGCTCGTGCACTGGTTCAGGCCACG

GCGCACCGAGTCCTCGCCCTTCGCAGAGTGATCACCCTCCCCGGAACCAGCCACG CTCGCTGCTGCGGGCCGATCAGCCGCGCGCACTCCCTGCAACTAGGGACAACTCA

GGCAACCACGCGCCTCACAAGCATGGCCGCCGTGGCATCCAACCCGCTCGTGAC

GGTGGGTGCGCAAGTGCCAGGGGCCTCGTCGTCACGGCGTGCATCCTCGAGGGA

TGCGATCCGGCAACTATATGTCGTTTATCTCCCCACCAATCACAGGATGAGCCCC

TGTCTAGA

SEQ ID NO: 131

Codon-optimized Coccomyxa C-169 THIC sequence with C. protothecoides transit peptide

ACTAGTATGGCCACCGCATCCACTTTCTCGGCGTTCAATGCCCGCTGCGGCGACC

TGCGTCGCTCGGCGGGCTCCGGGCCCCGGCGCCCAGCGAGGCCCCTCCCCGTGCG

CGGGCGCGCCGCGATCGCCGCTCCCGAGCGGCTGGACTACCTCGACAACGCGGA

GGAGGCGCGGCTGCAGCAGACGGACGCCTTCGCCGAGCTGAAGGCGCTGTCGAG

CCGCCAGTCCGTGAACCGGCCCCAAAAGGGCGAGCTGTCGTTCCGGCAGTCGCC

GACCTTTCAGGACTGCTTCCCTGGCTCGGAGAAGTGCTACCGCGAGGTGGAGCAC

GATGGCAAGACGCTCAAGGTGCCTTTTCGCCGGGTGCACCTGCAGGACGACAAC

GGCCATTTCGACCTGTACGACACCTCTGGCCCCCAGGGCGTGAACCCCCGCGAGG

GTCTGCCCAAGATCCGCTCCTCCTGGGTGGAGCCGCGCGAGGCGCGGGGCGACA

AGGTGCAAACGCAGCAGTACTACGCCAAGCAAGGCATCATCACCGAGGAGATGG

CCTTCTGCGCTGCGCGCGAGCGCATGGACCCCGAGTTCATCCGGTCTGAGGTGGC

CCGGGGTCGGGCCATCATCCCTGCGAACAAGCGCCATCTGGAGCTGGAGCCGAC

CGTCGTGGGCCGCAACTTTCTGGTCAAGGTGAACGCCAACATCGGCAACTCTGCG

GTCAGCTCGAGCATCGAGGAGGAGGTGGAGAAGCTGCAGTGGAGCACCATCTGG

GGTGCCGACACGGCCATGGACCTGAGCACCGGCAACAACATCCACGAGACGCGC

GAGTGGGTGATGCGCAACAGCCCGGTCCCTGTGGGTACGGTCCCTATCTACCAGT

GCCTGGAGAAGGCGGGCGGCATCGTGGAGAACATCACCTGGGAGCTGTTCCGGG

AGACGCTGATCGAGCAGGCCGAGCAGGGCGTCGACTACTTTACCATCCACGCGG

GGGTCCTGCTCCGCTACATCCCCCTGACGGCGAACCGCGTCACCGGCATCGTCAG

CCGCGGCGGCTCTATCCATGCCAAGCTCTGCCTGCTGGACCACACCGAGAACTTC

GCCTACATGCACTGGGACGAGATCCTGGACATCTGCGCCCAGTACGACATCACCC

TGTCGATCGGCGACGGCCTGCGCCCCGGCTGCATCGCGGACGCCAACGACGCTG

CCCAGTTTGCTGAGCTGAAGACCCAGGGCGAGCTGACCCGCCGCGCCTGGGCGA

AGGACGTGCAGGTGATGAACGAGGGTCCCGGCCATGTGCCGCTGCACAAGATCC

CCGAGAACATGGAGAAGCAGCTGGACTGGTGCAGCGAGGCGCCCTTCTACACGC

TCGGCCCCCTGGCGACGGACATCGCGCCTGCGTACGACCACATCACCTCCGCGAT

CGGCGCCGCCACGATCGGCGCGCTGGGGACCGCGCTGCTGTGTTACGTGACCCC

GAAGGAGCACCTGGGCCTCCCCGATCGCGACGACGTGAAGGCCGGGGTGATCGC

CTACAAGATCGCGGCGCACGCGGCTGACCTGGCGAAGGGTCACCCCTACGCTCA

GGAGTGGGATAACGCCCTCTCTAAGGCGCGCTTCGAGTTCCGGTGGTACGACCA

GTTCCACCTGTCGCTGGACCCCGTCACCGCCCGCCTGTTCCATGACGCGACCCTG

CCGCAGGAGCCCGCCAAGACCGCCCACTTCTGCTCCATGTGCGGCCCCAAGTTCT

GCTCCATGCAGATCACCCAGGACCTCCGCGAGTACGCCAAGAACCATCAAATGG

AGGAGGACGAGGCGATCCAAACCGGCATGGCCGAGATGTCCGAGCAGTTCAAGA

GCATCGGCGCGGAGGTCTACCTCGACGAGGCGGTGCTGGAGCACGCGTAACAAT

TG

SEQ ID NO: 132

Chlorella protothecoides actin promoter/5 'UTR ACTAGAGAGTTTAGGTCCAGCGTCCGTGGGGGGGGACGGGCTGGGAGCTTGGGC

CGGGAAGGGCAAGACGATGCAGTCCCTCTGGGGAGTCACAGCCGACTGTGTGTG

TTGCACTGTGCGGCCCGCAGCACTCACACGCAAAATGCCTGGCCGACAGGCAGG

CCCTGTCCAGTGCAACATCCACGGTCCCTCTCATCAGGCTCACCTTGCTCATTGAC

ATAACGGAATGCGTACCGCTCTTTCAGATCTGTCCATCCAGAGAGGGGAGCAGG

CTCCCCACCGACGCTGTCAAACTTGCTTCCTGCCCAACCGAAAACATTATTGTTT

GAGGGGGGGGGGGGGGGGGCAGATTGCATGGCGGGATATCTCGTGAGGAACAT

CACTGGGACACTGTGGAACACAGTGAGTGCAGTATGCAGAGCATGTATGCTAGG

GGTCAGCGCAGGAAGGGGGCCTTTCCCAGTCTCCCATGCCACTGCACCGTATCCA

CGACTCACCAGGACCAGCTTCTTGATCGGCTTCCGCTCCCGTGGACACCAGTGTG

TAGCCTCTGGACTCCAGGTATGCGTGCACCGCAAAGGCCAGCCGATCGTGCCGAT

TCCTGGGGTGGAGGATATGAGTCAGCCAACTTGGGGCTCAGAGTGCACACTGGG

GCACGATACGAAACAACATCTACACCGTGTCCTCCATGCTGACACACCACAGCTT

CGCTCCACCTGAATGTGGGCGCATGGGCCCGAATCACAGCCAATGTCGCTGCTGC

CATAATGTGATCCAGACCCTCTCCGCCCAGATGCCGAGCGGATCGTGGGCGCTGA

ATAGATTCCTGTTTCGATCACTGTTTGGGTCCTTTCCTTTTCGTCTCGGATGCGCG

CTGCGCGCGGGCAAGTTGCTTGACCCTGGGCTGGTACCAGGGTTGGAGGGTATTA

CCGCGTCAGGCCATTCCCAGCCCGGATTCAATTCAAAGTCTGGGCCACCACCCTC

CGCCGCTCTGTCTGATCACTCCACATTCGTGCATACACTACGTTCAAGTCCTGATC

CAGGCGTGTCTCGGGACAAGGTGTGCTTGAGTTTGAATCTCAAGGACCCACTCCA

GCACAGCTGCTGGTTGACCCCGCCCTCGCAA

SEQ ID NO: 133

Chlorella protothecoides EF1A 3'UTR

ACGGAGCGTCGTGCGGGAGGGAGTGTGCCGAGCGGGGAGTCCCGGTCTGTGCGA

GGCCCGGCAGCTGACGCTGGCGAGCCGTACGCCCCGAGGGTCCCCCTCCCCTGC

ACCCTCTTCCCCTTCCCTCTGACGGCCGCGCCTGTTCTTGCATGTTCAGCGAC

SEQ ID NO: 134

Relevant sequence of A. thaliana THIC expression cassette

GCTCTTCGCCGCCGCCACTCCTGCTCGAGCGCGCCCGCGCGTGCGCCGCCAGCGC

CTTGGCCTTTTCGCCGCGCTCGTGCGCGTCGCTGATGTCCATCACCAGGTCCATG

AGGTCTGCCTTGCGCCGGCTGAGCCACTGCTTCGTCCGGGCGGCCAAGAGGAGC

ATGAGGGAGGACTCCTGGTCCAGGGTCCTGACGTGGTCGCGGCTCTGGGAGCGG

GCCAGCATCATCTGGCTCTGCCGCACCGAGGCCGCCTCCAACTGGTCCTCCAGCA

GCCGCAGTCGCCGCCGACCCTGGCAGAGGAAGACAGGTGAGGGGGGTATGAATT

GTACAGAACAACCACGAGCCTTGTCTAGGCAGAATCCCTACCAGTCATGGCTTTA

CCTGGATGACGGCCTGCGAACAGCTGTCCAGCGACCCTCGCTGCCGCCGCTTCTC

CCGCACGCTTCTTTCCAGCACCGTGATGGCGCGAGCCAGCGCCGCACGCTGGCGC

TGCGCTTCGCCGATCTGAGGACAGTCGGGGAACTCTGATCAGTCTAAACCCCCTT

GCGCGTTAGTGTTGCCATCCTTTGCAGACCGGTGAGAGCCGACTTGTTGTGCGCC

ACCCCCCACACCACCTCCTCCCAGACCAATTCTGTCACCTTTTTGGCGAAGGCAT

CGGCCTCGGCCTGCAGAGAGGACAGCAGTGCCCAGCCGCTGGGGGTTGGCGGAT

GCACGCTCAGGTACCCTTTCTTGCGCTATGACACTTCCAGCAAAAGGTAGGGCGG

GCTGCGAGACGGCTTCCCGGCGCTGCATGCAACACCGATGATGCTTCGACCCCCC

GAAGCTCCTTCGGGGCTGCATGGGCGCTCCGATGCCGCTCCAGGGCGAGCGCTGT

TTAAATAGCCAGGCCCCCGATTGCAAAGACATTATAGCGAGCTACCAAAGCCAT ATTCAAACACCTAGATCACTACCACTTCTACACAGGCCACTCGAGCTTGTGATCG

CACTCCGCTAAGGGGGCGCCTCTTCCTCTTCGTTTCAGTCACAACCCGCAAACTC

TAGAATATCAATGATCGAGCAGGACGGCCTCCACGCCGGCTCCCCCGCCGCCTG

GGTGGAGCGCCTGTTCGGCTACGACTGGGCCCAGCAGACCATCGGCTGCTCCGA

CGCCGCCGTGTTCCGCCTGTCCGCCCAGGGCCGCCCCGTGCTGTTCGTGAAGACC

GACCTGTCCGGCGCCCTGAACGAGCTGCAGGACGAGGCCGCCCGCCTGTCCTGG

CTGGCCACCACCGGCGTGCCCTGCGCCGCCGTGCTGGACGTGGTGACCGAGGCC

GGCCGCGACTGGCTGCTGCTGGGCGAGGTGCCCGGCCAGGACCTGCTGTCCTCCC

ACCTGGCCCCCGCCGAGAAGGTGTCCATCATGGCCGACGCCATGCGCCGCCTGC

ACACCCTGGACCCCGCCACCTGCCCCTTCGACCACCAGGCCAAGCACCGCATCGA

GCGCGCCCGCACCCGCATGGAGGCCGGCCTGGTGGACCAGGACGACCTGGACGA

GGAGCACCAGGGCCTGGCCCCCGCCGAGCTGTTCGCCCGCCTGAAGGCCCGCAT

GCCCGACGGCGAGGACCTGGTGGTGACCCACGGCGACGCCTGCCTGCCCAACAT

CATGGTGGAGAACGGCCGCTTCTCCGGCTTCATCGACTGCGGCCGCCTGGGCGTG

GCCGACCGCTACCAGGACATCGCCCTGGCCACCCGCGACATCGCCGAGGAGCTG

GGCGGCGAGTGGGCCGACCGCTTCCTGGTGCTGTACGGCATCGCCGCCCCCGACT

CCCAGCGCATCGCCTTCTACCGCCTGCTGGACGAGTTCTTCTGACAATTGGCAGC

AGCAGCTCGGATAGTATCGACACACTCTGGACGCTGGTCGTGTGATGGACTGTTG

CCGCCACACTTGCTGCCTTGACCTGTGAATATCCCTGCCGCTTTTATCAAACAGCC

TCAGTGTGTTTGATCTTGTGTGTACGCGCTTTTGCGAGTTGCTAGCTGCTTGTGCT

ATTTGCGAATACCACCCCCAGCATCCCCTTCCCTCGTTTCATATCGCTTGCATCCC

AACCGCAACTTATCTACGCTGTCCTGCTATCCCTCAGCGCTGCTCCTGCTCCTGCT

CACTGCCCCTCGCACAGCCTTGGTTTGGGCTCCGCCTGTATTCTCCTGGTACTGCA

ACCTGTAAACCAGCACTGCAATGCTGATGCACGGGAAGTAGTGGGATGGGAACA

CAAATGGAGGATCCACTAGAGAGTTTAGGTCCAGCGTCCGTGGGGGGGGACGGG

CTGGGAGCTTGGGCCGGGAAGGGCAAGACGATGCAGTCCCTCTGGGGAGTCACA

GCCGACTGTGTGTGTTGCACTGTGCGGCCCGCAGCACTCACACGCAAAATGCCTG

GCCGACAGGCAGGCCCTGTCCAGTGCAACATCCACGGTCCCTCTCATCAGGCTCA

CCTTGCTCATTGACATAACGGAATGCGTACCGCTCTTTCAGATCTGTCCATCCAG

AGAGGGGAGCAGGCTCCCCACCGACGCTGTCAAACTTGCTTCCTGCCCAACCGA

AAACATTATTGTTTGAGGGGGGGGGGGGGGGGGCAGATTGCATGGCGGGATATC

TCGTGAGGAACATCACTGGGACACTGTGGAACACAGTGAGTGCAGTATGCAGAG

CATGTATGCTAGGGGTCAGCGCAGGAAGGGGGCCTTTCCCAGTCTCCCATGCCAC

TGCACCGTATCCACGACTCACCAGGACCAGCTTCTTGATCGGCTTCCGCTCCCGT

GGACACCAGTGTGTAGCCTCTGGACTCCAGGTATGCGTGCACCGCAAAGGCCAG

CCGATCGTGCCGATTCCTGGGGTGGAGGATATGAGTCAGCCAACTTGGGGCTCA

GAGTGCACACTGGGGCACGATACGAAACAACATCTACACCGTGTCCTCCATGCT

GACACACCACAGCTTCGCTCCACCTGAATGTGGGCGCATGGGCCCGAATCACAG

CCAATGTCGCTGCTGCCATAATGTGATCCAGACCCTCTCCGCCCAGATGCCGAGC

GGATCGTGGGCGCTGAATAGATTCCTGTTTCGATCACTGTTTGGGTCCTTTCCTTT

CTCCCTCCGTCACCATCCTGCGCGCGGGCAAGTTGCTTGACCCTGGGCTGGTACC

AGGGTTGGAGGGTATTACCGCGTCAGGCCATTCCCAGCCCGGATTCAATTCAAAG

TCTGGGCCACCACCCTCCGCCGCTCTGTCTGATCACTCCACATTCGTGCATACACT

ACGTTCAAGTCCTGATCCAGGCGTGTCTCGGGACAAGGTGTGCTTGAGTTTGAAT

CTCAAGGACCCACTCCAGCACAGCTGCTGGTTGACCCCGCCCTCGCAAACTAGTA

TGGCCACCGCATCCACTTTCTCGGCGTTCAATGCCCGCTGCGGCGACCTGCGTCG

CTCGGCGGGCTCCGGGCCCCGGCGCCCAGCGAGGCCCCTCCCCGTGCGCGGGCG

CGCCGTCCAGGCCGCGGCCACCCGCTTCAAGAAGGAGACGACGACCACCCGCGC

CACGCTGACGTTCGACCCCCCCACGACCAACTCCGAGCGCGCCAAGCAGCGCAA GCACACCATCGACCCCTCCTCCCCCGACTTCCAGCCCATCCCCTCCTTCGAGGAG

TGCTTCCCCAAGTCCACGAAGGAGCACAAGGAGGTGGTGCACGAGGAGTCCGGC

CACGTCCTGAAGGTGCCCTTCCGCCGCGTGCACCTGTCCGGCGGCGAGCCCGCCT

TCGACAACTACGACACGTCCGGCCCCCAGAACGTCAACGCCCACATCGGCCTGG

CGAAGCTGCGCAAGGAGTGGATCGACCGCCGCGAGAAGCTGGGCACGCCCCGCT

ACACGCAGATGTACTACGCGAAGCAGGGCATCATCACGGAGGAGATGCTGTACT

GCGCGACGCGCGAGAAGCTGGACCCCGAGTTCGTCCGCTCCGAGGTCGCGCGGG

GCCGCGCCATCATCCCCTCCAACAAGAAGCACCTGGAGCTGGAGCCCATGATCG

TGGGCCGCAAGTTCCTGGTGAAGGTGAACGCGAACATCGGCAACTCCGCCGTGG

CCTCCTCCATCGAGGAGGAGGTCTACAAGGTGCAGTGGGCCACCATGTGGGGCG

CCGACACCATCATGGACCTGTCCACGGGCCGCCACATCCACGAGACGCGCGAGT

GGATCCTGCGCAACTCCGCGGTCCCCGTGGGCACCGTCCCCATCTACCAGGCGCT

GGAGAAGGTGGACGGCATCGCGGAGAACCTGAACTGGGAGGTGTTCCGCGAGAC

GCTGATCGAGCAGGCCGAGCAGGGCGTGGACTACTTCACGATCCACGCGGGCGT

GCTGCTGCGCTACATCCCCCTGACCGCCAAGCGCCTGACGGGCATCGTGTCCCGC

GGCGGCTCCATCCACGCGAAGTGGTGCCTGGCCTACCACAAGGAGAACTTCGCC

TACGAGCACTGGGACGACATCCTGGACATCTGCAACCAGTACGACGTCGCCCTGT

CCATCGGCGACGGCCTGCGCCCCGGCTCCATCTACGACGCCAACGACACGGCCC

AGTTCGCCGAGCTGCTGACCCAGGGCGAGCTGACGCGCCGCGCGTGGGAGAAGG

ACGTGCAGGTGATGAACGAGGGCCCCGGCCACGTGCCCATGCACAAGATCCCCG

AGAACATGCAGAAGCAGCTGGAGTGGTGCAACGAGGCGCCCTTCTACACCCTGG

GCCCCCTGACGACCGACATCGCGCCCGGCTACGACCACATCACCTCCGCCATCGG

CGCGGCCAACATCGGCGCCCTGGGCACCGCCCTGCTGTGCTACGTGACGCCCAA

GGAGCACCTGGGCCTGCCCAACCGCGACGACGTGAAGGCGGGCGTCATCGCCTA

CAAGATCGCCGCCCACGCGGCCGACCTGGCCAAGCAGCACCCCCACGCCCAGGC

GTGGGACGACGCGCTGTCCAAGGCGCGCTTCGAGTTCCGCTGGATGGACCAGTTC

GCGCTGTCCCTGGACCCCATGACGGCGATGTCCTTCCACGACGAGACGCTGCCCG

CGGACGGCGCGAAGGTCGCCCACTTCTGCTCCATGTGCGGCCCCAAGTTCTGCTC

CATGAAGATCACGGAGGACATCCGCAAGTACGCCGAGGAGAACGGCTACGGCTC

CGCCGAGGAGGCCATCCGCCAGGGCATGGACGCCATGTCCGAGGAGTTCAACAT

CGCCAAGAAGACGATCTCCGGCGAGCAGCACGGCGAGGTCGGCGGCGAGATCTA

CCTGCCCGAGTCCTACGTCAAGGCCGCGCAGAAGTGATACGTACTCGAGACGGA

GCGTCGTGCGGGAGGGAGTGTGCCGAGCGGGGAGTCCCGGTCTGTGCGAGGCCC

GGCAGCTGACGCTGGCGAGCCGTACGCCCCGAGGGTCCCCCTCCCCTGCACCCTC

TTCCCCTTCCCTCTGACGGCCGCGCCTGTTCTTGCATGTTCAGCGACGAGCTCTTG

TTTTCCAGAAGGAGTTGCTCCTTGAGCCTTTCATTCTCAGCCTCGATAACCTCCAA

AGCCGCTCTAATTGTGGAGGGGGTTCGAATTTAAAAGCTTGGAATGTTGGTTCGT

GCGTCTGGAACAAGCCCAGACTTGTTGCTCACTGGGAAAAGGACCATCAGCTCC

AAAAAACTTGCCGCTCAAACCGCGTACCTCTGCTTTCGCGCAATCTGCCCTGTTG

AAATCGCCACCACATTCATATTGTGACGCTTGAGCAGTCTGTAATTGCCTCAGAA

TGTGGAATCATCTGCCCCCTGTGCGAGCCCATGCCAGGCATGTCGCGGGCGAGG

ACACCCGCCACTCGTACAGCAGACCATTATGCTACCTCACAATAGTTCATAACAG

TGACCATATTTCTCGAAGCTCCCCAACGAGCACCTCCATGCTCTGAGTGGCCACC

CCCCGGCCCTGGTGCTTGCGGAGGGCAGGTCAACCGGCATGGGGCTACCGAAAT

CCCCGACCGGATCCCACCACCCCCGCGATGGGAAGAATCTCTCCCCGGGATGTG

GGCCCACCACCAGCACAACCTGCTGGCCCAGGCGAGCGTCAAACCATACCACAC

AAATATCCTTGGCATCGGCCCTGAATTCCTTCTGCCGCTCTGCTACCCGGTGCTTC

TGTCCGAAGCAGGGGTTGCTAGGGATCGCTCCGAGTCCGCAAACCCTTGTCGCGT

GGCGGGGCTTGTTCGAGCTTGAAGAGC SEQ ID NO: 135

Codon-optimized A. thaliana THIC with C. protothecoides transit peptide

ATGGCCACCGCATCCACTTTCTCGGCGTTCAATGCCCGCTGCGGCGACCTGCGTC

GCTCGGCGGGCTCCGGGCCCCGGCGCCCAGCGAGGCCCCTCCCCGTGCGCGGGC

GCGCCGTCCAGGCCGCGGCCACCCGCTTCAAGAAGGAGACGACGACCACCCGCG

CCACGCTGACGTTCGACCCCCCCACGACCAACTCCGAGCGCGCCAAGCAGCGCA

AGCACACCATCGACCCCTCCTCCCCCGACTTCCAGCCCATCCCCTCCTTCGAGGA

GTGCTTCCCCAAGTCCACGAAGGAGCACAAGGAGGTGGTGCACGAGGAGTCCGG

CCACGTCCTGAAGGTGCCCTTCCGCCGCGTGCACCTGTCCGGCGGCGAGCCCGCC

TTCGACAACTACGACACGTCCGGCCCCCAGAACGTCAACGCCCACATCGGCCTG

GCGAAGCTGCGCAAGGAGTGGATCGACCGCCGCGAGAAGCTGGGCACGCCCCGC

TACACGCAGATGTACTACGCGAAGCAGGGCATCATCACGGAGGAGATGCTGTAC

TGCGCGACGCGCGAGAAGCTGGACCCCGAGTTCGTCCGCTCCGAGGTCGCGCGG

GGCCGCGCCATCATCCCCTCCAACAAGAAGCACCTGGAGCTGGAGCCCATGATC

GTGGGCCGCAAGTTCCTGGTGAAGGTGAACGCGAACATCGGCAACTCCGCCGTG

GCCTCCTCCATCGAGGAGGAGGTCTACAAGGTGCAGTGGGCCACCATGTGGGGC

GCCGACACCATCATGGACCTGTCCACGGGCCGCCACATCCACGAGACGCGCGAG

TGGATCCTGCGCAACTCCGCGGTCCCCGTGGGCACCGTCCCCATCTACCAGGCGC

TGGAGAAGGTGGACGGCATCGCGGAGAACCTGAACTGGGAGGTGTTCCGCGAGA

CGCTGATCGAGCAGGCCGAGCAGGGCGTGGACTACTTCACGATCCACGCGGGCG

TGCTGCTGCGCTACATCCCCCTGACCGCCAAGCGCCTGACGGGCATCGTGTCCCG

CGGCGGCTCCATCCACGCGAAGTGGTGCCTGGCCTACCACAAGGAGAACTTCGC

CTACGAGCACTGGGACGACATCCTGGACATCTGCAACCAGTACGACGTCGCCCT

GTCCATCGGCGACGGCCTGCGCCCCGGCTCCATCTACGACGCCAACGACACGGC

CCAGTTCGCCGAGCTGCTGACCCAGGGCGAGCTGACGCGCCGCGCGTGGGAGAA

GGACGTGCAGGTGATGAACGAGGGCCCCGGCCACGTGCCCATGCACAAGATCCC

CGAGAACATGCAGAAGCAGCTGGAGTGGTGCAACGAGGCGCCCTTCTACACCCT

GGGCCCCCTGACGACCGACATCGCGCCCGGCTACGACCACATCACCTCCGCCATC

GGCGCGGCCAACATCGGCGCCCTGGGCACCGCCCTGCTGTGCTACGTGACGCCC

AAGGAGCACCTGGGCCTGCCCAACCGCGACGACGTGAAGGCGGGCGTCATCGCC

TACAAGATCGCCGCCCACGCGGCCGACCTGGCCAAGCAGCACCCCCACGCCCAG

GCGTGGGACGACGCGCTGTCCAAGGCGCGCTTCGAGTTCCGCTGGATGGACCAG

TTCGCGCTGTCCCTGGACCCCATGACGGCGATGTCCTTCCACGACGAGACGCTGC

CCGCGGACGGCGCGAAGGTCGCCCACTTCTGCTCCATGTGCGGCCCCAAGTTCTG

CTCCATGAAGATCACGGAGGACATCCGCAAGTACGCCGAGGAGAACGGCTACGG

CTCCGCCGAGGAGGCCATCCGCCAGGGCATGGACGCCATGTCCGAGGAGTTCAA

CATCGCCAAGAAGACGATCTCCGGCGAGCAGCACGGCGAGGTCGGCGGCGAGAT

CTACCTGCCCGAGTCCTACGTCAAGGCCGCGCAGAAGTGA

SEQ ID NO: 136

A. thaliana amino acid sequence (with native transit peptide sequence)

MAASVHCTLMSVVCNNKNHSARPKLPNSSLLPGFDVVVQAAATRFKKETTTTRATL

TFDPPTTNSERAKQRKHTIDPSSPDFQPIPSFEECFPKSTKEH EVVHEESGHVLKVPF

RRVHLSGGEPAFDNYDTSGPQNVNAHIGLAKLRKEWIDRREKLGTPRYTQMYYAKQ

GIITEEMLYCATREKLDPEFVRSEVARGRAIIPSNKKHLELEPMIVGRKFLVKVNAN IG

NSAVASSIEEEVYKVQWATMWGADTIMDLSTGRHIHETREWILRNSAVPVGTVPIYQ

ALEKVDGIAENLNWEVFRETLIEQAEQGVDYFTIHAGVLLRYIPLTAKRLTGIVSRG G

SIHAKWCLAYHKENFAYEHWDDILDICNQYDVALSIGDGLRPGSIYDANDTAQFAEL LTQGELTRRAWEKDVQVMNEGPGHVPMHKIPENMQ QLEWCNEAPFYTLGPLTTD

IAPGYDHITSAIGAANIGALGTALLCYVTPKEHLGLPNRDDVKAGVIAYKIAAHAAD

LAKQHPHAQAWDDALSKARFEFRWMDQFALSLDPMTAMSFHDETLPADGAKVAH

FCSMCGPKFCSMKITEDIRKYAEENGYGSAEEAIRQGMDAMSEEFNIAKKTISGEQH

GEVGGEIYLPESYVKAAQK

SEQ ID NO: 137

Relevant sequence of Synechocystis sp. thiC expression cassette

GCTCTTCGCCGCCGCCACTCCTGCTCGAGCGCGCCCGCGCGTGCGCCGCCAGCGC

AGGTCTGCCTTGCGCCGGCTGAGCCACTGCTTCGTCCGGGCGGCCAAGAGGAGC

ATGAGGGAGGACTCCTGGTCCAGGGTCCTGACGTGGTCGCGGCTCTGGGAGCGG

GCCAGCATCATCTGGCTCTGCCGCACCGAGGCCGCCTCCAACTGGTCCTCCAGCA

GCCGCAGTCGCCGCCGACCCTGGCAGAGGAAGACAGGTGAGGGGGGTATGAATT

GTACAGAACAACCACGAGCCTTGTCTAGGCAGAATCCCTACCAGTCATGGCTTTA

CCTGGATGACGGCCTGCGAACAGCTGTCCAGCGACCCTCGCTGCCGCCGCTTCTC

CCGCACGCTTCTTTCCAGCACCGTGATGGCGCGAGCCAGCGCCGCACGCTGGCGC

TGCGCTTCGCCGATCTGAGGACAGTCGGGGAACTCTGATCAGTCTAAACCCCCTT

GCGCGTTAGTGTTGCCATCCTTTGCAGACCGGTGAGAGCCGACTTGTTGTGCGCC

ACCCCCCACACCACCTCCTCCCAGACCAATTCTGTCACCTTTTTGGCGAAGGCAT

CGGCCTCGGCCTGCAGAGAGGACAGCAGTGCCCAGCCGCTGGGGGTTGGCGGAT

GCACGCTCAGGTACCCTTTCTTGCGCTATGACACTTCCAGCAAAAGGTAGGGCGG

GCTGCGAGACGGCTTCCCGGCGCTGCATGCAACACCGATGATGCTTCGACCCCCC

GAAGCTCCTTCGGGGCTGCATGGGCGCTCCGATGCCGCTCCAGGGCGAGCGCTGT

TTAAATAGCCAGGCCCCCGATTGCAAAGACATTATAGCGAGCTACCAAAGCCAT

ATTCAAACACCTAGATCACTACCACTTCTACACAGGCCACTCGAGCTTGTGATCG

CACTCCGCTAAGGGGGCGCCTCTTCCTCTTCGTTTCAGTCACAACCCGCAAACTC

TAGAATATCAATGATCGAGCAGGACGGCCTCCACGCCGGCTCCCCCGCCGCCTG

GGTGGAGCGCCTGTTCGGCTACGACTGGGCCCAGCAGACCATCGGCTGCTCCGA

CGCCGCCGTGTTCCGCCTGTCCGCCCAGGGCCGCCCCGTGCTGTTCGTGAAGACC

GACCTGTCCGGCGCCCTGAACGAGCTGCAGGACGAGGCCGCCCGCCTGTCCTGG

CTGGCCACCACCGGCGTGCCCTGCGCCGCCGTGCTGGACGTGGTGACCGAGGCC

GGCCGCGACTGGCTGCTGCTGGGCGAGGTGCCCGGCCAGGACCTGCTGTCCTCCC

ACCTGGCCCCCGCCGAGAAGGTGTCCATCATGGCCGACGCCATGCGCCGCCTGC

ACACCCTGGACCCCGCCACCTGCCCCTTCGACCACCAGGCCAAGCACCGCATCGA

GCGCGCCCGCACCCGCATGGAGGCCGGCCTGGTGGACCAGGACGACCTGGACGA

GGAGCACCAGGGCCTGGCCCCCGCCGAGCTGTTCGCCCGCCTGAAGGCCCGCAT

GCCCGACGGCGAGGACCTGGTGGTGACCCACGGCGACGCCTGCCTGCCCAACAT

CATGGTGGAGAACGGCCGCTTCTCCGGCTTCATCGACTGCGGCCGCCTGGGCGTG

GCCGACCGCTACCAGGACATCGCCCTGGCCACCCGCGACATCGCCGAGGAGCTG

GGCGGCGAGTGGGCCGACCGCTTCCTGGTGCTGTACGGCATCGCCGCCCCCGACT

CCCAGCGCATCGCCTTCTACCGCCTGCTGGACGAGTTCTTCTGACAATTGGCAGC

AGCAGCTCGGATAGTATCGACACACTCTGGACGCTGGTCGTGTGATGGACTGTTG

CCGCCACACTTGCTGCCTTGACCTGTGAATATCCCTGCCGCTTTTATCAAACAGCC

TCAGTGTGTTTGATCTTGTGTGTACGCGCTTTTGCGAGTTGCTAGCTGCTTGTGCT

ATTTGCGAATACCACCCCCAGCATCCCCTTCCCTCGTTTCATATCGCTTGCATCCC

AACCGCAACTTATCTACGCTGTCCTGCTATCCCTCAGCGCTGCTCCTGCTCCTGCT

CACTGCCCCTCGCACAGCCTTGGTTTGGGCTCCGCCTGTATTCTCCTGGTACTGCA

ACCTGTAAACCAGCACTGCAATGCTGATGCACGGGAAGTAGTGGGATGGGAACA CAAATGGAGGATCCACTAGAGAGTTTAGGTCCAGCGTCCGTGGGGGGGGACGGG

CTGGGAGCTTGGGCCGGGAAGGGCAAGACGATGCAGTCCCTCTGGGGAGTCACA

GCCGACTGTGTGTGTTGCACTGTGCGGCCCGCAGCACTCACACGCAAAATGCCTG

GCCGACAGGCAGGCCCTGTCCAGTGCAACATCCACGGTCCCTCTCATCAGGCTCA

CCTTGCTCATTGACATAACGGAATGCGTACCGCTCTTTCAGATCTGTCCATCCAG

AGAGGGGAGCAGGCTCCCCACCGACGCTGTCAAACTTGCTTCCTGCCCAACCGA

AAACATTATTGTTTGAGGGGGGGGGGGGGGGGGCAGATTGCATGGCGGGATATC

TCGTGAGGAACATCACTGGGACACTGTGGAACACAGTGAGTGCAGTATGCAGAG

CATGTATGCTAGGGGTCAGCGCAGGAAGGGGGCCTTTCCCAGTCTCCCATGCCAC

TGCACCGTATCCACGACTCACCAGGACCAGCTTCTTGATCGGCTTCCGCTCCCGT

GGACACCAGTGTGTAGCCTCTGGACTCCAGGTATGCGTGCACCGCAAAGGCCAG

CCGATCGTGCCGATTCCTGGGGTGGAGGATATGAGTCAGCCAACTTGGGGCTCA

GAGTGCACACTGGGGCACGATACGAAACAACATCTACACCGTGTCCTCCATGCT

GACACACCACAGCTTCGCTCCACCTGAATGTGGGCGCATGGGCCCGAATCACAG

CCAATGTCGCTGCTGCCATAATGTGATCCAGACCCTCTCCGCCCAGATGCCGAGC

GGATCGTGGGCGCTGAATAGATTCCTGTTTCGATCACTGTTTGGGTCCTTTCCTTT

CTCCCTCCGTCACCATCCTGCGCGCGGGCAAGTTGCTTGACCCTGGGCTGGTACC

AGGGTTGGAGGGTATTACCGCGTCAGGCCATTCCCAGCCCGGATTCAATTCAAAG

TCTGGGCCACCACCCTCCGCCGCTCTGTCTGATCACTCCACATTCGTGCATACACT

ACGTTCAAGTCCTGATCCAGGCGTGTCTCGGGACAAGGTGTGCTTGAGTTTGAAT

CTCAAGGACCCACTCCAGCACAGCTGCTGGTTGACCCCGCCCTCGCAAACTAGTA

TGGCCACCGCATCCACTTTCTCGGCGTTCAATGCCCGCTGCGGCGACCTGCGTCG

CTCGGCGGGCTCCGGGCCCCGGCGCCCAGCGAGGCCCCTCCCCGTGCGCGGGCG

CGCCATGCGCACCGCGTGGGTCGCCAAGCGCCAGGGCCAGACGAACGTCTCCCA

GATGCACTACGCGCGCAAGGGCGTGATCACCGAGGAGATGGACTACGTGGCGAA

GCGCGAGAACCTGCCCGTGGAGCTGATCAAGGACGAGGTGGCGCGGGGCCGCAT

GATCATCCCCGCCAACATCAACCACACGAACCTGGAGCCCATGGCCATCGGCAT

CGCCTCCAAGTGCAAGGTCAACGCGAACATCGGCGCGTCCCCCAACTCCTCCAAC

ATCGACGAGGAGGTGGAGAAGCTGCTGCTGTCCGTGAAGTACGGCGCGGACACG

GTGATGGACCTGTCCACCGGCGGCGGCGACCTGGACGTGATCCGCACCGCCATC

ATCAACGCCTCCCCCGTGCCCATCGGCACGGTGCCCATCTACCAGGCCCTGGAGT

CCGTGCACGGCTCCATCGAGAACCTGACGCCCGACGACTTCCTGCACATCATCGA

GAAGCACGCCCAGCAGGGCGTGGACTACATGACGATCCACGCGGGCCTGCTGAT

CGAGTACCTGCCCCTGGTGAAGTCCCGCATCACGGGCATCGTGTCCCGCGGCGGC

GGCATCATCGCCAAGTGGATGCTGCACCACCACAAGCAGAACCCCCTGTACACG

CACTTCGACGAGATCATCGAGATCTTCAAGAAGTACGACGTGTCCTTCTCCCTGG

GCGACTCCCTGCGCCCCGGCTGCACCCACGACGCCTCCGACGACGCCCAGCTGTC

CGAGCTGAAGACGCTGGGCCAGCTGACGCGCCGCGCCTGGGAGCACGACGTCCA

GGTCATGGTGGAGGGCCCCGGCCACGTCCCCATCGACCAGATCGAGTTCAACGT

CAAGAAGCAGATGGAGGAGTGCTCCGAGGCCCCCTTCTACGTGCTGGGCCCCCT

GGTCACCGACATCGCGCCCGGCTACGACCACATCACCTCCGCGATCGGCGCCGCC

ATCGCGGGCTGGCACGGCACCGCGATGCTGTGCTACGTGACGCCCAAGGAGCAC

CTGGGCCTGCCCAACGCGGAGGACGTGCGCAACGGCCTGATCGCCTACAAGATC

GCCGCGCACGCCGCGGACATCGCCCGCCACCGCCCCGGCGCGCGCGACCGCGAC

GACGAGCTGTCCAAGGCGCGCTACAACTTCGACTGGAACCGCCAGTTCGAGCTG

TCCCTGGACCCCGAGCGCGCGAAGGAGTACCACGACGAGACGCTGCCCGCGGAC

ATCTACAAGACCGCGGAGTTCTGCTCCATGTGCGGCCCCAAGTTCTGCCCCATGC

AGACCAAGGTGGACGCCGAGATGCTGGAGGAGCTGGAGGTCTTCCTGGCCAAGG

ACAAGGAGATGGTGTCCCAGCGCTGATACGTACTCGAGACGGAGCGTCGTGCGG GAGGGAGTGTGCCGAGCGGGGAGTCCCGGTCTGTGCGAGGCCCGGCAGCTGACG

CTGGCGAGCCGTACGCCCCGAGGGTCCCCCTCCCCTGCACCCTCTTCCCCTTCCCT

CTGACGGCCGCGCCTGTTCTTGCATGTTCAGCGACGAGCTCTTGTTTTCCAGAAG

GAGTTGCTCCTTGAGCCTTTCATTCTCAGCCTCGATAACCTCCAAAGCCGCTCTAA

TTGTGGAGGGGGTTCGAATTTAAAAGCTTGGAATGTTGGTTCGTGCGTCTGGAAC

AAGCCCAGACTTGTTGCTCACTGGGAAAAGGACCATCAGCTCCAAAAAACTTGC

CGCTCAAACCGCGTACCTCTGCTTTCGCGCAATCTGCCCTGTTGAAATCGCCACC

ACATTCATATTGTGACGCTTGAGCAGTCTGTAATTGCCTCAGAATGTGGAATCAT

CTGCCCCCTGTGCGAGCCCATGCCAGGCATGTCGCGGGCGAGGACACCCGCCAC

TCGTACAGCAGACCATTATGCTACCTCACAATAGTTCATAACAGTGACCATATTT

CTCGAAGCTCCCCAACGAGCACCTCCATGCTCTGAGTGGCCACCCCCCGGCCCTG

GTGCTTGCGGAGGGCAGGTCAACCGGCATGGGGCTACCGAAATCCCCGACCGGA

TCCCACCACCCCCGCGATGGGAAGAATCTCTCCCCGGGATGTGGGCCCACCACCA

GCACAACCTGCTGGCCCAGGCGAGCGTCAAACCATACCACACAAATATCCTTGG

CATCGGCCCTGAATTCCTTCTGCCGCTCTGCTACCCGGTGCTTCTGTCCGAAGCAG

GGGTTGCTAGGGATCGCTCCGAGTCCGCAAACCCTTGTCGCGTGGCGGGGCTTGT

TCGAGCTTGAAGAGCCTCTAGAGTCGACCTGCAGGCATGCAAGCTTGGCGTAATC

ATGGTCATAGCTGTTTCCTGTGTGAAATTGTTATCCGCTCACAATTCCACACAAC

ATACGAGCCGGAAGCATAAAGTGTAAAGCCTGGGGTGCCTAATGAGTGAGCTAA

CTCACATTAATTGCGTTGCGCTCACTGCCCGCTTTCCAGTCGGGAAACCTGTCGT

GCCAGCTGCATTAATGAATCGGCCAACGCGCGGGGAGAGGCGGTTTGCGTATTG

GGCGCTCTTC

SEQ ID NO: 138

Codon-optimized Synechosystis sp. thiC sequence with C. protothecoides transit peptide

ATGGCCACCGCATCCACTTTCTCGGCGTTCAATGCCCGCTGCGGCGACCTGCGTC

GCTCGGCGGGCTCCGGGCCCCGGCGCCCAGCGAGGCCCCTCCCCGTGCGCGGGC

GCGCCATGCGCACCGCGTGGGTCGCCAAGCGCCAGGGCCAGACGAACGTCTCCC

AGATGCACTACGCGCGCAAGGGCGTGATCACCGAGGAGATGGACTACGTGGCGA

AGCGCGAGAACCTGCCCGTGGAGCTGATCAAGGACGAGGTGGCGCGGGGCCGCA

TGATCATCCCCGCCAACATCAACCACACGAACCTGGAGCCCATGGCCATCGGCAT

CGCCTCCAAGTGCAAGGTCAACGCGAACATCGGCGCGTCCCCCAACTCCTCCAAC

ATCGACGAGGAGGTGGAGAAGCTGCTGCTGTCCGTGAAGTACGGCGCGGACACG

GTGATGGACCTGTCCACCGGCGGCGGCGACCTGGACGTGATCCGCACCGCCATC

ATCAACGCCTCCCCCGTGCCCATCGGCACGGTGCCCATCTACCAGGCCCTGGAGT

CCGTGCACGGCTCCATCGAGAACCTGACGCCCGACGACTTCCTGCACATCATCGA

GAAGCACGCCCAGCAGGGCGTGGACTACATGACGATCCACGCGGGCCTGCTGAT

CGAGTACCTGCCCCTGGTGAAGTCCCGCATCACGGGCATCGTGTCCCGCGGCGGC

GGCATCATCGCCAAGTGGATGCTGCACCACCACAAGCAGAACCCCCTGTACACG

CACTTCGACGAGATCATCGAGATCTTCAAGAAGTACGACGTGTCCTTCTCCCTGG

GCGACTCCCTGCGCCCCGGCTGCACCCACGACGCCTCCGACGACGCCCAGCTGTC

CGAGCTGAAGACGCTGGGCCAGCTGACGCGCCGCGCCTGGGAGCACGACGTCCA

GGTCATGGTGGAGGGCCCCGGCCACGTCCCCATCGACCAGATCGAGTTCAACGT

CAAGAAGCAGATGGAGGAGTGCTCCGAGGCCCCCTTCTACGTGCTGGGCCCCCT

GGTCACCGACATCGCGCCCGGCTACGACCACATCACCTCCGCGATCGGCGCCGCC

ATCGCGGGCTGGCACGGCACCGCGATGCTGTGCTACGTGACGCCCAAGGAGCAC

CTGGGCCTGCCCAACGCGGAGGACGTGCGCAACGGCCTGATCGCCTACAAGATC

GCCGCGCACGCCGCGGACATCGCCCGCCACCGCCCCGGCGCGCGCGACCGCGAC

GACGAGCTGTCCAAGGCGCGCTACAACTTCGACTGGAACCGCCAGTTCGAGCTG TCCCTGGACCCCGAGCGCGCGAAGGAGTACCACGACGAGACGCTGCCCGCGGAC ATCTACAAGACCGCGGAGTTCTGCTCCATGTGCGGCCCCAAGTTCTGCCCCATGC AGACCAAGGTGGACGCCGAGATGCTGGAGGAGCTGGAGGTCTTCCTGGCCAAGG ACAAGGAGATGGTGTCCCAGCGCTGA

SEQ ID NO: 139

Synechosystis sp. thiC amino acid sequence (with native transit peptide sequence)

MRTAWVAKRQGQTNVSQMHYARKGVITEEMDYVAKRENLPVELIKDEVARGRMII

PANINHTNLEPMAIGIASKCKVNANIGASPNSSNIDEEVEKLLLSVKYGADTVMDLS T

GGGDLDVIRTAIINASPVPIGTVPIYQALESVHGSIENLTPDDFLHIIEKHAQQGVD YM

TIHAGLLIEYLPLVKSRITGIVSRGGGIIAKWMLHHHKQNPLYTHFDEIIEIFKKYD VSF

SLGDSLRPGCTHDASDDAQLSELKTLGQLTRRAWEHDVQVMVEGPGHVPIDQIEFN

VKKQMEECSEAPFYVLGPLVTDIAPGYDHITSAIGAAIAGWHGTAMLCYVTPKEHLG

LPNAEDVRNGLIAYKIAAHAADIARHRPGARDRDDELSKARYNFDWNRQFELSLDP

ERAKEYHDETLPADIYKTAEFCSMCGPKFCPMQTKVDAEMLEELEVFLAKDKEMVS

QR

SEQ ID NO: 140

Codon-optimized neoR gene

CTTTCTTGCGCTATGACACTTCCAGCAAAAGGTAGGGCGGGCTGCGAGACGGCTT

CCCGGCGCTGCATGCAACACCGATGATGCTTCGACCCCCCGAAGCTCCTTCGGGG

CTGCATGGGCGCTCCGATGCCGCTCCAGGGCGAGCGCTGTTTAAATAGCCAGGCC

CCCGATTGCAAAGACATTATAGCGAGCTACCAAAGCCATATTCAAACACCTAGA

TCACTACCACTTCTACACAGGCCACTCGAGCTTGTGATCGCACTCCGCTAAGGGG

GCGCCTCTTCCTCTTCGTTTCAGTCACAACCCGCAAAC

SEQ ID NO: 141

5' THIC Coccomyxa C-169 confirmation primer 5 ' - GGGTGATCGCCTAC AAGA-3 '

SEQ ID NO: 142

3' THIC confirmation primer

5 ' - ACGTCGCGACCCATGCTTCC-3 ' SEQ ID NO: 143

5' THIC A. thaliana confirmation primer 5'- GCGTCATCGCCTAC AAGA-3' SEQ ID NO: 144

5' thiC Synechosystis sp. confirmation primer 5 ' -CGATGCTGTGCTACGTGA-3 ' SEQ ID NO: 145

C callophylla TE; UTEX 1450 SAD transit peptide underlined atggcttccgcggcat tcaccatgtc ggcgtgcccc gcgatgactg gcagggcccc tggggcacgtcgctccggac ggccagtcgc cacccgcctg agggggcgcg cccccaaggc caacggctccgccgtgtccc tgaagtccgg ctccctggac acccaggagg acacctcctc ctcctcctcccccccccgca ccttcatcaa ccagctgccc gactggtcca tgctgctgtc cgccatcaccaccgtgttcg tggccgccga gaagcagtgg accatgctgg accgcaagtc caagcgccccgacaccctga tggacccctt cggcgtggac cgcgtggtgc aggacggcgt ggtgttccgccagtccttct ccatccgctc ctacgagatc ggcgccgacc gcaccgcctc catcgagaccctgatgaaca tcttccagga gacctccctg aaccactgca agtccatcgg cctgctgaacgacggcttcg gccgcacccc cgagatgtgc aagcgcgacc tgatctgggt ggtgaccaagatgcacatcg aggtgaaccg ctaccccacc tggggcgaca ccatcgaggt gaacacctgggtgtccgagt ccggcaagac cggcatgggc cgcgactggc tgatctccga ctgccacaccggcgagatcc tgatccgcgc cacctccgtg tgcgccatga tgaaccagac cacccgccgcttctccaagt tcccctacga ggtgcgccag gagctggccc cccacttcgt ggactccgcccccgtgatcg aggactacca gaagctgcac aagctggacg tgaagaccgg cgactccatctgcaacggcc tgaccccccg ctggaacgac ctggacgtga accagcacgt gaacaacgtgaagtacatcg gctggattct ggagtccgtg cccaaggagg tgttcgagac ccaggagctgtgcggcctga ccctggagta ccgccgcgag tgcggccgcg actccgtgct gaagtccgtgaccgccatgg acccctccaa ggagggcgac cgctccctgt accagcacct gctgcgcctggaggacggca ccgacatcgc caagggccgc accaagtggc gccccaagaa cgccggcaccaacggcgcca tctccaccgg caagacctcc aacggcaact ccatctcctg a

SEQ ID NO: 146

C callophylla TE; UTEX 1450 SAD transit peptide underlined

MASAAFTMSACPAMTGRAPGARRSGRPVATRLRGRAPKANGSAVSLKSGSLDTQEDT SSS

SSPPRTFINQLPDWSMLLSAITTVFVAAEKQWTMLDRKSKRPDTLMDPFGVDRVVQD GVV

FRQSFSIRSYEIGADRTASIETLMNIFQETSLNHCKSIGLLNDGFGRTPEMCKRDLI WVV

TKMHIEVNRYPTWGDTIEVNTWVSESGKTGMGRDWLISDCHTGEILIRATSVCAMMN QTT

RRFSKFPYEVRQELAPHFVDSAPVIEDYQKLHKLDVKTGDSICNGLTPRWNDLDVNQ HVN

NVKYIGWILESVPKEVFETQELCGLTLEYRRECGRDSVLKSVTAMDPSKEGDRSLYQ HLL

RLEDGTDIAKGRTKWRPKNAGTNGAISTGKTSNGNSIS

SEQ ID NO: 147

R communis SAD nt sequence; UTEX 250 SAD transit peptide underlined atggccaccgcatccactttctcggcgttcaatgcccgctgcggcgacctgcgtcgctcg gcgggctccgggccccggcgcccagc gaggcccctccccgtgcgcgggcgcgccgcctccaccctgaagtccggctccaaggaggt ggagaacctgaagaagcccttcatg cccccccgcgaggtgcacgtgcaggtgacccactccatgcccccccagaagatcgagatc ttcaagtccctggacaactgggccga ggagaacatcctggtgcacctgaagcccgtggagaagtgctggcagccccaggacttcct gcccgaccccgcctccgacggcttcg acgagcaggtgcgcgagctgcgcgagcgcgccaaggagatccccgacgactacttcgtgg tgctggtgggcgacatgatcaccga ggaggccctgcccacctaccagaccatgctgaacaccctggacggcgtgcgcgacgagac cggcgcctcccccacctcctgggcc atctggacccgcgcctggaccgccgaggagaaccgccacggcgacctgctgaacaagtac ctgtacctgtccggccgcgtggaca tgcgccagatcgagaagaccatccagtacctgatcggctccggcatggacccccgcaccg agaactccccctacctgggcttcatct acacctccttccaggagcgcgccaccttcatctcccacggcaacaccgcccgccaggcca aggagcacggcgacatcaagctggc ccagatctgcggcaccatcgccgccgacgagaagcgccacgagaccgcctacaccaagat cgtggagaagctgttcgagatcgac cccgacggcaccgtgctggccttcgccgacatgatgcgcaagaagatctccatgcccgcc cacctgatgtacgacggccgcgacga caacctgttcgaccacttctccgccgtggcccagcgcctgggcgtgtacaccgccaagga ctacgccgacatcctggagttcctggtg ggccgctggaaggtggacaagctgaccggcctgtccgccgagggccagaaggcccaggac tacgtgtgccgcctgcccccccgc atccgccgcctggaggagcgcgcccagggccgcgccaaggaggcccccaccatgcccttc tcctggatcttcgaccgccaggtga agctgatggactacaaggaccacgacggcgactacaaggaccacgacatcgactacaagg acgacgacgacaagtga

SEQ ID NO: 148

R communis SAD aa sequence; UTEX 250 SAD transit peptide underlined

MATASTFSAFNARCGDLRRSAGSGPRRPARPLPVRGRAASTLKSGSKEVENLKKPFM PPR EVHVQVTHSMPPQKIEIFKSLD AEENILVHLKPVEKCWQPQDFLPDPASDGFDEQVRE

LRERAKEIPDDYFVVLVGDMITEEALPTYQTMLNTLDGVRDETGASPTSWAIWTRAW TAE

ENRHGDLLNKYLYLSGRVDMRQIEKTIQYLIGSGMDPRTENSPYLGFIYTSFQERAT HS

HGNTARQAKEHGDIKLAQICGTIAADEKRHETAYTKIVEKLFEIDPDGTVLAFADMM RKK

ISMPAHLMYDGRDDNLFDHFSAVAQRLGVYTAKDYADILEFLVGRWKVDKLTGLSAE GQK

AQDYVCRLPPRIRRLEERAQGRAKEAPTMPFSWIFDRQVKLMDYKDHDGDYKDHDID YKD

DDDK

SEQ ID NO: 149

Nucleotide sequence of transforming DNA contained in pSZ1503_[KASII_btub-y.inv- nr_KASII] gctcttcccgcaccggctggctccaccccaacttgaacctcgagaaccccgcgcctggcg tcgaccccgtcgtgctcgtggggc cgcggaaggagcgcgccgaagacctggacgtcgtcctctccaactcctttggctttggcg ggcacaattcgtgcgtcggtacc

|ctttcttgcgctatgacacttccagcaaaaggtagggcgggctgcgagacggcttc ccggcgctgcatgcaacaccgatgatgcttcg|

[accccccgaagctccttcggggctgcatgggcgctccgatgccgctccagggcgag cgctgtttaaatagccaggcccccgattgq

|aaagacattatagcgagctaccaaagccatattcaaacacctagatcactaccact tctacacaggccactcgagcttgtgatcgcactc|

|cgctaagggggcgcctcttcctcttcgtttcagtcacaacccgcaaac|ggcgcgc cArGctec?gcaggcc?tecfgf?cc^c?gg ccggcttcgccgccaagatcagcgcctccatgacgaacgagacgtccgaccgccccctgg tgcacttcacccccaacaagggct ggatgaacgaccccaacggcctgtggtacgacgagaaggacgccaagtggcacctgtact tccagtacaacccgaacgacac cgtctgggggacgcccttgttctggggccacgccacgtccgacgacctgaccaactggga ggaccagcccatcgccatcgcccc ga gcgcaacgactccggcgccttctccggctccatggtggtggactacaacaacacctccgg cttcttcaacgacaccatcgac ccgcgccagcgctgcgtggccatctggacctacaacaccccggagtccgaggagcagtac atctcctacagcctggacggcgg ctacaccttcaccgagtaccagaagaaccccgtgctggccgccaactccacccagttccg cgacccgaaggtcttctggtacgag ccctcccagaagtggatcatgaccgcggccaagtcccaggactacaagatcgagatctac tcctccgacgacctgaagtcctgg aagctggagtccgcgttcgccaacgagggcttcctcggctaccagtacgagtgccccggc ctgatcgaggtccccaccgagcag gaccccagcaagtcctactgggtgatgttcatctccatcaaccccggcgccccggccggc ggctccttcaaccagtacttcgtcgg cagcttcaacggcacccacttcgaggccttcgacaaccagtcccgcgtggtggacttcgg caaggactactacgccctgcagacc ttcttcaacaccgacccgacctacgggagcgccctgggcatcgcgtgggcctccaactgg gagtactccgccttcgtgcccacca acccctggcgctcctccatgtccctcgtgcgcaagttctccctcaacaccgagtaccagg ccaacccggagacggagctgatcaa cctgaaggccgagccgatcctgaacatcagcaacgccggcccctggagccggttcgccac caacaccacgttgacgaaggcc aacagctacaacgtcgacctgtccaacagcaccggcaccctggagttcgagctggtgtac gccgtcaacaccacccagacgatc tccaagtccgtgttcgcggacctctccctctggttcaagggcctggaggaccccgaggag tacctccgcatgggcttcgaggtgtc cgcgtcctccttcttcctggaccgcgggaacagcaaggtgaagttcgtgaaggagaaccc ctacttcaccaaccgcatgagcgtg aacaaccagcccttcaagagcgagaacgacctgtcctactacaaggtgtacggcttgctg gaccagaacatcctggagctgtact tcaacgacggcgacgtcgtgtccaccaacacctacttcatgaccaccgggaacgccctgg gctccgtgaacatgacgacgggg

^tegacaacc½?tetocafcgflcaagftecfl^g?gcgc^ag^teaflgTGAcaa ttggcagcagcagctcggatagtatcgac acactctggacgctggtcgtgtgatggactgttgccgccacacttgctgccttgacctgt gaatatccctgccgcttttatcaaacagcct cagtgtgtttgatcttgtgtgtacgcgcttttgcgagttgctagctgcttgtgctatttg cgaataccacccccagcatccccttccctcgttt catatcgcttgcatcccaaccgcaacttatctacgctgtcctgctatccctcagcgctgc tcctgctcctgctcactgcccctcgcacagc cttggtttgggctccgcctgtattctcctggtactgcaacctgtaaaccagcactgcaat gctgatgcacgggaagtagtgggatggga acacaaatggaggatcgtagagctcatcttccgaaagtacgacgagtgagcgagctgatt ctctttgagcggggtcgggtggttc ggggagagtgcgcggaaaggcgcagagacgtgcggccggccgtgtccctccgtcttcccc tggttggtgctatagtaacctgc ctgtgtcgcgtgcgcgtcgggaagagc

SEQ ID NO: 150

B. braunii malate dehydrogenase 5'UTR aattggaaaccccgcgcaagaccgggttgtttggccgcctgaccggaaagggggggcctg tcccgaagggggtctatctcttgggg gatgtcgggcgcggaaagtcgatgttgatggacctcttcttcgaccatgtcggggtcgag gccaagagccgcgtccatttcgccgagt tcatgatggaggtgaatgaccgcatcgccaccgaacgcgccaagaagcgggcgaccgatc gcccccgtcgctgcagcccttgccg aggaagtccggctgctggcgttcgacgagatgatggtgacgaacagcccggacgcgatga tcctgtcgcggctgttcaccgcgctg atcgaggcgggggtgacgatcgtcaccacctccaaccggccgcccagggatctctataag aacgggctcaaccgcgagcatttcct gcccttcatcgcgctgatcgaggcgcggctggacgtgctggcgctgaacggcccgaccga ctatcggcgcgaccggctggggcg gctggacacgtggttggtgcccaatggccccaaggcgacgattaccttgtcggcggcgtt cttccgcctgaccgactatccggtcgag gatgccgcgcatgtgccctctgaggacctgaaggtgggcgggcgcgtgctgaatgtcccc aaggcgctgaagggcgtcgcggtctt ctcgttcaagcggttgtgcggcgaagcgcggggggcggcggactatctggcggtcgcgcg gggcttccacaccgtcatcctggtcg gaatccccaagctgggggcggagaaccgcaacgaggcggggcgcttcgtccagctgatcg acgcgctctacgaacataaggtcaa gctgctcgccgcagccgatgccagcccgccgaactctatgaaaccggcgacggccggttc gagtttgagcgcagatcagccggttg gaagagatgcgctccgaggattatctggcccaaggccatggctcggaggggccttgatca ggccttaatgcacttcgcaaccattatc gtttaaaatcttaaactctgtggaataacggttccccgacgccgcaatacacgtacgtcc actacggagtaggattgga

SEQ ID NO: 151

Chlamydomonas RBCS2 promoter cgcttagaagatttcgataaggcgccagaaggagcgcagccaaaccaggatgatgtttga tggggtatttgagcacttgcaacccttat ccggaagccccctggcccacaaaggctaggcgccaatgcaagcagttcgcatgcagcccc tggagcggtgccctcctgataaacc ggccagggggcctatgttctttacttttttacaagagaagtcactcaacatcttaaacgg tcttaagaagtctatccgg

SEQ ID NO: 152

CMV-Hyg-CMV BamHI-SacII cassette from pCAMBIA ggatccccgggaattcggcgcgccgggcccaacatggtggagcacgacactctcgtctac tccaagaatatcaaagatacagtctca gaagaccaaagggctattgagacttttcaacaaagggtaatatcgggaaacctcctcgga ttccattgcccagctatctgtcacttcatca aaaggacagtagaaaaggaaggtggcacctacaaatgccatcattgcgataaaggaaagg ctatcgttcaagatgcctctgccgaca gtggtcccaaagatggacccccacccacgaggagcatcgtggaaaaagaagacgttccaa ccacgtcttcaaagcaagtggattgat gtgataacatggtggagcacgacactctcgtctactccaagaatatcaaagatacagtct cagaagaccaaagggctattgagacttttc aacaaagggtaatatcgggaaacctcctcggattccattgcccagctatctgtcacttca tcaaaaggacagtagaaaaggaaggtgg cacctacaaatgccatcattgcgataaaggaaaggctatcgttcaagatgcctctgccga cagtggtcccaaagatggacccccaccc acgaggagcatcgtggaaaaagaagacgttccaaccacgtcttcaaagcaagtggattga tgtgatatctccactgacgtaagggatg acgcacaatcccactatccttcgcaagaccttcctctatataaggaagttcatttcattt ggagaggacacgctgaaatcaccagtctctct ctacaaatctatctctctcgagctttcgcagatcccggggggcaatgagatatgaaaaag cctgaactcaccgcgacgtctgtcgagaa gtttctgatcgaaaagttcgacagcgtctccgacctgatgcagctctcggagggcgaaga atctcgtgctttcagcttcgatgtaggag ggcgtggatatgtcctgcgggtaaatagctgcgccgatggtttctacaaagatcgttatg tttatcggcactttgcatcggccgcgctccc gattccggaagtgcttgacattggggagtttagcgagagcctgacctattgcatctcccg ccgtgcacagggtgtcacgttgcaagacc tgcctgaaaccgaactgcccgctgttctacaaccggtcgcggaggctatggatgcgatcg ctgcggccgatcttagccagacgagcg ggttcggcccattcggaccgcaaggaatcggtcaatacactacatggcgtgatttcatat gcgcgattgctgatccccatgtgtatcact ggcaaactgtgatggacgacaccgtcagtgcgtccgtcgcgcaggctctcgatgagctga tgctttgggccgaggactgccccgaa gtccggcacctcgtgcacgcggatttcggctccaacaatgtcctgacggacaatggccgc ataacagcggtcattgactggagcgag gcgatgttcggggattcccaatacgaggtcgccaacatcttcttctggaggccgtggttg gcttgtatggagcagcagacgcgctactt cgagcggaggcatccggagcttgcaggatcgccacgactccgggcgtatatgctccgcat tggtcttgaccaactctatcagagcttg gttgacggcaatttcgatgatgcagcttgggcgcagggtcgatgcgacgcaatcgtccga tccggagccgggactgtcgggcgtaca caaatcgcccgcagaagcgcggccgtctggaccgatggctgtgtagaagtactcgccgat agtggaaaccgacgccccagcactc gtccgagggcaaagaaatagagtagatgccgaccggatctgtcgatcgacaagctcgagt ttctccataataatgtgtgagtagttccc agataagggaattagggttcctatagggtttcgctcatgtgttgagcatataagaaaccc ttagtatgtatttgtatttgtaaaatacttctatc aataaaatttctaattcctaaaaccaaaatccagtactaaaatccagatcccccgaatta attcggcgttaattcagtacattaaaaacgtcc gcaatgtgttattaagttgtctaagcgtcaatttgtttacaccacaatatatcctgccac cagccagccaacagctccccgaccggcagct cggcacaaaatcaccactcgatacaggcagcccatcagtccgggacggcgtcagcgggag agccgttgtaaggcggcagactttg ctcatgttaccgatgctattcggaagaacggcaactaagctgccgggtttgaaacacgga tgatctcgcggagggtagcatgttgattg taacgatgacagagcgttgctgcctgtgatcaccgcgg

SEQ ID NO: 153

CAACCACGTCTTCAAAGCAA SEQ ID NO: 154 AGCAATCGCGCATATGAAAT SEQ ID NO: 155

Endogenous Chlorella protothecoides actin promoter and 5'UTR

GAATTCGAGTTTAGGTCCAGCGTCCGTGGGGGGGGACGGGCTGGGAGCTTGGGC

CGGGAAGGGCAAGACGATGCAGTCCCTCTGGGGAGTCACAGCCGACTGTGTGTG

TTGCACTGTGCGGCCCGCAGCACTCACACGCAAAATGCCTGGCCGACAGGCAGG

CCCTGTCCAGTGCAACATCCACGGTCCCTCTCATCAGGCTCACCTTGCTCATTGAC

ATAACGGAATGCGTACCGCTCTTTCAGATCTGTCCATCCAGAGAGGGGAGCAGG

CTCCCCACCGACGCTGTCAAACTTGCTTCCTGCCCAACCGAAAACATTATTGTTT

GAGGGGGGGGGGGGGGGGGCAGATTGCATGGCGGGATATCTCGTGAGGAACAT

CACTGGGACACTGTGGAACACAGTGAGTGCAGTATGCAGAGCATGTATGCTAGG

GGTCAGCGCAGGAAGGGGGCCTTTCCCAGTCTCCCATGCCACTGCACCGTATCCA

CGACTCACCAGGACCAGCTTCTTGATCGGCTTCCGCTCCCGTGGACACCAGTGTG

TAGCCTCTGGACTCCAGGTATGCGTGCACCGCAAAGGCCAGCCGATCGTGCCGAT

TCCTGGGGTGGAGGATATGAGTCAGCCAACTTGGGGCTCAGAGTGCACACTGGG

GCACGATACGAAACAACATCTACACCGTGTCCTCCATGCTGACACACCACAGCTT

CGCTCCACCTGAATGTGGGCGCATGGGCCCGAATCACAGCCAATGTCGCTGCTGC

CATAATGTGATCCAGACCCTCTCCGCCCAGATGCCGAGCGGATCGTGGGCGCTGA

ATAGATTCCTGTTTCGATCACTGTTTGGGTCCTTTCCTTTTCGTCTCGGATGCGCG

CTGCGCGCGGGCAAGTTGCTTGACCCTGGGCTGGTACCAGGGTTGGAGGGTATTA

CCGCGTCAGGCCATTCCCAGCCCGGATTCAATTCAAAGTCTGGGCCACCACCCTC

CGCCGCTCTGTCTGATCACTCCACATTCGTGCATACACTACGTTCAAGTCCTGATC

CAGGCGTGTCTCGGGACAAGGTGTGCTTGAGTTTGAATCTCAAGGACCCACTCCA

GCACAGCTGCTGGTTGACCCCGCCCTCGCAACTCCCTACCATGrCrGCrGgtaggtc ca gggatctttgccatgcacacaggaccccgtttgtgggggtccccggtgcatgctgtcgct gtgcaggcgccggtgtggggcctgggc cccgcgggagctcaactcctccccatatgcctgccgtccctcccacccaccgcgacctgg ccccctttgcagAGGAAGGCGA

AGTCAGCGCCATCGTGTGCGATAATGGATCCGG

SEQ ID NO: 156

Endogenous Chlorella protothecoides EFla promoter and 5'UTR

GAATTCGCCCTTGAGTTTAGGTCCAGCGTCCGTGGGGGGGGCGTGAGACTCCCCC

CTGACCTTCGTATGGCAGGGACTCCTACTTGCCAAGTAATCAGTTGACAATGCCA

CTTCAATGCTCGTTGTGGTACACTGACGCGGGTCTAACATACTGGGAAGCATGAA

TTGCCGACATGGACTCAGTTGGAGACAGTAACAGCTCTTTGTGTTCTATCTTCAG

GAACACATTTGGCAGCGCACCCATACAGTGGCGCACACGCAGCTGTACCTGATG

TGGCTCTATTCCCACATGTTTCAACTTGATCCAAAAGTCACTCAGACTCTCAGCA

GCTAGACTTGATCGCATCTTTGGCCATGAAGATGCTTGCGCAACTCTAGGAATGG GACGAGAAAAGAGCCTGCTCTGATCGGATATTTCCATTCTCTGGATGGGACTGAG

ATGATTCTGAAGAAATGCTGCTCGACTTATTTGGAAGAACAGCACCTGACGCATG

CTTTGAGGCTGCTGTGGCTGGGATGTGCTGTATTTGTCAGCATTGAGCATCTACG

GGTAGATGGCCATAACCACGCGCTGCCTATCATGCGGTGGGTTGTGTGGAAAAC

GTACAATGGACAGAAATCAATCCCATTGCGAGCCTAGCGTGCAGCCATGCGCTC

CCTCTGTAGCCCCGCTCCAAGACAAAGCCAGCCAATGCCAGAACCCACATAGAG

AGGGTATCTTCCTAATGACCTCGCCCATCATTTCCTCCAAATTAACTATAATGCCT

TGATTGTGGAGTTGGCTTTGGCTTGCAGCTGCTCGCGCTGGCACTTTTGTAGGCA

GCACAGGGTATGCCAGCGCCGAACTTTGTGCCCTTGAGCAGGCCACAAGGGCAC

AAGACTACACCATGCAGCTGGTATACTTGGAACTGATACCATTCTTACCAAGCAA

GGCACAGCACAGCCTGCACCGACTCACTTTGCTTGAGCGGGGCACAGCGCCGCG

ACTGATCCTGCGAGCTGTGGGGAGTTCCGACTGTTCTGGACCTCGGTCTCTGAAA

GATGTGTACGATGGGATCAAGTCATTCAAGTATGCTCTTCACATGAGCAATCGGG

GGAGACACGGTGGCCCTAAAGGTGTTCATCTGATTCAAGTGTAGTGGGGGGGTG

CTGTTTGTCCCGGGGCGCCCCCCGCTCCCCGACCCCGGAGAAGGGCCCCAGAGG

ACTCGGCCGCCCACAGAGGAATAACCGGGCGTGGCTCGGCCCTGCGCCTCCCTCT

TTCAATATTTCACCTGGTGTTCAGTGCACGGACACGTAAAGAACTAGATACAATG

GCCGAGGGAA GACGgtgagagcttggcgttggtggaccgggcagcatcagaaactcctcttccccgcccg ccttgaaa ctcactgtaactccctcctcttccccctcgcagCArCrGrCrArCGTTArCgtgagtgaa agggactgccatgtgtcgggt cgttgaccacggtcggctcgggcgctgctgcccgcgtcgcgaacgttccctgcaaacgcc gcgcagccgtccctttttctgccgccg ccccaccccctcgctccccccttcaatcacaccgcagrGCGGACArGrCGArrCCGGCAA GrCCACC

SEQ ID NO: 157

Endogenous Chlorella protothecoides beta-tubulin promoter (isoform A)

GAATTCCCTGCAGGAAGAAGGCCGGCAGCAGCTGGTACTTGTCCTTCACCTCCTT

GATCGGCTGGGTGAGCTTGGCCGGGTCGCAGTCGTCGATGCCGGCATCGCCCAG

CACGCTGTGCGGGGAGCCGGCATCGACAACCTTGGCACTGCTCACCTTGGTCACC

GGCATGGGGTCATGGCGCTGCAGACCAGCGGCCTGTCAGCATGCTGCAGGCATC

TGTGTTTTGTAGTAGATACTTTCTGATGCATCACCACACGTTTGGAAGGTCCCCA

AGCCCCTTCAACAGTCTCGACATATGACACTCGCGCCCTCTTCCTCGTCCCGTGG

CCTGATGAGGGTACGCAGGTACCGCAGCTGCGCCCCGTCCCGCCAGTTGCCCTGG

CCCCGCCGGGCCCAATCTGTTCATTGCCGCTCCCTGGCAGCCGTGAACTTCACAC

TACCGCTCTCTGTGACCTTCAGCACAGCAGGAATCGCCATTTCACCGGCGGTCGT

TGCTGCGGAGCCTCAGCTGATCTCGCCTGCGAGACCCCACAGTTTGAATTTGCGG

TCCCCACACAACCTCTGACGCC

SEQ ID NO: 158

Endogenous Chlorella protothecoides beta-tubulin promoter (isoform B)

GAATTCCCTCAGGAAGAAGGCCGGCAGCAGCTGGTACTTGTCCTTCACCTCCTTG

ATCGGCTGGGTGAGCTTCGCAGGATCGCAGTCGTCGATGCCGGCATCGCCCAGC

ACGCTGTGCGGGGAGCCGGCATCNACAACCTTGGCACTGCTCCCCTTGGTCACCG

GCATGGGGTCATGGCGCTGCAGCCCAGCGGCCTGTCAGCATGCTGCAGGCATCT

GTGTATTGTAGTAGGTACTTCCTGATGCATCAACACACGTTTGGAAGCTCCCCAA

GCCCCTTCAACAGTCTCGACGTATGACACTCGCGCCCTCTTCCTCGCCCCGTGGC

CTGATGAGGGTACGCAGGTACCACAGCTGCGCCCCGTCCCGCCAGTTGCCCTGGC

CCGGCCGGGCCCAATCTGTTCATTGCCGCTCCCTGGTAGCCGTGAACTCACATTA

CCGCTCTCTGTGACCTTCAGCACAGCAGGAATCGCCATTTCACCGGCGGTCGTTG CTGCGGAGCCTCAGCTGATCTCGCCTGCGAGACCCCACAGTTTGAATTTGCGGTC CCCACACAACCTCTGACGCC

SEQ ID NO: 159

Acyl ACP desaturase atcaaaggcatagattcacatttgttggcattgcagagcaatcatcgcgcaggacgaaca tcgctcaccaagcacgtactgggcatcc ggaggcctccgcaaattcctgcaacaggactcgctgatcagttcgcccaaggtctacgac gctccctatcggcgctagacttcaacac atatttcactgtcacagcctcggcATGCATCAGGCCTCAGTCTCCACCATGAAGACCATC CAGTC

TCGGCACGCCGGTCCCATCGGACATGTGCAGTCGGGTCGCCGATCGGCGGGGCG

CGCGGGATCCCGCATGGCGACCCCCGTGGCCGCAGCTACCGTCGCAGCCCCTCGC

TCGGCCCTCAACCTCTCCCCCACCATCATTCGACAGGAGGTGCTCCACTCCGCCA

GCGCCCAGCAACTAGACTGCGTGGCCTCCCTGGCGCCCGTCTTCGAGTCCCAGAT

CCTCCCCCTCCTGACGCCCGTGGACGAGATGTGGCAGCCCACCGACTTCCTCCCC

GCCTCGAACTCGGAGGCATTCTTCGACCAGATCGGCGACCTGCGGGCGCGATCG

GCGGCCATCCCCGACGACCTGCTGGTCTGCCTGGTGGGGGACATGATCACGGAG

GAGGCCCTGCCCACCTACATGGCCATGCTGAACACCCTGGACGTCGTGCGCGATG

AGACAGGGCACAGCCAGCACCCCTACGCCAAGTGGACCAGGGCTTGGATCGCGG

AGGAGAACCGCCATGGCGACCTGCTGAACAAGTACATGTGGCTGACGGGGCGGG

TGGGACATGCTGGCGGTGGAGCGCACCATCCAGCCATGCTGGCGGTGGAGCGCA

CCATCCAGCGCCTCATCTCATCGGGCATGGACCCGGGCACGGAGAACCACCCCT

ACCACGCCTTTGTGTTCACCAGCTTCCAGGAGCGCGCCACCAAGCTGAGCCACGG

CTCCACCGCCCGCCTGGCGGTCGCCGCCGGGGACGAGGCCCTGGCCAAGATCTG

CGGGACCATTGCGCGGGACGAGTCGCGCCACGAGGCGGCGTACACGCGGACCAT

GGATGCCATCTTCCAGCGCGACCCCAGCGGGGCCATGGTGGCGTTTGCGCACATG

ATGATGCGCAAGATCACCATGCCCGCCCACCTCATGGACGACGGCCAGCACGGC

GCGCGCAACGGGGGGGCGCAACTTGTTCGACGACTTTGCGGCAGTGGCGGAGCG

GGCAGGGGTGTACACCGCCGGCGACTACATCGGCATCCTGCGCCACCTCATCCG

GCGCTGGGACGTGGAGGG

SEQ ID NO: 160

Acyl ACP desaturase

MHQASVSTMKTIQSRHAGPIGHVQSGRRSAGRAGSRMATPVAAATVAAPRSALNLS PTIIRQEVLHSASAQQLDCVASLAPVFESQILPLLTPVDEMWQPTDFLPASNSEAFFDQ IGDLRARSAAIPDDLLVCLVGDMITEEALPTYMAMLNTLDVVRDETGHSQHPYAKW TRAWIAEENRHGDLLNKYMWLTGRVGHAGGGAHHPAMLAVERTIQRLISSGMDPG TENHPYHAFVFTSFQERATKLSHGSTARLAVAAGDEALAKICGTIARDESRHEAAYT RTMDAIFQRDPSGAMVAFAHMMMRKITMPAHLMDDGQHGARNGGAQLVRRLCGS GGAGRGVHRRRLHRHPAPPHPALGRGG

SEQ ID NO: 161

Geranyl geranyl diphosphate synthase attatacatcggcatcgtctcaggtttcacgatctgcatgctatctatgggactgtgact ccgccggccaggttgtggtgcgcgagaatc ctccccgctcctgccttctcatttccctgacgggagtcgccgctgagcaccgggcggatc ATGGGCGTCGGCACACT

CCAAACCCCATATACATGTGGTCGTGCATTCACGCATAGCGCACGGTATGTCCCG

CGACGCGCGGCTCGAAGCCGTGGCCATCCGACGCGCTGCACGGCCGAGGTGAGG

GCACGCCCCTCCGCCAATGGCGCGCAGCCCATGACCGCCTTCGACTTCCGGCAGT

ACATGCAGCAGCGCGCCGCGCTGGTGGACGCAGCGCTGGACCTGGCAGTGCCGC TGCAGTACCCCGAGAAGATCAACGAGGCCATGCGGTACAGCCTGCTGGCCGGGG

GCAAGCGCGTGCGCCCCGCGCTCTGCCTCGCTGCCTGCGAGCTCGTGGGCGGCCC

CCTGGAGGCGGCCATGCCCGCCGCCTGCGCCATGGAGATGATCCACACCATGAG

CCTCATCCACGACGACCTCCCCGCCATGGACAACGACGACTTCCGGCGCGGCCA

GCCCGCCAACCACAAGGCCTATGGCGAGGAGATTGCGATCCTGGCGGGCGACGC

GCTGCTGTCGCTGAGCTTTGAGCACATCGCGCGCGAGACGCGAGGCGTGGACCC

GGTGCGCGTCCTGGCCGCCATCTCGGAGTGGCGCGCGGTGGGCAGCCGCGGGCT

GGTGGCGGGGCAGGTGGTGGACCTGGGTTTCGAGGGCGGCGGCGTGGGGCTGGC

CCCGCTGCGCTACATCCACGAGCACAAAACCGCGGCGCTGCTGGAGGCGGCGGT

GGTGTCCGGCGCGCTGCTGGGCGGCGCGGAGGAGGCGGACCTGGAGCGCCTGCG

CACCTACAACCGCGCCATCGGCCTCGCTTTCCAGGTGGTGGGGGACATCCTGGAC

ATCCCGGGGACCAGCGAGGAGCTGGGCAAGACCGCGGGCAAGGACCTGAGCTCC

CCCAAAACCCCCTACCCGTCCCTGGTGGGGCTGGCCAGGTCCAAAAAAATTGCG

GACGAACTGATTGAGGACGCGAAAACCCAACTCACCCAGTACGAGCCGGCCCGA

GCGGCGCCCCTCGTAACCCTGGCCGAAAACATTTGAaaccggaagaactgactgggg gcccccc ctgcccccagatacggcggggctcctccatccagttttgggatgggaggagcgacaaccg accccgtaaccctgtgacgcgtttgcc ttgcatacgtacgcatgccttgaaacccatccatgaccctcaacaatacctggttgtgtg tagcttggtcctgaaaaaaaaaaaaaaaaa aaaaaaaaaaa

SEQ ID NO: 162

Geranyl geranyl diphosphate synthase

MGVGTLQTPYTCGRAFTHSARYVPRRAARSRGHPTRCTAEVRARPSANGAQPMTAF

DFRQYMQQRAALVDAALDLAVPLQYPEKINEAMRYSLLAGGKRVRPALCLAACEL

VGGPLEAAMPAACAMEMIHTMSLIHDDLPAMDNDDFRRGQPANHKAYGEEIAILAG

DALLSLSFEHIARETRGVDPVRVLAAISEWRAVGSRGLVAGQVVDLGFEGGGVGLA

PLRYIHEHKTAALLEAAVVSGALLGGAEEADLERLRTYNRAIGLAFQVVGDILDIPG T

SEELGKTAGKDLSSPKTPYPSLVGLARSKiQADELIEDAKTQLTQYEPARAAPLVTL A

ENI

SEQ ID NO: 163

Geranyl geranyl diphosphate synthase cagatgccATGCGCCCTCGGGCCGCGGGCCTGAGGGTCCACGCAGCGTCCTCGGTGG

CCCAGACGCACCAGGCCGCCCCCCCGGCGGACAGGAGGTTCGACGACTACCAGC

CCCGCACCGCCATCCTCTTCCCCGGCCAGGGCGCGCAGAGCGTGGGCATGGCGG

GAGAGCTGGCGAAGGCCGTCCCCGCCGCCGCGGCGCTGTTCGACGCCGCCTCCG

ACCAGCTCGGCTATGACCTGCTCCGCGTGTGCGTTGAGGGCCCCAAGGCGCGCCT

GGACAGCACCGCCGTCAGCCAGCCCGCCATCTACGTGGCCAGCCTGGCGGCGGT

GGAGAAGCTGCGCGCGGAGGGCGGGGAGGAGGCACTGGCCGCCATCGACGTCG

CTGCCGGTCTGTCCTTGGGCGAGTACACCGCGCTGGCCTTTGCCGGCGCCTTCTC

CTTCGCCGACGGGCTGCGCCTGGTGGCCCTGCGCGGCGCCAGCATGCAGGCCGC

CGCCGACGCCGCACCCTCGGGCATGGTCTCCGTCATCGGTCTGCCCTCCGACGCG

GTGGCCGCGCTGTGCGAGGCCGCCAACGCGCAGGTGGCCCCCGACCAGGCCGTG

CGCATCGCCAACTACCTCTGCGACGGCAACTACGCCGTCAGCGGTGGGCTGGAG

GGCTGCGCGGCGGTGGAGGGCCTGGCCAAGGCCCACAAGGCGCGCATGACGGTG

CGCCTGGCGGTGGCGGGCGCCTTCCACACCCCCTTCATGCAGCCGGCGGTGGAG

GCGCTGAGCGCGGGCGCTGGCGGACACGCCGCTGGTCGCGCCGCGCATCCCCGT

GGTCAGCAACGGGACGCC SEQ ID NO: 164

Geranyl geranyl diphosphate synthase

MRPRAAGLRVHAASSVAQTHQAAPPADRRFDDYQPRTAILFPGQGAQSVGMAGEL

AKAVPAAAALFDAASDQLGYDLLRVCVEGPKARLDSTAVSQPAIYVASLAAVEKLR

AEGGEEALAAIDVAAGLSLGEYTALAFAGAFSFADGLRLVALRGASMQAAADAAPS

GMVSVIGLPSDAVAALCEAANAQVAPDQAVRIANYLCDGNYAVSGGLEGCAAVEG

LAKAHKARMTVRLAVAGAFHTPFMQPAVEALSAGAGGHAAGRAAHPRGQQRDA

SEQ ID NO: 165

Gylceraldehyde 3 -phosphate dehydrogenase cDNA sequence

TGTCCATCTCCCCCCACCCTCCATCCAACCATCGTCGACGGCATGCAGGCGCTGT

GTTCTCACCCCGCGTCCCTCACGGCGCGTGCGGTACCCCATGGGCGGGCCAGCCC

AGCACAGCGGGTGTCCAGCGCCGGCCCGGCCTACACCGGCCTGTCCCGGCACAC

CCTGGGCTGCCCCAGCACCCCCACCCTCCAGTCCCGCGCCGCGGTCCAGACCCGC

GGCTCCTCCTCCGGCTCCACCACGCGCATGACCACCACCGCCCAGCGCAAGATCA

AGGTGGCCATCAACGGGTTCGGCCGCATCGGCCGCCAGTTCCTGCGCTGCGTGGA

GGGGCGCGAGGACTCGCTGCTGGAGATCGTGGCCGTGAACGACTCCGGCGGCGT

GAAGCAGGCCAGCCACCTGCTCAAGTACGACTCCACCATGGGCACCTTCAACGC

CGACATCAAGATCTCGGGCGAGGGCACCTTCTCCGTCAACGGCCGCGACATCCG

CGTCGTCTCCTCCCGCGACCCCCTGGCCCTGCCCTGGGGCGAGCTGGGCGTGGAC

CTGGTGATCGAGGGGACGGGAGTGTTTGTGGACCGCAAGGGTGCCAGCAAGCAC

CTGCAGGCGGGGGCCAAGAAGGTCATCATCACCGCGCCGGCCAAGGGCTCCGAC

GTGCCCACCTACGTCATGGGCGTGAACGCGGACCAGTACTCCAACTCCGACGAC

ATCATCTCCAACGCCTCCTGCACCACCAACTGCCTGGCGCCCTTTGTCAAGGTGC

TCAACGACCGCTTCGGCATCGTGA

SEQ ID NO: 166

Glyceraldehyde 3 -phosphate dehydrogenase

MQALCSHPASLTARAVPHGRASPAQRVSSAGPAYTGLSRHTLGCPSTPTLQSRAAVQ TRGSSSGSTTRMTTTAQRKIKVAINGFGRIGRQFLRCVEGREDSLLEIVAVNDSGGVK QASHLLKYDSTMGTFNADI ISGEGTFSVNGRDIRVVSSRDPLALPWGELGVDLVIEG TGVFVDRKGASKHLQAGAKKVIITAPAKGSDVPTYVMGVNADQYSNSDDIISNASCT TNCLAPFVKVLNDRFGIV

SEQ ID NO: 167

Oxygen Evolving Complex protein (OEE33) cDNA sequence gatgttgagaatagtagcttgctgccttgtcgccatgcagagcgtgtgcgcgcagtcggt ttcatgcaagggggccttcacccagtccc tgcggaccccccgatgcagcaggagccagctcgtctgccgggctgatggcaaggccggag ccttcatcaagaccgtaaagagcgg tgctgccgctctggctgcctccctcctcctgtctgggggtgcgggcgcactgacctttga tgagctgcagggcctgacctacctgcag gtgaagggctctggcatcgccaacacctgccccaccctgtctggcggctcctccaacatc aaggacctgaagagcgggacctactcc gtcaacaagatgtgcctggagcccacgtccttcaaggtcaaggaggaggcacagttcaag aacggcgaggccgactttgtgcccac caagctcgtcacgcgtctgacctacaccctggacgagatctctggccagatgaagatcga cggcagcggcggcgtggagttcaagg aggaggatggcatcgactatgctgcagtcaccgtgcagcttccgggcggggagcgcgtgc ccttcctcttcaccatcaaggagcttg acgccaaggggactgccgacggcttcaagggcgagttcaccgtgccctcctaccgtgggt cctccttcctggaccccaagggccgc ggcgcctccaccggctacgacaacgccgtggccctgcccgccgccggcgattccgaggag ttggagaaggagaacaacaagtcc accaaggctctgaagggggaggccatcttctccatcgccaaggtggacgccgggacaggg gaggtggcgggcatctttgagt SEQ ID NO: 168

Oxygen Evolving Complex protein (OEE33)

MQSVCAQSVSCKGAFTQSLRTPRCSRSQLVCRADGKAGAFIKTVKSGAAALAASLL LS GG AGALTFDELQGLT YLQ VKGS GI ANTCPTLS GGS SNIKDLKS GT YS VNKMCLEP TSFKVKEEAQFKNGEADFVPTKLVTRLTYTLDEISGQMKIDGSGGVEFKEEDGIDYA AVTVQLPGGERVPFLFTIKELDAKGTADGFKGEFTVPSYRGSSFLDPKGRGASTGYD NAVALPAAGDSEELEKENNKSTKALKGEAIFSIAKVDAGTGEVAGIFE

SEQ ID NO: 169

Clp protease cDNA sequence ataatcggaacccagctgcacgcaccatcagtgcggcagcatgcagaccgtcgcagccag ctatggcgtattggcgccctccggct ccagcgtgacccggggctcgaccagcagcaagcagcacttcaccaccctcactccctttt ccggcttcaggcgcctgaatcatgtgga tcgggcggggcaggcggggtctgggagcccccagaccctgcagcaggccgtgggcaaggc cgtgcgccggtcgcggggccgc accaccagcgccgtgcgcgtgacccgcatgatgtttgagcggttcaccgagaaggccatc aaggtggtcatgctcgcgcaggagga ggctcgccgtctgggccacaacttcgtggggacggagcaaatcctgctggggttgattgg ggagtccacaggcatcgccgccaagg tcctcaagtcgatgggcgtcacgctgaaagatgcgcgtgtggaggtcgagaagatcatcg gccgggggagcggctttgtggccgtg gagatccccttcaccccccgcgccaagcgtgtgctggagctgtccctggaggaggctcgc cagctcggccacaactacattggcac ggagcacatcctgctgggcctgctgcgcgagggtgagggcgtggcctcccgcgtgctgga gaccctgggcgccgacccccagaa gatccgcactcaggtggtacgcatggtgggtgagtcgcaggagcccgtgggcaccacggt gggcggagggtccaccggctccaa caagatgcccaccctggaggagtacggcaccaacctgaccgcccaggccg

SEQ ID NO: 170

Clp protease

MQTVAASYGVLAPSGSSVTRGSTSSKQHFTTLTPFSGFRRLNHVDRAGQAGSGSPQT

LQQAVGKAVRRSRGRTTSAVRVTRMMFERFTEKAIKVVMLAQEEARRLGHNFVGT

EQILLGLIGESTGIAAKVLKSMGVTL DARVEVEKIIGRGSGFVAVEIPFTPRAKRVLE

LSLEEARQLGHNYIGTEHILLGLLREGEGVASRVLETLGADPQKIRTQVVRMVGESQ

EPVGTTVGGGSTGSNKMPTLEEYGTNLTAQA

SEQ ID NO: 171

TCCGGTGTGTTGTAAGTCCA SEQ ID NO: 172 TTGTCGGAATGTCATATCAA SEQ ID NO: 173 AACGCCTTTGTACAACTGCA SEQ ID NO: 174

CTGACCCGACCTATGGGAGCGCTCTTGGC SEQ ID NO: 175 CTTGACTTCCCTCACCTGGAATTTGTCG

SEQ ID NO: 176

GTGGCCATATGGACTTACAA SEQ ID NO: 177

CAAGGGCTGGATGAATGACCCCAATGGACTGTGGTACGACG SEQ ID NO: 178

CACCCGTCGTCATGTTCACGGAGCCCAGTGCG SEQ ID NO: 179

KE858 Homologous recombination construct SZ725

GCCCTTTGTCATCGTTGGCATGCTTTTTGCGTATGTACCATATGTTGAATGTATAA

TACGAACGGTTGACCGTCTGAGATGCGAGCTTTGGGTCTTGTCAAATGCGTGGCC

GCACGGCTCCCTCGCACCCAGCCCCGAGGCGTCGCGCACCTGGCGAGGAGCAGA

CCCACGCCAAGAAAGTCTAGTCCAGCATGTAACAACATCAGGCAATGTGACGTT

TTCGGTTCCCGATTTCTCTGCCGCTCTTTGACGGCAGGCACGGGCGAGCAACCGG

CGGCGCTCGCGTCAGGCACGATGGATGCGGCGCTGCCCACCTGTCAATGTACCCC

ACCAGTCTGTCGATCGCTACAAGCAACCTTGTGCTCCACATTCCCACTTGCAGAC

AGTCTAGTCGATTTTGCCAAGCTGGATGTGAGGATTGGCCATATCTTGGAGGCCA

AGATTCACCCGGATGCTGATGGGTACGTACGCGAGCCAGGCAGGCAGCTGCGTT

GACTTTCTGATTGGCACAAAGCTTTGGCTACTCTCAATACCAACCACGTGCCCCT

TCTGCACACCTGCTTCCTTCTGATGACCACTCGCCACGCATGTCGCAGTCTGTACG

TCGAGCAGATCGACCTCGGCGAGGAGGGGGGCCCTCGCACCATCGTGAGTGGCC

TGGTCCGGCACGTGACCCTGGAGGACCTTGTCGGCCGGCGGGTGGTGGTGCTGG

CCAACCTCAAGCCTCGGAGCATGCGCGGGGTCAAATCGGCTGGGATGCTGCTCT

GCGCCGCCAACGCGGATCACACCGCGGTGGAGCCGCTGCGGGTCCCGGACGCCG

CCGTGACGGGGGAGCGGGTCTGGGCGGGGGACGAGGCACTCCTGTCCACGGAGC

CTGCCACACCCAATCAGGTAAGGACACGTTATTGGTGCGCATGGTGCGAATGCGT

GGTCTGACCTGCTGTGGGTATGTGTTGTGGGATTGGAAACCGAATGAGGGCCGTT

CAGGATTGAGCCCTTGGCCCCACCCTGCTCATCCTCTCACGCCCGCAGGTCCAGA

AGAAGAAAATCTGGGAGGCAGTACAGCCGCTGCTGAGAGTGAACGCCCAGGGG

ATCGCTACTGTGGCAGGAGAGGCTATGGTGACCAGTGCGGGGCCACTGACCGCG

CCCACGCTGGTTGACGCCGCGATTTCCTGACGCGAGCGACTGATTCTTGACCTTT

CACCATTTCAGTACTGTAGGACCCCCAAAATAGTGTGATCACGCTCGCAAGGCAC

CTGTGTGATCACGGGGAAGGGCGAATTCCTTTCTTGCGCTATGACACTTCCAGCA

AAAGGTAGGGCGGGCTGCGAGACGGCTTCCCGGCGCTGCATGCAACACCGATGA

TGCTTCGACCCCCCGAAGCTCCTTCGGGGCTGCATGGGCGCTCCGATGCCGCTCC

AGGGCGAGCGCTGTTTAAATAGCCAGGCCCCCGATTGCAAAGACATTATAGCGA

GCTACCAAAGCCATATTCAAACACCTAGATCACTACCACTTCTACACAGGCCACT

CGAGCTTGTGATCGCACTCCGCTAAGGGGGCGCCTCTTCCTCTTCGTTTCAGTCAC

AACCCGCAAACGGCGCGCCATGCTGCTGCAGGCCTTCCTGTTCCTGCTGGCCGGC

TTCGCCGCCAAGATCAGCGCCTCCATGACGAACGAGACGTCCGACCGCCCCCTG GTGCACTTCACCCCCAACAAGGGCTGGATGAACGACCCCAACGGCCTGTGGTAC

GACGAGAAGGACGCCAAGTGGCACCTGTACTTCCAGTACAACCCGAACGACACC

GTCTGGGGGACGCCCTTGTTCTGGGGCCACGCCACGTCCGACGACCTGACCAACT

GGGAGGACCAGCCCATCGCCATCGCCCCGAAGCGCAACGACTCCGGCGCCTTCT

CCGGCTCCATGGTGGTGGACTACAACAACACCTCCGGCTTCTTCAACGACACCAT

CGACCCGCGCCAGCGCTGCGTGGCCATCTGGACCTACAACACCCCGGAGTCCGA

GGAGCAGTACATCTCCTACAGCCTGGACGGCGGCTACACCTTCACCGAGTACCA

GAAGAACCCCGTGCTGGCCGCCAACTCCACCCAGTTCCGCGACCCGAAGGTCTTC

TGGTACGAGCCCTCCCAGAAGTGGATCATGACCGCGGCCAAGTCCCAGGACTAC

AAGATCGAGATCTACTCCTCCGACGACCTGAAGTCCTGGAAGCTGGAGTCCGCGT

TCGCCAACGAGGGCTTCCTCGGCTACCAGTACGAGTGCCCCGGCCTGATCGAGGT

CCCCACCGAGCAGGACCCCAGCAAGTCCTACTGGGTGATGTTCATCTCCATCAAC

CCCGGCGCCCCGGCCGGCGGCTCCTTCAACCAGTACTTCGTCGGCAGCTTCAACG

GCACCCACTTCGAGGCCTTCGACAACCAGTCCCGCGTGGTGGACTTCGGCAAGG

ACTACTACGCCCTGCAGACCTTCTTCAACACCGACCCGACCTACGGGAGCGCCCT

GGGCATCGCGTGGGCCTCCAACTGGGAGTACTCCGCCTTCGTGCCCACCAACCCC

TGGCGCTCCTCCATGTCCCTCGTGCGCAAGTTCTCCCTCAACACCGAGTACCAGG

CCAACCCGGAGACGGAGCTGATCAACCTGAAGGCCGAGCCGATCCTGAACATCA

GCAACGCCGGCCCCTGGAGCCGGTTCGCCACCAACACCACGTTGACGAAGGCCA

ACAGCTACAACGTCGACCTGTCCAACAGCACCGGCACCCTGGAGTTCGAGCTGG

TGTACGCCGTCAACACCACCCAGACGATCTCCAAGTCCGTGTTCGCGGACCTCTC

CCTCTGGTTCAAGGGCCTGGAGGACCCCGAGGAGTACCTCCGCATGGGCTTCGA

GGTGTCCGCGTCCTCCTTCTTCCTGGACCGCGGGAACAGCAAGGTGAAGTTCGTG

AAGGAGAACCCCTACTTCACCAACCGCATGAGCGTGAACAACCAGCCCTTCAAG

AGCGAGAACGACCTGTCCTACTACAAGGTGTACGGCTTGCTGGACCAGAACATC

CTGGAGCTGTACTTCAACGACGGCGACGTCGTGTCCACCAACACCTACTTCATGA

CCACCGGGAACGCCCTGGGCTCCGTGAACATGACGACGGGGGTGGACAACCTGT

TCTACATCGACAAGTTCCAGGTGCGCGAGGTCAAGTGATTAATTAACTCGAGGCA

GCAGCAGCTCGGATAGTATCGACACACTCTGGACGCTGGTCGTGTGATGGACTGT

TGCCGCCACACTTGCTGCCTTGACCTGTGAATATCCCTGCCGCTTTTATCAAACAG

CCTCAGTGTGTTTGATCTTGTGTGTACGCGCTTTTGCGAGTTGCTAGCTGCTTGTG

CTATTTGCGAATACCACCCCCAGCATCCCCTTCCCTCGTTTCATATCGCTTGCATC

CCAACCGCAACTTATCTACGCTGTCCTGCTATCCCTCAGCGCTGCTCCTGCTCCTG

CTCACTGCCCCTCGCACAGCCTTGGTTTGGGCTCCGCCTGTATTCTCCTGGTACTG

CAACCTGTAAACCAGCACTGCAATGCTGATGCACGGGAAGTAGTGGGATGGGAA

CACAAATGGAAAGCTT

SEQ ID NO: 180

Homologous recombination construct SZ726

AGTACTGAAATGGTGAGAAGTCGTACTGAAATCAAGGATGAACAATGAAAATGG

TGCTGTGGTGGCTTCTCAAAGGTCAAGAATCAGTCGCTCGCGTCAGGAAATCGCG

GCGTCAACCAGCGTGGGCGCGGTCAGTGGCCCCGCACTGGTCACCATAGCCTCTC

CTGCCACAGTAGCGATCCCCTGGGCGTTCACTCTCAGCAGCGGCTGTACTGCCTC

CCAGATTTTCTTCTTCTGGACCTGCGGGCGTGAGAGGATGAGCAGGGTGGGGCCA

AGGGCTCAATCCTGAACGGCCCTCATTCGGTTTCCAATCCCACAACACATACCCA

CAGCAGGTCAGACCACGCATTCGCACCATGCGCACCAAATAACGTGTCCTTACCT

GATTGGGTGTGGCAGGCTCCGTGGACAGGAGTGCCTCGTCCCCCGCCCAGACCC

GCTCCCCCGTCACGGCGGCGTCCGGGACCCGCAGCGGCTCCACCGCGGTGTGATC CGCGTTGGCGGCGCAGAGCAGCATCCCAGCCGATTTGACCCCGCGCATGCTCCG

AGGCTTGAGGTTGGCCAGCACCACCACCCGCCGGCCGACAAGGTCCTCCAGGGT

CACGTGCCGGACCAGGCCACTCACGATGGTGCGAGGGCCCCCCTCCTCGCCGAG

GTCGATCTGCTCGACGTACAGACTGCGACATGCGTGGCGAGTGGTCATCAGAAG

GAAGCAGGTGTGCAGAAGGGGCACGTGGTTGGTATTGAGAGTAGCCAAAGCTTT

GTGCCAATCAGAAAGTCAACGCAGCTGCCTGCCTGGCTCGCGTACAATTCCTTTC

TTGCGCTATGACACTTCCAGCAAAAGGTAGGGCGGGCTGCGAGACGGCTTCCCG

GCGCTGCATGCAACACCGATGATGCTTCGACCCCCCGAAGCTCCTTCGGGGCTGC

ATGGGCGCTCCGATGCCGCTCCAGGGCGAGCGCTGTTTAAATAGCCAGGCCCCC

GATTGCAAAGACATTATAGCGAGCTACCAAAGCCATATTCAAACACCTAGATCA

CTACCACTTCTACACAGGCCACTCGAGCTTGTGATCGCACTCCGCTAAGGGGGCG

CCTCTTCCTCTTCGTTTCAGTCACAACCCGCAAACGGCGCGCCATGCTGCTGCAG

GCCTTCCTGTTCCTGCTGGCCGGCTTCGCCGCCAAGATCAGCGCCTCCATGACGA

ACGAGACGTCCGACCGCCCCCTGGTGCACTTCACCCCCAACAAGGGCTGGATGA

ACGACCCCAACGGCCTGTGGTACGACGAGAAGGACGCCAAGTGGCACCTGTACT

TCCAGTACAACCCGAACGACACCGTCTGGGGGACGCCCTTGTTCTGGGGCCACGC

CACGTCCGACGACCTGACCAACTGGGAGGACCAGCCCATCGCCATCGCCCCGAA

GCGCAACGACTCCGGCGCCTTCTCCGGCTCCATGGTGGTGGACTACAACAACACC

TCCGGCTTCTTCAACGACACCATCGACCCGCGCCAGCGCTGCGTGGCCATCTGGA

CCTACAACACCCCGGAGTCCGAGGAGCAGTACATCTCCTACAGCCTGGACGGCG

GCTACACCTTCACCGAGTACCAGAAGAACCCCGTGCTGGCCGCCAACTCCACCCA

GTTCCGCGACCCGAAGGTCTTCTGGTACGAGCCCTCCCAGAAGTGGATCATGACC

GCGGCCAAGTCCCAGGACTACAAGATCGAGATCTACTCCTCCGACGACCTGAAG

TCCTGGAAGCTGGAGTCCGCGTTCGCCAACGAGGGCTTCCTCGGCTACCAGTACG

AGTGCCCCGGCCTGATCGAGGTCCCCACCGAGCAGGACCCCAGCAAGTCCTACT

GGGTGATGTTCATCTCCATCAACCCCGGCGCCCCGGCCGGCGGCTCCTTCAACCA

GTACTTCGTCGGCAGCTTCAACGGCACCCACTTCGAGGCCTTCGACAACCAGTCC

CGCGTGGTGGACTTCGGCAAGGACTACTACGCCCTGCAGACCTTCTTCAACACCG

ACCCGACCTACGGGAGCGCCCTGGGCATCGCGTGGGCCTCCAACTGGGAGTACT

CCGCCTTCGTGCCCACCAACCCCTGGCGCTCCTCCATGTCCCTCGTGCGCAAGTTC

TCCCTCAACACCGAGTACCAGGCCAACCCGGAGACGGAGCTGATCAACCTGAAG

GCCGAGCCGATCCTGAACATCAGCAACGCCGGCCCCTGGAGCCGGTTCGCCACC

AACACCACGTTGACGAAGGCCAACAGCTACAACGTCGACCTGTCCAACAGCACC

GGCACCCTGGAGTTCGAGCTGGTGTACGCCGTCAACACCACCCAGACGATCTCCA

AGTCCGTGTTCGCGGACCTCTCCCTCTGGTTCAAGGGCCTGGAGGACCCCGAGGA

GTACCTCCGCATGGGCTTCGAGGTGTCCGCGTCCTCCTTCTTCCTGGACCGCGGG

AACAGCAAGGTGAAGTTCGTGAAGGAGAACCCCTACTTCACCAACCGCATGAGC

GTGAACAACCAGCCCTTCAAGAGCGAGAACGACCTGTCCTACTACAAGGTGTAC

GGCTTGCTGGACCAGAACATCCTGGAGCTGTACTTCAACGACGGCGACGTCGTGT

CCACCAACACCTACTTCATGACCACCGGGAACGCCCTGGGCTCCGTGAACATGAC

GACGGGGGTGGACAACCTGTTCTACATCGACAAGTTCCAGGTGCGCGAGGTCAA

GTGATTAATTAACTCGAGGCAGCAGCAGCTCGGATAGTATCGACACACTCTGGA

CGCTGGTCGTGTGATGGACTGTTGCCGCCACACTTGCTGCCTTGACCTGTGAATA

TCCCTGCCGCTTTTATCAAACAGCCTCAGTGTGTTTGATCTTGTGTGTACGCGCTT

TTGCGAGTTGCTAGCTGCTTGTGCTATTTGCGAATACCACCCCCAGCATCCCCTTC

CCTCGTTTCATATCGCTTGCATCCCAACCGCAACTTATCTACGCTGTCCTGCTATC

CCTCAGCGCTGCTCCTGCTCCTGCTCACTGCCCCTCGCACAGCCTTGGTTTGGGCT

CCGCCTGTATTCTCCTGGTACTGCAACCTGTAAACCAGCACTGCAATGCTGATGC

ACGGGAAGTAGTGGGATGGGAACACAAATGGAAAGCTTGAGCTCGGTACCCGTA

CCCATCAGCATCCGGGTGAATCTTGGCCTCCAAGATATGGCCAATCCTCACATCC AGCTTGGCAAAATCGACTAGACTGTCTGCAAGTGGGAATGTGGAGCACAAGGTT GCTTGTAGCGATCGACAGACTGGTGGGGTACATTGACAGGTGGGCAGCGCCGCA TCCATCGTGCCTGACGCGAGCGCCGCCGGTTGCTCGCCCGTGCCTGCCGTCAAAG AGCGGCAGAGAAATCGGGAACCGAAAACGTCACATTGCCTGATGTTGTTACATG CTGGACTAGACTTTCTTGGCGTGGGTCTGCTCCTCGCCAGGTGCGCGACGCCTCG GGGCTGGGTGCGAGGGAGCCGTGCGGCCACGCATTTGACAAGACCCAAAGCTCG CATCTCAGACGGTCAACCGTTCGTATTATACATTCAACATATGGTACATACGCAA AAAGCATG

SEQ ID NO: 181

Homologous recombination: Targeting cassette for disruption of Prototheca moriformis stearoyl ACP desaturase coding region (β-tubulin driven suc2 cassette)

TTTGGCCCCGCTTTCCAGCTCCGGATCTGCTGGCGTCCGCCGCGAGACGTGACAT CGCACGTCGCCGGGAGCGCCAGCTTGATCACTTGGCAGGGGGCCGTGCTCTACA AATACCAGGCCCCGCGGCGGTCAGTTCGCACATCCAATACCTGCCGAGCCATCTT

TGGAATTCGCCGAATTCAGCTCGATCAGTCGCTTCTGCAACTGATCTCGGCCGTT

CGCAGACTGCCTTTTCTCAGCTTGTCAGGTAGCGAGTTGTTGTTTTATATTTATTC

GATTTCATCTGTGTTGCATGTCTTGTTCGTGCTGTGCGTTCTTTCTGGGCCGCGCT

GTCGGGTCGCATGGGCTAGCTGTACTCATGTTAGTCATGCCGGTCCGACCTTGTT

CGAGGAAGGCCCCACACTGAGCGTGCCCTCTTTCTACACCCCTTGTGCAGAAATT

AGATAGAAAGCAGAATTCCTTTCTTGCGCTATGACACTTCCAGCAAAAGGTAGG

GCGGGCTGCGAGACGGCTTCCCGGCGCTGCATGCAACACCGATGATGCTTCGAC

CCCCCGAAGCTCCTTCGGGGCTGCATGGGCGCTCCGATGCCGCTCCAGGGCGAGC

GCTGTTTAAATAGCCAGGCCCCCGATTGCAAAGACATTATAGCGAGCTACCAAA

GCCATATTCAAACACCTAGATCACTACCACTTCTACACAGGCCACTCGAGCTTGT

GATCGCACTCCGCTAAGGGGGCGCCTCTTCCTCTTCGTTTCAGTCACAACCCGCA

AACGGCGCGCCATGCTGCTGCAGGCCTTCCTGTTCCTGCTGGCCGGCTTCGCCGC

CAAGATCAGCGCCTCCATGACGAACGAGACGTCCGACCGCCCCCTGGTGCACTTC

ACCCCCAACAAGGGCTGGATGAACGACCCCAACGGCCTGTGGTACGACGAGAAG

GACGCCAAGTGGCACCTGTACTTCCAGTACAACCCGAACGACACCGTCTGGGGG

ACGCCCTTGTTCTGGGGCCACGCCACGTCCGACGACCTGACCAACTGGGAGGAC

CAGCCCATCGCCATCGCCCCGAAGCGCAACGACTCCGGCGCCTTCTCCGGCTCCA

TGGTGGTGGACTACAACAACACCTCCGGCTTCTTCAACGACACCATCGACCCGCG

CCAGCGCTGCGTGGCCATCTGGACCTACAACACCCCGGAGTCCGAGGAGCAGTA

CATCTCCTACAGCCTGGACGGCGGCTACACCTTCACCGAGTACCAGAAGAACCCC

GTGCTGGCCGCCAACTCCACCCAGTTCCGCGACCCGAAGGTCTTCTGGTACGAGC

CCTCCCAGAAGTGGATCATGACCGCGGCCAAGTCCCAGGACTACAAGATCGAGA

TCTACTCCTCCGACGACCTGAAGTCCTGGAAGCTGGAGTCCGCGTTCGCCAACGA

GGGCTTCCTCGGCTACCAGTACGAGTGCCCCGGCCTGATCGAGGTCCCCACCGAG

CAGGACCCCAGCAAGTCCTACTGGGTGATGTTCATCTCCATCAACCCCGGCGCCC

CGGCCGGCGGCTCCTTCAACCAGTACTTCGTCGGCAGCTTCAACGGCACCCACTT

CGAGGCCTTCGACAACCAGTCCCGCGTGGTGGACTTCGGCAAGGACTACTACGC

CCTGCAGACCTTCTTCAACACCGACCCGACCTACGGGAGCGCCCTGGGCATCGCG

TGGGCCTCCAACTGGGAGTACTCCGCCTTCGTGCCCACCAACCCCTGGCGCTCCT

CCATGTCCCTCGTGCGCAAGTTCTCCCTCAACACCGAGTACCAGGCCAACCCGGA

GACGGAGCTGATCAACCTGAAGGCCGAGCCGATCCTGAACATCAGCAACGCCGG

CCCCTGGAGCCGGTTCGCCACCAACACCACGTTGACGAAGGCCAACAGCTACAA

CGTCGACCTGTCCAACAGCACCGGCACCCTGGAGTTCGAGCTGGTGTACGCCGTC AACACCACCCAGACGATCTCCAAGTCCGTGTTCGCGGACCTCTCCCTCTGGTTCA

AGGGCCTGGAGGACCCCGAGGAGTACCTCCGCATGGGCTTCGAGGTGTCCGCGT

CCTCCTTCTTCCTGGACCGCGGGAACAGCAAGGTGAAGTTCGTGAAGGAGAACC

CCTACTTCACCAACCGCATGAGCGTGAACAACCAGCCCTTCAAGAGCGAGAACG

ACCTGTCCTACTACAAGGTGTACGGCTTGCTGGACCAGAACATCCTGGAGCTGTA

CTTCAACGACGGCGACGTCGTGTCCACCAACACCTACTTCATGACCACCGGGAAC

GCCCTGGGCTCCGTGAACATGACGACGGGGGTGGACAACCTGTTCTACATCGAC

AAGTTCCAGGTGCGCGAGGTCAAGTGATTAATTAACTCGAGGCAGCAGCAGCTC

GGATAGTATCGACACACTCTGGACGCTGGTCGTGTGATGGACTGTTGCCGCCACA

CTTGCTGCCTTGACCTGTGAATATCCCTGCCGCTTTTATCAAACAGCCTCAGTGTG

TTTGATCTTGTGTGTACGCGCTTTTGCGAGTTGCTAGCTGCTTGTGCTATTTGCGA

ATACCACCCCCAGCATCCCCTTCCCTCGTTTCATATCGCTTGCATCCCAACCGCAA

CTTATCTACGCTGTCCTGCTATCCCTCAGCGCTGCTCCTGCTCCTGCTCACTGCCC

CTCGCACAGCCTTGGTTTGGGCTCCGCCTGTATTCTCCTGGTACTGCAACCTGTAA

ACCAGCACTGCAATGCTGATGCACGGGAAGTAGTGGGATGGGAACACAAATGGA

CCGACACGCCCCCGGCCCAGGTCCAGTTCTCCTGGGTCTTCCAGAGGCCCGTCGC

CATGTAAAGTGGCAGAGATTGGCGCCTGATTCGATTTGGATCCAAGGATCTCCAA

TCGGTGATGGGGACTGAGTGCCCAACTACCACCCTTGCACTATCGTCCTCGCACT

ATTTATTCCCACCTTCTGCTCGCCCTGCCGGGCGATTGCGGGCGTTTCTGCCCTTG

ACGTATCAATTTCGCCCCTGCTGGCGCGAGGATTCTTCATTCTAATAAGAACTCA

CTCCCGCCAGCTCTGTACTTTTCCTGCGGGGCCCCTGCATGGCTTGTTCCCAATGC

TTGCTCGATCGACGGCGCCCATTGCCCACGGCGCTGCCGCATCCATGTGAAGAAA

CACGGAAGAGTGCGAAGACTGGAAGTGAATTAAGAGTATAAGAAGAGGTACCA

CTGCTCGTGTCGCCACGGTGGTCAAGCCGCCCCATCTGCGATCCACCAGGCCCAT

CCGCGGACTCGCGATCAGCCTGCTGGATCCGGACTGCCGACCTGACCGCTCGCAT

CCACCATTACAACCCTCCAATTGGACACCACTCCCACGTCCTAAAGTTCACCATG

CAAGCTGATCGATCGCATTCGCCGATGCACTCGCCTGCCACAGAGGTGTGCGCTT

CGGACTAGCGTGCAGGCGCCCCGAGGCCACCAGCATGCACCGATGGAAGCGGGC

ACGGCCGCTGCTCCAGGTCGCTGGCTCGCTCAGACCCATAGCAACCTCCGCTGCG

TCCCTAAATGTCACACAGAGCGTCTTTGATGGGTACGGATGGGAGAGAATCTGAT

TGGGCATTGCTGGTGCAGTGCAGGAAGATGGCAAGTGCACAGTCAGTCATGCTG

TACAAACTGGTGCCTCGTAGTATTGACTCGTATAGTGCATAGTATCATGCATGGT

CGTTACTTGCAA

SEQ ID NO: 182

Homologous recombination: Targeting cassette for disruption of Prototheca moriformis stearoyl ACP desaturase coding region (suc2 cassette alone)

TTTGGCCCCGCTTTCCAGCTCCGGATCTGCTGGCGTCCGCCGCGAGACGTGA

CATCGCACGTCGCCGGGAGCGCCAGCTTGATCACTTGGCAGGGGGCCGTGCT

CTACAAATACCAGGCCCCGCGGCGGTCAGTTCGCACATCCAATACCTGCCGA

GCCATCTTGCCTACACTTTTTATCGACTCCTCTACTCTGTTCGCGAGAGCGCTC

GGTCCAGGCTTGGAATTCGCCGAATTCAGCTCGATCAGTCGCTTCTGCAACT

GATCTCGGCCGTTCGCAGACTGCCTTTTCTCAGCTTGTCAGGTAGCGAGTTGT

TGTTTTATATTTATTCGATTTCATCTGTGTTGCATGTCTTGTTCGTGCTGTGCGT

TCTTTCTGGGCCGCGCTGTCGGGTCGCATGGGCTAGCTGTACTCATGTTAGTC

ATGCCGGTCCGACCTTGTTCGAGGAAGGCCCCACACTGAGCGTGCCCTCTTT CTACACCCCTTGTGCAGAAATTAGATAGAAAGCAATGCTGCTGCAGGCCTTC

CTGTTCCTGCTGGCCGGCTTCGCCGCCAAGATCAGCGCCTCCATGACGAACG

AGACGTCCGACCGCCCCCTGGTGCACTTCACCCCCAACAAGGGCTGGATGA

ACGACCCCAACGGCCTGTGGTACGACGAGAAGGACGCCAAGTGGCACCTGT

ACTTCCAGTACAACCCGAACGACACCGTCTGGGGGACGCCCTTGTTCTGGGG

CCACGCCACGTCCGACGACCTGACCAACTGGGAGGACCAGCCCATCGCCAT

CGCCCCGAAGCGCAACGACTCCGGCGCCTTCTCCGGCTCCATGGTGGTGGAC

TACAACAACACCTCCGGCTTCTTCAACGACACCATCGACCCGCGCCAGCGCT

GCGTGGCCATCTGGACCTACAACACCCCGGAGTCCGAGGAGCAGTACATCT

CCTACAGCCTGGACGGCGGCTACACCTTCACCGAGTACCAGAAGAACCCCG

TGCTGGCCGCCAACTCCACCCAGTTCCGCGACCCGAAGGTCTTCTGGTACGA

GCCCTCCCAGAAGTGGATCATGACCGCGGCCAAGTCCCAGGACTACAAGAT

CGAGATCTACTCCTCCGACGACCTGAAGTCCTGGAAGCTGGAGTCCGCGTTC

GCCAACGAGGGCTTCCTCGGCTACCAGTACGAGTGCCCCGGCCTGATCGAG

GTCCCCACCGAGCAGGACCCCAGCAAGTCCTACTGGGTGATGTTCATCTCCA

TCAACCCCGGCGCCCCGGCCGGCGGCTCCTTCAACCAGTACTTCGTCGGCAG

CTTCAACGGCACCCACTTCGAGGCCTTCGACAACCAGTCCCGCGTGGTGGAC

TTCGGCAAGGACTACTACGCCCTGCAGACCTTCTTCAACACCGACCCGACCT

ACGGGAGCGCCCTGGGCATCGCGTGGGCCTCCAACTGGGAGTACTCCGCCTT

CGTGCCCACCAACCCCTGGCGCTCCTCCATGTCCCTCGTGCGCAAGTTCTCCC

TCAACACCGAGTACCAGGCCAACCCGGAGACGGAGCTGATCAACCTGAAGG

CCGAGCCGATCCTGAACATCAGCAACGCCGGCCCCTGGAGCCGGTTCGCCA

CCAACACCACGTTGACGAAGGCCAACAGCTACAACGTCGACCTGTCCAACA

GCACCGGCACCCTGGAGTTCGAGCTGGTGTACGCCGTCAACACCACCCAGA

CGATCTCCAAGTCCGTGTTCGCGGACCTCTCCCTCTGGTTCAAGGGCCTGGAG

GACCCCGAGGAGTACCTCCGCATGGGCTTCGAGGTGTCCGCGTCCTCCTTCTT

CCTGGACCGCGGGAACAGCAAGGTGAAGTTCGTGAAGGAGAACCCCTACTT

CACCAACCGCATGAGCGTGAACAACCAGCCCTTCAAGAGCGAGAACGACCT

GTCCTACTACAAGGTGTACGGCTTGCTGGACCAGAACATCCTGGAGCTGTAC

TTCAACGACGGCGACGTCGTGTCCACCAACACCTACTTCATGACCACCGGGA

ACGCCCTGGGCTCCGTGAACATGACGACGGGGGTGGACAACCTGTTCTACAT

CGACAAGTTCCAGGTGCGCGAGGTCAAGTGACCGACACGCCCCCGGCCCAG

GTCCAGTTCTCCTGGGTCTTCCAGAGGCCCGTCGCCATGTAAAGTGGCAGAG

ATTGGCGCCTGATTCGATTTGGATCCAAGGATCTCCAATCGGTGATGGGGAC

TGAGTGCCCAACTACCACCCTTGCACTATCGTCCTCGCACTATTTATTCCCAC

CTTCTGCTCGCCCTGCCGGGCGATTGCGGGCGTTTCTGCCCTTGACGTATCAA

TTTCGCCCCTGCTGGCGCGAGGATTCTTCATTCTAATAAGAACTCACTCCCGC

CAGCTCTGTACTTTTCCTGCGGGGCCCCTGCATGGCTTGTTCCCAATGCTTGCT

CGATCGACGGCGCCCATTGCCCACGGCGCTGCCGCATCCATGTGAAGAAAC

ACGGAAGAGTGCGAAGACTGGAAGTGAATTAAGAGTATAAGAAGAGGTAC

CAAGGGATTCTCAGGTGCTCTTAGGAACGGCTTTTCCTTCGCCAAGAGAAAC

TGCTACTGCTCGTGTCGCCACGGTGGTCAAGCCGCCCCATCTGCGATCCACC

AGGCCCATCCGCGGACTCGCGATCAGCCTGCTGGATCCGGACTGCCGACCTG ACCGCTCGCATCCACCATTACAACCCTCCAATTGGACACCACTCCCACGTCC

TAAAGTTCACCATGCAAGCTGATCGATCGCATTCGCCGATGCACTCGCCTGC

CACAGAGGTGTGCGCTTCGGACTAGCGTGCAGGCGCCCCGAGGCCACCAGC

ATGCACCGATGGAAGCGGGCACGGCCGCTGCTCCAGGTCGCTGGCTCGCTC

AGACCCATAGCAACCTCCGCTGCGTCCCTAAATGTCACACAGAGCGTCTTTG

ATGGGTACGGATGGGAGAGAATCTGATTGGGCATTGCTGGTGCAGTGCAGG

AAGATGGCAAGTGCACAGTCAGTCATGCTGTACAAACTGGTGCCTCGTAGTA

TTGACTCGTATAGTGCATAGTATCATGCATGGTCGTTACTTGCAA

SEQ ID NO: 183

Cuphea wrightii acyl-ACP thioesterase Genbank ACCESSION U56103

VERSION U56103.1 GM 336005

MVAAAASSAFFSVPTPGTSPKPGKFGNWPSSLSVPFKPDNGGFH

VKANASAHPKANGSAVNLKSGSLETPPRSFINQLPDLSMLLSKITTVFGAAEKQWKR P GMLVEPFGVDRIFQDGVFFRQSFSIRSYEIGVDRTASIETLMNIFQETSLNHCKSIGL LNDGFGRTPEMCKRDLI VVTKIQVEVNRYPTWGDTIEVNTWVSESGKNGMGRDWLIS DCRTGEILIRATSVWAMMNQNTRRLSKFPYEVRQEIAPHFVDSAPVIEDDRKLHKLDV KTGDSIRDGLTPRWNDLDVNQHVNNVKYIGWILKSVPIEVFETQELCGVTLEYRRECG RDSVLESVTTMDPAKEGDRCVYQHLLRLEDGADITIGRTEWRPKNAGANGAISSGKTS NGNSVS

SEQ ID NO: 184

Cuphea wrightii acyl-ACP thioesterase Genbank ACCESSION U56104

VERSION U56104.1 Gl:1336007

MWAAAASSAFFPVPAPRPTPKPGKFGNWPSSLSQPFKPKSNPN

GRFQVKANVSPHPKANGSAVSLKSGSLNTLEDPPSSPPPRTFLNQLPD SRLRTAITT

VFVAAEKQFTRLDRKSKRPDMLVD FGSETIVQDGLVFRERFSIRSYEIGADRTASIE

TLMNHLQDTSLNHCKSVGLLNDGFGRTPEMCTRDLIWVLTKMQIWNRYPT GDTVEI

NSWFSQSGKIGMGREWLISDCNTGEILVRATSAWAMMNQKTRRFSKLPCEVRQEIAP H

FVDAPPVIEDNDRKLHKFDVKTGDSICKGLTPG NDFDVNQHVSNVKYIGWILESMPT

EVLETQELCSLTLEYRRECGRESWESVTSMNPSKVGDRSQYQHLLRLEDGADIMKGR

TEWRPKNAGTNRAIST

SEQ ID NO: 185

Cuphea wrightii beta-ketoacyl-ACP synthase II Genbank ACCESSION No. U67317

VERSION U67317.1

MAAAASMVASPFCTWLVASCMSTSFDNDPRSPSVKRFPRRKRVL

SQRGSTFQCLVASCIDPCDQYRSSASLSFLGDNGFASLFGSKPFMSNRGHRRLRRAS H

SGEAMAVALQPAQEAGTKKKPVIKQRRVWTGMGVVTPLGHEPDVFYNNLLDGVSGIS

EIETFDCTQFPTRIAGEIKSFSTDGWVAPKLSKRMDKFMLYLLTAGKKALADGGITD E

VMKELDKRKCGVLIGSGMGGMKVFNDAIEALRVSYKKMNPFCVPFATTNMGSAMLAM D

LGWMGPNYSISTACATSNFCILNAANHIIRGEADMMLCGGSDAVIIPIGLGGFVACR A

LSQRNSDPTKASRPWDSNRDGFVMGEGAGVLLLEELEHAKKRGATIYAEFLGGSFTC D

AYHMTEPHPEGAGVILCIEKALAQAGVSKEDVNYINAHATSTSAGDIKEYQALARCF G

QNSELRVNSTKSMIGHLLGAAGGVEAVTWQAIRTGWIHPNLNLEDPDKAVDAKLLVG

PKKERLNVKVGLSNSFGFGGHNSSILFAPCNV SEQ ID NO: 186

Cuphea hookeriana 3-ketoacyl-ACP synthase (Kas4)

Genbank ACCESSION AF060519 VERSION AF060519.2 Gl:30995500

MATASCMVASPFCTWLVAACMPTSSDNDPRSLSHKRLRLSRRRR

TLSSHCSLRGSTFQCLDPCNQQRFLGDNGFASLFGSKPLRSNRGHLRLGRTSHSGEV M

AVAMQPAQEVSTNKKPATKQRRVWTGMGWTPLGHDPDVYYNNLLDGISGISEIENF

DCSQFPTRIAGEIKSFSTDGWVAPKFSERMDKFMLYMLTAGKKALADGGITEDAMKE L

NKRKCGVLIGSGLGGMKVFSDSIEALRTSYKKISPFCVPFSTTNMGSAILAMDLGWM G

PNYSISTACATSNFCILNAANHI IKGEADMMLCGGSDAAVLPVGLGGFVACRALSQRN

NDPTKASRPWDSNRDGFVMGEGAGVLLLEELEHAKKRGATIYAEFLGGSFTCDAYHM T

EPHPEGAGVILCIEKALAQSGVSREDVNYINAHATSTPAGDIKEYQALAHCFGQNSE L

RVNSTKSMIGHLLGGAGGVEAVAWQAIRTGWIHPNINLEDPDEGVDAKLLVGPKKEK

LKVKVGLSNSFGFGGHNSSILFAPCN

SEQ ID NO: 187

oleoyl-acyl carrier protein thioesterase [Carthamus tinctorius] (safflower).

Genbank accession no. AAA33019 VERSION AAA33019.1 Gl:404026

MLSKGAPAAP AVAAMYNASA KDTTFALTHS RSIGSVSIRR RYNVFLCNSS SSSRKVSPLL AVATGEQPSG VASLREADKE KSLGNRLRLG SLTEDGLSYK EKFVIRCYEV GINKTATIET IANLLQEVGG NHAQGVGFST DGFATTTTMR KLHLIWVTAR MHIEIYRYPA WSDVIEIETW VQGEGKVGTR RDWILKDYAN GEVIGRATSK WVMMNEDTRR LQKVSDDVRE EYLVFCPRTL RLAFPEENNN SMKKIPKLED PAEYSRLGLV PRRSDLDMNK HVNNVTYIGW ALESIPPEII DTHELQAITL DYRRECQRDD IVDSLTSREP LGNAAGVKFK EINGSVSPKK DEQDLSRFMH LLRSAGSGLE INRCRTEWRK KPAKR

SEQ ID NO: 188

Arabidopsis thaliana (thale cress) Genbank accession no: NP_566464 VERSION NP_566464.1

MTKEVCSNIG LWLLLTLLIG NYWNLEASH HVYKRLTQST NTKSPSVNQP YRTGFHFQPP

KNWMNDPNGP MIYKGIYHLF YQWNPKGAVW GNIVWAHSTS TDLINWDPHP PAIFPSAPFD

INGCWSGSAT ILPNGKPVIL YTGIDPKNQQ VQNIAEPKNL SDPYLREWKK SPLNPLMAPD

AVNGINASSF RDPTTAWLGQ DKKWRVIIGS KIHRRGLAIT YTSKDFLKWE KSPEPLHYDD

GSGMWECPDF FPVTRFGSNG VETSSFGEPN EILKHVLKIS LDDTKHDYYT IGTYDRVKDK

FVPDNGFKMD GTAPRYDYGK YYASKTFFDS AKNRRILWGW TNESSSVEDD VEKGWSGIQT

IPRKIWLDRS GKQLIQWPVR EVERLRTKQV KNLRNKVLKS GSRLEVYGVT AAQADVEVLF

KVRDLEKADV IEPSWTDPQL ICSKMNVSVK SGLGPFGLMV LASKNLEEYT SVYFRIFKAR

QNSNKYWLM CSDQSRSSLK EDNDKTTYGA FVDINPHQPL SLRALIDHSV VESFGGKGRA

CITSRVYPKL AIGKSSHLFA FNYGYQSVDV LNLNAWSMNS AQIS

SEQ ID NO: 189

Codon optimized coding region of Cocos nucifera FatB3-B

1 ACTAGTATGG TGGCCTCCGT GGCCGCCTCC TCCTCCTTCT TCCCCGTGCC CTCCTCCTCC

61 TCCTCCGCCT CCGCCAAGGC CTCCCGCGGC ATCCCCGACG GCCTGGACGT GCGCGGCATC 121 GTGGCCAAGC CCGCCTCCTC CTCCGGCTGG ATGCAGGCCA AGGCCTCCGC CCGCGCCATC 181 CCCAAGATCG ACGACACCAA GGTGGGCCTG CGCACCGACG TGGAGGAGGA CGCCGCCTCC 241 ACCGCCCGCC GCACCTCCTA CGGGCGCGCC AACCAGCTGC CCGACTGGTC CATGCTGCTG 301 GCCGCCATCC GCACCATCTT CTCCGCCGCC GAGAAGCAGT GGACCCTGCT GGACTCCAAG 361 AAGCGCGGCG CCGACGCCGT GGCCGACGCC TCCGGCGTGG GCAAGATGGT GAAGAACGGC 421 CTGGTGTACC GCCAGAACTT CTCCATCCGC TCCTACGAGA TCGGCGTGGA CAAGCGCGCC 481 TCCGTGGAGG CCCTGATGAA CCACTTCCAG GAGACCTCCC TGAACCACTG CAAGTGCATC 541 GGCCTGATGC ACGGCGGCTT CGGCTGCACC CCCGAGATGA CCCGCCGCAA CCTGATCTGG 601 GTGGTGGCCA AGATGCTGGT GCACGTGGAG CGCTACCCCT GGTGGGGCGA CGTGGTGCAG 661 ATCAACACCT GGATCTCCTC CTCCGGCAAG AACGGCATGG GCCGCGACTG GCACGTGCAC 721 GACTGCCAGA CCGGCCTGCC CATCATGCGC GGCACCTCCG TGTGGGTGAT GATGGACAAG

781 CACACCCGCC GCCTGTCCAA GCTGCCCGAG GAGGTGCGCG CCGAGATCAC CCCCTTCTTC

841 TCCGAGCGCG ACGCCGTGCT GGACGACAAC GGCCGCAAGC TGCCCAAGTT CGACGACGAC

901 TCCGCCGCCC ACGTGCGCCG CGGCCTGACC CCCCGCTGGC ACGACTTCGA CGTGAACCAG

961 CACGTGAACA ACGTGAAGTA CGTGGGCTGG ATTCTGGAGT CCGTGCCCGT GTGGATGCTG

1021 GACGGCTACG AGGTGGCCAC CATGTCCCTG GAGTACCGCC GCGAGTGCCG CATGGACTCC

1081 GTGGTGCAGT CCCTGACCGC CGTGTCCTCC GACCACGCCG ACGGCTCCCC CATCGTGTGC

1141 CAGCACCTGC TGCGCCTGGA GGACGGCACC GAGATCGTGC GCGGCCAGAC CGAGTGGCGC

1201 CCCAAGCAGC AGGCCCGCGA CCTGGGCAAC ATGGGCCTGC ACCCCACCGA GTCCAAGTGA

1261 TTAATTAACT CGAG

SEQ ID NO: 190

Aspergillus awamori, preproglucoamylase G1 , Genbank accession no. AAB59296

VERSION AAB59296.1

MSFRSLLALS GLVCTGLANV ISKRATLDSW LSNEATVART AILNNIGADG AWVSGADSGI WASPSTDNP DYFYTWTRDS GLVLKTLVDL FRNGDTSLLS TIENYISAQA IVQGISNPSG DLSSGAGLGE PKFNVDETAY TGSWGRPQRD GPALRATA I GFGQWLLDNG YTSTATDIVW PLVRNDLSYV AQYWNQTGYD LWEEVNGSSF FTI VQHRAL VEGSAFATAV GSSCSWCDSQ APEILCYLQS FWTGSFILAN FDSSRSGKDA NTLLGSIHTF DPEAACDDST FQPCSPRALA NHKEWDSFR SIYTLNDGLS DSEAVAVGRY PEDTYYNGNP WFLCTLAAAE QLYDALYQWD KQGSLEVTDV SLDFFKALYS DAATGTYSSS SSTYSSIVDA VKTFADGFVS IVETHAASNG SMSEQYDKSD GEQLSARDLT WSYAALLTAN NRRNSWPAS WGETSASSVP GTCAATSAIG TYSSVTVTSW PSIVATGGTT TTATPTGSGS VTSTSKTTAT ASKTSTSTSS TSCTTPTAVA VTFDLTATTT YGENIYLVGS ISQLGDWETS DGIALSADKY TSSDPLWYVT VTLPAGESFE YKFIRIESDD SVEWESDPNR EYTVPQACGT STATVTDTWR

SEQ ID NO: 191

Rhizopus oryzae (Rhizopus delemar) Glucoamylase 1 ;

1 ,4-alpha-D-glucan glucohydrolase, Genbank accession no. P07683

VERSION P07683.2

MQLFNLPLKV SFFLVLSYFS LLVSAASIPS SASVQLDSYN YDGSTFSGKI YVKNIAYSKK VTVIYADGSD NWNNNGNTIA ASYSAPISGS NYEYWTFSAS INGIKEFYIK YEVSGKTYYD NNNSANYQVS TSKPTTTTAT ATTTTAPSTS TTTPPSRSEP ATFPTGNSTI SSWIKKQEGI SRFAMLRNIN PPGSATGFIA ASLSTAGPDY YYAWTRDAAL TSNVIVYEYN TTLSGNKTIL NVLKDYVTFS VKTQSTSTVC NCLGEPKFNP DASGYTGAWG RPQNDGPAER ATTFILFADS YLTQTKDASY VTGTLKPAIF KDLDYWNVW SNGCFDLWEE VNGVHFYTLM VMRKGLLLGA DFAKRNGDST RASTYSSTAS TIANKISSFW VSSNNWIQVS QSVTGGVSKK GLDVSTLLAA NLGSVDDGFF TPGSEKILAT AVAVEDSFAS LYPINKNLPS YLGNSIGRYP EDTYNGNGNS QGNSWFLAVT GYAELYYRAI KEWIGNGGVT VSSISLPFFK KFDSSATSGK KYTVGTSDFN NLAQNIALAA DRFLSTVQLH AHNNGSLAEE FDRTTGLSTG ARDLTWSHAS LITASYAKAG APAA

SEQ ID NO: 192

Chlamydomonas reinhardtii hydroxymethylpyrimidine phosphate synthase, Genbank accession no. XP_001697756, VERSION XP_001697756.1

MQLFARSPAG VRVQQHAQRK SAKVAGRGRL NVRAEAIA P PAPAEQEKEL QRLEKGNAFE ELKAMATSPR QSVNRPQKAE DLTFRQAPTI ADCFPDSEKM YKEVKYDDEI TLRVPFRRIH LTTGTDFDVY DTSGPQNVDP RVGLPKLRKP WVERREANGT HARAGVTQMA LARAGIISEE MRFAAEREGL DPEFVRSELA RGRAIIPANK CHLELEPCVI GRNF TKVNS NFGNSAVTSS IEEEVEKLQW STIWGADTV DLSTGHNIFE TREWVMRNSP VPVGTVPIYE ALERADGQVE GITWELFRQV LLDQAEQGVD YWTIHAGVLL RHVPLTANRI TGIVSRGGSI HAKLCLMEHK ENFAYEHWDE ILDICAKYDI TLSIGDGLRP GCIADANDAA QFAELKTQGE LTRRAWEKNV QVMNEGPGHV PLNKIPENMA KQLEWCSEAP FYTLGPLTTD IAPGYDHITS AIGAATIGAL GTALLCYVTP KEHLGLPNRD DVKAGVIAYK IAAHAADLAK GHPAAASWDL ELSKARFEFR WRDQFALSLD PVTAQAYHDA TLPQEPAKTA HFCSMCGPKF CSMNITQELR QMVQAEQAQE AAAAAAAADD ADLLAAAAEG MKEMSEKFKQ SGAQLYH

SEQ ID NO: 193

thiamine biosynthesis protein ThiC [Salmonella enterica subsp. enterica serovar Typhimurium str. LT2], Genbank accession no. NP_463033, VERSION NP_463033.1

MSTTTLTRRE QRAKAQHFID TLEGTAFPNS KRIYVTGSQH DIRVPMREIQ LSPTLIGGSK DNPQFEENEA VPVYDTSGPY GDPEVAINVQ QGLAKLRQPW IDARNDSEEL DDRSSAYTRE RLADDGLDDL RFTGLLTPKR AKAGKRVTQL HYARKGIVTP EMEFIAIREN MGRERIRSEV LRHQHPGMNF GARLPENITP EFVRDEVAAG RAI IPANINH PESEPMIIGR NFLVKV A I GNSAVTSSIE EEVEKLVWST RWGADTVMDL STGRYIHETR EWILRNSPVP IGTVPIYQAL EKVNGIAEDL TWEAFRDTLL EQAEQGVDYF TIHAGVLLRY VPMTAKRLTG IVSRGGSIMA KWCLSHHKEN FLFEHFREIC EICAAYDVSL SLGDGLRPGS IQDANDEAQF SELHTLGELT KIAWEYDVQV MIEGPGHVP HMIQRNMTEE LESCHEAPFY TLGPLTTDIA PGYDHFTSGI GAAMIGWFGC AMLCYVTPKE HLGLPNKEDV KQGLITYKIA AHAADLAKGH PGAQIRDNAM SKARFEFRWE DQFNLALDPF TARAYHDETL PQESGKVAHF CSMCGPKFCS MKISQEVRDY AAAQTIEIGM AD SEDFRAK GGEIYLKREE A

SEQ ID NO: 194

aminoglycoside-3'-0-phosphotransferase [Cloning vector pTAP6].Genbank accession no. ADJ58594, VERSION ADJ58594.1

MIEQDGLHAG SPAAWVERLF GYDWAQQTIG CSDAAVFRLS AQGRPVLFVK TDLSGALNEL QDEAARLSWL ATTGVPCAAV LDWTEAGRD WLLLGEVPGQ DLLSSHLAPA EKVSIMADAM RRLHTLDPAT CPFDHQAKHR IERARTRMEA GLVDQDDLDE EHQGLAPAEL FARLKARMPD GEDLWTHGD ACLPNIMVEN GRFSGFIDCG RLGVADRYQD IALATRDIAE ELGGEWADRF LVLYGIAAPD SQRIAFYRLL DEFF

SEQ ID NO: 195

acyl ACP thioesterase [Brassica napus (rape)].Genbank accession no. CAA52070,

VERSION CAA52070.1

MLKLSCNVTN NLHTFSFFSD SSLFIPVNRR TIAVSSSQLR KPALDPLRAV ISADQGSISP VNSCTPADRL RAGRLMEDGY SYKEKFIVRS YEVGINKTAT VETIANLLQE VACNHVQKCG FSTDGFATTL TMRKLHLIWV TARMHIEIYK YPAWSDWEI ETWCQSEGRI GTRRDWILRD SATNEVIGRA TSKWVMMNQD TRRLQRVTDE VRDEYLVFCP REPRLAFPEE NNSSLKKIPK LEDPAQYSML ELKPRRADLD MNQHVNNVTY IGWVLESIPQ EI IDTHELQV ITLDYRRECQ QDDIVDSLTT SEIPDDPISK FTGTNGSAMS SIQGHNESQF LHMLRLSENG QEINRGRTQW RKKSSR

SEQ ID NO: 196

acyl-ACP thioesterase [Garcinia mangostana], Genbank accession no. AAB51525

VERSION AAB51525.1

MVATAATSSF FPLTSPSGDA KSGNPGKGSV SFGSMKSKSA ASSRGLQVKA NAQAPTKING STDDAQLPAP RTFINQLPDW SMLLAAITTV FLAAEKQWMM LDWKPRRPDM LIDTFGLGRI VQDGLVFRQN FSIRSYEIGA DRTASIETVM NHLQETALNH VKTAGLLGDG FGSTPEMSKR NLIWWTKMQ VEVDRYPTWG DWQVDTWVS ASGKNGMRRD WLLRDGNTGE TLTRASSVWV MMNKLTRRLS KIPEEVREEI GSYFVNSDPV VEEDGRKVTK LDDNTADFVR KGLTPKWNDL DINQHVNNVK YIGWILESAP QPILETRELS AVTLEYRREC GRDSVLRSLT AVSGGGVGDL GHAGNVECQH VLRLEDGAEI VRGRTEWRPK YINNFSIMGQ IPTDAS

SEQ ID NO: 197 Cuphea hookeriana 8:0- and 10:0-ACP specific thioesterase (FatB2) mRNA, complete cds., Genbank accession no. U39834, VERSION U39834.1

MVAAAASSAF FPVPAPGASP KPGKFGNWPS SLSPSFKPKS IPNGGFQVKA NDSAHPKANG SAVSLKSGSL NTQEDTSSSP PPRTFLHQLP DWSRLLTAIT TVFVKSKRPD MHDRKSKRPD MLVDSFGLES TVQDGLVFRQ SFSIRSYEIG TDRTASIETL MNHLQETSLN HCKSTGILLD GFGRTLEMCK RDLIWWIK QIKVNRYPAW GDTVEINTRF SRLGKIGMGR DWLISDCNTG EILVRATSAY AMMNQKTRRL SKLPYEVHQE IVPLFVDSPV IEDSDLKVHK FKVKTGDSIQ KGLTPGWNDL DVNQHVSNVK YIGWILESMP TEVLETQELC SLALEYRREC GRDSVLESVT AMDPSKVGVR SQYQHLLRLE DGTAIVNGAT EWRPKNAGAN GAISTGKTSN GNSVS

SEQ ID NO: 198

acyl-ACP thioesterase FatA [Ricinus communis] (castor bean) Genbank ACCESSION ABS30422 VERSION ABS30422.1

MLKVPCCNAT DPIQSLSSQC RFLTHFNNRP YFTRRPSIPT FFSSKNSSAS LQAWSDISS

VESAACDSLA NRLRLGKLTE DGFSYKEKFI VRSYEVGINK TATVETIANL LQEVGCNHAQ

SVGFSTDGFA TTTSMRKMHL IWVTAR HIE IYKYPAWSDV VEVETWCQSE GRIGTRRDWI

LTDYATGQII GRATSKWVM NQDTRRLQKV TDDVREEYLV FCPRELRLAF PEENNRSSKK

ISKLEDPAQY SKLGLVPRRA DLDMNQHVNN VTYIGWVLES IPQEIIDTHE LQTITLDYRR

ECQHDDIVDS LTSVEPSENL EAVSELRGTN GSATTTAGDE DCRNFLHLLR LSGDGLEINR GRTEWRKKSA R

SEQ ID NO: 199

stearoyl-acyl-carrier protein desaturase [Ricinus communis] Genbank ACCESSION AAA74692 VERSION AAA74692.1

FRQITKNQKK KVRKKTMALK LNPFLSQTQK LPSFALPPMA STRSPKFYMA STLKSGSKEV

ENLKKPFMPP REVHVQVTHS MPPQKIEIFK SLDNWAEENI LVHLKPVEKC WQPQDFLPDP

ASDGFDEQVR ELRERAKEIP DDYFWLVGD MITEEALPTY QTMLNTLDGV RDETGASPTS

WAIWTRAWTA EENRHGDLLN KYLYLSGRVD MRQIEKTIQY LIGSGMDPRT ENSPYLGFIY

TSFQERATFI SHGNTARQAK EHGDIKLAQI CGTIAADEKR HETAYTKIVE KLFEIDPDGT

VLAFAD MRK KISMPAHLMY DGRDDNLFDH FSAVAQRLGV YTAKDYADIL EFLVGRWKVD

KLTGLSAEGQ KAQDYVCRLP PRIRRLEERA QGRAKEAPTM PFSWIFDRQV KL

SEQ ID NO: 200

stearoyl-ACP desaturase [Olea europaea] Genbank ACCESSION AAB67840 VERSION

AAB67840.1

MALKLCFPPH KMPSFPDARI RSHRVF AST IHSPSMEVGK VKKPFTPPRE VHVQVTHSLA

PEKREIFNSL NNWAQE ILV LLKDVDKCWQ PSDFLPDSAS EGFDEQVMEL RKRCKEIPDD

YFIVLVGDMI TEEALPTYQT MLNTLDGVRD ETGASLTPWA IWTRAWTAEE NRHGDLLNKY

LYLSGRVDMK QIEKTIQYLI GSGMDPRTEN NPYLGFIYTS FQERATFISH GNTARLAKEH

GDLKLAQICG I IAADEKRHE TAYTKIVEKL FEIDPDGTVL ALADMMRKKV SMPAHLMYDG

QDDNLFENFS SVAQRLGVYT AKDYADILEF LVGRWDIEKL TGLSGEGRKA QDYVCTLPPR IRRLEERAQS RVKKASATPF SWIFGREINL

SEQ ID NO: 201

Myristoyl-acyl carrier protein thioesterase, chloroplastic; Cuphea hookeriana, Genbank ACCESSION Q39513 VERSION Q39513.1

MVATAASSAF FPLPSADTSS RPGKLGNKPS SLSPLKPKST PNGGLQVKAN ASAPPKINGS

PVGLKSGGLK TQEDAHSAPP PRTFINQLPD WSMLLAAITT VFLAAEKQWM MLDWKPKRPD

MLVDPFGLGS IVQDGLVFRQ NFSIRSYEIG ADRTASIETV MNHLQETALN HVKIAGLSND GFGRTPEMYK RDLIWWAK QVMVNRYPTW GDTVEVNTWV AKSGKNGMRR DWLISDCNTG

EILTRASSVW VMMNQKTRRL SKIPDEVRNE IEPHFVDSPP VIEDDDRKLP KLDEKTADSI

RKGLTPRWND LDVNQHVNNV KYIGWILEST PPEVLETQEL CSLTLEYRRE CGRESVLESL

TAMDPSGGGY GSQFQHLLRL EDGGEIVKGR TEWRPKNGVI NGWPTGESS PGDYS

SEQ ID NO: 202

thioesterase FatBM [Cuphea hookeriana], Genbank ACCESSION AAC72882 VERSION

AAC72882.1

MVATAASSAF FPVSSPVTSS RPGKPGNGSS SFSPIKPKFV ANGGLQVKAN ASAPPKINGS

SVGLKSCSLK TQEDTPSAPA PRTFINQLPD WS LLAAITT VFLAAEKQWM LDWKPKRPD

MLVDPFGLGS IVQHGLVFRQ NFSIRSYEIG ADRTASIETV MNHLQETALN HVKSAGLMND

GFGRTPEMYK KDLIWWAKM QVMVNRYPTW GDTVEVNTWV AKSGKNGMRR DWLISDCNTG

EILTRASSVW VMMNQKTRRL SKIPDEVRHE IEPHFVDSPP VIEDDDRKLP KLDDKTADSI

RKGLTPKWND LDVNQHVNNV KYIGWILEST PQEVLETQEL CSLTLEYRRE CGRESVLESL

TAADPSGKGF GSQFQHLLRL EDGGEIVKGR TEWRPKTAGI NGAIPSGETS PGDS

SEQ ID NO: 203

Uc FatB2 [Umbellularia californica], Genbank ACCESSION AAC49001 VERSION AAC49001.1

MVTTSLASAF FSMKAVMLAP DGSGIKPRSS GLQVRAGKEQ NSCKMINGTK VKDTEGLKGR

STLHGWSMPL ELITTIFSAA EKQWTNLVSK PPQLLDDHLG LHGLVFRRTF AIRCSEVGPD

RSTSIVAVMN YLQEAACNHA ESLGLLGDGF GETLEMSRRD LIWWRRTHV WERYPAWGD

TVEVEAWIGA AGNIGMRRHF LVRDCKTGHI LARCTSVSVM MNMRTRRLSK IPQEVRGEID

PLFIEKFAVK EGEIKKLQKF NDSTADYIQG GWTPRWNDLD VNQHVNNIKY VGWIFKSVPD

SIYENHHLSS ITLEYRRECT RGRALQSLTT VCGGSSEAGI ICEHLLQLED GSEVLRGRTD WRPKRTDSFE GISERFPQQE PHN

SEQ ID NO: 204

palmitoyl-ACP thioesterase [Elaeis guineensis], (African oil palm). Genbank ACCESSION

ABD83939 VERSION ABD83939.1

MVASIAASAF FPTPSFSPTA SAKASKTIGE GSENLDARGI IAKPTSSSAA MQGKVMAQAV SKINGAKVGL KAESQKAEED AAPSSAPRTF YNQLPDWSVL LAAVTTIFLA AEKQWTLLDW KPRRPDMLTD AFSLGKIVQD GLVFKQNFSI RSYEIGADRT ASIETLMNHL QETALNHVRS AGLMGDGFGA TPEMSKRNLI WWTKVRVLI EHYPSWGDW EVDTWVGPAG KNGMRRDWHV RDHRTGQTIL RATRVWVMMN KNTRKLSKVP EEVRAEIGPY FVERAAIVDE DSRKLPKLDE DTTDYVKKGL TPRWSDLDVN QHVNNVKYIG WILESAPISI LENHELASMT LEYRRECGRD SVLQSLTAVA NDCTGGLPEA SIECQHLLQL ECGAEIVRGR TQWRPRRASG PTSAGSA

SEQ ID NO: 205

palmitoyl-acyl carrier protein thioesterase [Elaeis guineensis] (African oil palm) Genbank ACCESSION AAD42220 VERSION AAD42220.2

MVASIVAWAF FPTPSFSPTA SAKASKTIGE GSENLNVRGI IAKPTSSSAA KQGKVMAQAV PKINGAKVGL KAESQKAEED AAPSSAPRTF YNQLPDWSVL LAAVTTIFLA AEKQWTLLDW KPRRPDMLTG AFSLGKIVQD GLVFRQNFSI RSYEIGADRT ASIETLMNHL QETALNHVRN AGLLGDGFGA TPEMSKRNLI WWTKMQVLI EHYPSWGDW EVDTWVGASG KNGMRRDWHV RDYRTGQTIL RATSIWVMMD KHTRKLSKMP EEVRAEIGPY FMEHAAIVDE DSRKLPKLDD DTADYIKWGL TPRWSDLDVN QHVNNVKYIG WILESAPISI LENHELASMT LEYRRECGRD SVLQSLTAVA NDCTGGLPEA SIECQHLLQL ECGAEIVRGR TQWRPRRASG PTSAGSA

SEQ ID NO: 206 palmitoyl-ACP thioesterase [Elaeis guineensis]. Genbank ACCESSION AAL15645 VERSION AAL 15645.1

FYNQLPDWSV LLAAVTTIFL AAEKQWTLLD WKPRRPDMLA DAFGLGKIVQ DGLVFKQNFS IRSYEIGADR TASIETLMNH LQETALNHVR SAGLMGDGFG ATPEMSKRNL IWWTKMRVL IKHYPSWGDV VEVDTWVGPT GKNGMRRDWH VRDHRTGQTI LRATSVWVMM NKNTRKLSKV PEEVRAELGP YFVERAAIVD EDSRKLPKLD EDTTDYIKKG LTPRWSDLDV NQHVNNVKYI GWILESAPIS FLENHELAS SLEYRRECGR DSVLQSLTAV SNDLTDGLPE AGIECQHLLQ LECGTELVKG RTEWRPKHSL ALRNMGPTPG GSA

SEQ ID NO: 207

FATB [Populus tomentosa]. Genbank ACCESSION ABC47311 VERSION ABC47311.1

MVATAATSSF FPVPSPPGDA KSSKVGSGSA SLGGIKSKSA SSGALQVKAN AQAPPKINGS PVGLTASVET AKKEDWSSP APRTFINQLP DWSMLLAAIT TMFLAAEKQW MMLDWKPKRA DMLIDPFGIG RIVQDGLVFS QNFSIRSYEI GADRTASIET LMNHLQETAL NHVKTAGLLG DGFGSTPEMS KRNLIWWTR MQILVDRYPT WGDWHVDTW VSASGKNGMR RDWLVRDAKT GETLTRASSL WVMMNKVTRR LSKIPEDVRG EIEPYFLNSD PWNEDSTKL PKLDDKTADY IRKGLTPRWN DLDVNQHVNN VKYIGWILES APPPILESHE LAAITLEYRR ECGRDSVLQS LTAVSGAGIG NLGGPGKVEC QHLLRHEDGA EIVRGRTEWR PKHANNFGMM GGQ PADESG A

SEQ ID NO: 208

FATB (fatty acyl-ACP thioesterases B); acyl carrier/ acyl-[acyl-carrier-protein] hydrolase [Arabidopsis thaliana]. Genbank ACCESSION NPJ72327 VERSION NP_172327.1

MVATSATSSF FPVPSSSLDP NGKGNKIGST NLAGLNSAPN SGRMKVKPNA QAPPKINGKK VGLPGSVDIV RTDTETSSHP APRTFINQLP DWSMLLAAIT TIFLAAEKQW MMLDWKPRRS DMLVDPFGIG RIVQDGLVFR QNFSIRSYEI GADRSASIET VMNHLQETAL NHVKTAGLLG DGFGSTPEMF KKNLIWWTR MQVWDKYPT WGDWEVDTW VSQSGKNGMR RDWLVRDCNT GETLTRASSV WVMMNKLTRR LSKIPEEVRG EIEPYFVNSD PVLAEDSRKL TKIDDKTADY VRSGLTPRWS DLDVNQHVNN VKYIGWILES APVGIMERQK LKSMTLEYRR ECGRDSVLQS LTAVTGCDIG NLATAGDVEC QHLLRLQDGA EWRGRTEWS SKTPTTTWGT AP

SEQ ID NO: 209

acyl-(acyl carrier protein) thioesterase [Arabidopsis thaliana]. Genbank ACCESSION CAA85387 VERSION CAA85387.1

MVATSATSSF FPVPSSSLDP NGKGNKIGST NLAGLNSAPN SGRMKVKPNA QAPPKINGKR VGLPGSVDIV RTDTETSSHP APRTFINQLP DWSMLLAAIT TIFLAAEKQW MMLDWKPRRS DMLVDPFGIG RIVQDGLVFR QNFSIRSYEI GADRSASIET VMNHLQETAL NHVKTAGLLG DGFGSTPEMF KKNLIWWTR MQVWDKYPT WGDWEVDTW VSQSGKNGMR RDWLVRDCNT GETLTRASSV WVMMNKLTRR LSKIPEEVRG EIEPYFVNSD PVLAEDSRKL TKIDDKTADY VRSGLTPRWS DLDVNQHVNN VKYIGWILES APVGIMERQK LKSMTLEYRR ECGRDSVLQS LTAVTGCDIG NLATAGDVEC QHLLRLQDGA EWRGRTEWS SKTPTTTWGT AP

SEQ ID NO: 210

acyl-(acyl carrier protein) thioesterase [Arabidopsis thaliana]. Genbank ACCESSION CAA85388 VERSION CAA85388.1

MVATSATSSF FPVPSSSLDP NGKGNKIGST NLAGLNSTPN SGRMKVKPNA QAPPKINGKR VGLPGSVDIV RTDTETSSHP APRTFINQLP DWSMLLAAIT TIFLAAEKQW MMLDWKPRRS DMLVDPFGIG RIVQDGLVFR QNFSIRSYEI GADRSASIET VMNHLQETAL NHVKTAGLLG DGFGSTPEMF KKNLIWWTR MQVWDKYPT WGDWEVDTW VSQSGKNGMR RDWLVRDCNT GETLTRASSV WVMMNKLTRR LSKIPEEVRG EIEPYFVNSD PVLAEDSRKL TKIDDKTADY VRSGLTPRWS DLDVNQHVNN VKYIGWILES APVGIMERQK LKSMTLEYRR ECGRDSVLQS LTAVTGCDIG NLATAGDVEC QHLLRLQDGA EWRGRTEWS SKTPTTTWGT AP

SEQ ID NO: 211

Gossypium hirsutum Genbank ACCESSION Q9SQI3

MVATAVTSAF FPVTSSPDSS DSKNKKLGSI KSKPSVSSGS LQVKANAQAP PKINGTVAST TPVEGSKNDD GASSPPPRTF INQLPDWSML LAAITTIFLA AEKQWMMLDW KPRRPDMVID PFGIGKIVQD GLVFSQNFSI RSYEIGADQT ASIETLMNHL QETAINHCRS AGLLGEGFGA TPEMCKK LI WWTRMQVW DRYPTWGDW QVDTWVSASG KNGMRRDWLV SNSETGEILT RATSVWVMMN KLTRRLSKIP EEVRGEIEPF FMNSDPVLAE DSQKLVKLDD STAEHVCKGL TPKWSDLDVN QHVNNVKYIG WILESAPLPI LESHELSALT LEYRRECGRD SVLQSLTTVS DSNTENAVNV GEFNCQHLLR LDDGAEIVRG RTRWRPKHAK SSANMDQITA KRA

SEQ ID NO: 212

acyl-ACP thioesterase [Cuphea lanceolata]. Genbank ACCESSION CAA54060 VERSION CAA54060.1

MVATAASSAF FPLPSPDTSS RPGKLGNGSS SLSPLKPKFV ANAGLKVKAS ASAPPKINGS SVGLKSGSLK TQEDTPSVPP PRTFINQLPD WSMLLAAITT VFLAAEKQWM MLDWKPKRPD MLVDPFGLGS IVQGGLVFRQ NFSIRSYEIG ADRTASIETV MNHLQETALN HVKSAGLLND GFGRTPEMFK RDLIWWAKM QVMVNRYPTW GDTVEVNTWV AKSGKNGMRR DWLISDCNTG EILTRASSVW VMMNQKTRKL SKIPDEVRHE IEPHFIDCAP VIEDDDRKLR KLDEKTADSI RKGLTPKWND LDVNQHVNNV KYIGWILEST PQEVLETQEL SSLTLEYRRE CGRESVLESL TAVDSSGKGF GSQFQHLLRL EDGGEIVKGR TEWRPKTAGV NGAIASGETS HGDS

SEQ ID NO: 213

acyl-acyl carrier protein thioesterase [Cuphea calophylla subsp. mesostemon], Genbank ACCESSION ABB71581 VERSION ABB71581.1

MVATAASSAF FPVPSPDTSS RPGKLGNGSS SLSPLKPKLM ANGGLQVKAN ASAPPKINGS

SVGLKSGSLK TQEDTPSAPP PRTFINQLPD WSMLLAAITT VFLAAEKQWM MLDWKPKRPD

MLVDPFGLGR IVQDGLVFRQ NFSIRSYEIG ADRTASIETV MNHLQETALN HVKSAGLLND

GFGRTPEMYK RDLIWWAKM QVMVNRYPTW GDTVEVNTWV AKSGKNGMRR DWLISDCNTG

EILTRASSVW VMMNQKTRRL SKIPDEVRHE IEPHFVDSAP VIEDDDRKLP KLDEKTADSI

RKGLTPKWND LDVNQHVNNV KYIGWILEST PPEVLETQEL CSLTLEYRRE CGRESVLESL

TAVDPSGKGS GSQFQHLLRL EDGGEIVKGR TEWRPKTAGI NGPIASGETS PGDS

SEQ ID NO: 214

palmitoyl-acyl carrier protein thioesterase [Gossypium hirsutum](upland cotton). Genbank ACCESSION AAD01982 VERSION AAD01982.1

TAVTSAFFPV TSSPDSSDSK NKKLGSIKSK PSVSSGSLQV KANAQAPPKI NGTVASTTPV

EGSKNDDGAS SPPPRTFINQ LPDWSMLLAA ITTIFLAAEK QWMMLDWKPR RPDMVIDPFG

IGKIVQDGLV FSQNFSIRSY EIGADQTASI ETLMNHLQET AINHCRSAGL LGEGFGATPE

MCKKNLIWW TRMQVWDRY PTWGDWQVD TWVSASGKNG MRRDWLVSNS ETGEILTRAT

SVWVMMNKLT RRLSKIPEEV RGEIEPFFMN SDPVLAEDSQ KLVKLDDSTA EHVCKGLTPK

WSDLDVNQHV NNVKYIGWIL ESAPLPILES HELSALTLEY RRECGRDSVL QSLTTVSDSN TENAVNVGEF NCQHLLRLDD GAEIVRGRTR WRPKHAKSSA NMDQITAKRA

SEQ ID NO: 215

hypothetical protein VITISV_008781 [Vitis vinifera]. Genbank ACCESSION CAN81819 VERSION CAN81819.1 MVATAATSAF FAVASPSSDP DAKPSTKPGX GSAILRGIKS RNAPSGSLQV KANAQAPPKI NGTTVGYTSS AEGVKIEDD SSPPPRTFIN QLPDWSMLLA AITTIFLAAE KQW MLDWKP RRSDMLIDPF GLGKIVQDGL VFRQNFSIRS YEIGADRTAS IETLMNHLQE TALNHVRTAG LLGDGFGSTP EMSIRNLIWV VTRMQVWDR YPTWGDWQV DTWVCASGKN GMRRDWI IRD CKTGETLTRA SSV VMMNKQ TRRLSKIPDA VRAEIEPYFM DSAPIVDEDG RKLPKLDDST ADYIRTGLTP RWSDLDVNQH VNNVKYIGWI LESAPLPILE SHELSSMTLE YRRECGRDSV LQSLTAVSGT GVGNLLDCGN VECQHLLRLE EGAEIVEGKD

SEQ ID NO: 216

palmitoyl-ACP thioesterase [Brassica juncea]. Genbank ACCESSION ABI18986 VERSION ABM 8986.1

MVATSATSLF FPLPSSSLDP NXKTNNRVTS TNFAGLGPTP NSGGRMKVKP NAQAPPKING

KKVGLPGSVE IETSQQQQPA PRTFINQLPD WSMLLAAITT VFLAAEKQWM MLDWKPRRSD

MIMEPFGLGR IVQDGLVFRQ NFSIRSYEIG ADRSASIETV MNHLQETALN XVKTAGLLGD

GFGSTPEMVK KXLIWWTR QVWDTYPTW GDWEVDTWV SKSGKNGMRR DWLVRDGNTG

QILTRASSVW VMMNKLTRRL SKIPEEVRGE IEPYFVDFDP VLAEDSRKLT KLDDKTADYV

RSGLTPRWSD LDVNQHVNNV KYIGWILESA PVGMMESQKL KSMTLEYRRE CGRDSVLQSL

TAVSGCDIGN LGTAGEVECQ HLLRLQDGAE WRGRTEWSS KTPTTTWDIT P

SEQ ID NO: 217

chloroplast stearoyl/oleoyi specific acyl-acyl carrier protein thioesterase precursor [Madhuca longifolia var. latifolia]. Genbank ACCESSION AAX51637 VERSION AAX51637.1

INQLPDWSML LAAITTIFLA AEKQWMMLDW KPKRPDMLID PFGLGKIVQD GLVFRQNFSI RSYEIGADRT ASIETLMNHL QETALNHVKT AGLLGDGFGV TPEMCKKNLI WWAKMQVLV DRYPTWGDW QVDTWVAASG KNGMRRDWLV RDFETGDILT KASSVWVMMN RETRRLSKIP DEVRLEIGSY FVDSPPVLDE DGRKLPKLDE STADHIRTGL TPRWNDLDVN QHVNNVKYIG WILESAPQPI LENHELASMT LEYRRECGKD SVLQSLTGVT SGGVGGLADS GHVECQHLLR LEGGAEIVKG RTEWRPKYAN RLGCLDQLPA GST

SEQ ID NO: 218

hypothetical protein Osl_08261 [Oryza sativa Indica Group]. Genbank ACCESSION EAY86877 VERSION EAY86877.1

MAGSLAASAF FPGPGSSPAA SARSSKSAAV TGELPENLSV RGIVAKPNPP PAAMQVKAQA QTLPKVNGTK VNLKTVKPDM EETVPYSAPK TFYNQLPDWS MLLAAITTIF LAAEKQWTLL DWKPKKPDML VDTFGFGRII QDGMVFRQNF MIRSYEIGAD RTASIETLMN HLQETALNHV RTAGLLGDGF GATPEMSKRN LIWWSKIQL LVEQYPAWGD TVQVDTWVAA AGKNGMRRDW HVRDYNSGRT ILRATSVWVM MHKKTRRLSK MPDEVRAEIG PYFNDRSAIT EEQSEKLAKT GNKVGDDATE QFIRKGLTPR WGDLDVNQHV NNVKYIGWIL ESAPISVLEK HELASMTLDY RKECGRDSVL QSLTTVSGEC TSIGADKQAS AIQCDHLLQL ESGADIVKAH TEWRPKRSHA AAENA

SEQ ID NO: 219

Os11g0659500 [Oryza sativa Japonica Group]. Genbank ACCESSION NP_001068400 VERSION NP_001068400.1

MAGSLAASAF FPGPGSSPAA SARSSKNAAV TGELPENLSV RGIVAKPNPP PAAMQVKAQA QTLPKVNGTK VNLKTVKPDM EETVPYSAPK TFYNQLPDWS MLLAAITTIF LAAEKQWTLL DWKPKKPDML VDTFGFGRII QDGMVFRQNF MIRSYEIGAD RTASIETLMN HLQETALNHV RTAGLLGDGF GATPEMSKRN LIWWSKIQL LVEQYPAWGD TVQVDTWVAA AGKNGMRRDW HVRDYNSGRT ILRATSVWVM MHKKTRRLSK MPDEVRAEIG PYFNDRSAIT EEQSEKLAKT GNKVGDDATE QFIRKGLTPR WGDLDVNQHV NNVKYIGWIL ESAPISVLEK HELASMTLDY RKECGRDSVL QSLTTVSGEC TSIGADKQAS AIQCDHLLQL ESGADIVKAH TEWRPKRSHA AAENA

SEQ ID NO: 220

hypothetical protein Osl_21596 [Oryza sativa Indica Group]. Genbank ACCESSION EAY99617 VERSION EAY99617.1

MAGSLAASAF FPVPGSSPAA SARSSKNTTG ELPENLSVRG IVAKPNPSPG AMQVKAQAQA

LPKVNGTKVN LKTTSPDKED IIPYTAPKTF YNQLPDWSML LAAVTTIFLA AEKQWTLLDW

KPKKPDMLAD TFGFGRIIQD GLVFRQNFLI RSYEIGADRT ASIETLMNHL QETALNHVKT

AGLLGDGFGA TPEMSKRNLI WWSKIQLLV ERYPSWGDMV QVDTWVAAAG KNGMRRDWHV

RDYNSGQTIL RATSVWVMMN KNTRRLSKMP DEVRAEIGPY FNGRSAISEE QGEKLPKPGT

TFDGAATKQF TRKGLTPKWS DLDVNQHVNN VKYIGWILES APISILEKHE LASMTLDYRK

ECGRDSVLQS LTTVSGECDD GNTESSIQCD HLLQLESGAD IVKAHTEWRP KRAQGEGNMG FFPAESA

SEQ ID NO: 221

acyl-(ACP) thioesterase type B [Cuphea lanceolata]. Genbank ACCESSION CAB60830 VERSION CAB60830.1

MVAAAATSAF FPVPAPGTSP KPGKSGNWPS SLSPTFKPKS IPNAGFQVKA NASAHPKANG SAVNLKSGSL NTQEDTSSSP PPRAFLNQLP DWSMLLTAIT TVFVAAEKQW TMLDRKSKRP DMLVDSVGLK SIVRDGLVSR QSFLIRSYEI GADRTASIET LMNHLQETSI NHCKSLGLLN DGFGRTPGMC KNDLIWVLTK MQIMVNRYPT WGDTVEINTW FSQSGKIGMA SDWLISDCNT GEILIRATSV WAMMNQKTRR FSRLPYEVRQ ELTPHFVDSP HVIEDNDQKL HKFDVKTGDS IRKGLTPRWN DLDVNQHVSN VKYIGWILES MPIEVLETQE LCSLTVEYRR ECGMDSVLES VTAVDPSENG GRSQYKHLLR LEDGTDIVKS RTEWRPKNAG TNGAISTSTA KTSNGNSAS

SEQ ID NO: 222

acyl-ACP thioesterase [Myristica fragrans]. Genbank ACCESSION AAB71730 VERSION AAB71730.1

PDWSMLLAAI TTIFLAAEKQ WTNLDWKPRR PDMLVDFDPF SLGRFVQDGL IFRQNFSIRS YEIGADRTAS IETLMNHLQE TALNHVRCIG LLDDGFGSTP EMTRRDLIWV VTRMQVLVDR YPSWGDVIEV DSWVTPSGKN GMKREWFLRD CKTGEILTRA TSVWVMMNKR TRRLSKIPEE VRVEIEPYFV EHGVLDEDSR KLPKLNDNTA NYIRRGLAPR WSDLDVNQHV NNVKYIGWIL ESVPSSLLES HELYGMTLEY RKECGKDGLL QSLTAVASDY GGGSLEAGVE CDHLLRLEDG SEIMRGKTEW RPKRAANTTY FGSVDDIPPH PIYIYIYIYI YIYIYWVGSS CSGSSTTMSR TR

SEQ ID NO: 223

Cinnamomum camphora acyl-ACP thioesterase. Genbank ACCESSION U31813 VERSION U31813.1

MATTSLASAFCSMKAVMLARDGRGMKPRSSDLQLRAGNAQTSLK

MINGTKFSYTESLKKLPDWSMLFAVITTIFSAAEKQWTNLEWKPKPNPPQLLDDHFG P HGLVFRRTFAIRSYEVGPDRSTSIVAVMNHLQEAALNHAKSVGILGDGFGTTLEMSKR DLIWWKRTHVAVERYPAWGDTVEVECWVGASGNNGRRHDFLVRDCKTGEILTRCTSL SVMMNTRTRRLSKIPEEVRGEIGPAFIDNVAVKDEEIKKPQKLNDSTADYIQGGLTPR WNDLDINQHVNNIKYVDWILETVPDSIFESHHISSFTIEYRRECTMDSVLQSLTTVSG GSSEAGLVCEHLLQLEGGSEVLRAKTEWRPKLTDSFRGISVIPAESSV

SEQ ID NO: 224

acyl-ACP thioesterase [Myristica fragrans]. Genbank ACCESSION AAB71729 VERSION AAB71729.1

MVATSAASAF FPVASPSPVK PSMMLGGGGG SDNLDARGIK SRPASSGGLQ VKANAHTVPK INGNKAGLLT PMESTKDEDI VAAPTVAPKR TFINQLPDWS MLLAAITTIF LAAEKQWTNL DWKPRRPDML VDFDPFSLGR FVQDGLIFRQ NFSIRSYEIG ADRTASIETL MNHLQETALN HVRCIGLLDD GFGSTPEMTR RDLIWWTRM QVLVDRYPSW GDVIEVDSWV TPSGKNGMKR EWFLRDCKTG EILTRATSVW VMMNKRTRRL SKIPEEVRVE IEPYFVEHGV LDEDSRKLPK LNDNTANYIR RGLAPRWSDL DVNQHVNNVK YIGWILESVP SSLLESHELY GMTLEYRKEC GKDGLLQSLT AVASDYGGGS LEAGVECDHL LRLEDGSEIM RGKTEWRPKR AANTTYFGSV DDIPPANNA

SEQ ID NO: 225

pSZ1420

1 CACTGGCAGC AGCCACTGGT AACAGGATTA GCAGAGCGAG GTATGTAGGC GGTGCTACAG

61 AGTTCTTGAA GTGGTGGCCT AACTACGGCT ACACTAGAAG GACAGTATTT GGTATCTGCG

121 CTCTGCTGAA GCCAGTTACC TTCGGAAAAA GAGTTGGTAG CTCTTGATCC GGCAAACAAA

181 CCACCGCTGG TAGCGGTGGT TTTTTTGTTT GCAAGCAGCA GATTACGCGC AGAAAAAAAG

241 GATCTCAAGA AGATCCTTTG ATCTTTTCTA CGGGGTCTGA CGCTCAGTGG AACGAAAACT

301 CACGTTAAGG GATTTTGGTC ATGAGATTAT CAAAAAGGAT CTTCACCTAG ATCCTTTTAA

361 ATTAAAAATG AAGTTTTAAA TCAATCTAAA GTATATATGA GTAAACTTGG TCTGACAGTT

421 ACCAATGCTT AATCAGTGAG GCACCTATCT CAGCGATCTG TCTATTTCGT TCATCCATAG

481 TTGCCTGACT CCCCGTCGTG TAGATAACTA CGATACGGGA GGGCTTACCA TCTGGCCCCA

541 GTGCTGCAAT GATACCGCGA GACCCACGCT CACCGGCTCC AGATTTATCA GCAATAAACC

601 AGCCAGCCGG AAGGGCCGAG CGCAGAAGTG GTCCTGCAAC TTTATCCGCC TCCATCCAGT

661 CTATTAATTG TTGCCGGGAA GCTAGAGTAA GTAGTTCGCC AGTTAATAGT TTGCGCAACG

721 TTGTTGCCAT TGCTACAGGC ATCGTGGTGT CACGCTCGTC GTTTGGTATG GCTTCATTCA

781 GCTCCGGTTC CCAACGATCA AGGCGAGTTA CATGATCCCC CATGTTGTGC AAAAAAGCGG

841 TTAGCTCCTT CGGTCCTCCG ATCGTTGTCA GAAGTAAGTT GGCCGCAGTG TTATCACTCA

901 TGGTTATGGC AGCACTGCAT AATTCTCTTA CTGTCATGCC ATCCGTAAGA TGCTTTTCTG

961 TGACTGGTGA GTACTCAACC AAGTCATTCT GAGAATAGTG TATGCGGCGA CCGAGTTGCT

1021 CTTGCCCGGC GTCAATACGG GATAATACCG CGCCACATAG CAGAACTTTA AAAGTGCTCA

1081 TCATTGGAAA ACGTTCTTCG GGGCGAAAAC TCTCAAGGAT CTTACCGCTG TTGAGATCCA

1141 GTTCGATGTA ACCCACTCGT GCACCCAACT GATCTTCAGC ATCTTTTACT TTCACCAGCG

1201 TTTCTGGGTG AGCAAAAACA GGAAGGCAAA ATGCCGCAAA AAAGGGAATA AGGGCGACAC

1261 GGAAATGTTG AATACTCATA CTCTTCCTTT TTCAATATTA TTGAAGCATT TATCAGGGTT

1321 ATTGTCTCAT GAGCGGATAC ATATTTGAAT GTATTTAGAA AAATAAACAA ATAGGGGTTC

1381 CGCGCACATT TCCCCGAAAA GTGCCACCTG ACGTCTAAGA AACCATTATT ATCATGACAT

1441 TAACCTATAA AAATAGGCGT ATCACGAGGC CCTTTCGTCT CGCGCGTTTC GGTGATGACG

1501 GTGAAAACCT CTGACACATG CAGCTCCCGG AGACGGTCAC AGCTTGTCTG TAAGCGGATG

1561 CCGGGAGCAG ACAAGCCCGT CAGGGCGCGT CAGCGGGTGT TGGCGGGTGT CGGGGCTGGC

1621 TTAACTATGC GGCATCAGAG CAGATTGTAC TGAGAGTGCA CCATATGCGG TGTGAAATAC

1681 CGCACAGATG CGTAAGGAGA AAATACCGCA TCAGGCGCCA TTCGCCATTC AGGCTGCGCA

1741 ACTGTTGGGA AGGGCGATCG GTGCGGGCCT CTTCGCTATT ACGCCAGCTG GCGAAAGGGG

1801 GATGTGCTGC AAGGCGATTA AGTTGGGTAA CGCCAGGGTT TTCCCAGTCA CGACGTTGTA

1861 AAACGACGGC CAGTGAATTG ATGCATGCTC TTCGCCGCCG CCACTCCTGC TCGAGCGCGC

1921 CCGCGCGTGC GCCGCCAGCG CCTTGGCCTT TTCGCCGCGC TCGTGCGCGT CGCTGATGTC

1981 CATCACCAGG TCCATGAGGT CTGCCTTGCG CCGGCTGAGC CACTGCTTCG TCCGGGCGGC

2041 CAAGAGGAGC ATGAGGGAGG ACTCCTGGTC CAGGGTCCTG ACGTGGTCGC GGCTCTGGGA

2101 GCGGGCCAGC ATCATCTGGC TCTGCCGCAC CGAGGCCGCC TCCAACTGGT CCTCCAGCAG

2161 CCGCAGTCGC CGCCGACCCT GGCAGAGGAA GACAGGTGAG GGGGGTATGA ATTGTACAGA

2221 ACAACCACGA GCCTTGTCTA GGCAGAATCC CTACCAGTCA TGGCTTTACC TGGATGACGG

2281 CCTGCGAACA GCTGTCCAGC GACCCTCGCT GCCGCCGCTT CTCCCGCACG CTTCTTTCCA

2341 GCACCGTGAT GGCGCGAGCC AGCGCCGCAC GCTGGCGCTG CGCTTCGCCG ATCTGAGGAC

2401 AGTCGGGGAA CTCTGATCAG TCTAAACCCC CTTGCGCGTT AGTGTTGCCA TCCTTTGCAG

2461 ACCGGTGAGA GCCGACTTGT TGTGCGCCAC CCCCCACACC ACCTCCTCCC AGACCAATTC

2521 TGTCACCTTT TTGGCGAAGG CATCGGCCTC GGCCTGCAGA GAGGACAGCA GTGCCCAGCC

2581 GCTGGGGGTT GGCGGATGCA CGCTCAGGTA CCCTTTCTTG CGCTATGACA CTTCCAGCAA

2641 AAGGTAGGGC GGGCTGCGAG ACGGCTTCCC GGCGCTGCAT GCAACACCGA TGATGCTTCG

2701 ACCCCCCGAA GCTCCTTCGG GGCTGCATGG GCGCTCCGAT GCCGCTCCAG GGCGAGCGCT 2761 GTTTAAATAG CCAGGCCCCC GATTGCAAAG ACATTATAGC GAGCTACCAA AGCCATATTC

2821 AAACACCTAG ATCACTACCA CTTCTACACA GGCCACTCGA GCTTGTGATC GCACTCCGCT

2881 AAGGGGGCGC CTCTTCCTCT TCGTTTCAGT CACAACCCGC AAACTCTAGA ATATCAATGC

2941 TGCTGCAGGC CTTCCTGTTC CTGCTGGCCG GCTTCGCCGC CAAGATCAGC GCCTCCATGA

3001 CGAACGAGAC GTCCGACCGC CCCCTGGTGC ACTTCACCCC CAACAAGGGC TGGATGAACG 3061 ACCCCAACGG CCTGTGGTAC GACGAGAAGG ACGCCAAGTG GCACCTGTAC TTCCAGTACA 3121 ACCCGAACGA CACCGTCTGG GGGACGCCCT TGTTCTGGGG CCACGCCACG TCCGACGACC 3181 TGACCAACTG GGAGGACCAG CCCATCGCCA TCGCCCCGAA GCGCAACGAC TCCGGCGCCT 3241 TCTCCGGCTC CATGGTGGTG GACTACAACA ACACCTCCGG CTTCTTCAAC GACACCATCG 3301 ACCCGCGCCA GCGCTGCGTG GCCATCTGGA CCTACAACAC CCCGGAGTCC GAGGAGCAGT 3361 ACATCTCCTA CAGCCTGGAC GGCGGCTACA CCTTCACCGA GTACCAGAAG AACCCCGTGC 3421 TGGCCGCCAA CTCCACCCAG TTCCGCGACC CGAAGGTCTT CTGGTACGAG CCCTCCCAGA 3481 AGTGGATCAT GACCGCGGCC AAGTCCCAGG ACTACAAGAT CGAGATCTAC TCCTCCGACG 3541 ACCTGAAGTC CTGGAAGCTG GAGTCCGCGT TCGCCAACGA GGGCTTCCTC GGCTACCAGT 3601 ACGAGTGCCC CGGCCTGATC GAGGTCCCCA CCGAGCAGGA CCCCAGCAAG TCCTACTGGG 3661 TGATGTTCAT CTCCATCAAC CCCGGCGCCC CGGCCGGCGG CTCCTTCAAC CAGTACTTCG 3721 TCGGCAGCTT CAACGGCACC CACTTCGAGG CCTTCGACAA CCAGTCCCGC GTGGTGGACT 3781 TCGGCAAGGA CTACTACGCC CTGCAGACCT TCTTCAACAC CGACCCGACC TACGGGAGCG 3841 CCCTGGGCAT CGCGTGGGCC TCCAACTGGG AGTACTCCGC CTTCGTGCCC ACCAACCCCT 3901 GGCGCTCCTC CATGTCCCTC GTGCGCAAGT TCTCCCTCAA CACCGAGTAC CAGGCCAACC

3961 CGGAGACGGA GCTGATCAAC CTGAAGGCCG AGCCGATCCT GAACATCAGC AACGCCGGCC

4021 CCTGGAGCCG GTTCGCCACC AACACCACGT TGACGAAGGC CAACAGCTAC AACGTCGACC 4081 TGTCCAACAG CACCGGCACC CTGGAGTTCG AGCTGGTGTA CGCCGTCAAC ACCACCCAGA 4141 CGATCTCCAA GTCCGTGTTC GCGGACCTCT CCCTCTGGTT CAAGGGCCTG GAGGACCCCG 4201 AGGAGTACCT CCGCATGGGC TTCGAGGTGT CCGCGTCCTC CTTCTTCCTG GACCGCGGGA 4261 ACAGCAAGGT GAAGTTCGTG AAGGAGAACC CCTACTTCAC CAACCGCATG AGCGTGAACA 4321 ACCAGCCCTT CAAGAGCGAG AACGACCTGT CCTACTACAA GGTGTACGGC TTGCTGGACC 4381 AGAACATCCT GGAGCTGTAC TTCAACGACG GCGACGTCGT GTCCACCAAC ACCTACTTCA 4441 TGACCACCGG GAACGCCCTG GGCTCCGTGA ACATGACGAC GGGGGTGGAC AACCTGTTCT 4501 ACATCGACAA GTTCCAGGTG CGCGAGGTCA AGTGACAATT GGCAGCAGCA GCTCGGATAG 4561 TATCGACACA CTCTGGACGC TGGTCGTGTG ATGGACTGTT GCCGCCACAC TTGCTGCCTT 4621 GACCTGTGAA TATCCCTGCC GCTTTTATCA AACAGCCTCA GTGTGTTTGA TCTTGTGTGT 4681 ACGCGCTTTT GCGAGTTGCT AGCTGCTTGT GCTATTTGCG AATACCACCC CCAGCATCCC 4741 CTTCCCTCGT TTCATATCGC TTGCATCCCA ACCGCAACTT ATCTACGCTG TCCTGCTATC 4801 CCTCAGCGCT GCTCCTGCTC CTGCTCACTG CCCCTCGCAC AGCCTTGGTT TGGGCTCCGC 4861 CTGTATTCTC CTGGTACTGC AACCTGTAAA CCAGCACTGC AATGCTGATG CACGGGAAGT 4921 AGTGGGATGG GAACACAAAT GGAGGATCCC GCGTCTCGAA CAGAGCGCGC AGAGGAACGC

4981 TGAAGGTCTC GCCTCTGTCG CACCTCAGCG CGGCATACAC CACAATAACC ACCTGACGAA 5041 TGCGCTTGGT TCTTCGTCCA TTAGCGAAGC GTCCGGTTCA CACACGTGCC ACGTTGGCGA 5101 GGTGGCAGGT GACAATGATC GGTGGAGCTG ATGGTCGAAA CGTTCACAGC CTAGGGATAT 5161 CGAATTCCTT TCTTGCGCTA TGACACTTCC AGCAAAAGGT AGGGCGGGCT GCGAGACGGC 5221 TTCCCGGCGC TGCATGCAAC ACCGATGATG CTTCGACCCC CCGAAGCTCC TTCGGGGCTG 5281 CATGGGCGCT CCGATGCCGC TCCAGGGCGA GCGCTGTTTA AATAGCCAGG CCCCCGATTG 5341 CAAAGACATT ATAGCGAGCT ACCAAAGCCA TATTCAAACA CCTAGATCAC TACCACTTCT 5401 ACACAGGCCA CTCGAGCTTG TGATCGCACT CCGCTAAGGG GGCGCCTCTT CCTCTTCGTT 5461 TCAGTCACAA CCCGCAAACA CTAGTATGGC CACCGCATCC ACTTTCTCGG CGTTCAATGC 5521 CCGCTGCGGC GACCTGCGTC GCTCGGCGGG CTCCGGGCCC CGGCGCCCAG CGAGGCCCCT 5581 CCCCGTGCGC GGGCGCGCCA CCCAGGAGGA CGCCCACTCC GCCCCCCCCC CCCGCACCTT 5641 CATCAACCAG CTGCCCGACT GGTCCATGCT GCTGGCCGCC ATCACCACCG TGTTCCTGGC

5701 CGCCGAGAAG CAGTGGATGA TGCTGGACTG GAAGCCCAAG CGCCCCGACA TGCTGGTGGA

5761 CCCCTTCGGC CTGGGCTCCA TCGTGCAGGA CGGCCTGGTG TTCCGCCAGA ACTTCTCCAT 5821 CCGCTCCTAC GAGATCGGCG CCGACCGCAC CGCCTCCATC GAGACCGTGA TGAACCACCT 5881 GCAGGAGACC GCCCTGAACC ACGTGAAGAT CGCCGGCCTG TCCAACGACG GCTTCGGCCG 5941 CACCCCCGAG ATGTACAAGC GCGACCTGAT CTGGGTGGTG GCCAAGATGC AGGTGATGGT

6001 GAACCGCTAC CCCACCTGGG GCGACACCGT GGAGGTGAAC ACCTGGGTGG CCAAGTCCGG 6061 CAAGAACGGC ATGCGCCGCG ACTGGCTGAT CTCCGACTGC AACACCGGCG AGATCCTGAC 6121 CCGCGCCTCC TCCGTGTGGG TGATGATGAA CCAGAAGACC CGCCGCCTGT CCAAGATCCC 6181 CGACGAGGTG CGCAACGAGA TCGAGCCCCA CTTCGTGGAC TCCCCCCCCG TGATCGAGGA 6241 CGACGACCGC AAGCTGCCCA AGCTGGACGA GAAGACCGCC GACTCCATCC GCAAGGGCCT 6301 GACCCCCCGC TGGAACGACC TGGACGTGAA CCAGCACGTG AACAACGTGA AGTACATCGG 6361 CTGGATCCTG GAGTCCACCC CCCCCGAGGT GCTGGAGACC CAGGAGCTGT GCTCCCTGAC 6421 CCTGGAGTAC CGCCGCGAGT GCGGCCGCGA GTCCGTGCTG GAGTCCCTGA CCGCCATGGA

6481 CCCCTCCGGC GGCGGCTACG GCTCCCAGTT CCAGCACCTG CTGCGCCTGG AGGACGGCGG

6541 CGAGATCGTG AAGGGCCGCA CCGAGTGGCG CCCCAAGAAC GGCGTGATCA ACGGCGTGGT

6601 GCCCACCGGC GAGTCCTCCC CCGGCGACTA CTCCATGGAC TACAAGGACC ACGACGGCGA

6661 CTACAAGGAC CACGACATCG ACTACAAGGA CGACGACGAC AAGTGAATCG ATAGATCTCT

6721 TAAGGCAGCA GCAGCTCGGA TAGTATCGAC ACACTCTGGA CGCTGGTCGT GTGATGGACT

6781 GTTGCCGCCA CACTTGCTGC CTTGACCTGT GAATATCCCT GCCGCTTTTA TCAAACAGCC

6841 TCAGTGTGTT TGATCTTGTG TGTACGCGCT TTTGCGAGTT GCTAGCTGCT TGTGCTATTT

6901 GCGAATACCA CCCCCAGCAT CCCCTTCCCT CGTTTCATAT CGCTTGCATC CCAACCGCAA

6961 CTTATCTACG CTGTCCTGCT ATCCCTCAGC GCTGCTCCTG CTCCTGCTCA CTGCCCCTCG

7021 CACAGCCTTG GTTTGGGCTC CGCCTGTATT CTCCTGGTAC TGCAACCTGT AAACCAGCAC

7081 TGCAATGCTG ATGCACGGGA AGTAGTGGGA TGGGAACACA AATGGAAAGC TTAATTAAGA

7141 GCTCTTGTTT TCCAGAAGGA GTTGCTCCTT GAGCCTTTCA TTCTCAGCCT CGATAACCTC

7201 CAAAGCCGCT CTAATTGTGG AGGGGGTTCG AATTTAAAAG CTTGGAATGT TGGTTCGTGC

7261 GTCTGGAACA AGCCCAGACT TGTTGCTCAC TGGGAAAAGG ACCATCAGCT CCAAAAAACT

7321 TGCCGCTCAA ACCGCGTACC TCTGCTTTCG CGCAATCTGC CCTGTTGAAA TCGCCACCAC

7381 ATTCATATTG TGACGCTTGA GCAGTCTGTA ATTGCCTCAG AATGTGGAAT CATCTGCCCC

7441 CTGTGCGAGC CCATGCCAGG CATGTCGCGG GCGAGGACAC CCGCCACTCG TACAGCAGAC

7501 CATTATGCTA CCTCACAATA GTTCATAACA GTGACCATAT TTCTCGAAGC TCCCCAACGA

7561 GCACCTCCAT GCTCTGAGTG GCCACCCCCC GGCCCTGGTG CTTGCGGAGG GCAGGTCAAC

7621 CGGCATGGGG CTACCGAAAT CCCCGACCGG ATCCCACCAC CCCCGCGATG GGAAGAATCT

7681 CTCCCCGGGA TGTGGGCCCA CCACCAGCAC AACCTGCTGG CCCAGGCGAG CGTCAAACCA

7741 TACCACACAA ATATCCTTGG CATCGGCCCT GAATTCCTTC TGCCGCTCTG CTACCCGGTG

7801 CTTCTGTCCG AAGCAGGGGT TGCTAGGGAT CGCTCCGAGT CCGCAAACCC TTGTCGCGTG

7861 GCGGGGCTTG TTCGAGCTTG AAGAGCCTCT AGAGTCGACC TGCAGGCATG CAAGCTTGGC

7921 GTAATCATGG TCATAGCTGT TTCCTGTGTG AAATTGTTAT CCGCTCACAA TTCCACACAA

7981 CATACGAGCC GGAAGCATAA AGTGTAAAGC CTGGGGTGCC TAATGAGTGA GCTAACTCAC

8041 ATTAATTGCG TTGCGCTCAC TGCCCGCTTT CCAGTCGGGA AACCTGTCGT GCCAGCTGCA

8101 TTAATGAATC GGCCAACGCG CGGGGAGAGG CGGTTTGCGT ATTGGGCGCT CTTCCGCTTC

8161 CTCGCTCACT GACTCGCTGC GCTCGGTCGT TCGGCTGCGG CGAGCGGTAT CAGCTCACTC

8221 AAAGGCGGTA ATACGGTTAT CCACAGAATC AGGGGATAAC GCAGGAAAGA ACATGTGAGC

8281 AAAAGGCCAG CAAAAGGCCA GGAACCGTAA AAAGGCCGCG TTGCTGGCGT TTTTCCATAG

8341 GCTCCGCCCC CCTGACGAGC ATCACAAAAA TCGACGCTCA AGTCAGAGGT GGCGAAACCC

8401 GACAGGACTA TAAAGATACC AGGCGTTTCC CCCTGGAAGC TCCCTCGTGC GCTCTCCTGT

8461 TCCGACCCTG CCGCTTACCG GATACCTGTC CGCCTTTCTC CCTTCGGGAA GCGTGGCGCT

8521 TTCTCATAGC TCACGCTGTA GGTATCTCAG TTCGGTGTAG GTCGTTCGCT CCAAGCTGGG

8581 CTGTGTGCAC GAACCCCCCG TTCAGCCCGA CCGCTGCGCC TTATCCGGTA ACTATCGTCT

8641 TGAGTCCAAC CCGGTAAGAC ACGACTTATC GC

SEQ ID NO: 226

pSZ1417

1 CACTGGCAGC AGCCACTGGT AACAGGATTA GCAGAGCGAG GTATGTAGGC GGTGCTACAG

61 AGTTCTTGAA GTGGTGGCCT AACTACGGCT ACACTAGAAG GACAGTATTT GGTATCTGCG

121 CTCTGCTGAA GCCAGTTACC TTCGGAAAAA GAGTTGGTAG CTCTTGATCC GGCAAACAAA

181 CCACCGCTGG TAGCGGTGGT TTTTTTGTTT GCAAGCAGCA GATTACGCGC AGAAAAAAAG

241 GATCTCAAGA AGATCCTTTG ATCTTTTCTA CGGGGTCTGA CGCTCAGTGG AACGAAAACT

301 CACGTTAAGG GATTTTGGTC ATGAGATTAT CAAAAAGGAT CTTCACCTAG ATCCTTTTAA

361 ATTAAAAATG AAGTTTTAAA TCAATCTAAA GTATATATGA GTAAACTTGG TCTGACAGTT

421 ACCAATGCTT AATCAGTGAG GCACCTATCT CAGCGATCTG TCTATTTCGT TCATCCATAG

481 TTGCCTGACT CCCCGTCGTG TAGATAACTA CGATACGGGA GGGCTTACCA TCTGGCCCCA

541 GTGCTGCAAT GATACCGCGA GACCCACGCT CACCGGCTCC AGATTTATCA GCAATAAACC

601 AGCCAGCCGG AAGGGCCGAG CGCAGAAGTG GTCCTGCAAC TTTATCCGCC TCCATCCAGT

661 CTATTAATTG TTGCCGGGAA GCTAGAGTAA GTAGTTCGCC AGTTAATAGT TTGCGCAACG

721 TTGTTGCCAT TGCTACAGGC ATCGTGGTGT CACGCTCGTC GTTTGGTATG GCTTCATTCA

781 GCTCCGGTTC CCAACGATCA AGGCGAGTTA CATGATCCCC CATGTTGTGC AAAAAAGCGG

841 TTAGCTCCTT CGGTCCTCCG ATCGTTGTCA GAAGTAAGTT GGCCGCAGTG TTATCACTCA

901 TGGTTATGGC AGCACTGCAT AATTCTCTTA CTGTCATGCC ATCCGTAAGA TGCTTTTCTG

961 TGACTGGTGA GTACTCAACC AAGTCATTCT GAGAATAGTG TATGCGGCGA CCGAGTTGCT

1021 CTTGCCCGGC GTCAATACGG GATAATACCG CGCCACATAG CAGAACTTTA AAAGTGCTCA 1081 TCATTGGAAA ACGTTCTTCG GGGCGAAAAC TCTCAAGGAT CTTACCGCTG TTGAGATCCA

1141 GTTCGATGTA ACCCACTCGT GCACCCAACT GATCTTCAGC ATCTTTTACT TTCACCAGCG

1201 TTTCTGGGTG AGCAAAAACA GGAAGGCAAA ATGCCGCAAA AAAGGGAATA AGGGCGACAC

1261 GGAAATGTTG AATACTCATA CTCTTCCTTT TTCAATATTA TTGAAGCATT TATCAGGGTT

1321 ATTGTCTCAT GAGCGGATAC ATATTTGAAT GTATTTAGAA AAATAAACAA ATAGGGGTTC

1381 CGCGCACATT TCCCCGAAAA GTGCCACCTG ACGTCTAAGA AACCATTATT ATCATGACAT

1441 TAACCTATAA AAATAGGCGT ATCACGAGGC CCTTTCGTCT CGCGCGTTTC GGTGATGACG

1501 GTGAAAACCT CTGACACATG CAGCTCCCGG AGACGGTCAC AGCTTGTCTG TAAGCGGATG

1561 CCGGGAGCAG ACAAGCCCGT CAGGGCGCGT CAGCGGGTGT TGGCGGGTGT CGGGGCTGGC

1621 TTAACTATGC GGCATCAGAG CAGATTGTAC TGAGAGTGCA CCATATGCGG TGTGAAATAC

1681 CGCACAGATG CGTAAGGAGA AAATACCGCA TCAGGCGCCA TTCGCCATTC AGGCTGCGCA

1741 ACTGTTGGGA AGGGCGATCG GTGCGGGCCT CTTCGCTATT ACGCCAGCTG GCGAAAGGGG

1801 GATGTGCTGC AAGGCGATTA AGTTGGGTAA CGCCAGGGTT TTCCCAGTCA CGACGTTGTA

1861 AAACGACGGC CAGTGAATTG ATGCATGCTC TTCGCCGCCG CCACTCCTGC TCGAGCGCGC

1921 CCGCGCGTGC GCCGCCAGCG CCTTGGCCTT TTCGCCGCGC TCGTGCGCGT CGCTGATGTC

1981 CATCACCAGG TCCATGAGGT CTGCCTTGCG CCGGCTGAGC CACTGCTTCG TCCGGGCGGC

2041 CAAGAGGAGC ATGAGGGAGG ACTCCTGGTC CAGGGTCCTG ACGTGGTCGC GGCTCTGGGA

2101 GCGGGCCAGC ATCATCTGGC TCTGCCGCAC CGAGGCCGCC TCCAACTGGT CCTCCAGCAG

2161 CCGCAGTCGC CGCCGACCCT GGCAGAGGAA GACAGGTGAG GGGGGTATGA ATTGTACAGA

2221 ACAACCACGA GCCTTGTCTA GGCAGAATCC CTACCAGTCA TGGCTTTACC TGGATGACGG

2281 CCTGCGAACA GCTGTCCAGC GACCCTCGCT GCCGCCGCTT CTCCCGCACG CTTCTTTCCA

2341 GCACCGTGAT GGCGCGAGCC AGCGCCGCAC GCTGGCGCTG CGCTTCGCCG ATCTGAGGAC

2401 AGTCGGGGAA CTCTGATCAG TCTAAACCCC CTTGCGCGTT AGTGTTGCCA TCCTTTGCAG

2461 ACCGGTGAGA GCCGACTTGT TGTGCGCCAC CCCCCACACC ACCTCCTCCC AGACCAATTC

2521 TGTCACCTTT TTGGCGAAGG CATCGGCCTC GGCCTGCAGA GAGGACAGCA GTGCCCAGCC

2581 GCTGGGGGTT GGCGGATGCA CGCTCAGGTA CCCTTTCTTG CGCTATGACA CTTCCAGCAA

2641 AAGGTAGGGC GGGCTGCGAG ACGGCTTCCC GGCGCTGCAT GCAACACCGA TGATGCTTCG

2701 ACCCCCCGAA GCTCCTTCGG GGCTGCATGG GCGCTCCGAT GCCGCTCCAG GGCGAGCGCT

2761 GTTTAAATAG CCAGGCCCCC GATTGCAAAG ACATTATAGC GAGCTACCAA AGCCATATTC

2821 AAACACCTAG ATCACTACCA CTTCTACACA GGCCACTCGA GCTTGTGATC GCACTCCGCT

2881 AAGGGGGCGC CTCTTCCTCT TCGTTTCAGT CACAACCCGC AAACGGCGCG CCATGCTGCT

2941 GCAGGCCTTC CTGTTCCTGC TGGCCGGCTT CGCCGCCAAG ATCAGCGCCT CCATGACGAA

3001 CGAGACGTCC GACCGCCCCC TGGTGCACTT CACCCCCAAC AAGGGCTGGA TGAACGACCC 3061 CAACGGCCTG TGGTACGACG AGAAGGACGC CAAGTGGCAC CTGTACTTCC AGTACAACCC 3121 GAACGACACC GTCTGGGGGA CGCCCTTGTT CTGGGGCCAC GCCACGTCCG ACGACCTGAC 3181 CAACTGGGAG GACCAGCCCA TCGCCATCGC CCCGAAGCGC AACGACTCCG GCGCCTTCTC 3241 CGGCTCCATG GTGGTGGACT ACAACAACAC CTCCGGCTTC TTCAACGACA CCATCGACCC 3301 GCGCCAGCGC TGCGTGGCCA TCTGGACCTA CAACACCCCG GAGTCCGAGG AGCAGTACAT 3361 CTCCTACAGC CTGGACGGCG GCTACACCTT CACCGAGTAC CAGAAGAACC CCGTGCTGGC 3421 CGCCAACTCC ACCCAGTTCC GCGACCCGAA GGTCTTCTGG TACGAGCCCT CCCAGAAGTG 3481 GATCATGACC GCGGCCAAGT CCCAGGACTA CAAGATCGAG ATCTACTCCT CCGACGACCT 3541 GAAGTCCTGG AAGCTGGAGT CCGCGTTCGC CAACGAGGGC TTCCTCGGCT ACCAGTACGA 3601 GTGCCCCGGC CTGATCGAGG TCCCCACCGA GCAGGACCCC AGCAAGTCCT ACTGGGTGAT 3661 GTTCATCTCC ATCAACCCCG GCGCCCCGGC CGGCGGCTCC TTCAACCAGT ACTTCGTCGG 3721 CAGCTTCAAC GGCACCCACT TCGAGGCCTT CGACAACCAG TCCCGCGTGG TGGACTTCGG 3781 CAAGGACTAC TACGCCCTGC AGACCTTCTT CAACACCGAC CCGACCTACG GGAGCGCCCT 3841 GGGCATCGCG TGGGCCTCCA ACTGGGAGTA CTCCGCCTTC GTGCCCACCA ACCCCTGGCG 3901 CTCCTCCATG TCCCTCGTGC GCAAGTTCTC CCTCAACACC GAGTACCAGG CCAACCCGGA

3961 GACGGAGCTG ATCAACCTGA AGGCCGAGCC GATCCTGAAC ATCAGCAACG CCGGCCCCTG

4021 GAGCCGGTTC GCCACCAACA CCACGTTGAC GAAGGCCAAC AGCTACAACG TCGACCTGTC 4081 CAACAGCACC GGCACCCTGG AGTTCGAGCT GGTGTACGCC GTCAACACCA CCCAGACGAT 4141 CTCCAAGTCC GTGTTCGCGG ACCTCTCCCT CTGGTTCAAG GGCCTGGAGG ACCCCGAGGA 4201 GTACCTCCGC ATGGGCTTCG AGGTGTCCGC GTCCTCCTTC TTCCTGGACC GCGGGAACAG 4261 CAAGGTGAAG TTCGTGAAGG AGAACCCCTA CTTCACCAAC CGCATGAGCG TGAACAACCA 4321 GCCCTTCAAG AGCGAGAACG ACCTGTCCTA CTACAAGGTG TACGGCTTGC TGGACCAGAA 4381 CATCCTGGAG CTGTACTTCA ACGACGGCGA CGTCGTGTCC ACCAACACCT ACTTCATGAC 4441 CACCGGGAAC GCCCTGGGCT CCGTGAACAT GACGACGGGG GTGGACAACC TGTTCTACAT 4501 CGACAAGTTC CAGGTGCGCG AGGTCAAGTG ACAATTGGCA GCAGCAGCTC GGATAGTATC 4561 GACACACTCT GGACGCTGGT CGTGTGATGG ACTGTTGCCG CCACACTTGC TGCCTTGACC 4621 TGTGAATATC CCTGCCGCTT TTATCAAACA GCCTCAGTGT GTTTGATCTT GTGTGTACGC 4681 GCTTTTGCGA GTTGCTAGCT GCTTGTGCTA TTTGCGAATA CCACCCCCAG CATCCCCTTC 4741 CCTCGTTTCA TATCGCTTGC ATCCCAACCG CAACTTATCT ACGCTGTCCT GCTATCCCTC

4801 AGCGCTGCTC CTGCTCCTGC TCACTGCCCC TCGCACAGCC TTGGTTTGGG CTCCGCCTGT

4861 ATTCTCCTGG TACTGCAACC TGTAAACCAG CACTGCAATG CTGATGCACG GGAAGTAGTG

4921 GGATGGGAAC ACAAATGGAG GATCCCGCGT CTCGAACAGA GCGCGCAGAG GAACGCTGAA

4981 GGTCTCGCCT CTGTCGCACC TCAGCGCGGC ATACACCACA ATAACCACCT GACGAATGCG 5041 CTTGGTTCTT CGTCCATTAG CGAAGCGTCC GGTTCACACA CGTGCCACGT TGGCGAGGTG 5101 GCAGGTGACA ATGATCGGTG GAGCTGATGG TCGAAACGTT CACAGCCTAG GGATATCGAA 5161 TTCGGCCGAC AGGACGCGCG TCAAAGGTGC TGGTCGTGTA TGCCCTGGCC GGCAGGTCGT 5221 TGCTGCTGCT GGTTAGTGAT TCCGCAACCC TGATTTTGGC GTCTTATTTT GGCGTGGCAA 5281 ACGCTGGCGC CCGCGAGCCG GGCCGGCGGC GATGCGGTGC CCCACGGCTG CCGGAATCCA 5341 AGGGAGGCAA GAGCGCCCGG GTCAGTTGAA GGGCTTTACG CGCAAGGTAC AGCCGCTCCT 5401 GCAAGGCTGC GTGGTGGAAT TGGACGTGCA GGTCCTGCTG AAGTTCCTCC ACCGCCTCAC 5461 CAGCGGACAA AGCACCGGTG TATCAGGTCC GTGTCATCCA CTCTAAAGAA CTCGACTACG 5521 ACCTACTGAT GGCCCTAGAT TCTTCATCAA AAACGCCTGA GACACTTGCC CAGGATTGAA 5581 ACTCCCTGAA GGGACCACCA GGGGCCCTGA GTTGTTCCTT CCCCCCGTGG CGAGCTGCCA 5641 GCCAGGCTGT ACCTGTGATC GAGGCTGGCG GGAAAATAGG CTTCGTGTGC TCAGGTCATG

5701 GGAGGTGCAG GACAGCTCAT GAAACGCCAA CAATCGCACA ATTCATGTCA AGCTAATCAG

5761 CTATTTCCTC TTCACGAGCT GTAATTGTCC CAAAATTCTG GTCTACCGGG GGTGATCCTT 5821 CGTGTACGGG CCCTTCCCTC AACCCTAGGT ATGCGCGCAT GCGGTCGCCG CGCAACTCGC 5881 GCGAGGGCCG AGGGTTTGGG ACGGGCCGTC CCGAAATGCA GTTGCACCCG GATGCGTGGC 5941 ACCTTTTTTG CGATAATTTA TGCAATGGAC TGCTCTGCAA AATTCTGGCT CTGTCGCCAA

6001 CCCTAGGATC AGCGGCGTAG GATTTCGTAA TCATTCGTCC TGATGGGGAG CTACCGACTA 6061 CCCTAATATC AGCCCGACTG CCTGACGCCA GCGTCCACTT TTGTGCACAC ATTCCATTCG 6121 TGCCCAAGAC ATTTCATTGT GGTGCGAAGC GTCCCCAGTT ACGCTCACCT GTTTCCCGAC 6181 CTCCTTACTG TTCTGTCGAC AGAGCGGGCC CACAGGCCGG TCGCAGCCAC TAGTATGGTG 6241 GCCACCGCCG CCTCCTCCGC CTTCTTCCCC CTGCCCTCCG CCGACACCTC CTCCCGCCCC 6301 GGCAAGCTGG GCAACAAGCC CTCCTCCCTG TCCCCCCTGA AGCCCAAGTC CACCCCCAAC 6361 GGCGGCCTGC AGGTGAAGGC CAACGCCTCC GCCCCCCCCA AGATCAACGG CTCCCCCGTG 6421 GGCCTGAAGT CCGGCGGCCT GAAGGGGCGC GCCACCCAGG AGGACGCCCA CTCCGCCCCC 6481 CCCCCCCGCA CCTTCATCAA CCAGCTGCCC GACTGGTCCA TGCTGCTGGC CGCCATCACC 6541 ACCGTGTTCC TGGCCGCCGA GAAGCAGTGG ATGATGCTGG ACTGGAAGCC CAAGCGCCCC 6601 GACATGCTGG TGGACCCCTT CGGCCTGGGC TCCATCGTGC AGGACGGCCT GGTGTTCCGC 6661 CAGAACTTCT CCATCCGCTC CTACGAGATC GGCGCCGACC GCACCGCCTC CATCGAGACC 6721 GTGATGAACC ACCTGCAGGA GACCGCCCTG AACCACGTGA AGATCGCCGG CCTGTCCAAC 6781 GACGGCTTCG GCCGCACCCC CGAGATGTAC AAGCGCGACC TGATCTGGGT GGTGGCCAAG 6841 ATGCAGGTGA TGGTGAACCG CTACCCCACC TGGGGCGACA CCGTGGAGGT GAACACCTGG 6901 GTGGCCAAGT CCGGCAAGAA CGGCATGCGC CGCGACTGGC TGATCTCCGA CTGCAACACC

6961 GGCGAGATCC TGACCCGCGC CTCCTCCGTG TGGGTGATGA TGAACCAGAA GACCCGCCGC 7021 CTGTCCAAGA TCCCCGACGA GGTGCGCAAC GAGATCGAGC CCCACTTCGT GGACTCCCCC 7081 CCCGTGATCG AGGACGACGA CCGCAAGCTG CCCAAGCTGG ACGAGAAGAC CGCCGACTCC 7141 ATCCGCAAGG GCCTGACCCC CCGCTGGAAC GACCTGGACG TGAACCAGCA CGTGAACAAC 7201 GTGAAGTACA TCGGCTGGAT CCTGGAGTCC ACCCCCCCCG AGGTGCTGGA GACCCAGGAG 7261 CTGTGCTCCC TGACCCTGGA GTACCGCCGC GAGTGCGGCC GCGAGTCCGT GCTGGAGTCC 7321 CTGACCGCCA TGGACCCCTC CGGCGGCGGC TACGGCTCCC AGTTCCAGCA CCTGCTGCGC 7381 CTGGAGGACG GCGGCGAGAT CGTGAAGGGC CGCACCGAGT GGCGCCCCAA GAACGGCGTG 7441 ATCAACGGCG TGGTGCCCAC CGGCGAGTCC TCCCCCGGCG ACTACTCCAT GGACTACAAG 7501 GACCACGACG GCGACTACAA GGACCACGAC ATCGACTACA AGGACGACGA CGACAAGTGA 7561 ATCGATAGAT CTCTTAAGGC AGCAGCAGCT CGGATAGTAT CGACACACTC TGGACGCTGG 7621 TCGTGTGATG GACTGTTGCC GCCACACTTG CTGCCTTGAC CTGTGAATAT CCCTGCCGCT 7681 TTTATCAAAC AGCCTCAGTG TGTTTGATCT TGTGTGTACG CGCTTTTGCG AGTTGCTAGC

7741 TGCTTGTGCT ATTTGCGAAT ACCACCCCCA GCATCCCCTT CCCTCGTTTC ATATCGCTTG 7801 CATCCCAACC GCAACTTATC TACGCTGTCC TGCTATCCCT CAGCGCTGCT CCTGCTCCTG 7861 CTCACTGCCC CTCGCACAGC CTTGGTTTGG GCTCCGCCTG TATTCTCCTG GTACTGCAAC 7921 CTGTAAACCA GCACTGCAAT GCTGATGCAC GGGAAGTAGT GGGATGGGAA CACAAATGGA 7981 AAGCTTAATT AAGAGCTCTT GTTTTCCAGA AGGAGTTGCT CCTTGAGCCT TTCATTCTCA

8041 GCCTCGATAA CCTCCAAAGC CGCTCTAATT GTGGAGGGGG TTCGAATTTA AAAGCTTGGA 8101 ATGTTGGTTC GTGCGTCTGG AACAAGCCCA GACTTGTTGC TCACTGGGAA AAGGACCATC 8161 AGCTCCAAAA AACTTGCCGC TCAAACCGCG TACCTCTGCT TTCGCGCAAT CTGCCCTGTT 8221 GAAATCGCCA CCACATTCAT ATTGTGACGC TTGAGCAGTC TGTAATTGCC TCAGAATGTG 8281 GAATCATCTG CCCCCTGTGC GAGCCCATGC CAGGCATGTC GCGGGCGAGG ACACCCGCCA 8341 CTCGTACAGC AGACCATTAT GCTACCTCAC AATAGTTCAT AACAGTGACC ATATTTCTCG 8401 AAGCTCCCCA ACGAGCACCT CCATGCTCTG AGTGGCCACC CCCCGGCCCT GGTGCTTGCG

8461 GAGGGCAGGT CAACCGGCAT GGGGCTACCG AAATCCCCGA CCGGATCCCA CCACCCCCGC

8521 GATGGGAAGA ATCTCTCCCC GGGATGTGGG CCCACCACCA GCACAACCTG CTGGCCCAGG

8581 CGAGCGTCAA ACCATACCAC ACAAATATCC TTGGCATCGG CCCTGAATTC CTTCTGCCGC

8641 TCTGCTACCC GGTGCTTCTG TCCGAAGCAG GGGTTGCTAG GGATCGCTCC GAGTCCGCAA

8701 ACCCTTGTCG CGTGGCGGGG CTTGTTCGAG CTTGAAGAGC CTCTAGAGTC GACCTGCAGG

8761 CATGCAAGCT TGGCGTAATC ATGGTCATAG CTGTTTCCTG TGTGAAATTG TTATCCGCTC

8821 ACAATTCCAC ACAACATACG AGCCGGAAGC ATAAAGTGTA AAGCCTGGGG TGCCTAATGA

8881 GTGAGCTAAC TCACATTAAT TGCGTTGCGC TCACTGCCCG CTTTCCAGTC GGGAAACCTG

8941 TCGTGCCAGC TGCATTAATG AATCGGCCAA CGCGCGGGGA GAGGCGGTTT GCGTATTGGG

9001 CGCTCTTCCG CTTCCTCGCT CACTGACTCG CTGCGCTCGG TCGTTCGGCT GCGGCGAGCG

9061 GTATCAGCTC ACTCAAAGGC GGTAATACGG TTATCCACAG AATCAGGGGA TAACGCAGGA

9121 AAGAACATGT GAGCAAAAGG CCAGCAAAAG GCCAGGAACC GTAAAAAGGC CGCGTTGCTG

9181 GCGTTTTTCC ATAGGCTCCG CCCCCCTGAC GAGCATCACA AAAATCGACG CTCAAGTCAG

9241 AGGTGGCGAA ACCCGACAGG ACTATAAAGA TACCAGGCGT TTCCCCCTGG AAGCTCCCTC

9301 GTGCGCTCTC CTGTTCCGAC CCTGCCGCTT ACCGGATACC TGTCCGCCTT TCTCCCTTCG

9361 GGAAGCGTGG CGCTTTCTCA TAGCTCACGC TGTAGGTATC TCAGTTCGGT GTAGGTCGTT

9421 CGCTCCAAGC TGGGCTGTGT GCACGAACCC CCCGTTCAGC CCGACCGCTG CGCCTTATCC

9481 GGTAACTATC GTCTTGAGTC CAACCCGGTA AGACACGACT TATCG

SEQ ID NO: 227

pSZ1119

1 CACTGGCAGC AGCCACTGGT AACAGGATTA GCAGAGCGAG GTATGTAGGC GGTGCTACAG

61 AGTTCTTGAA GTGGTGGCCT AACTACGGCT ACACTAGAAG GACAGTATTT GGTATCTGCG

121 CTCTGCTGAA GCCAGTTACC TTCGGAAAAA GAGTTGGTAG CTCTTGATCC GGCAAACAAA

181 CCACCGCTGG TAGCGGTGGT TTTTTTGTTT GCAAGCAGCA GA TACGCGC AGAAAAAAAG

241 GATCTCAAGA AGATCCTTTG ATCTTTTCTA CGGGGTCTGA CGCTCAGTGG AACGAAAACT

301 CACGTTAAGG GATTTTGGTC ATGAGATTAT CAAAAAGGAT CTTCACCTAG ATCCTTTTAA

361 ATTAAAAATG AAGTTTTAAA TCAATCTAAA GTATATATGA GTAAACTTGG TCTGACAGTT

421 ACCAATGCTT AATCAGTGAG GCACCTATCT CAGCGATCTG TCTATTTCGT TCATCCATAG

481 TTGCCTGACT CCCCGTCGTG TAGATAACTA CGATACGGGA GGGCTTACCA TCTGGCCCCA

541 GTGCTGCAAT GATACCGCGA GACCCACGCT CACCGGCTCC AGAT A CA GCAATAAACC

601 AGCCAGCCGG AAGGGCCGAG CGCAGAAGTG GTCCTGCAAC TTTATCCGCC TCCATCCAGT

661 CTATTAATTG TTGCCGGGAA GCTAGAGTAA GTAGTTCGCC AGTTAATAGT TTGCGCAACG

721 TTGTTGCCAT TGCTACAGGC ATCGTGGTGT CACGCTCGTC GTTTGGTATG GCTTCATTCA

781 GCTCCGGTTC CCAACGATCA AGGCGAGTTA CATGATCCCC CATGTTGTGC AAAAAAGCGG

841 TTAGCTCCTT CGGTCCTCCG ATCGTTGTCA GAAGTAAGTT GGCCGCAGTG T ATCACTCA

901 TGGTTATGGC AGCACTGCAT AATTCTCTTA CTGTCATGCC ATCCGTAAGA TGCTTTTCTG

961 TGACTGGTGA GTACTCAACC AAGTCATTCT GAGAATAGTG TATGCGGCGA CCGAGTTGCT

1021 CTTGCCCGGC GTCAATACGG GATAATACCG CGCCACATAG CAGAACTTTA AAAGTGCTCA

1081 TCATTGGAAA ACGTTCTTCG GGGCGAAAAC TCTCAAGGAT CTTACCGCTG TTGAGATCCA

1141 GTTCGATGTA ACCCACTCGT GCACCCAACT GATCTTCAGC ATCTTTTACT TTCACCAGCG

1201 TTTCTGGGTG AGCAAAAACA GGAAGGCAAA ATGCCGCAAA AAAGGGAATA AGGGCGACAC

1261 GGAAATGTTG AATACTCATA CTCTTCCTTT TTCAATA TA TTGAAGCATT TATCAGGGTT

1321 ATTGTCTCAT GAGCGGATAC ATATTTGAAT GTATTTAGAA AAATAAACAA ATAGGGGTTC

1381 CGCGCACATT TCCCCGAAAA GTGCCACCTG ACGTCTAAGA AACCATTATT ATCATGACAT

1441 TAACCTATAA AAATAGGCGT ATCACGAGGC CCTTTCGTCT CGCGCGTTTC GGTGATGACG

1501 GTGAAAACCT CTGACACATG CAGCTCCCGG AGACGGTCAC AGCTTGTCTG TAAGCGGATG

1561 CCGGGAGCAG ACAAGCCCGT CAGGGCGCGT CAGCGGGTGT TGGCGGGTGT CGGGGCTGGC

1621 TTAAC ATGC GGCATCAGAG CAGATTG AC TGAGAGTGCA CCATATGCGG TGTGAAATAC

1681 CGCACAGATG CGTAAGGAGA AAATACCGCA TCAGGCGCCA TTCGCCATTC AGGCTGCGCA

1741 ACTGTTGGGA AGGGCGATCG GTGCGGGCCT CTTCGCTATT ACGCCAGCTG GCGAAAGGGG

1801 GATGTGCTGC AAGGCGATTA AGTTGGGTAA CGCCAGGGTT TTCCCAGTCA CGACGTTGTA

1861 AAACGACGGC CAGTGAATTG ATGCATGCTC TTCGCCGCCG CCACTCCTGC TCGAGCGCGC

1921 CCGCGCGTGC GCCGCCAGCG CCTTGGCCTT TTCGCCGCGC TCGTGCGCGT CGCTGATGTC

1981 CATCACCAGG TCCATGAGGT CTGCCTTGCG CCGGCTGAGC CACTGCTTCG TCCGGGCGGC

2041 CAAGAGGAGC ATGAGGGAGG ACTCCTGGTC CAGGGTCCTG ACGTGGTCGC GGCTCTGGGA

2101 GCGGGCCAGC ATCATCTGGC TCTGCCGCAC CGAGGCCGCC TCCAACTGGT CCTCCAGCAG

2161 CCGCAGTCGC CGCCGACCCT GGCAGAGGAA GACAGGTGAG GGGGGTATGA ATTGTACAGA 2221 ACAACCACGA GCCTTGTCTA GGCAGAATCC CTACCAGTCA TGGCTTTACC TGGATGACGG

2281 CCTGCGAACA GCTGTCCAGC GACCCTCGCT GCCGCCGCTT CTCCCGCACG CTTCTTTCCA

2341 GCACCGTGAT GGCGCGAGCC AGCGCCGCAC GCTGGCGCTG CGCTTCGCCG ATCTGAGGAC

2401 AGTCGGGGAA CTCTGATCAG TCTAAACCCC CTTGCGCGTT AGTGTTGCCA TCCTTTGCAG

2461 ACCGGTGAGA GCCGACTTGT TGTGCGCCAC CCCCCACACC ACCTCCTCCC AGACCAATTC

2521 TGTCACCTTT TTGGCGAAGG CATCGGCCTC GGCCTGCAGA GAGGACAGCA GTGCCCAGCC

2581 GCTGGGGGTT GGCGGATGCA CGCTCAGGTA CCCTTTCTTG CGCTATGACA CTTCCAGCAA

2641 AAGGTAGGGC GGGCTGCGAG ACGGCTTCCC GGCGCTGCAT GCAACACCGA TGATGCTTCG

2701 ACCCCCCGAA GCTCCTTCGG GGCTGCATGG GCGCTCCGAT GCCGCTCCAG GGCGAGCGCT

2761 GTTTAAATAG CCAGGCCCCC GATTGCAAAG ACATTATAGC GAGCTACCAA AGCCATATTC

2821 AAACACCTAG ATCACTACCA CTTCTACACA GGCCACTCGA GCTTGTGATC GCACTCCGCT

2881 AAGGGGGCGC CTCTTCCTCT TCGTTTCAGT CACAACCCGC AAACGGCGCG CCATGCTGCT

2941 GCAGGCCTTC CTGTTCCTGC TGGCCGGCTT CGCCGCCAAG ATCAGCGCCT CCATGACGAA

3001 CGAGACGTCC GACCGCCCCC TGGTGCACTT CACCCCCAAC AAGGGCTGGA TGAACGACCC 3061 CAACGGCCTG TGGTACGACG AGAAGGACGC CAAGTGGCAC CTGTACTTCC AGTACAACCC 3121 GAACGACACC GTCTGGGGGA CGCCCTTGTT CTGGGGCCAC GCCACGTCCG ACGACCTGAC 3181 CAACTGGGAG GACCAGCCCA TCGCCATCGC CCCGAAGCGC AACGACTCCG GCGCCTTCTC 3241 CGGCTCCATG GTGGTGGACT ACAACAACAC CTCCGGCTTC TTCAACGACA CCATCGACCC 3301 GCGCCAGCGC TGCGTGGCCA TCTGGACCTA CAACACCCCG GAGTCCGAGG AGCAGTACAT 3361 CTCCTACAGC CTGGACGGCG GCTACACCTT CACCGAGTAC CAGAAGAACC CCGTGCTGGC 3421 CGCCAACTCC ACCCAGTTCC GCGACCCGAA GGTCTTCTGG TACGAGCCCT CCCAGAAGTG 3481 GATCATGACC GCGGCCAAGT CCCAGGACTA CAAGATCGAG ATCTACTCCT CCGACGACCT 3541 GAAGTCCTGG AAGCTGGAGT CCGCGTTCGC CAACGAGGGC TTCCTCGGCT ACCAGTACGA 3601 GTGCCCCGGC CTGATCGAGG TCCCCACCGA GCAGGACCCC AGCAAGTCCT ACTGGGTGAT 3661 GTTCATCTCC ATCAACCCCG GCGCCCCGGC CGGCGGCTCC TTCAACCAGT ACTTCGTCGG 3721 CAGCTTCAAC GGCACCCACT TCGAGGCCTT CGACAACCAG TCCCGCGTGG TGGACTTCGG 3781 CAAGGACTAC TACGCCCTGC AGACCTTCTT CAACACCGAC CCGACCTACG GGAGCGCCCT 3841 GGGCATCGCG TGGGCCTCCA ACTGGGAGTA CTCCGCCTTC GTGCCCACCA ACCCCTGGCG 3901 CTCCTCCATG TCCCTCGTGC GCAAGTTCTC CCTCAACACC GAGTACCAGG CCAACCCGGA

3961 GACGGAGCTG ATCAACCTGA AGGCCGAGCC GATCCTGAAC ATCAGCAACG CCGGCCCCTG

4021 GAGCCGGTTC GCCACCAACA CCACGTTGAC GAAGGCCAAC AGCTACAACG TCGACCTGTC 4081 CAACAGCACC GGCACCCTGG AGTTCGAGCT GGTGTACGCC GTCAACACCA CCCAGACGAT 4141 CTCCAAGTCC GTGTTCGCGG ACCTCTCCCT CTGGTTCAAG GGCCTGGAGG ACCCCGAGGA 4201 GTACCTCCGC ATGGGCTTCG AGGTGTCCGC GTCCTCCTTC TTCCTGGACC GCGGGAACAG 4261 CAAGGTGAAG TTCGTGAAGG AGAACCCCTA CTTCACCAAC CGCATGAGCG TGAACAACCA 4321 GCCCTTCAAG AGCGAGAACG ACCTGTCCTA CTACAAGGTG TACGGCTTGC TGGACCAGAA 4381 CATCCTGGAG CTGTACTTCA ACGACGGCGA CGTCGTGTCC ACCAACACCT ACTTCATGAC 4441 CACCGGGAAC GCCCTGGGCT CCGTGAACAT GACGACGGGG GTGGACAACC TGTTCTACAT 4501 CGACAAGTTC CAGGTGCGCG AGGTCAAGTG ACAATTGGCA GCAGCAGCTC GGATAGTATC 4561 GACACACTCT GGACGCTGGT CGTGTGATGG ACTGTTGCCG CCACACTTGC TGCCTTGACC 4621 TGTGAATATC CCTGCCGCTT TTATCAAACA GCCTCAGTGT GTTTGATCTT GTGTGTACGC 4681 GCTTTTGCGA GTTGCTAGCT GCTTGTGCTA TTTGCGAATA CCACCCCCAG CATCCCCTTC 4741 CCTCGTTTCA TATCGCTTGC ATCCCAACCG CAACTTATCT ACGCTGTCCT GCTATCCCTC 4801 AGCGCTGCTC CTGCTCCTGC TCACTGCCCC TCGCACAGCC TTGGTTTGGG CTCCGCCTGT 4861 ATTCTCCTGG TACTGCAACC TGTAAACCAG CACTGCAATG CTGATGCACG GGAAGTAGTG 4921 GGATGGGAAC ACAAATGGAG GATCCCGCGT CTCGAACAGA GCGCGCAGAG GAACGCTGAA

4981 GGTCTCGCCT CTGTCGCACC TCAGCGCGGC ATACACCACA ATAACCACCT GACGAATGCG 5041 CTTGGTTCTT CGTCCATTAG CGAAGCGTCC GGTTCACACA CGTGCCACGT TGGCGAGGTG 5101 GCAGGTGACA ATGATCGGTG GAGCTGATGG TCGAAACGTT CACAGCCTAG GGATATCGAA 5161 TTCGGCCGAC AGGACGCGCG TCAAAGGTGC TGGTCGTGTA TGCCCTGGCC GGCAGGTCGT 5221 TGCTGCTGCT GGTTAGTGAT TCCGCAACCC TGATTTTGGC GTCTTATTTT GGCGTGGCAA 5281 ACGCTGGCGC CCGCGAGCCG GGCCGGCGGC GATGCGGTGC CCCACGGCTG CCGGAATCCA 5341 AGGGAGGCAA GAGCGCCCGG GTCAGTTGAA GGGCTTTACG CGCAAGGTAC AGCCGCTCCT 5401 GCAAGGCTGC GTGGTGGAAT TGGACGTGCA GGTCCTGCTG AAGTTCCTCC ACCGCCTCAC 5461 CAGCGGACAA AGCACCGGTG TATCAGGTCC GTGTCATCCA CTCTAAAGAG CTCGACTACG 5521 ACCTACTGAT GGCCCTAGAT TCTTCATCAA AAACGCCTGA GACACTTGCC CAGGATTGAA 5581 ACTCCCTGAA GGGACCACCA GGGGCCCTGA GTTGTTCCTT CCCCCCGTGG CGAGCTGCCA 5641 GCCAGGCTGT ACCTGTGATC GAGGCTGGCG GGAAAATAGG CTTCGTGTGC TCAGGTCATG

5701 GGAGGTGCAG GACAGCTCAT GAAACGCCAA CAATCGCACA ATTCATGTCA AGCTAATCAG 5761 CTATTTCCTC TTCACGAGCT GTAATTGTCC CAAAATTCTG GTCTACCGGG GGTGATCCTT 5821 CGTGTACGGG CCCTTCCCTC AACCCTAGGT ATGCGCGCAT GCGGTCGCCG CGCAACTCGC 5881 GCGAGGGCCG AGGGTTTGGG ACGGGCCGTC CCGAAATGCA GTTGCACCCG GATGCGTGGC

5941 ACCTTTTTTG CGATAATTTA TGCAATGGAC TGCTCTGCAA AATTCTGGCT CTGTCGCCAA

6001 CCCTAGGATC AGCGGCGTAG GATTTCGTAA TCATTCGTCC TGATGGGGAG CTACCGACTA

6061 CCCTAATATC AGCCCGACTG CCTGACGCCA GCGTCCACTT TTGTGCACAC ATTCCATTCG

6121 TGCCCAAGAC ATTTCATTGT GGTGCGAAGC GTCCCCAGTT ACGCTCACCT GTTTCCCGAC

6181 CTCCTTACTG TTCTGTCGAC AGAGCGGGCC CACAGGCCGG TCGCAGCCAC TAGTATGGCC

6241 ACCGCATCCA CTTTCTCGGC GTTCAATGCC CGCTGCGGCG ACCTGCGTCG CTCGGCGGGC

6301 TCCGGGCCCC GGCGCCCAGC GAGGCCCCTC CCCGTGCGCG GGCGCGCCCA GCTGCCCGAC

6361 TGGAGCATGC TGCTGGCCGC GATCACCACC CTGTTCCTGG CGGCCGAGAA GCAGTGGATG

6421 ATGCTGGACT GGAAGCCCAA GCGCCCCGAC ATGCTGGTGG ACCCCTTCGG CCTGGGCCGC

6481 TTCGTGCAGG ACGGCCTGGT GTTCCGCAAC AACTTCAGCA TCCGCAGCTA CGAGATCGGC

6541 GCGGACCGCA CCGCCAGCAT CGAGACCCTG ATGAACCACC TGCAGGAGAC CGCCCTGAAC

6601 CACGTGAAGA GCGTGGGCCT GCTGGAGGAC GGCCTGGGCA GCACCCGCGA GATGAGCCTG

6661 CGCAACCTGA TCTGGGTGGT GACCAAGATG CAGGTGGCGG TGGACCGCTA CCCCACCTGG

6721 GGCGACGAGG TGCAGGTGAG CAGCTGGGCG ACCGCCATCG GCAAGAACGG CATGCGCCGC

6781 GAGTGGATCG TGACCGACTT CCGCACCGGC GAGACCCTGC TGCGCGCCAC CAGCGTGTGG

6841 GTGATGATGA ACAAGCTGAC CCGCCGCATC AGCAAGATCC CCGAGGAGGT GTGGCACGAG

6901 ATCGGCCCCA GCTTCATCGA CGCGCCCCCC CTGCCCACCG TGGAGGACGA CGGCCGCAAG

6961 CTGACCCGCT TCGACGAGAG CAGCGCCGAC TTCATCCGCA AGGGCCTGAC CCCCCGCTGG 7021 AGCGACCTGG ACATCAACCA GCACGTGAAC AACGTGAAGT ACATCGGCTG GCTGCTGGAG 7081 AGCGCGCCCC CCGAGATCCA CGAGAGCCAC GAGATCGCCA GCCTGACCCT GGAGTACCGC 7141 CGCGAGTGCG GCCGCGACAG CGTGCTGAAC AGCGCCACCA AGGTGAGCGA CAGCAGCCAG 7201 CTGGGCAAGA GCGCCGTGGA GTGCAACCAC CTGGTGCGCC TGCAGAACGG CGGCGAGATC 7261 GTGAAGGGCC GCACCGTGTG GCGCCCCAAG CGCCCCCTGT ACAACGACGG CGCCGTGGTG 7321 GACGTGCCCG CCAAGACCAG CGATGACGAT GACAAGCTGG GATGACTCGA GGCAGCAGCA 7381 GCTCGGATAG TATCGACACA CTCTGGACGC TGGTCGTGTG ATGGACTGTT GCCGCCACAC 7441 TTGCTGCCTT GACCTGTGAA TATCCCTGCC GCTTTTATCA AACAGCCTCA GTGTGTTTGA 7501 TCTTGTGTGT ACGCGCTTTT GCGAGTTGCT AGCTGCTTGT GCTATTTGCG AATACCACCC 7561 CCAGCATCCC CTTCCCTCGT TTCATATCGC TTGCATCCCA ACCGCAACTT ATCTACGCTG 7621 TCCTGCTATC CCTCAGCGCT GCTCCTGCTC CTGCTCACTG CCCCTCGCAC AGCCTTGGTT 7681 TGGGCTCCGC CTGTATTCTC CTGGTACTGC AACCTGTAAA CCAGCACTGC AATGCTGATG

7741 CACGGGAAGT AGTGGGATGG GAACACAAAT GGAAAGCTTG AGCTCTTGTT TTCCAGAAGG 7801 AGTTGCTCCT TGAGCCTTTC ATTCTCAGCC TCGATAACCT CCAAAGCCGC TCTAATTGTG 7861 GAGGGGGTTC GAATTTAAAA GCTTGGAATG TTGGTTCGTG CGTCTGGAAC AAGCCCAGAC 7921 TTGTTGCTCA CTGGGAAAAG GACCATCAGC TCCAAAAAAC TTGCCGCTCA AACCGCGTAC 7981 CTCTGCTTTC GCGCAATCTG CCCTGTTGAA ATCGCCACCA CATTCATATT GTGACGCTTG

8041 AGCAGTCTGT AATTGCCTCA GAATGTGGAA TCATCTGCCC CCTGTGCGAG CCCATGCCAG 8101 GCATGTCGCG GGCGAGGACA CCCGCCACTC GTACAGCAGA CCATTATGCT ACCTCACAAT 8161 AGTTCATAAC AGTGACCATA TTTCTCGAAG CTCCCCAACG AGCACCTCCA TGCTCTGAGT 8221 GGCCACCCCC CGGCCCTGGT GCTTGCGGAG GGCAGGTCAA CCGGCATGGG GCTACCGAAA 8281 TCCCCGACCG GATCCCACCA CCCCCGCGAT GGGAAGAATC TCTCCCCGGG ATGTGGGCCC 8341 ACCACCAGCA CAACCTGCTG GCCCAGGCGA GCGTCAAACC ATACCACACA AATATCCTTG 8401 GCATCGGCCC TGAATTCCTT CTGCCGCTCT GCTACCCGGT GCTTCTGTCC GAAGCAGGGG 8461 TTGCTAGGGA TCGCTCCGAG TCCGCAAACC CTTGTCGCGT GGCGGGGCTT GTTCGAGCTT 8521 GAAGAGCCTC TAGAGTCGAC CTGCAGGCAT GCAAGCTTGG CGTAATCATG GTCATAGCTG 8581 TTTCCTGTGT GAAATTGTTA TCCGCTCACA ATTCCACACA ACATACGAGC CGGAAGCATA 8641 AAGTGTAAAG CCTGGGGTGC CTAATGAGTG AGCTAACTCA CATTAATTGC GTTGCGCTCA 8701 CTGCCCGCTT TCCAGTCGGG AAACCTGTCG TGCCAGCTGC ATTAATGAAT CGGCCAACGC 8761 GCGGGGAGAG GCGGTTTGCG TATTGGGCGC TCTTCCGCTT CCTCGCTCAC TGACTCGCTG 8821 CGCTCGGTCG TTCGGCTGCG GCGAGCGGTA TCAGCTCACT CAAAGGCGGT AATACGGTTA 8881 TCCACAGAAT CAGGGGATAA CGCAGGAAAG AACATGTGAG CAAAAGGCCA GCAAAAGGCC

8941 AGGAACCGTA AAAAGGCCGC GTTGCTGGCG TTTTTCCATA GGCTCCGCCC CCCTGACGAG

9001 CATCACAAAA ATCGACGCTC AAGTCAGAGG TGGCGAAACC CGACAGGACT ATAAAGATAC 9061 CAGGCGTTTC CCCCTGGAAG CTCCCTCGTG CGCTCTCCTG TTCCGACCCT GCCGCTTACC 9121 GGATACCTGT CCGCCTTTCT CCCTTCGGGA AGCGTGGCGC TTTCTCATAG CTCACGCTGT 9181 AGGTATCTCA GTTCGGTGTA GGTCGTTCGC TCCAAGCTGG GCTGTGTGCA CGAACCCCCC 9241 GTTCAGCCCG ACCGCTGCGC CTTATCCGGT AACTATCGTC TTGAGTCCAA CCCGGTAAGA 9301 CACGACTTAT CGC

SEQ ID NO: 228 pSZ944

1 CTAAATTGTA AGCGTTAATA TTTTGTTAAA ATTCGCGTTA AATTTTTGTT AAATCAGCTC

61 ATTTTTTAAC CAATAGGCCG AAATCGGCAA AATCCCTTAT AAATCAAAAG AATAGACCGA

121 GATAGGGTTG AGTGTTGTTC CAGTTTGGAA CAAGAGTCCA CTATTAAAGA ACGTGGACTC

181 CAACGTCAAA GGGCGAAAAA CCGTCTATCA GGGCGATGGC CCACTACGTG AACCATCACC

241 CTAATCAAGT TTTTTGGGGT CGAGGTGCCG TAAAGCACTA AATCGGAACC CTAAAGGGAG

301 CCCCCGATTT AGAGCTTGAC GGGGAAAGCC GGCGAACGTG GCGAGAAAGG AAGGGAAGAA

361 AGCGAAAGGA GCGGGCGCTA GGGCGCTGGC AAGTGTAGCG GTCACGCTGC GCGTAACCAC

421 CACACCCGCC GCGCTTAATG CGCCGCTACA GGGCGCGTCC CATTCGCCAT TCAGGCTGCG

481 CAACTGTTGG GAAGGGCGAT CGGTGCGGGC CTCTTCGCTA TTACGCCAGC TGGCGAAAGG

541 GGGATGTGCT GCAAGGCGAT TAAGTTGGGT AACGCCAGGG TTTTCCCAGT CACGACGTTG

601 TAAAACGACG GCCAGTGAGC GCGCGTAATA CGACTCACTA TAGGGCGAAT TGCAGCTTTT

661 GTTCCCTTTA GTGAGGGTTA ATTGCGCGCT TGGCGTAATC ATGGTCATAG CTGTTTCCTG

721 TGTGAAATTG TTATCCGCTC ACAATTCCAC ACAACATACG AGCCGGAAGC ATAAAGTGTA

781 AAGCCTGGGG TGCCTAATGA GTGAGCTAAC TCACATTAAT TGCGTTGCGC TCACTGCCCG

841 CTTTCCAGTC GGGAAACCTG TCGTGCCAGC TGCATTAATG AATCGGCCAA CGCGCGGGGA

901 GAGGCGGTGC GTATTGGGCG CTCTTCCGCT CCCGTGATCA CACAGGTGCC TTGCGAGCGT

961 GATCACACTA TTTTGGGGGT CCTACAGTAC TGAAATGGTG AGAAGTCGTA CTGAAATCAA

1021 GGATGAACAA TGAAAATGGT GCTGTGGTGG CTTCTCAAAG GTCAAGAATC AGTCGCTCGC

1081 GTCAGGAAAT CGCGGCGTCA ACCAGCGTGG GCGCGGTCAG TGGCCCCGCA CTGGTCACCA

1141 TAGCCTCTCC TGCCACAGTA GCGATCCCCT GGGCGTTCAC TCTCAGCAGC GGCTGTACTG

1201 CCTCCCAGAT TTTCTTCTTC TGGACCTGCG GGCGTGAGAG GATGAGCAGG GTGGGGCCAA

1261 GGGCTCAATC CTGAACGGCC CTCATTCGGT TTCCAATCCC ACAACACATA CCCACAGCAG

1321 GTCAGACCAC GCATTCGCAC CATGCGCACC AATAACGTGT CCTTACCTGA TTGGGTGTGG

1381 CAGGCTCCGT GGACAGGAGT GCCTCGTCCC CCGCCCAGAC CCGCTCCCCC GTCACGGCGG

1441 CGTCCGGGAC CCGCAGCGGC TCCACCGCGG TGTGATCCGC GTTGGCGGCG CAGAGCAGCA

1501 TCCCAGCCGA TTTGACCCCG CGCATGCTCC GAGGCTTGAG GTTGGCCAGC ACCACCACCC

1561 GCCGGCCGAC AAGGTCCTCC AGGGTCACGT GCCGGACCAG GCCACTCACG ATGGTGCGAG

1621 GGCCCCCCTC CTCGCCGAGG TCGATCTGCT CGACGTACAG ACTGCGACAT GCGTGGCGAG

1681 TGGTCATCAG AAGGAAGCAG GTGTGCAGAA GGGGCACGTG GTTGGTATTG AGAGTAGCCA

1741 AAGCTTTGTG CCAATCAGAA AGTCAACGCA GCTGCCTGCC TGGCTCGCGT ACGGTACCCT

1801 TTCTTGCGCT ATGACACTTC CAGCAAAAGG TAGGGCGGGC TGCGAGACGG CTTCCCGGCG

1861 CTGCATGCAA CACCGATGAT GCTTCGACCC CCCGAAGCTC CTTCGGGGCT GCATGGGCGC

1921 TCCGATGCCG CTCCAGGGCG AGCGCTGTTT AAATAGCCAG GCCCCCGATT GCAAAGACAT

1981 TATAGCGAGC TACCAAAGCC ATATTCAAAC ACCTAGATCA CTACCACTTC TACACAGGCC

2041 ACTCGAGCTT GTGATCGCAC TCCGCTAAGG GGGCGCCTCT TCCTCTTCGT TTCAGTCACA

2101 ACCCGCAAAC TCTAGAATAT CAATGCTGCT GCAGGCCTTC CTGTTCCTGC TGGCCGGCTT

2161 CGCCGCCAAG ATCAGCGCCT CCATGACGAA CGAGACGTCC GACCGCCCCC TGGTGCACTT

2221 CACCCCCAAC AAGGGCTGGA TGAACGACCC CAACGGCCTG TGGTACGACG AGAAGGACGC

2281 CAAGTGGCAC CTGTACTTCC AGTACAACCC GAACGACACC GTCTGGGGGA CGCCCTTGTT

2341 CTGGGGCCAC GCCACGTCCG ACGACCTGAC CAACTGGGAG GACCAGCCCA TCGCCATCGC

2401 CCCGAAGCGC AACGACTCCG GCGCCTTCTC CGGCTCCATG GTGGTGGACT ACAACAACAC

2461 CTCCGGCTTC TTCAACGACA CCATCGACCC GCGCCAGCGC TGCGTGGCCA TCTGGACCTA

2521 CAACACCCCG GAGTCCGAGG AGCAGTACAT CTCCTACAGC CTGGACGGCG GCTACACCTT

2581 CACCGAGTAC CAGAAGAACC CCGTGCTGGC CGCCAACTCC ACCCAGTTCC GCGACCCGAA

2641 GGTCTTCTGG TACGAGCCCT CCCAGAAGTG GATCATGACC GCGGCCAAGT CCCAGGACTA

2701 CAAGATCGAG ATCTACTCCT CCGACGACCT GAAGTCCTGG AAGCTGGAGT CCGCGTTCGC

2761 CAACGAGGGC TTCCTCGGCT ACCAGTACGA GTGCCCCGGC CTGATCGAGG TCCCCACCGA

2821 GCAGGACCCC AGCAAGTCCT ACTGGGTGAT GTTCATCTCC ATCAACCCCG GCGCCCCGGC

2881 CGGCGGCTCC TTCAACCAGT ACTTCGTCGG CAGCTTCAAC GGCACCCACT TCGAGGCCTT

2941 CGACAACCAG TCCCGCGTGG TGGACTTCGG CAAGGACTAC TACGCCCTGC AGACCTTCTT

3001 CAACACCGAC CCGACCTACG GGAGCGCCCT GGGCATCGCG TGGGCCTCCA ACTGGGAGTA 3061 CTCCGCCTTC GTGCCCACCA ACCCCTGGCG CTCCTCCATG TCCCTCGTGC GCAAGTTCTC 3121 CCTCAACACC GAGTACCAGG CCAACCCGGA GACGGAGCTG ATCAACCTGA AGGCCGAGCC 3181 GATCCTGAAC ATCAGCAACG CCGGCCCCTG GAGCCGGTTC GCCACCAACA CCACGTTGAC 3241 GAAGGCCAAC AGCTACAACG TCGACCTGTC CAACAGCACC GGCACCCTGG AGTTCGAGCT 3301 GGTGTACGCC GTCAACACCA CCCAGACGAT CTCCAAGTCC GTGTTCGCGG ACCTCTCCCT 3361 CTGGTTCAAG GGCCTGGAGG ACCCCGAGGA GTACCTCCGC ATGGGCTTCG AGGTGTCCGC 3421 GTCCTCCTTC TTCCTGGACC GCGGGAACAG CAAGGTGAAG TTCGTGAAGG AGAACCCCTA 3481 CTTCACCAAC CGCATGAGCG TGAACAACCA GCCCTTCAAG AGCGAGAACG ACCTGTCCTA 3541 CTACAAGGTG TACGGCTTGC TGGACCAGAA CATCCTGGAG CTGTACTTCA ACGACGGCGA

3601 CGTCGTGTCC ACCAACACCT ACTTCATGAC CACCGGGAAC GCCCTGGGCT CCGTGAACAT

3661 GACGACGGGG GTGGACAACC TGTTCTACAT CGACAAGTTC CAGGTGCGCG AGGTCAAGTG

3721 ACAATTGGCA GCAGCAGCTC GGATAGTATC GACACACTCT GGACGCTGGT CGTGTGATGG

3781 ACTGTTGCCG CCACACTTGC TGCCTTGACC TGTGAATATC CCTGCCGCTT TTATCAAACA

3841 GCCTCAGTGT GTTTGATCTT GTGTGTACGC GCTTTTGCGA GTTGCTAGCT GCTTGTGCTA

3901 TTTGCGAATA CCACCCCCAG CATCCCCTTC CCTCGTTTCA TATCGCTTGC ATCCCAACCG 3961 CAACTTATCT ACGCTGTCCT GCTATCCCTC AGCGCTGCTC CTGCTCCTGC TCACTGCCCC

4021 TCGCACAGCC TTGGTTTGGG CTCCGCCTGT ATTCTCCTGG TACTGCAACC TGTAAACCAG 4081 CACTGCAATG CTGATGCACG GGAAGTAGTG GGATGGGAAC ACAAATGGAG GATCCCGCGT 4141 CTCGAACAGA GCGCGCAGAG GAACGCTGAA GGTCTCGCCT CTGTCGCACC TCAGCGCGGC 4201 ATACACCACA ATAACCACCT GACGAATGCG CTTGGTTCTT CGTCCATTAG CGAAGCGTCC 4261 GGTTCACACA CGTGCCACGT TGGCGAGGTG GCAGGTGACA ATGATCGGTG GAGCTGATGG 4321 TCGAAACGTT CACAGCCTAG GGATATCGAA TTCCTTTCTT GCGCTATGAC ACTTCCAGCA 4381 AAAGGTAGGG CGGGCTGCGA GACGGCTTCC CGGCGCTGCA TGCAACACCG ATGATGCTTC 4441 GACCCCCCGA AGCTCCTTCG GGGCTGCATG GGCGCTCCGA TGCCGCTCCA GGGCGAGCGC 4501 TGTTTAAATA GCCAGGCCCC CGATTGCAAA GACATTATAG CGAGCTACCA AAGCCATATT 4561 CAAACACCTA GATCACTACC ACTTCTACAC AGGCCACTCG AGCTTGTGAT CGCACTCCGC 4621 TAAGGGGGCG CCTCTTCCTC TTCGTTTCAG TCACAACCCG CAAACACTAG TATGGCCACC 4681 GCATCCACTT TCTCGGCGTT CAATGCCCGC TGCGGCGACC TGCGTCGCTC GGCGGGCTCC 4741 GGGCCCCGGC GCCCAGCGAG GCCCCTCCCC GTGCGCGGGC GCGCCCCCGA CTGGTCCATG 4801 CTGTTCGCCG TGATCACCAC CATCTTCTCC GCCGCCGAGA AGCAGTGGAC CAACCTGGAG 4861 TGGAAGCCCA AGCCCAACCC CCCCCAGCTG CTGGACGACC ACTTCGGCCC CCACGGCCTG 4921 GTGTTCCGCC GCACCTTCGC CATCCGCAGC TACGAGGTGG GCCCCGACCG CTCCACCAGC

4981 ATCGTGGCCG TGATGAACCA CCTGCAGGAG GCCGCCCTGA ACCACGCCAA GTCCGTGGGC 5041 ATCCTGGGCG ACGGCTTCGG CACCACCCTG GAGATGTCCA AGCGCGACCT GATCTGGGTG 5101 GTGAAGCGCA CCCACGTGGC CGTGGAGCGC TACCCCGCCT GGGGCGACAC CGTGGAGGTG 5161 GAGTGCTGGG TGGGCGCCTC CGGCAACAAC GGCCGCCGCC ACGACTTCCT GGTGCGCGAC 5221 TGCAAGACCG GCGAGATCCT GACCCGCTGC ACCTCCCTGA GCGTGATGAT GAACACCCGC 5281 ACCCGCCGCC TGAGCAAGAT CCCCGAGGAG GTGCGCGGCG AGATCGGCCC CGCCTTCATC 5341 GACAACGTGG CCGTGAAGGA CGAGGAGATC AAGAAGCCCC AGAAGCTGAA CGACTCCACC 5401 GCCGACTACA TCCAGGGCGG CCTGACCCCC CGCTGGAACG ACCTGGACAT CAACCAGCAC 5461 GTGAACAACA TCAAGTACGT GGACTGGATC CTGGAGACCG TGCCCGACAG CATCTTCGAG 5521 AGCCACCACA TCTCCTCCTT CACCATCGAG TACCGCCGCG AGTGCACCAT GGACAGCGTG 5581 CTGCAGTCCC TGACCACCGT GAGCGGCGGC TCCTCCGAGG CCGGCCTGGT GTGCGAGCAC 5641 CTGCTGCAGC TGGAGGGCGG CAGCGAGGTG CTGCGCGCCA AGACCGAGTG GCGCCCCAAG

5701 CTGACCGACT CCTTCCGCGG CATCAGCGTG ATCCCCGCCG AGTCCAGCGT GATGGACTAC

5761 AAGGACCACG ACGGCGACTA CAAGGACCAC GACATCGACT ACAAGGACGA CGACGACAAG 5821 TGACTCGAGG CAGCAGCAGC TCGGATAGTA TCGACACACT CTGGACGCTG GTCGTGTGAT 5881 GGACTGTTGC CGCCACACTT GCTGCCTTGA CCTGTGAATA TCCCTGCCGC TTTTATCAAA 5941 CAGCCTCAGT GTGTTTGATC TTGTGTGTAC GCGCTTTTGC GAGTTGCTAG CTGCTTGTGC

6001 TATTTGCGAA TACCACCCCC AGCATCCCCT TCCCTCGTTT CATATCGCTT GCATCCCAAC 6061 CGCAACTTAT CTACGCTGTC CTGCTATCCC TCAGCGCTGC TCCTGCTCCT GCTCACTGCC 6121 CCTCGCACAG CCTTGGTTTG GGCTCCGCCT GTATTCTCCT GGTACTGCAA CCTGTAAACC 6181 AGCACTGCAA TGCTGATGCA CGGGAAGTAG TGGGATGGGA ACACAAATGG AAAGCTTGAG 6241 CTCGTACCCA TCAGCATCCG GGTGAATCTT GGCCTCCAAG ATATGGCCAA TCCTCACATC 6301 CAGCTTGGCA AAATCGACTA GACTGTCTGC AAGTGGGAAT GTGGAGCACA AGGTTGCTTG 6361 TAGCGATCGA CAGACTGGTG GGGTACATTG ACAGGTGGGC AGCGCCGCAT CCATCGTGCC 6421 TGACGCGAGC GCCGCCGGTT GCTCGCCCGT GCCTGCCGTC AAAGAGCGGC AGAGAAATCG 6481 GGAACCGAAA ACGTCACATT GCCTGATGTT GTTACATGCT GGACTAGACT TTCTTGGCGT 6541 GGGTCTGCTC CTCGCCAGGT GCGCGACGCC TCGGGGCTGG GTGCGAGGGA GCCGTGCGGC 6601 CACGCATTTG ACAAGACCCA AAGCTCGCAT CTCAGACGGT CAACCGTTCG TATTATACAT 6661 TCAACATATG GTACATACGC AAAAAGCATG CCAACGATGA CAGCTCTTCG CCCACATGTG 6721 AGCAAAAGGC CAGCAAAAGG CCAGGAACCG TAAAAAGGCC GCGTTGCTGG CGTTTTTCCA 6781 TAGGCTCCGC CCCCCTGACG AGCATCACAA AAATCGACGC TCAAGTCAGA GGTGGCGAAA 6841 CCCGACAGGA CTATAAAGAT ACCAGGCGTT TCCCCCTGGA AGCTCCCTCG TGCGCTCTCC 6901 TGTTCCGACC CTGCCGCTTA CCGGATACCT GTCCGCCTTT CTCCCTTCGG GAAGCGTGGC 6961 GCTTTCTCAT AGCTCACGCT GTAGGTATCT CAGTTCGGTG TAGGTCGTTC GCTCCAAGCT 7021 GGGCTGTGTG CACGAACCCC CCGTTCAGCC CGACCGCTGC GCCTTATCCG GTAACTATCG 7081 TCTTGAGTCC AACCCGGTAA GACACGACTT ATCGCCACTG GCAGCAGCCA CTGGTAACAG 7141 GATTAGCAGA GCGAGGTATG TAGGCGGTGC TACAGAGTTC TTGAAGTGGT GGCCTAACTA 7201 CGGCTACACT AGAAGGACAG TATTTGGTAT CTGCGCTCTG CTGAAGCCAG TTACCTTCGG 7261 AAAAAGAGTT GGTAGCTCTT GATCCGGCAA ACAAACCACC GCTGGTAGCG GTGGTTTTTT 7321 TGTTTGCAAG CAGCAGATTA CGCGCAGAAA AAAAGGATCT CAAGAAGATC CTTTGATCTT 7381 TTCTACGGGG TCTGACGCTC AGTGGAACGA AAACTCACGT TAAGGGATTT TGGTCATGAG 7441 ATTATCAAAA AGGATCTTCA CCTAGATCCT TTTAAATTAA AAATGAAGTT TTAAATCAAT 7501 CTAAAGTATA TATGAGTAAA CTTGGTCTGA CAGTTACCAA TGCTTAATCA GTGAGGCACC 7561 TATCTCAGCG ATCTGTCTAT TTCGTTCATC CATAGTTGCC TGACTCCCCG TCGTGTAGAT 7621 AACTACGATA CGGGAGGGCT TACCATCTGG CCCCAGTGCT GCAATGATAC CGCGAGACCC 7681 ACGCTCACCG GCTCCAGATT TATCAGCAAT AAACCAGCCA GCCGGAAGGG CCGAGCGCAG 7741 AAGTGGTCCT GCAACTTTAT CCGCCTCCAT CCAGTCTATT AATTGTTGCC GGGAAGCTAG 7801 AG AAGTAGT TCGCCAGTTA ATAGTTTGCG CAACGTTGTT GCCATTGCTA CAGGCATCGT 7861 GGTGTCACGC TCGTCGTTTG GTATGGCTTC ATTCAGCTCC GGTTCCCAAC GATCAAGGCG 7921 AGTTACATGA TCCCCCATGT TGTGCAAAAA AGCGGTTAGC TCCTTCGGTC CTCCGATCGT 7981 TGTCAGAAGT AAGTTGGCCG CAGTGTTATC ACTCATGGTT ATGGCAGCAC TGCATAATTC 8041 TCTTACTGTC ATGCCATCCG TAAGATGCTT TTCTGTGACT GGTGAGTACT CAACCAAGTC 8101 AT CTGAGAA TAGTGTATGC GGCGACCGAG TTGCTCTTGC CCGGCGTCAA TACGGGATAA 8161 TACCGCGCCA CATAGCAGAA CTTTAAAAGT GCTCATCATT GGAAAACGTT CTTCGGGGCG 8221 AAAACTCTCA AGGATCTTAC CGCTGTTGAG ATCCAGTTCG ATGTAACCCA CTCGTGCACC 8281 CAACTGATCT TCAGCATCTT TTACTTTCAC CAGCGTTTCT GGGTGAGCAA AAACAGGAAG 8341 GCAAAATGCC GCAAAAAAGG GAATAAGGGC GACACGGAAA TGTTGAATAC TCATACTCTT 8401 CCTTTTTCAA TATTATTGAA GCATTTATCA GGGTTATTGT CTCATGAGCG GATACATATT 8461 TGAATGTATT TAGAAAAATA AACAAATAGG GGTTCCGCGC ACATTTCCCC GAAAAGTGCC 8521 AC

ID NO: 229

1 CACTGGCAGC AGCCACTGGT AACAGGATTA GCAGAGCGAG GTATGTAGGC GGTGCTACAG

61 AGTTCTTGAA GTGGTGGCCT AACTACGGCT ACACTAGAAG GACAGTATTT GGTATCTGCG

121 CTCTGCTGAA GCCAGTTACC TTCGGAAAAA GAGTTGGTAG CTCTTGATCC GGCAAACAAA

181 CCACCGCTGG TAGCGGTGGT TTTTTTGTTT GCAAGCAGCA GATTACGCGC AGAAAAAAAG

241 GATCTCAAGA AGATCCTTTG ATCTTTTCTA CGGGGTCTGA CGCTCAGTGG AACGAAAACT

301 CACGTTAAGG GATTTTGGTC ATGAGATTAT CAAAAAGGAT CTTCACCTAG ATCCTTTTAA

361 ATTAAAAATG AAGTTTTAAA TCAATCTAAA GTATATATGA GTAAACTTGG TCTGACAGTT

421 ACCAATGCTT AATCAGTGAG GCACCTATCT CAGCGATCTG TCTATTTCGT TCATCCATAG

481 TTGCCTGACT CCCCGTCGTG TAGATAACTA CGATACGGGA GGGCTTACCA TCTGGCCCCA

541 GTGCTGCAAT GATACCGCGA GACCCACGCT CACCGGCTCC AGATTTATCA GCAATAAACC

601 AGCCAGCCGG AAGGGCCGAG CGCAGAAGTG GTCCTGCAAC TTTATCCGCC TCCATCCAGT

661 CTATTAATTG TTGCCGGGAA GCTAGAGTAA GTAGTTCGCC AGTTAATAGT TTGCGCAACG

721 TTGTTGCCAT TGCTACAGGC ATCGTGGTGT CACGCTCGTC GTTTGGTATG GCTTCATTCA

781 GCTCCGGTTC CCAACGATCA AGGCGAGTTA CATGATCCCC CATGTTGTGC AAAAAAGCGG

841 TTAGCTCCTT CGGTCCTCCG ATCGTTGTCA GAAGTAAGTT GGCCGCAGTG TTATCACTCA

901 TGGTTATGGC AGCACTGCAT AATTCTCTTA CTGTCATGCC ATCCGTAAGA TGCTTTTCTG

961 TGACTGGTGA GTACTCAACC AAGTCATTCT GAGAATAGTG TATGCGGCGA CCGAGTTGCT

1021 CTTGCCCGGC GTCAATACGG GATAATACCG CGCCACATAG CAGAACTTTA AAAGTGCTCA

1081 TCATTGGAAA ACGTTCTTCG GGGCGAAAAC TCTCAAGGAT CTTACCGCTG TTGAGATCCA

1141 GTTCGATGTA ACCCACTCGT GCACCCAACT GATCTTCAGC ATCTTTTACT TTCACCAGCG

1201 TTTCTGGGTG AGCAAAAACA GGAAGGCAAA ATGCCGCAAA AAAGGGAATA AGGGCGACAC

1261 GGAAATGTTG AATACTCATA CTCTTCCTTT TTCAATATTA TTGAAGCATT TATCAGGGTT

1321 ATTGTCTCAT GAGCGGATAC ATATTTGAAT GTATTTAGAA AAATAAACAA ATAGGGGTTC

1381 CGCGCACATT TCCCCGAAAA GTGCCACCTG ACGTCTAAGA AACCATTATT ATCATGACAT

1441 TAACCTATAA AAATAGGCGT ATCACGAGGC CCTTTCGTCT CGCGCGTTTC GGTGATGACG

1501 GTGAAAACCT CTGACACATG CAGCTCCCGG AGACGGTCAC AGCTTGTCTG TAAGCGGATG

1561 CCGGGAGCAG ACAAGCCCGT CAGGGCGCGT CAGCGGGTGT TGGCGGGTGT CGGGGCTGGC

1621 TTAACTATGC GGCATCAGAG CAGATTGTAC TGAGAGTGCA CCATATGCGG TGTGAAATAC

1681 CGCACAGATG CGTAAGGAGA AAATACCGCA TCAGGCGCCA TTCGCCATTC AGGCTGCGCA

1741 ACTGTTGGGA AGGGCGATCG GTGCGGGCCT CTTCGCTATT ACGCCAGCTG GCGAAAGGGG

1801 GATGTGCTGC AAGGCGATTA AGTTGGGTAA CGCCAGGGTT TTCCCAGTCA CGACGTTGTA

1861 AAACGACGGC CAGTGAATTG ATGCATGCTC TTCGCCGCCG CCACTCCTGC TCGAGCGCGC

1921 CCGCGCGTGC GCCGCCAGCG CCTTGGCCTT TTCGCCGCGC TCGTGCGCGT CGCTGATGTC 1981 CATCACCAGG TCCATGAGGT CTGCCTTGCG CCGGCTGAGC CACTGCTTCG TCCGGGCGGC

2041 CAAGAGGAGC ATGAGGGAGG ACTCCTGGTC CAGGGTCCTG ACGTGGTCGC GGCTCTGGGA

2101 GCGGGCCAGC ATCATCTGGC TCTGCCGCAC CGAGGCCGCC TCCAACTGGT CCTCCAGCAG

2161 CCGCAGTCGC CGCCGACCCT GGCAGAGGAA GACAGGTGAG GGGGGTATGA ATTGTACAGA

2221 ACAACCACGA GCCTTGTCTA GGCAGAATCC CTACCAGTCA TGGCTTTACC TGGATGACGG

2281 CCTGCGAACA GCTGTCCAGC GACCCTCGCT GCCGCCGCTT CTCCCGCACG CTTCTTTCCA

2341 GCACCGTGAT GGCGCGAGCC AGCGCCGCAC GCTGGCGCTG CGCTTCGCCG ATCTGAGGAC

2401 AGTCGGGGAA CTCTGATCAG TCTAAACCCC CTTGCGCGTT AGTGTTGCCA TCCTTTGCAG

2461 ACCGGTGAGA GCCGACTTGT TGTGCGCCAC CCCCCACACC ACCTCCTCCC AGACCAATTC

2521 TGTCACCTTT TTGGCGAAGG CATCGGCCTC GGCCTGCAGA GAGGACAGCA GTGCCCAGCC

2581 GCTGGGGGTT GGCGGATGCA CGCTCAGGTA CCCTTTCTTG CGCTATGACA CTTCCAGCAA

2641 AAGGTAGGGC GGGCTGCGAG ACGGCTTCCC GGCGCTGCAT GCAACACCGA TGATGCTTCG

2701 ACCCCCCGAA GCTCCTTCGG GGCTGCATGG GCGCTCCGAT GCCGCTCCAG GGCGAGCGCT

2761 GTTTAAATAG CCAGGCCCCC GATTGCAAAG ACATTATAGC GAGCTACCAA AGCCATATTC

2821 AAACACCTAG ATCACTACCA CTTCTACACA GGCCACTCGA GCTTGTGATC GCACTCCGCT

2881 AAGGGGGCGC CTCTTCCTCT TCGTTTCAGT CACAACCCGC AAACGGCGCG CCATGCTGCT

2941 GCAGGCCTTC CTGTTCCTGC TGGCCGGCTT CGCCGCCAAG ATCAGCGCCT CCATGACGAA

3001 CGAGACGTCC GACCGCCCCC TGGTGCACTT CACCCCCAAC AAGGGCTGGA TGAACGACCC 3061 CAACGGCCTG TGGTACGACG AGAAGGACGC CAAGTGGCAC CTGTACTTCC AGTACAACCC 3121 GAACGACACC GTCTGGGGGA CGCCCTTGTT CTGGGGCCAC GCCACGTCCG ACGACCTGAC 3181 CAACTGGGAG GACCAGCCCA TCGCCATCGC CCCGAAGCGC AACGACTCCG GCGCCTTCTC 3241 CGGCTCCATG GTGGTGGACT ACAACAACAC CTCCGGCTTC TTCAACGACA CCATCGACCC 3301 GCGCCAGCGC TGCGTGGCCA TCTGGACCTA CAACACCCCG GAGTCCGAGG AGCAGTACAT 3361 CTCCTACAGC CTGGACGGCG GCTACACCTT CACCGAGTAC CAGAAGAACC CCGTGCTGGC 3421 CGCCAACTCC ACCCAGTTCC GCGACCCGAA GGTCTTCTGG TACGAGCCCT CCCAGAAGTG 3481 GATCATGACC GCGGCCAAGT CCCAGGACTA CAAGATCGAG ATCTACTCCT CCGACGACCT 3541 GAAGTCCTGG AAGCTGGAGT CCGCGTTCGC CAACGAGGGC TTCCTCGGCT ACCAGTACGA 3601 GTGCCCCGGC CTGATCGAGG TCCCCACCGA GCAGGACCCC AGCAAGTCCT ACTGGGTGAT 3661 GTTCATCTCC ATCAACCCCG GCGCCCCGGC CGGCGGCTCC TTCAACCAGT ACTTCGTCGG 3721 CAGCTTCAAC GGCACCCACT TCGAGGCCTT CGACAACCAG TCCCGCGTGG TGGACTTCGG 3781 CAAGGACTAC TACGCCCTGC AGACCTTCTT CAACACCGAC CCGACCTACG GGAGCGCCCT 3841 GGGCATCGCG TGGGCCTCCA ACTGGGAGTA CTCCGCCTTC GTGCCCACCA ACCCCTGGCG 3901 CTCCTCCATG TCCCTCGTGC GCAAGTTCTC CCTCAACACC GAGTACCAGG CCAACCCGGA

3961 GACGGAGCTG ATCAACCTGA AGGCCGAGCC GATCCTGAAC ATCAGCAACG CCGGCCCCTG

4021 GAGCCGGTTC GCCACCAACA CCACGTTGAC GAAGGCCAAC AGCTACAACG TCGACCTGTC 4081 CAACAGCACC GGCACCCTGG AGTTCGAGCT GGTGTACGCC GTCAACACCA CCCAGACGAT 4141 CTCCAAGTCC GTGTTCGCGG ACCTCTCCCT CTGGTTCAAG GGCCTGGAGG ACCCCGAGGA 4201 GTACCTCCGC ATGGGCTTCG AGGTGTCCGC GTCCTCCTTC TTCCTGGACC GCGGGAACAG 4261 CAAGGTGAAG TTCGTGAAGG AGAACCCCTA CTTCACCAAC CGCATGAGCG TGAACAACCA 4321 GCCCTTCAAG AGCGAGAACG ACCTGTCCTA CTACAAGGTG TACGGCTTGC TGGACCAGAA 4381 CATCCTGGAG CTGTACTTCA ACGACGGCGA CGTCGTGTCC ACCAACACCT ACTTCATGAC 4441 CACCGGGAAC GCCCTGGGCT CCGTGAACAT GACGACGGGG GTGGACAACC TGTTCTACAT 4501 CGACAAGTTC CAGGTGCGCG AGGTCAAGTG ACAATTGGCA GCAGCAGCTC GGATAGTATC 4561 GACACACTCT GGACGCTGGT CGTGTGATGG ACTGTTGCCG CCACACTTGC TGCCTTGACC 4621 TGTGAATATC CCTGCCGCTT TTATCAAACA GCCTCAGTGT GTTTGATCTT GTGTGTACGC 4681 GCTTTTGCGA GTTGCTAGCT GCTTGTGCTA TTTGCGAATA CCACCCCCAG CATCCCCTTC 4741 CCTCGTTTCA TATCGCTTGC ATCCCAACCG CAACTTATCT ACGCTGTCCT GCTATCCCTC 4801 AGCGCTGCTC CTGCTCCTGC TCACTGCCCC TCGCACAGCC TTGGTTTGGG CTCCGCCTGT 4861 ATTCTCCTGG TACTGCAACC TGTAAACCAG CACTGCAATG CTGATGCACG GGAAGTAGTG 4921 GGATGGGAAC ACAAATGGAG GATCCCGCGT CTCGAACAGA GCGCGCAGAG GAACGCTGAA 4981 GGTCTCGCCT CTGTCGCACC TCAGCGCGGC ATACACCACA ATAACCACCT GACGAATGCG 5041 CTTGGTTCTT CGTCCATTAG CGAAGCGTCC GGTTCACACA CGTGCCACGT TGGCGAGGTG 5101 GCAGGTGACA ATGATCGGTG GAGCTGATGG TCGAAACGTT CACAGCCTAG GGATATCGAA 5161 TTCGGCCGAC AGGACGCGCG TCAAAGGTGC TGGTCGTGTA TGCCCTGGCC GGCAGGTCGT 5221 TGCTGCTGCT GGTTAGTGAT TCCGCAACCC TGATTTTGGC GTCTTATTTT GGCGTGGCAA 5281 ACGCTGGCGC CCGCGAGCCG GGCCGGCGGC GATGCGGTGC CCCACGGCTG CCGGAATCCA 5341 AGGGAGGCAA GAGCGCCCGG GTCAGTTGAA GGGCTTTACG CGCAAGGTAC AGCCGCTCCT 5401 GCAAGGCTGC GTGGTGGAAT TGGACGTGCA GGTCCTGCTG AAGTTCCTCC ACCGCCTCAC 5461 CAGCGGACAA AGCACCGGTG TATCAGGTCC GTGTCATCCA CTCTAAAGAG CTCGACTACG 5521 ACCTACTGAT GGCCCTAGAT TCTTCATCAA AAACGCCTGA GACACTTGCC CAGGATTGAA 5581 ACTCCCTGAA GGGACCACCA GGGGCCCTGA GTTGTTCCTT CCCCCCGTGG CGAGCTGCCA 5641 GCCAGGCTGT ACCTGTGATC GAGGCTGGCG GGAAAATAGG CTTCGTGTGC TCAGGTCATG

5701 GGAGGTGCAG GACAGCTCAT GAAACGCCAA CAATCGCACA ATTCATGTCA AGCTAATCAG

5761 CTATTTCCTC TTCACGAGCT GTAATTGTCC CAAAATTCTG GTCTACCGGG GGTGATCCTT 5821 CGTGTACGGG CCCTTCCCTC AACCCTAGGT ATGCGCGCAT GCGGTCGCCG CGCAACTCGC 5881 GCGAGGGCCG AGGGTTTGGG ACGGGCCGTC CCGAAATGCA GTTGCACCCG GATGCGTGGC 5941 ACCTTTTTTG CGATAATTTA TGCAATGGAC TGCTCTGCAA AATTCTGGCT CTGTCGCCAA

6001 CCCTAGGATC AGCGGCGTAG GATTTCGTAA TCATTCGTCC TGATGGGGAG CTACCGACTA 6061 CCCTAATATC AGCCCGACTG CCTGACGCCA GCGTCCACTT TTGTGCACAC ATTCCATTCG 6121 TGCCCAAGAC ATTTCATTGT GGTGCGAAGC GTCCCCAGTT ACGCTCACCT GTTTCCCGAC 6181 CTCCTTACTG TTCTGTCGAC AGAGCGGGCC CACAGGCCGG TCGCAGCCAC TAGTATGGTG 6241 GTGGCCGCCG CCGCCAGCAG CGCCTTCTTC CCCGTGCCCG CCCCCCGCCC CACCCCCAAG 6301 CCCGGCAAGT TCGGCAACTG GCCCAGCAGC CTGAGCCAGC CCTTCAAGCC CAAGAGCAAC 6361 CCCAACGGCC GCTTCCAGGT GAAGGCCAAC GTGAGCCCCC ACGGGCGCGC CCCCAAGGCC 6421 AACGGCAGCG CCGTGAGCCT GAAGTCCGGC AGCCTGAACA CCCTGGAGGA CCCCCCCAGC 6481 AGCCCCCCCC CCCGCACCTT CCTGAACCAG CTGCCCGACT GGAGCCGCCT GCGCACCGCC 6541 ATCACCACCG TGTTCGTGGC CGCCGAGAAG CAGTTCACCC GCCTGGACCG CAAGAGCAAG 6601 CGCCCCGACA TGCTGGTGGA CTGGTTCGGC AGCGAGACCA TCGTGCAGGA CGGCCTGGTG 6661 TTCCGCGAGC GCTTCAGCAT CCGCAGCTAC GAGATCGGCG CCGACCGCAC CGCCAGCATC 6721 GAGACCCTGA TGAACCACCT GCAGGACACC AGCCTGAACC ACTGCAAGAG CGTGGGCCTG 6781 CTGAACGACG GCTTCGGCCG CACCCCCGAG ATGTGCACCC GCGACCTGAT CTGGGTGCTG 6841 ACCAAGATGC AGATCGTGGT GAACCGCTAC CCCACCTGGG GCGACACCGT GGAGATCAAC 6901 AGCTGGTTCA GCCAGAGCGG CAAGATCGGC ATGGGCCGCG AGTGGCTGAT CAGCGACTGC

6961 AACACCGGCG AGATCCTGGT GCGCGCCACC AGCGCCTGGG CCATGATGAA CCAGAAGACC 7021 CGCCGCTTCA GCAAGCTGCC CTGCGAGGTG CGCCAGGAGA TCGCCCCCCA CTTCGTGGAC 7081 GCCCCCCCCG TGATCGAGGA CAACGACCGC AAGCTGCACA AGTTCGACGT GAAGACCGGC 7141 GACAGCATCT GCAAGGGCCT GACCCCCGGC TGGAACGACT TCGACGTGAA CCAGCACGTG 7201 AGCAACGTGA AGTACATCGG CTGGATTCTG GAGAGCATGC CCACCGAGGT GCTGGAGACC 7261 CAGGAGCTGT GCAGCCTGAC CCTGGAGTAC CGCCGCGAGT GCGGCCGCGA GAGCGTGGTG 7321 GAGAGCGTGA CCAGCATGAA CCCCAGCAAG GTGGGCGACC GCAGCCAGTA CCAGCACCTG 7381 CTGCGCCTGG AGGACGGCGC CGACATCATG AAGGGCCGCA CCGAGTGGCG CCCCAAGAAC 7441 GCCGGCACCA ACCGCGCCAT CAGCACCTGA TTAATTAACT CGAGGCAGCA GCAGCTCGGA 7501 TAGTATCGAC ACACTCTGGA CGCTGGTCGT GTGATGGACT GTTGCCGCCA CACTTGCTGC 7561 CTTGACCTGT GAATATCCCT GCCGCTTTTA TCAAACAGCC TCAGTGTGTT TGATCTTGTG 7621 TGTACGCGCT TTTGCGAGTT GCTAGCTGCT TGTGCTATTT GCGAATACCA CCCCCAGCAT 7681 CCCCTTCCCT CGTTTCATAT CGCTTGCATC CCAACCGCAA CTTATCTACG CTGTCCTGCT

7741 ATCCCTCAGC GCTGCTCCTG CTCCTGCTCA CTGCCCCTCG CACAGCCTTG GTTTGGGCTC 7801 CGCCTGTATT CTCCTGGTAC TGCAACCTGT AAACCAGCAC TGCAATGCTG ATGCACGGGA 7861 AGTAGTGGGA TGGGAACACA AATGGAAAGC TTGAGCTCTT GTTTTCCAGA AGGAGTTGCT 7921 CCTTGAGCCT TTCATTCTCA GCCTCGATAA CCTCCAAAGC CGCTCTAATT GTGGAGGGGG 7981 TTCGAATTTA AAAGCTTGGA ATGTTGGTTC GTGCGTCTGG AACAAGCCCA GACTTGTTGC

8041 TCACTGGGAA AAGGACCATC AGCTCCAAAA AACTTGCCGC TCAAACCGCG TACCTCTGCT 8101 TTCGCGCAAT CTGCCCTGTT GAAATCGCCA CCACATTCAT ATTGTGACGC TTGAGCAGTC 8161 TGTAATTGCC TCAGAATGTG GAATCATCTG CCCCCTGTGC GAGCCCATGC CAGGCATGTC 8221 GCGGGCGAGG ACACCCGCCA CTCGTACAGC AGACCATTAT GCTACCTCAC AATAGTTCAT 8281 AACAGTGACC ATATTTCTCG AAGCTCCCCA ACGAGCACCT CCATGCTCTG AGTGGCCACC 8341 CCCCGGCCCT GGTGCTTGCG GAGGGCAGGT CAACCGGCAT GGGGCTACCG AAATCCCCGA 8401 CCGGATCCCA CCACCCCCGC GATGGGAAGA ATCTCTCCCC GGGATGTGGG CCCACCACCA 8461 GCACAACCTG CTGGCCCAGG CGAGCGTCAA ACCATACCAC ACAAATATCC TTGGCATCGG 8521 CCCTGAATTC CTTCTGCCGC TCTGCTACCC GGTGCTTCTG TCCGAAGCAG GGGTTGCTAG 8581 GGATCGCTCC GAGTCCGCAA ACCCTTGTCG CGTGGCGGGG CTTGTTCGAG CTTGAAGAGC 8641 CTCTAGAGTC GACCTGCAGG CATGCAAGCT TGGCGTAATC ATGGTCATAG CTGTTTCCTG 8701 TGTGAAATTG TTATCCGCTC ACAATTCCAC ACAACATACG AGCCGGAAGC ATAAAGTGTA 8761 AAGCCTGGGG TGCCTAATGA GTGAGCTAAC TCACATTAAT TGCGTTGCGC TCACTGCCCG 8821 CTTTCCAGTC GGGAAACCTG TCGTGCCAGC TGCATTAATG AATCGGCCAA CGCGCGGGGA 8881 GAGGCGGTTT GCGTATTGGG CGCTCTTCCG CTTCCTCGCT CACTGACTCG CTGCGCTCGG

8941 TCGTTCGGCT GCGGCGAGCG GTATCAGCTC ACTCAAAGGC GGTAATACGG TTATCCACAG

9001 AATCAGGGGA TAACGCAGGA AAGAACATGT GAGCAAAAGG CCAGCAAAAG GCCAGGAACC 9061 GTAAAAAGGC CGCGTTGCTG GCGTTTTTCC ATAGGCTCCG CCCCCCTGAC GAGCATCACA 9121 AAAATCGACG CTCAAGTCAG AGGTGGCGAA ACCCGACAGG ACTATAAAGA TACCAGGCGT 9181 TTCCCCCTGG AAGCTCCCTC GTGCGCTCTC CTGTTCCGAC CCTGCCGCTT ACCGGATACC 9241 TGTCCGCCTT TCTCCCTTCG GGAAGCGTGG CGCTTTCTCA TAGCTCACGC TGTAGGTATC 9301 TCAGTTCGGT GTAGGTCGTT CGCTCCAAGC TGGGCTGTGT GCACGAACCC CCCGTTCAGC 9361 CCGACCGCTG CGCCTTATCC GGTAACTATC GTCTTGAGTC CAACCCGGTA AGACACGACT 9421 TATCGC

SEQ ID NO: 230

pSZ1410

1 CACTGGCAGC AGCCACTGGT AACAGGATTA GCAGAGCGAG GTATGTAGGC GGTGCTACAG

61 AGTTCTTGAA GTGGTGGCCT AACTACGGCT ACACTAGAAG GACAGTATTT GGTATCTGCG

121 CTCTGCTGAA GCCAGTTACC TTCGGAAAAA GAGTTGGTAG CTCTTGATCC GGCAAACAAA

181 CCACCGCTGG TAGCGGTGGT TTTTTTGTTT GCAAGCAGCA GATTACGCGC AGAAAAAAAG

241 GATCTCAAGA AGATCCTTTG ATCTTTTCTA CGGGGTCTGA CGCTCAGTGG AACGAAAACT

301 CACGTTAAGG GATTTTGGTC ATGAGATTAT CAAAAAGGAT CTTCACCTAG ATCCTTTTAA

361 ATTAAAAATG AAGTTTTAAA TCAATCTAAA GTATATATGA GTAAACTTGG TCTGACAGTT

421 ACCAATGCTT AATCAGTGAG GCACCTATCT CAGCGATCTG TCTATTTCGT TCATCCATAG

481 TTGCCTGACT CCCCGTCGTG TAGATAACTA CGATACGGGA GGGCTTACCA TCTGGCCCCA

541 GTGCTGCAAT GATACCGCGA GACCCACGCT CACCGGCTCC AGATTTATCA GCAATAAACC

601 AGCCAGCCGG AAGGGCCGAG CGCAGAAGTG GTCCTGCAAC TTTATCCGCC TCCATCCAGT

661 CTATTAATTG TTGCCGGGAA GCTAGAGTAA GTAGTTCGCC AGTTAATAGT TTGCGCAACG

721 TTGTTGCCAT TGCTACAGGC ATCGTGGTGT CACGCTCGTC GTTTGGTATG GCTTCATTCA

781 GCTCCGGTTC CCAACGATCA AGGCGAGTTA CATGATCCCC CATGTTGTGC AAAAAAGCGG

841 TTAGCTCCTT CGGTCCTCCG ATCGTTGTCA GAAGTAAGTT GGCCGCAGTG TTATCACTCA

901 TGGTTATGGC AGCACTGCAT AATTCTCTTA CTGTCATGCC ATCCGTAAGA TGCTTTTCTG

961 TGACTGGTGA GTACTCAACC AAGTCATTCT GAGAATAGTG TATGCGGCGA CCGAGTTGCT

1021 CTTGCCCGGC GTCAATACGG GATAATACCG CGCCACATAG CAGAACTTTA AAAGTGCTCA

1081 TCATTGGAAA ACGTTCTTCG GGGCGAAAAC TCTCAAGGAT CTTACCGCTG TTGAGATCCA

1141 GTTCGATGTA ACCCACTCGT GCACCCAACT GATCTTCAGC ATCTTTTACT TTCACCAGCG

1201 TTTCTGGGTG AGCAAAAACA GGAAGGCAAA ATGCCGCAAA AAAGGGAATA AGGGCGACAC

1261 GGAAATGTTG AATACTCATA CTCTTCCTTT TTCAATATTA TTGAAGCATT TATCAGGGTT

1321 ATTGTCTCAT GAGCGGATAC ATATTTGAAT GTATTTAGAA AAATAAACAA ATAGGGGTTC

1381 CGCGCACATT TCCCCGAAAA GTGCCACCTG ACGTCTAAGA AACCATTATT ATCATGACAT

1441 TAACCTATAA AAATAGGCGT ATCACGAGGC CCTTTCGTCT CGCGCGTTTC GGTGATGACG

1501 GTGAAAACCT CTGACACATG CAGCTCCCGG AGACGGTCAC AGCTTGTCTG TAAGCGGATG

1561 CCGGGAGCAG ACAAGCCCGT CAGGGCGCGT CAGCGGGTGT TGGCGGGTGT CGGGGCTGGC

1621 TTAACTATGC GGCATCAGAG CAGATTGTAC TGAGAGTGCA CCATATGCGG TGTGAAATAC

1681 CGCACAGATG CGTAAGGAGA AAATACCGCA TCAGGCGCCA TTCGCCATTC AGGCTGCGCA

1741 ACTGTTGGGA AGGGCGATCG GTGCGGGCCT CTTCGCTATT ACGCCAGCTG GCGAAAGGGG

1801 GATGTGCTGC AAGGCGATTA AGTTGGGTAA CGCCAGGGTT TTCCCAGTCA CGACGTTGTA

1861 AAACGACGGC CAGTGAATTG ATGCATGCTC TTCCGCCTGG AGCTGGTGCA GAGCATGGGG

1921 CAGTTTGCGG AGGAGAGGGT GCTCCCCGTG CTGCACCCCG TGGACAAGCT GTGGCAGCCG

1981 CAGGACTTCC TGCCCGACCC CGAGTCGCCC GACTTCGAGG ACCAGGTGGC GGAGCTGCGC

2041 GCGCGCGCCA AGGACCTGCC CGACGAGTAC TTTGTGGTGC TGGTGGGCGA CATGATCACG

2101 GAGGAGGCGC TGCCGACCTA CATGGCCATG CTCAACACCT TGGACGGTGT GCGCGACGAC

2161 ACGGGCGCGG CTGACCACCC GTGGGCGCGC TGGACGCGGC AGTGGGTGGC CGAGGAGAAC

2221 CGGCACGGCG ACCTGCTGAA CAAGTACTGT TGGCTGACGG GGCGCGTCAA CATGCGGGCC

2281 GTGGAGGTGA CCATCAACAA CCTGATCAAG AGCGGCATGA ACCCGCAGAC GGACAACAAC

2341 CCTTACTTGG GCTTCGTCTA CACCTCCTTC CAGGAGCGCG CCACCAAGTA GGTACCCTTT

2401 CTTGCGCTAT GACACTTCCA GCAAAAGGTA GGGCGGGCTG CGAGACGGCT TCCCGGCGCT

2461 GCATGCAACA CCGATGATGC TTCGACCCCC CGAAGCTCCT TCGGGGCTGC ATGGGCGCTC

2521 CGATGCCGCT CCAGGGCGAG CGCTGTTTAA ATAGCCAGGC CCCCGATTGC AAAGACATTA

2581 TAGCGAGCTA CCAAAGCCAT ATTCAAACAC CTAGATCACT ACCACTTCTA CACAGGCCAC

2641 TCGAGCTTGT GATCGCACTC CGCTAAGGGG GCGCCTCTTC CTCTTCGTTT CAGTCACAAC

2701 CCGCAAACTC TAGAATATCA ATGCTGCTGC AGGCCTTCCT GTTCCTGCTG GCCGGCTTCG

2761 CCGCCAAGAT CAGCGCCTCC ATGACGAACG AGACGTCCGA CCGCCCCCTG GTGCACTTCA

2821 CCCCCAACAA GGGCTGGATG AACGACCCCA ACGGCCTGTG GTACGACGAG AAGGACGCCA

2881 AGTGGCACCT GTACTTCCAG TACAACCCGA ACGACACCGT CTGGGGGACG CCCTTGTTCT

2941 GGGGCCACGC CACGTCCGAC GACCTGACCA ACTGGGAGGA CCAGCCCATC GCCATCGCCC

3001 CGAAGCGCAA CGACTCCGGC GCCTTCTCCG GCTCCATGGT GGTGGACTAC AACAACACCT 3061 CCGGCTTCTT CAACGACACC ATCGACCCGC GCCAGCGCTG CGTGGCCATC TGGACCTACA 3121 ACACCCCGGA GTCCGAGGAG CAGTACATCT CCTACAGCCT GGACGGCGGC TACACCTTCA 3181 CCGAGTACCA GAAGAACCCC GTGCTGGCCG CCAACTCCAC CCAGTTCCGC GACCCGAAGG

3241 TCTTCTGGTA CGAGCCCTCC CAGAAGTGGA TCATGACCGC GGCCAAGTCC CAGGACTACA

3301 AGATCGAGAT CTACTCCTCC GACGACCTGA AGTCCTGGAA GCTGGAGTCC GCGTTCGCCA

3361 ACGAGGGCTT CCTCGGCTAC CAGTACGAGT GCCCCGGCCT GATCGAGGTC CCCACCGAGC

3421 AGGACCCCAG CAAGTCCTAC TGGGTGATGT TCATCTCCAT CAACCCCGGC GCCCCGGCCG

3481 GCGGCTCCTT CAACCAGTAC TTCGTCGGCA GCTTCAACGG CACCCACTTC GAGGCCTTCG

3541 ACAACCAGTC CCGCGTGGTG GACTTCGGCA AGGACTACTA CGCCCTGCAG ACCTTCTTCA

3601 ACACCGACCC GACCTACGGG AGCGCCCTGG GCATCGCGTG GGCCTCCAAC TGGGAGTACT

3661 CCGCCTTCGT GCCCACCAAC CCCTGGCGCT CCTCCATGTC CCTCGTGCGC AAGTTCTCCC

3721 TCAACACCGA GTACCAGGCC AACCCGGAGA CGGAGCTGAT CAACCTGAAG GCCGAGCCGA

3781 TCCTGAACAT CAGCAACGCC GGCCCCTGGA GCCGGTTCGC CACCAACACC ACGTTGACGA

3841 AGGCCAACAG CTACAACGTC GACCTGTCCA ACAGCACCGG CACCCTGGAG TTCGAGCTGG

3901 TGTACGCCGT CAACACCACC CAGACGATCT CCAAGTCCGT GTTCGCGGAC CTCTCCCTCT

3961 GGTTCAAGGG CCTGGAGGAC CCCGAGGAGT ACCTCCGCAT GGGCTTCGAG GTGTCCGCGT

4021 CCTCCTTCTT CCTGGACCGC GGGAACAGCA AGGTGAAGTT CGTGAAGGAG AACCCCTACT 4081 TCACCAACCG CATGAGCGTG AACAACCAGC CCTTCAAGAG CGAGAACGAC CTGTCCTACT 4141 ACAAGGTGTA CGGCTTGCTG GACCAGAACA TCCTGGAGCT GTACTTCAAC GACGGCGACG 4201 TCGTGTCCAC CAACACCTAC TTCATGACCA CCGGGAACGC CCTGGGCTCC GTGAACATGA 4261 CGACGGGGGT GGACAACCTG TTCTACATCG ACAAGTTCCA GGTGCGCGAG GTCAAGTGAC 4321 AATTGGCAGC AGCAGCTCGG ATAGTATCGA CACACTCTGG ACGCTGGTCG TGTGATGGAC 4381 TGTTGCCGCC ACACTTGCTG CCTTGACCTG TGAATATCCC TGCCGCTTTT ATCAAACAGC 4441 CTCAGTGTGT TTGATCTTGT GTGTACGCGC TTTTGCGAGT TGCTAGCTGC TTGTGCTATT 4501 TGCGAATACC ACCCCCAGCA TCCCCTTCCC TCGTTTCATA TCGCTTGCAT CCCAACCGCA 4561 ACTTATCTAC GCTGTCCTGC TATCCCTCAG CGCTGCTCCT GCTCCTGCTC ACTGCCCCTC 4621 GCACAGCCTT GGTTTGGGCT CCGCCTGTAT TCTCCTGGTA CTGCAACCTG TAAACCAGCA 4681 CTGCAATGCT GATGCACGGG AAGTAGTGGG ATGGGAACAC AAATGGAGGA TCCCGCGTCT 4741 CGAACAGAGC GCGCAGAGGA ACGCTGAAGG TCTCGCCTCT GTCGCACCTC AGCGCGGCAT 4801 ACACCACAAT AACCACCTGA CGAATGCGCT TGGTTCTTCG TCCATTAGCG AAGCGTCCGG 4861 TTCACACACG TGCCACGTTG GCGAGGTGGC AGGTGACAAT GATCGGTGGA GCTGATGGTC 4921 GAAACGTTCA CAGCCTAGGG ATATCGAATT CGGCCGACAG GACGCGCGTC AAAGGTGCTG

4981 GTCGTGTATG CCCTGGCCGG CAGGTCGTTG CTGCTGCTGG TTAGTGATTC CGCAACCCTG 5041 ATTTTGGCGT CTTATTTTGG CGTGGCAAAC GCTGGCGCCC GCGAGCCGGG CCGGCGGCGA 5101 TGCGGTGCCC CACGGCTGCC GGAATCCAAG GGAGGCAAGA GCGCCCGGGT CAGTTGAAGG 5161 GCTTTACGCG CAAGGTACAG CCGCTCCTGC AAGGCTGCGT GGTGGAATTG GACGTGCAGG 5221 TCCTGCTGAA GTTCCTCCAC CGCCTCACCA GCGGACAAAG CACCGGTGTA TCAGGTCCGT 5281 GTCATCCACT CTAAAGAACT CGACTACGAC CTACTGATGG CCCTAGATTC TTCATCAAAA 5341 ACGCCTGAGA CACTTGCCCA GGATTGAAAC TCCCTGAAGG GACCACCAGG GGCCCTGAGT 5401 TGTTCCTTCC CCCCGTGGCG AGCTGCCAGC CAGGCTGTAC CTGTGATCGA GGCTGGCGGG 5461 AAAATAGGCT TCGTGTGCTC AGGTCATGGG AGGTGCAGGA CAGCTCATGA AACGCCAACA 5521 ATCGCACAAT TCATGTCAAG CTAATCAGCT ATTTCCTCTT CACGAGCTGT AATTGTCCCA 5581 AAATTCTGGT CTACCGGGGG TGATCCTTCG TGTACGGGCC CTTCCCTCAA CCCTAGGTAT 5641 GCGCGCATGC GGTCGCCGCG CAACTCGCGC GAGGGCCGAG GGTTTGGGAC GGGCCGTCCC

5701 GAAATGCAGT TGCACCCGGA TGCGTGGCAC CTTTTTTGCG ATAATTTATG CAATGGACTG

5761 CTCTGCAAAA TTCTGGCTCT GTCGCCAACC CTAGGATCAG CGGCGTAGGA TTTCGTAATC 5821 ATTCGTCCTG ATGGGGAGCT ACCGACTACC CTAATATCAG CCCGACTGCC TGACGCCAGC 5881 GTCCACTTTT GTGCACACAT TCCATTCGTG CCCAAGACAT TTCATTGTGG TGCGAAGCGT 5941 CCCCAGTTAC GCTCACCTGT TTCCCGACCT CCTTACTGTT CTGTCGACAG AGCGGGCCCA

6001 CAGGCCGGTC GCAGCCACTA GTATGGTGGT GGCCGCCGCC GCCAGCAGCG CCTTCTTCCC 6061 CGTGCCCGCC CCCCGCCCCA CCCCCAAGCC CGGCAAGTTC GGCAACTGGC CCAGCAGCCT 6121 GAGCCAGCCC TTCAAGCCCA AGAGCAACCC CAACGGCCGC TTCCAGGTGA AGGCCAACGT 6181 GAGCCCCCAC GGGCGCGCCC CCAAGGCCAA CGGCAGCGCC GTGAGCCTGA AGTCCGGCAG 6241 CCTGAACACC CTGGAGGACC CCCCCAGCAG CCCCCCCCCC CGCACCTTCC TGAACCAGCT 6301 GCCCGACTGG AGCCGCCTGC GCACCGCCAT CACCACCGTG TTCGTGGCCG CCGAGAAGCA 6361 GTTCACCCGC CTGGACCGCA AGAGCAAGCG CCCCGACATG CTGGTGGACT GGTTCGGCAG 6421 CGAGACCATC GTGCAGGACG GCCTGGTGTT CCGCGAGCGC TTCAGCATCC GCAGCTACGA 6481 GATCGGCGCC GACCGCACCG CCAGCATCGA GACCCTGATG AACCACCTGC AGGACACCAG 6541 CCTGAACCAC TGCAAGAGCG TGGGCCTGCT GAACGACGGC TTCGGCCGCA CCCCCGAGAT 6601 GTGCACCCGC GACCTGATCT GGGTGCTGAC CAAGATGCAG ATCGTGGTGA ACCGCTACCC 6661 CACCTGGGGC GACACCGTGG AGATCAACAG CTGGTTCAGC CAGAGCGGCA AGATCGGCAT 6721 GGGCCGCGAG TGGCTGATCA GCGACTGCAA CACCGGCGAG ATCCTGGTGC GCGCCACCAG 6781 CGCCTGGGCC ATGATGAACC AGAAGACCCG CCGCTTCAGC AAGCTGCCCT GCGAGGTGCG 6841 CCAGGAGATC GCCCCCCACT TCGTGGACGC CCCCCCCGTG ATCGAGGACA ACGACCGCAA

6901 GCTGCACAAG TTCGACGTGA AGACCGGCGA CAGCATCTGC AAGGGCCTGA CCCCCGGCTG

6961 GAACGACTTC GACGTGAACC AGCACGTGAG CAACGTGAAG TACATCGGCT GGATTCTGGA

7021 GAGCATGCCC ACCGAGGTGC TGGAGACCCA GGAGCTGTGC AGCCTGACCC TGGAGTACCG

7081 CCGCGAGTGC GGCCGCGAGA GCGTGGTGGA GAGCGTGACC AGCATGAACC CCAGCAAGGT

7141 GGGCGACCGC AGCCAGTACC AGCACCTGCT GCGCCTGGAG GACGGCGCCG ACATCATGAA

7201 GGGCCGCACC GAGTGGCGCC CCAAGAACGC CGGCACCAAC CGCGCCATCA GCACCTGATT

7261 AATTAACTCG AGGCAGCAGC AGCTCGGATA GTATCGACAC ACTCTGGACG CTGGTCGTGT

7321 GATGGACTGT TGCCGCCACA CTTGCTGCCT TGACCTGTGA ATATCCCTGC CGCTTTTATC

7381 AAACAGCCTC AGTGTGTTTG ATCTTGTGTG TACGCGCTTT TGCGAGTTGC TAGCTGCTTG

7441 TGCTATTTGC GAATACCACC CCCAGCATCC CCTTCCCTCG TTTCATATCG CTTGCATCCC

7501 AACCGCAACT TATCTACGCT GTCCTGCTAT CCCTCAGCGC TGCTCCTGCT CCTGCTCACT

7561 GCCCCTCGCA CAGCCTTGGT TTGGGCTCCG CCTGTATTCT CCTGGTACTG CAACCTGTAA

7621 ACCAGCACTG CAATGCTGAT GCACGGGAAG TAGTGGGATG GGAACACAAA TGGAAAGCTT

7681 GAGCTCCAGC CACGGCAACA CCGCGCGCCT TGCGGCCGAG CACGGCGACA AGAACCTGAG

7741 CAAGATCTGC GGGCTGATCG CCAGCGACGA GGGCCGGCAC GAGATCGCCT ACACGCGCAT

7801 CGTGGACGAG TTCTTCCGCC TCGACCCCGA GGGCGCCGTC GCCGCCTACG CCAACATGAT

7861 GCGCAAGCAG ATCACCATGC CCGCGCACCT CATGGACGAC ATGGGCCACG GCGAGGCCAA

7921 CCCGGGCCGC AACCTCTTCG CCGACTTCTC CGCGGTCGCC GAGAAGATCG ACGTCTACGA

7981 CGCCGAGGAC TACTGCCGCA TCCTGGAGCA CCTCAACGCG CGCTGGAAGG TGGACGAGCG

8041 CCAGGTCAGC GGCCAGGCCG CCGCGGACCA GGAGTACGTC CTGGGCCTGC CCCAGCGCTT

8101 CCGGAAACTC GCCGAGAAGA CCGCCGCCAA GCGCAAGCGC GTCGCGCGCA GGCCCGTCGC

8161 CTTCTCCTGG AGAAGAGCCT CTAGAGTCGA CCTGCAGGCA TGCAAGCTTG GCGTAATCAT

8221 GGTCATAGCT GTTTCCTGTG TGAAATTGTT ATCCGCTCAC AATTCCACAC AACATACGAG

8281 CCGGAAGCAT AAAGTGTAAA GCCTGGGGTG CCTAATGAGT GAGCTAACTC ACATTAATTG

8341 CGTTGCGCTC ACTGCCCGCT TTCCAGTCGG GAAACCTGTC GTGCCAGCTG CATTAATGAA

8401 TCGGCCAACG CGCGGGGAGA GGCGGTTTGC GTATTGGGCG CTCTTCCGCT TCCTCGCTCA

8461 CTGACTCGCT GCGCTCGGTC GTTCGGCTGC GGCGAGCGGT ATCAGCTCAC TCAAAGGCGG

8521 TAATACGGTT ATCCACAGAA TCAGGGGATA ACGCAGGAAA GAACATGTGA GCAAAAGGCC

8581 AGCAAAAGGC CAGGAACCGT AAAAAGGCCG CGTTGCTGGC GTTTTTCCAT AGGCTCCGCC

8641 CCCCTGACGA GCATCACAAA AATCGACGCT CAAGTCAGAG GTGGCGAAAC CCGACAGGAC

8701 TATAAAGATA CCAGGCGTTT CCCCCTGGAA GCTCCCTCGT GCGCTCTCCT GTTCCGACCC

8761 TGCCGCTTAC CGGATACCTG TCCGCCTTTC TCCCTTCGGG AAGCGTGGCG CTTTCTCATA

8821 GCTCACGCTG TAGGTATCTC AGTTCGGTGT AGGTCGTTCG CTCCAAGCTG GGCTGTGTGC

8881 ACGAACCCCC CGTTCAGCCC GACCGCTGCG CCTTATCCGG TAACTATCGT CTTGAGTCCA

8941 ACCCGGTAAG ACACGACTTA TCGC

SEQ ID NO: 231

pSZ1413

1 CACTGGCAGC AGCCACTGGT AACAGGATTA GCAGAGCGAG GTATGTAGGC GGTGCTACAG 61 AGTTCTTGAA GTGGTGGCCT AACTACGGCT ACACTAGAAG GACAGTATTT GGTATCTGCG 121 CTCTGCTGAA GCCAGTTACC TTCGGAAAAA GAGTTGGTAG CTCTTGATCC GGCAAACAAA 181 CCACCGCTGG TAGCGGTGGT TTTTTTGTTT GCAAGCAGCA GAT ACGCGC AGAAAAAAAG 241 GATCTCAAGA AGATCCTTTG ATCTTTTCTA CGGGGTCTGA CGCTCAGTGG AACGAAAACT 301 CACGTTAAGG GATTTTGGTC ATGAGATTAT CAAAAAGGAT CTTCACCTAG ATCCTTTTAA 361 ATTAAAAATG AAGTTTTAAA TCAATCTAAA GTATATATGA GTAAACTTGG TCTGACAGTT 421 ACCAATGCTT AATCAGTGAG GCACCTATCT CAGCGATCTG TCTATTTCGT TCATCCATAG 481 TTGCCTGACT CCCCGTCGTG TAGATAACTA CGATACGGGA GGGCTTACCA TCTGGCCCCA 541 GTGCTGCAAT GATACCGCGA GACCCACGCT CACCGGCTCC AGATTTATCA GCAATAAACC 601 AGCCAGCCGG AAGGGCCGAG CGCAGAAGTG GTCCTGCAAC TTTATCCGCC TCCATCCAGT 661 CTATTAATTG TTGCCGGGAA GCTAGAGTAA GTAGTTCGCC AGTTAATAGT TTGCGCAACG 721 TTGTTGCCAT TGCTACAGGC ATCGTGGTGT CACGCTCGTC GTTTGGTATG GCTTCATTCA 781 GCTCCGGTTC CCAACGATCA AGGCGAGTTA CATGATCCCC CATGTTGTGC AAAAAAGCGG 841 TTAGCTCCTT CGGTCCTCCG ATCGTTGTCA GAAGTAAGTT GGCCGCAGTG TTATCACTCA 901 TGGTTATGGC AGCACTGCAT AATTCTCTTA CTGTCATGCC ATCCGTAAGA TGCTTTTCTG 961 TGACTGGTGA GTACTCAACC AAGTCATTCT GAGAATAGTG TATGCGGCGA CCGAGTTGCT 1021 CTTGCCCGGC GTCAATACGG GATAATACCG CGCCACATAG CAGAACTTTA AAAGTGCTCA 1081 TCATTGGAAA ACGTTCTTCG GGGCGAAAAC TCTCAAGGAT CTTACCGCTG TTGAGATCCA 1141 GTTCGATGTA ACCCACTCGT GCACCCAACT GATCTTCAGC ATCTTTTACT TTCACCAGCG 1201 TTTCTGGGTG AGCAAAAACA GGAAGGCAAA ATGCCGCAAA AAAGGGAATA AGGGCGACAC

1261 GGAAATGTTG AATACTCATA CTCTTCCTTT TTCAATATTA TTGAAGCATT TATCAGGGTT

1321 ATTGTCTCAT GAGCGGATAC ATATTTGAAT GTATTTAGAA AAATAAACAA ATAGGGGTTC

1381 CGCGCACATT TCCCCGAAAA GTGCCACCTG ACGTCTAAGA AACCATTATT ATCATGACAT

1441 TAACCTATAA AAATAGGCGT ATCACGAGGC CCTTTCGTCT CGCGCGTTTC GGTGATGACG

1501 GTGAAAACCT CTGACACATG CAGCTCCCGG AGACGGTCAC AGCTTGTCTG TAAGCGGATG

1561 CCGGGAGCAG ACAAGCCCGT CAGGGCGCGT CAGCGGGTGT TGGCGGGTGT CGGGGCTGGC

1621 TTAACTATGC GGCATCAGAG CAGATTGTAC TGAGAGTGCA CCATATGCGG TGTGAAATAC

1681 CGCACAGATG CGTAAGGAGA AAATACCGCA TCAGGCGCCA TTCGCCATTC AGGCTGCGCA

1741 ACTGTTGGGA AGGGCGATCG GTGCGGGCCT CTTCGCTATT ACGCCAGCTG GCGAAAGGGG

1801 GATGTGCTGC AAGGCGATTA AGTTGGGTAA CGCCAGGGTT TTCCCAGTCA CGACGTTGTA

1861 AAACGACGGC CAGTGAATTG ATGCATGCTC TTCCGCCTGG AGCTGGTGCA GAGCATGGGG

1921 CAGTTTGCGG AGGAGAGGGT GCTCCCCGTG CTGCACCCCG TGGACAAGCT GTGGCAGCCG

1981 CAGGACTTCC TGCCCGACCC CGAGTCGCCC GACTTCGAGG ACCAGGTGGC GGAGCTGCGC

2041 GCGCGCGCCA AGGACCTGCC CGACGAGTAC TTTGTGGTGC TGGTGGGCGA CATGATCACG

2101 GAGGAGGCGC TGCCGACCTA CATGGCCATG CTCAACACCT TGGACGGTGT GCGCGACGAC

2161 ACGGGCGCGG CTGACCACCC GTGGGCGCGC TGGACGCGGC AGTGGGTGGC CGAGGAGAAC

2221 CGGCACGGCG ACCTGCTGAA CAAGTACTGT TGGCTGACGG GGCGCGTCAA CATGCGGGCC

2281 GTGGAGGTGA CCATCAACAA CCTGATCAAG AGCGGCATGA ACCCGCAGAC GGACAACAAC

2341 CCTTACTTGG GCTTCGTCTA CACCTCCTTC CAGGAGCGCG CCACCAAGTA GGTACCCTTT

2401 CTTGCGCTAT GACACTTCCA GCAAAAGGTA GGGCGGGCTG CGAGACGGCT TCCCGGCGCT

2461 GCATGCAACA CCGATGATGC TTCGACCCCC CGAAGCTCCT TCGGGGCTGC ATGGGCGCTC

2521 CGATGCCGCT CCAGGGCGAG CGCTGTTTAA ATAGCCAGGC CCCCGATTGC AAAGACATTA

2581 TAGCGAGCTA CCAAAGCCAT ATTCAAACAC CTAGATCACT ACCACTTCTA CACAGGCCAC

2641 TCGAGCTTGT GATCGCACTC CGCTAAGGGG GCGCCTCTTC CTCTTCGTTT CAGTCACAAC

2701 CCGCAAACTC TAGAATATCA ATGCTGCTGC AGGCCTTCCT GTTCCTGCTG GCCGGCTTCG

2761 CCGCCAAGAT CAGCGCCTCC ATGACGAACG AGACGTCCGA CCGCCCCCTG GTGCACTTCA

2821 CCCCCAACAA GGGCTGGATG AACGACCCCA ACGGCCTGTG GTACGACGAG AAGGACGCCA

2881 AGTGGCACCT GTACTTCCAG TACAACCCGA ACGACACCGT CTGGGGGACG CCCTTGTTCT

2941 GGGGCCACGC CACGTCCGAC GACCTGACCA ACTGGGAGGA CCAGCCCATC GCCATCGCCC

3001 CGAAGCGCAA CGACTCCGGC GCCTTCTCCG GCTCCATGGT GGTGGACTAC AACAACACCT 3061 CCGGCTTCTT CAACGACACC ATCGACCCGC GCCAGCGCTG CGTGGCCATC TGGACCTACA 3121 ACACCCCGGA GTCCGAGGAG CAGTACATCT CCTACAGCCT GGACGGCGGC TACACCTTCA 3181 CCGAGTACCA GAAGAACCCC GTGCTGGCCG CCAACTCCAC CCAGTTCCGC GACCCGAAGG 3241 TCTTCTGGTA CGAGCCCTCC CAGAAGTGGA TCATGACCGC GGCCAAGTCC CAGGACTACA 3301 AGATCGAGAT CTACTCCTCC GACGACCTGA AGTCCTGGAA GCTGGAGTCC GCGTTCGCCA 3361 ACGAGGGCTT CCTCGGCTAC CAGTACGAGT GCCCCGGCCT GATCGAGGTC CCCACCGAGC 3421 AGGACCCCAG CAAGTCCTAC TGGGTGATGT TCATCTCCAT CAACCCCGGC GCCCCGGCCG 3481 GCGGCTCCTT CAACCAGTAC TTCGTCGGCA GCTTCAACGG CACCCACTTC GAGGCCTTCG 3541 ACAACCAGTC CCGCGTGGTG GACTTCGGCA AGGACTACTA CGCCCTGCAG ACCTTCTTCA 3601 ACACCGACCC GACCTACGGG AGCGCCCTGG GCATCGCGTG GGCCTCCAAC TGGGAGTACT 3661 CCGCCTTCGT GCCCACCAAC CCCTGGCGCT CCTCCATGTC CCTCGTGCGC AAGTTCTCCC 3721 TCAACACCGA GTACCAGGCC AACCCGGAGA CGGAGCTGAT CAACCTGAAG GCCGAGCCGA 3781 TCCTGAACAT CAGCAACGCC GGCCCCTGGA GCCGGTTCGC CACCAACACC ACGTTGACGA 3841 AGGCCAACAG CTACAACGTC GACCTGTCCA ACAGCACCGG CACCCTGGAG TTCGAGCTGG 3901 TGTACGCCGT CAACACCACC CAGACGATCT CCAAGTCCGT GTTCGCGGAC CTCTCCCTCT

3961 GGTTCAAGGG CCTGGAGGAC CCCGAGGAGT ACCTCCGCAT GGGCTTCGAG GTGTCCGCGT

4021 CCTCCTTCTT CCTGGACCGC GGGAACAGCA AGGTGAAGTT CGTGAAGGAG AACCCCTACT 4081 TCACCAACCG CATGAGCGTG AACAACCAGC CCTTCAAGAG CGAGAACGAC CTGTCCTACT 4141 ACAAGGTGTA CGGCTTGCTG GACCAGAACA TCCTGGAGCT GTACTTCAAC GACGGCGACG 4201 TCGTGTCCAC CAACACCTAC TTCATGACCA CCGGGAACGC CCTGGGCTCC GTGAACATGA 4261 CGACGGGGGT GGACAACCTG TTCTACATCG ACAAGTTCCA GGTGCGCGAG GTCAAGTGAC 4321 AATTGGCAGC AGCAGCTCGG ATAGTATCGA CACACTCTGG ACGCTGGTCG TGTGATGGAC 4381 TGTTGCCGCC ACACTTGCTG CCTTGACCTG TGAATATCCC TGCCGCTTTT ATCAAACAGC 4441 CTCAGTGTGT TTGATCTTGT GTGTACGCGC TTTTGCGAGT TGCTAGCTGC TTGTGCTATT 4501 TGCGAATACC ACCCCCAGCA TCCCCTTCCC TCGTTTCATA TCGCTTGCAT CCCAACCGCA 4561 ACTTATCTAC GCTGTCCTGC TATCCCTCAG CGCTGCTCCT GCTCCTGCTC ACTGCCCCTC 4621 GCACAGCCTT GGTTTGGGCT CCGCCTGTAT TCTCCTGGTA CTGCAACCTG TAAACCAGCA 4681 CTGCAATGCT GATGCACGGG AAGTAGTGGG ATGGGAACAC AAATGGAGGA TCCCGCGTCT 4741 CGAACAGAGC GCGCAGAGGA ACGCTGAAGG TCTCGCCTCT GTCGCACCTC AGCGCGGCAT 4801 ACACCACAAT AACCACCTGA CGAATGCGCT TGGTTCTTCG TCCATTAGCG AAGCGTCCGG 4861 TTCACACACG TGCCACGTTG GCGAGGTGGC AGGTGACAAT GATCGGTGGA GCTGATGGTC

4921 GAAACGTTCA CAGCCTAGGG ATATCGAATT CGGCCGACAG GACGCGCGTC AAAGGTGCTG

4981 GTCGTGTATG CCCTGGCCGG CAGGTCGTTG CTGCTGCTGG TTAGTGATTC CGCAACCCTG 5041 ATTTTGGCGT CTTATTTTGG CGTGGCAAAC GCTGGCGCCC GCGAGCCGGG CCGGCGGCGA 5101 TGCGGTGCCC CACGGCTGCC GGAATCCAAG GGAGGCAAGA GCGCCCGGGT CAGTTGAAGG 5161 GCTTTACGCG CAAGGTACAG CCGCTCCTGC AAGGCTGCGT GGTGGAATTG GACGTGCAGG 5221 TCCTGCTGAA GTTCCTCCAC CGCCTCACCA GCGGACAAAG CACCGGTGTA TCAGGTCCGT 5281 GTCATCCACT CTAAAGAACT CGACTACGAC CTACTGATGG CCCTAGATTC TTCATCAAAA 5341 ACGCCTGAGA CACTTGCCCA GGATTGAAAC TCCCTGAAGG GACCACCAGG GGCCCTGAGT 5401 TGTTCCTTCC CCCCGTGGCG AGCTGCCAGC CAGGCTGTAC CTGTGATCGA GGCTGGCGGG 5461 AAAATAGGCT TCGTGTGCTC AGGTCATGGG AGGTGCAGGA CAGCTCATGA AACGCCAACA 5521 ATCGCACAAT TCATGTCAAG CTAATCAGCT ATTTCCTCTT CACGAGCTGT AATTGTCCCA 5581 AAATTCTGGT CTACCGGGGG TGATCCTTCG TGTACGGGCC CTTCCCTCAA CCCTAGGTAT 5641 GCGCGCATGC GGTCGCCGCG CAACTCGCGC GAGGGCCGAG GGTTTGGGAC GGGCCGTCCC

5701 GAAATGCAGT TGCACCCGGA TGCGTGGCAC CTTTTTTGCG ATAATTTATG CAATGGACTG

5761 CTCTGCAAAA TTCTGGCTCT GTCGCCAACC CTAGGATCAG CGGCGTAGGA TTTCGTAATC 5821 ATTCGTCCTG ATGGGGAGCT ACCGACTACC CTAATATCAG CCCGACTGCC TGACGCCAGC 5881 GTCCACTTTT GTGCACACAT TCCATTCGTG CCCAAGACAT TTCATTGTGG TGCGAAGCGT 5941 CCCCAGTTAC GCTCACCTGT TTCCCGACCT CCTTACTGTT CTGTCGACAG AGCGGGCCCA

6001 CAGGCCGGTC GCAGCCACTA GTATGGCTAT CAAGACGAAC AGGCAGCCTG TGGAGAAGCC 6061 TCCGTTCACG ATCGGGACGC TGCGCAAGGC CATCCCCGCG CACTGTTTCG AGCGCTCGGC 6121 GCTTCGTGGG CGCGCCCCCA AGGCCAACGG CAGCGCCGTG AGCCTGAAGT CCGGCAGCCT 6181 GAACACCCTG GAGGACCCCC CCAGCAGCCC CCCCCCCCGC ACCTTCCTGA ACCAGCTGCC 6241 CGACTGGAGC CGCCTGCGCA CCGCCATCAC CACCGTGTTC GTGGCCGCCG AGAAGCAGTT 6301 CACCCGCCTG GACCGCAAGA GCAAGCGCCC CGACATGCTG GTGGACTGGT TCGGCAGCGA 6361 GACCATCGTG CAGGACGGCC TGGTGTTCCG CGAGCGCTTC AGCATCCGCA GCTACGAGAT 6421 CGGCGCCGAC CGCACCGCCA GCATCGAGAC CCTGATGAAC CACCTGCAGG ACACCAGCCT 6481 GAACCACTGC AAGAGCGTGG GCCTGCTGAA CGACGGCTTC GGCCGCACCC CCGAGATGTG 6541 CACCCGCGAC CTGATCTGGG TGCTGACCAA GATGCAGATC GTGGTGAACC GCTACCCCAC 6601 CTGGGGCGAC ACCGTGGAGA TCAACAGCTG GTTCAGCCAG AGCGGCAAGA TCGGCATGGG 6661 CCGCGAGTGG CTGATCAGCG ACTGCAACAC CGGCGAGATC CTGGTGCGCG CCACCAGCGC 6721 CTGGGCCATG ATGAACCAGA AGACCCGCCG CTTCAGCAAG CTGCCCTGCG AGGTGCGCCA 6781 GGAGATCGCC CCCCACTTCG TGGACGCCCC CCCCGTGATC GAGGACAACG ACCGCAAGCT 6841 GCACAAGTTC GACGTGAAGA CCGGCGACAG CATCTGCAAG GGCCTGACCC CCGGCTGGAA 6901 CGACTTCGAC GTGAACCAGC ACGTGAGCAA CGTGAAGTAC ATCGGCTGGA TTCTGGAGAG

6961 CATGCCCACC GAGGTGCTGG AGACCCAGGA GCTGTGCAGC CTGACCCTGG AGTACCGCCG 7021 CGAGTGCGGC CGCGAGAGCG TGGTGGAGAG CGTGACCAGC ATGAACCCCA GCAAGGTGGG 7081 CGACCGCAGC CAGTACCAGC ACCTGCTGCG CCTGGAGGAC GGCGCCGACA TCATGAAGGG 7141 CCGCACCGAG TGGCGCCCCA AGAACGCCGG CACCAACCGC GCCATCAGCA CCTGA TAAT 7201 TAACTCGAGG CAGCAGCAGC TCGGATAGTA TCGACACACT CTGGACGCTG GTCGTGTGAT 7261 GGACTGTTGC CGCCACACTT GCTGCCTTGA CCTGTGAATA TCCCTGCCGC TTTTATCAAA 7321 CAGCCTCAGT GTGTTTGATC TTGTGTGTAC GCGCTTTTGC GAGTTGCTAG CTGCTTGTGC 7381 TATTTGCGAA TACCACCCCC AGCATCCCCT TCCCTCGTTT CATATCGCTT GCATCCCAAC 7441 CGCAACTTAT CTACGCTGTC CTGCTATCCC TCAGCGCTGC TCCTGCTCCT GCTCACTGCC 7501 CCTCGCACAG CCTTGGTTTG GGCTCCGCCT GTATTCTCCT GGTACTGCAA CCTGTAAACC 7561 AGCACTGCAA TGCTGATGCA CGGGAAGTAG TGGGATGGGA ACACAAATGG AAAGCTTGAG 7621 CTCCAGCCAC GGCAACACCG CGCGCCTTGC GGCCGAGCAC GGCGACAAGA ACCTGAGCAA 7681 GATCTGCGGG CTGATCGCCA GCGACGAGGG CCGGCACGAG ATCGCCTACA CGCGCATCGT

7741 GGACGAGTTC TTCCGCCTCG ACCCCGAGGG CGCCGTCGCC GCCTACGCCA ACATGATGCG 7801 CAAGCAGATC ACCATGCCCG CGCACCTCAT GGACGACATG GGCCACGGCG AGGCCAACCC 7861 GGGCCGCAAC CTCTTCGCCG ACTTCTCCGC GGTCGCCGAG AAGATCGACG TCTACGACGC 7921 CGAGGACTAC TGCCGCATCC TGGAGCACCT CAACGCGCGC TGGAAGGTGG ACGAGCGCCA 7981 GGTCAGCGGC CAGGCCGCCG CGGACCAGGA GTACGTCCTG GGCCTGCCCC AGCGCTTCCG

8041 GAAACTCGCC GAGAAGACCG CCGCCAAGCG CAAGCGCGTC GCGCGCAGGC CCGTCGCCTT 8101 CTCCTGGAGA AGAGCCTCTA GAGTCGACCT GCAGGCATGC AAGCTTGGCG TAATCATGGT 8161 CATAGCTGTT TCCTGTGTGA AATTGTTATC CGCTCACAAT TCCACACAAC ATACGAGCCG 8221 GAAGCATAAA GTGTAAAGCC TGGGGTGCCT AATGAGTGAG CTAACTCACA TTAATTGCGT 8281 TGCGCTCACT GCCCGCTTTC CAGTCGGGAA ACCTGTCGTG CCAGCTGCAT TAATGAATCG 8341 GCCAACGCGC GGGGAGAGGC GGTTTGCGTA TTGGGCGCTC TTCCGCTTCC TCGCTCACTG 8401 ACTCGCTGCG CTCGGTCGTT CGGCTGCGGC GAGCGGTATC AGCTCACTCA AAGGCGGTAA 8461 TACGGTTATC CACAGAATCA GGGGATAACG CAGGAAAGAA CATGTGAGCA AAAGGCCAGC 8521 AAAAGGCCAG GAACCGTAAA AAGGCCGCGT TGCTGGCGTT TTTCCATAGG CTCCGCCCCC

8581 CTGACGAGCA TCACAAAAAT CGACGCTCAA GTCAGAGGTG GCGAAACCCG ACAGGACTAT

8641 AAAGATACCA GGCGTTTCCC CCTGGAAGCT CCCTCGTGCG CTCTCCTGTT CCGACCCTGC

8701 CGCTTACCGG ATACCTGTCC GCCTTTCTCC CTTCGGGAAG CGTGGCGCTT TCTCATAGCT

8761 CACGCTGTAG GTATCTCAGT TCGGTGTAGG TCGTTCGCTC CAAGCTGGGC TGTGTGCACG

8821 AACCCCCCGT TCAGCCCGAC CGCTGCGCCT TATCCGGTAA CTATCGTCTT GAGTCCAACC

8881 CGGTAAGACA CGACTTATCG C

SEQ ID NO: 232

pSZ1491

1 CACTGGCAGC AGCCACTGGT AACAGGATTA GCAGAGCGAG GTATGTAGGC GGTGCTACAG

61 AGTTCTTGAA GTGGTGGCCT AACTACGGCT ACACTAGAAG GACAGTATTT GGTATCTGCG

121 CTCTGCTGAA GCCAGTTACC TTCGGAAAAA GAGTTGGTAG CTCTTGATCC GGCAAACAAA

181 CCACCGCTGG TAGCGGTGGT TTTTTTGTTT GCAAGCAGCA GATTACGCGC AGAAAAAAAG

241 GATCTCAAGA AGATCCTTTG ATCTTTTCTA CGGGGTCTGA CGCTCAGTGG AACGAAAACT

301 CACGTTAAGG GATTTTGGTC ATGAGATTAT CAAAAAGGAT CTTCACCTAG ATCCTTTTAA

361 ATTAAAAATG AAGTTTTAAA TCAATCTAAA GTATATATGA GTAAACTTGG TCTGACAGTT

421 ACCAATGCTT AATCAGTGAG GCACCTATCT CAGCGATCTG TCTATTTCGT TCATCCATAG

481 TTGCCTGACT CCCCGTCGTG TAGATAACTA CGATACGGGA GGGCTTACCA TCTGGCCCCA

541 GTGCTGCAAT GATACCGCGA GACCCACGCT CACCGGCTCC AGATTTATCA GCAATAAACC

601 AGCCAGCCGG AAGGGCCGAG CGCAGAAGTG GTCCTGCAAC TTTATCCGCC TCCATCCAGT

661 CTATTAATTG TTGCCGGGAA GCTAGAGTAA GTAGTTCGCC AGTTAATAGT TTGCGCAACG

721 TTGTTGCCAT TGCTACAGGC ATCGTGGTGT CACGCTCGTC GTTTGGTATG GCTTCATTCA

781 GCTCCGGTTC CCAACGATCA AGGCGAGTTA CATGATCCCC CATGTTGTGC AAAAAAGCGG

841 TTAGCTCCTT CGGTCCTCCG ATCGTTGTCA GAAGTAAGTT GGCCGCAGTG TTATCACTCA

901 TGGTTATGGC AGCACTGCAT AATTCTCTTA CTGTCATGCC ATCCGTAAGA TGCTTTTCTG

961 TGACTGGTGA GTACTCAACC AAGTCATTCT GAGAATAGTG TATGCGGCGA CCGAGTTGCT

1021 CTTGCCCGGC GTCAATACGG GATAATACCG CGCCACATAG CAGAACTTTA AAAGTGCTCA

1081 TCATTGGAAA ACGTTCTTCG GGGCGAAAAC TCTCAAGGAT CTTACCGCTG TTGAGATCCA

1141 GTTCGATGTA ACCCACTCGT GCACCCAACT GATCTTCAGC ATCTTTTACT TTCACCAGCG

1201 TTTCTGGGTG AGCAAAAACA GGAAGGCAAA ATGCCGCAAA AAAGGGAATA AGGGCGACAC

1261 GGAAATGTTG AATACTCATA CTCTTCCTTT TTCAATATTA TTGAAGCATT TATCAGGGTT

1321 ATTGTCTCAT GAGCGGATAC ATATTTGAAT GTATTTAGAA AAATAAACAA ATAGGGGTTC

1381 CGCGCACATT TCCCCGAAAA GTGCCACCTG ACGTCTAAGA AACCATTATT ATCATGACAT

1441 TAACCTATAA AAATAGGCGT ATCACGAGGC CCTTTCGTCT CGCGCGTTTC GGTGATGACG

1501 GTGAAAACCT CTGACACATG CAGCTCCCGG AGACGGTCAC AGCTTGTCTG TAAGCGGATG

1561 CCGGGAGCAG ACAAGCCCGT CAGGGCGCGT CAGCGGGTGT TGGCGGGTGT CGGGGCTGGC

1621 TTAACTATGC GGCATCAGAG CAGATTGTAC TGAGAGTGCA CCATATGCGG TGTGAAATAC

1681 CGCACAGATG CGTAAGGAGA AAATACCGCA TCAGGCGCCA TTCGCCATTC AGGCTGCGCA

1741 ACTGTTGGGA AGGGCGATCG GTGCGGGCCT CTTCGCTATT ACGCCAGCTG GCGAAAGGGG

1801 GATGTGCTGC AAGGCGATTA AGTTGGGTAA CGCCAGGGTT TTCCCAGTCA CGACGTTGTA

1861 AAACGACGGC CAGTGAATTG ATGCATGCTC TTCGCCGCCG CCACTCCTGC TCGAGCGCGC

1921 CCGCGCGTGC GCCGCCAGCG CCTTGGCCTT TTCGCCGCGC TCGTGCGCGT CGCTGATGTC

1981 CATCACCAGG TCCATGAGGT CTGCCTTGCG CCGGCTGAGC CACTGCTTCG TCCGGGCGGC

2041 CAAGAGGAGC ATGAGGGAGG ACTCCTGGTC CAGGGTCCTG ACGTGGTCGC GGCTCTGGGA

2101 GCGGGCCAGC ATCATCTGGC TCTGCCGCAC CGAGGCCGCC TCCAACTGGT CCTCCAGCAG

2161 CCGCAGTCGC CGCCGACCCT GGCAGAGGAA GACAGGTGAG GGGGGTATGA ATTGTACAGA

2221 ACAACCACGA GCCTTGTCTA GGCAGAATCC CTACCAGTCA TGGCTTTACC TGGATGACGG

2281 CCTGCGAACA GCTGTCCAGC GACCCTCGCT GCCGCCGCTT CTCCCGCACG CTTCTTTCCA

2341 GCACCGTGAT GGCGCGAGCC AGCGCCGCAC GCTGGCGCTG CGCTTCGCCG ATCTGAGGAC

2401 AGTCGGGGAA CTCTGATCAG TCTAAACCCC CTTGCGCGTT AGTGTTGCCA TCCTTTGCAG

2461 ACCGGTGAGA GCCGACTTGT TGTGCGCCAC CCCCCACACC ACCTCCTCCC AGACCAATTC

2521 TGTCACCTTT TTGGCGAAGG CATCGGCCTC GGCCTGCAGA GAGGACAGCA GTGCCCAGCC

2581 GCTGGGGGTT GGCGGATGCA CGCTCAGGTA CCCTTTCTTG CGCTATGACA CTTCCAGCAA

2641 AAGGTAGGGC GGGCTGCGAG ACGGCTTCCC GGCGCTGCAT GCAACACCGA TGATGCTTCG

2701 ACCCCCCGAA GCTCCTTCGG GGCTGCATGG GCGCTCCGAT GCCGCTCCAG GGCGAGCGCT

2761 GTTTAAATAG CCAGGCCCCC GATTGCAAAG ACATTATAGC GAGCTACCAA AGCCATATTC

2821 AAACACCTAG ATCACTACCA CTTCTACACA GGCCACTCGA GCTTGTGATC GCACTCCGCT

2881 AAGGGGGCGC CTCTTCCTCT TCGTTTCAGT CACAACCCGC AAACTCTAGA ATATCAATGC 2941 TGCTGCAGGC CTTCCTGTTC CTGCTGGCCG GCTTCGCCGC CAAGATCAGC GCCTCCATGA

3001 CGAACGAGAC GTCCGACCGC CCCCTGGTGC ACTTCACCCC CAACAAGGGC TGGATGAACG 3061 ACCCCAACGG CCTGTGGTAC GACGAGAAGG ACGCCAAGTG GCACCTGTAC TTCCAGTACA 3121 ACCCGAACGA CACCGTCTGG GGGACGCCCT TGTTCTGGGG CCACGCCACG TCCGACGACC 3181 TGACCAACTG GGAGGACCAG CCCATCGCCA TCGCCCCGAA GCGCAACGAC TCCGGCGCCT 3241 TCTCCGGCTC CATGGTGGTG GACTACAACA ACACCTCCGG CTTCTTCAAC GACACCATCG 3301 ACCCGCGCCA GCGCTGCGTG GCCATCTGGA CCTACAACAC CCCGGAGTCC GAGGAGCAGT 3361 ACATCTCCTA CAGCCTGGAC GGCGGCTACA CCTTCACCGA GTACCAGAAG AACCCCGTGC 3421 TGGCCGCCAA CTCCACCCAG TTCCGCGACC CGAAGGTCTT CTGGTACGAG CCCTCCCAGA 3481 AGTGGATCAT GACCGCGGCC AAGTCCCAGG ACTACAAGAT CGAGATCTAC TCCTCCGACG 3541 ACCTGAAGTC CTGGAAGCTG GAGTCCGCGT TCGCCAACGA GGGCTTCCTC GGCTACCAGT 3601 ACGAGTGCCC CGGCCTGATC GAGGTCCCCA CCGAGCAGGA CCCCAGCAAG TCCTACTGGG 3661 TGATGTTCAT CTCCATCAAC CCCGGCGCCC CGGCCGGCGG CTCCTTCAAC CAGTACTTCG 3721 TCGGCAGCTT CAACGGCACC CACTTCGAGG CCTTCGACAA CCAGTCCCGC GTGGTGGACT 3781 TCGGCAAGGA CTACTACGCC CTGCAGACCT TCTTCAACAC CGACCCGACC TACGGGAGCG 3841 CCCTGGGCAT CGCGTGGGCC TCCAACTGGG AGTACTCCGC CTTCGTGCCC ACCAACCCCT 3901 GGCGCTCCTC CATGTCCCTC GTGCGCAAGT TCTCCCTCAA CACCGAGTAC CAGGCCAACC

3961 CGGAGACGGA GCTGATCAAC CTGAAGGCCG AGCCGATCCT GAACATCAGC AACGCCGGCC

4021 CCTGGAGCCG GTTCGCCACC AACACCACGT TGACGAAGGC CAACAGCTAC AACGTCGACC 4081 TGTCCAACAG CACCGGCACC CTGGAGTTCG AGCTGGTGTA CGCCGTCAAC ACCACCCAGA 4141 CGATCTCCAA GTCCGTGTTC GCGGACCTCT CCCTCTGGTT CAAGGGCCTG GAGGACCCCG 4201 AGGAGTACCT CCGCATGGGC TTCGAGGTGT CCGCGTCCTC CTTCTTCCTG GACCGCGGGA 4261 ACAGCAAGGT GAAGTTCGTG AAGGAGAACC CCTACTTCAC CAACCGCATG AGCGTGAACA 4321 ACCAGCCCTT CAAGAGCGAG AACGACCTGT CCTACTACAA GGTGTACGGC TTGCTGGACC 4381 AGAACATCCT GGAGCTGTAC TTCAACGACG GCGACGTCGT GTCCACCAAC ACCTACTTCA 4441 TGACCACCGG GAACGCCCTG GGCTCCGTGA ACATGACGAC GGGGGTGGAC AACCTGTTCT 4501 ACATCGACAA GTTCCAGGTG CGCGAGGTCA AGTGACAATT GGCAGCAGCA GCTCGGATAG 4561 TATCGACACA CTCTGGACGC TGGTCGTGTG ATGGACTGTT GCCGCCACAC TTGCTGCCTT 4621 GACCTGTGAA TATCCCTGCC GCTTTTATCA AACAGCCTCA GTGTGTTTGA TCTTGTGTGT 4681 ACGCGCTTTT GCGAGTTGCT AGCTGCTTGT GCTATTTGCG AATACCACCC CCAGCATCCC 4741 CTTCCCTCGT TTCATATCGC TTGCATCCCA ACCGCAACTT ATCTACGCTG TCCTGCTATC 4801 CCTCAGCGCT GCTCCTGCTC CTGCTCACTG CCCCTCGCAC AGCCTTGGTT TGGGCTCCGC 4861 CTGTATTCTC CTGGTACTGC AACCTGTAAA CCAGCACTGC AATGCTGATG CACGGGAAGT 4921 AGTGGGATGG GAACACAAAT GGAGGATCCC GCGTCTCGAA CAGAGCGCGC AGAGGAACGC

4981 TGAAGGTCTC GCCTCTGTCG CACCTCAGCG CGGCATACAC CACAATAACC ACCTGACGAA 5041 TGCGCTTGGT TCTTCGTCCA TTAGCGAAGC GTCCGGTTCA CACACGTGCC ACGTTGGCGA 5101 GGTGGCAGGT GACAATGATC GGTGGAGCTG ATGGTCGAAA CGTTCACAGC CTAGGGATAT 5161 CGAATTCGGC CGACAGGACG CGCGTCAAAG GTGCTGGTCG TGTATGCCCT GGCCGGCAGG 5221 TCGTTGCTGC TGCTGGTTAG TGATTCCGCA ACCCTGATTT TGGCGTCTTA TTTTGGCGTG 5281 GCAAACGCTG GCGCCCGCGA GCCGGGCCGG CGGCGATGCG GTGCCCCACG GCTGCCGGAA 5341 TCCAAGGGAG GCAAGAGCGC CCGGGTCAGT TGAAGGGCTT TACGCGCAAG GTACAGCCGC 5401 TCCTGCAAGG CTGCGTGGTG GAATTGGACG TGCAGGTCCT GCTGAAGTTC CTCCACCGCC 5461 TCACCAGCGG ACAAAGCACC GGTGTATCAG GTCCGTGTCA TCCACTCTAA AGAACTCGAC 5521 TACGACCTAC TGATGGCCCT AGATTCTTCA TCAAAAACGC CTGAGACACT TGCCCAGGAT 5581 TGAAACTCCC TGAAGGGACC ACCAGGGGCC CTGAGTTGTT CCTTCCCCCC GTGGCGAGCT 5641 GCCAGCCAGG CTGTACCTGT GATCGAGGCT GGCGGGAAAA TAGGCTTCGT GTGCTCAGGT

5701 CATGGGAGGT GCAGGACAGC TCATGAAACG CCAACAATCG CACAATTCAT GTCAAGCTAA

5761 TCAGCTATTT CCTCTTCACG AGCTGTAATT GTCCCAAAAT TCTGGTCTAC CGGGGGTGAT 5821 CCTTCGTGTA CGGGCCCTTC CCTCAACCCT AGGTATGCGC GCATGCGGTC GCCGCGCAAC 5881 TCGCGCGAGG GCCGAGGGTT TGGGACGGGC CGTCCCGAAA TGCAGTTGCA CCCGGATGCG 5941 TGGCACCTTT TTTGCGATAA TTTATGCAAT GGACTGCTCT GCAAAATTCT GGCTCTGTCG

6001 CCAACCCTAG GATCAGCGGC GTAGGATTTC GTAATCATTC GTCCTGATGG GGAGCTACCG 6061 AC ACCCTAA TATCAGCCCG ACTGCCTGAC GCCAGCGTCC ACTTTTGTGC ACACATTCCA 6121 TTCGTGCCCA AGACATTTCA TTGTGGTGCG AAGCGTCCCC AGTTACGCTC ACCTGTTTCC 6181 CGACCTCCTT ACTGTTCTGT CGACAGAGCG GGCCCACAGG CCGGTCGCAG CCCATATGGC 6241 TTCCGCGGCA TTCACCATGT CGGCGTGCCC CGCGATGACT GGCAGGGCCC CTGGGGCACG 6301 TCGCTCCGGA CGGCCAGTCG CCACCCGCCT GAGGTACGTA TTCCAGTGCC TGGTGGCCAG 6361 CTGCATCGAC CCCTGCGACC AGTACCGCAG CAGCGCCAGC CTGAGCTTCC TGGGCGACAA 6421 CGGCTTCGCC AGCCTGTTCG GCAGCAAGCC CTTCATGAGC AACCGCGGCC ACCGCCGCCT 6481 GCGCCGCGCC AGCCACAGCG GCGAGGCCAT GGCCGTGGCC CTGCAGCCCG CCCAGGAGGC 6541 CGGCACCAAG AAGAAGCCCG TGATCAAGCA GCGCCGCGTG GTGGTGACCG GCATGGGCGT 6601 GGTGACCCCC CTGGGCCACG AGCCCGACGT GTTCTACAAC AACCTGCTGG ACGGCGTGAG 6661 CGGCATCAGC GAGATCGAGA CCTTCGACTG CACCCAGTTC CCCACCCGCA TCGCCGGCGA 6721 GATCAAGAGC TTCAGCACCG ACGGCTGGGT GGCCCCCAAG CTGAGCAAGC GCATGGACAA 6781 GTTCATGCTG TACCTGCTGA CCGCCGGCAA GAAGGCCCTG GCCGACGGCG GCATCACCGA 6841 CGAGGTGATG AAGGAGCTGG ACAAGCGCAA GTGCGGCGTG CTGATCGGCA GCGGCATGGG 6901 CGGCATGAAG GTGTTCAACG ACGCCATCGA GGCCCTGCGC GTGAGCTACA AGAAGATGAA 6961 CCCCTTCTGC GTGCCCTTCG CCACCACCAA CATGGGCAGC GCCATGCTGG CCATGGACCT 7021 GGGCTGGATG GGCCCCAACT ACAGCATCAG CACCGCCTGC GCCACCAGCA ACTTCTGCAT 7081 CCTGAACGCC GCCAACCACA TCATCCGCGG CGAGGCCGAC ATGATGCTGT GCGGCGGCAG 7141 CGACGCCGTG ATCATCCCCA TCGGCCTGGG CGGCTTCGTG GCCTGCCGCG CCCTGAGCCA 7201 GCGCAACAGC GACCCCACCA AGGCCAGCCG CCCCTGGGAC AGCAACCGCG ACGGCTTCGT 7261 GATGGGCGAG GGCGCCGGCG TGCTGCTGCT GGAGGAGCTG GAGCACGCCA AGAAGCGCGG 7321 CGCCACCATC TACGCCGAGT TCCTGGGCGG CAGCTTCACC TGCGACGCCT ACCACATGAC 7381 CGAGCCCCAC CCCGAGGGCG CCGGCGTGAT CCTGTGCATC GAGAAGGCCC TGGCCCAGGC 7441 CGGCGTGAGC AAGGAGGACG TGAACTACAT CAACGCCCAC GCCACCAGCA CCAGCGCCGG 7501 CGACATCAAG GAGTACCAGG CCCTGGCCCG CTGCTTCGGC CAGAACAGCG AGCTGCGCGT 7561 GAACAGCACC AAGAGCATGA TCGGCCACCT GCTGGGCGCC GCCGGCGGCG TGGAGGCCGT 7621 GACCGTGGTG CAGGCCATCC GCACCGGCTG GATTCACCCC AACCTGAACC TGGAGGACCC 7681 CGACAAGGCC GTGGACGCCA AGCTGCTGGT GGGCCCCAAG AAGGAGCGCC TGAACGTGAA 7741 GGTGGGCCTG AGCAACAGCT TCGGCTTCGG CGGCCACAAC AGCAGCATCC TGTTCGCCCC 7801 CTGCAACGTG TGAATGCATA CGGAGCGTCG TGCGGGAGGG AGTGTGCCGA GCGGGGAGTC 7861 CCGGTCTGTG CGAGGCCCGG CAGCTGACGC TGGCGAGCCG TACGCCCCGA GGGTCCCCCT 7921 CCCCTCCCCC CTCTTCCCCT TCCCTCTGAC GGCCGCGCCT GTTTTTGCAT GTTCAGCGAC 7981 CTTAAGGATC TAAGTAAGAT TCGAAGCGCT CGACCGTGCC GGACGGACTG CAGCCCCATG 8041 TCGTAGTGAC CGCCAATGTA AGTGGGCTGG CGTTTCCCTG TACGTGACTC AACGTCACTG 8101 CACGCGCACC ACCCTCTCGA CCGGCAGGAC CAGGCATCGC GAGATACAGC GCGAGCCAGA 8161 CACGGAGTGC CGAGCTATGC GCACGCTCCA ACTAGATATC GAATTCGGCC GACAGGACGC 8221 GCGTCAAAGG TGCTGGTCGT GTATGCCCTG GCCGGCAGGT CGTTGCTGCT GCTGGTTAGT 8281 GATTCCGCAA CCCTGATTTT GGCGTCTTAT TTTGGCGTGG CAAACGCTGG CGCCCGCGAG 8341 CCGGGCCGGC GGCGATGCGG TGCCCCACGG CTGCCGGAAT CCAAGGGAGG CAAGAGCGCC 8401 CGGGTCAGTT GAAGGGCTTT ACGCGCAAGG TACAGCCGCT CCTGCAAGGC TGCGTGGTGG 8461 AATTGGACGT GCAGGTCCTG CTGAAGTTCC TCCACCGCCT CACCAGCGGA CAAAGCACCG 8521 GTGTATCAGG TCCGTGTCAT CCACTCTAAA GAACTCGACT ACGACCTACT GATGGCCCTA 8581 GATTCTTCAT CAAAAACGCC TGAGACACTT GCCCAGGATT GAAACTCCCT GAAGGGACCA 8641 CCAGGGGCCC TGAGTTGTTC CTTCCCCCCG TGGCGAGCTG CCAGCCAGGC TGTACCTGTG 8701 ATCGAGGCTG GCGGGAAAAT AGGCTTCGTG TGCTCAGGTC ATGGGAGGTG CAGGACAGCT 8761 CATGAAACGC CAACAATCGC ACAATTCATG TCAAGCTAAT CAGCTATTTC CTCTTCACGA 8821 GCTGTAATTG TCCCAAAATT CTGGTCTACC GGGGGTGATC CTTCGTGTAC GGGCCCTTCC 8881 CTCAACCCTA GGTATGCGCG CATGCGGTCG CCGCGCAACT CGCGCGAGGG CCGAGGGTTT 8941 GGGACGGGCC GTCCCGAAAT GCAGTTGCAC CCGGATGCGT GGCACCTTTT TTGCGATAAT 9001 TTATGCAATG GACTGCTCTG CAAAATTCTG GCTCTGTCGC CAACCCTAGG ATCAGCGGCG 9061 TAGGATTTCG TAATCATTCG TCCTGATGGG GAGCTACCGA CTACCCTAAT ATCAGCCCGA 9121 CTGCCTGACG CCAGCGTCCA CTTTTGTGCA CACATTCCAT TCGTGCCCAA GACATTTCAT 9181 TGTGGTGCGA AGCGTCCCCA GTTACGCTCA CCTGTTTCCC GACCTCCTTA CTGTTCTGTC 9241 GACAGAGCGG GCCCACAGGC CGGTCGCAGC CACTAGTATG GCCACCGCAT CCACTTTCTC 9301 GGCGTTCAAT GCCCGCTGCG GCGACCTGCG TCGCTCGGCG GGCTCCGGGC CCCGGCGCCC 9361 AGCGAGGCCC CTCCCCGTGC GCGGGCGCGC CCCCAAGGCC AACGGCAGCG CCGTGAGCCT 9421 GAAGTCCGGC AGCCTGAACA CCCTGGAGGA CCCCCCCAGC AGCCCCCCCC CCCGCACCTT 9481 CCTGAACCAG CTGCCCGACT GGAGCCGCCT GCGCACCGCC ATCACCACCG TGTTCGTGGC 9541 CGCCGAGAAG CAGTTCACCC GCCTGGACCG CAAGAGCAAG CGCCCCGACA TGCTGGTGGA 9601 CTGGTTCGGC AGCGAGACCA TCGTGCAGGA CGGCCTGGTG TTCCGCGAGC GCTTCAGCAT 9661 CCGCAGCTAC GAGATCGGCG CCGACCGCAC CGCCAGCATC GAGACCCTGA TGAACCACCT 9721 GCAGGACACC AGCCTGAACC ACTGCAAGAG CGTGGGCCTG CTGAACGACG GCTTCGGCCG 9781 CACCCCCGAG ATGTGCACCC GCGACCTGAT CTGGGTGCTG ACCAAGATGC AGATCGTGGT 9841 GAACCGCTAC CCCACCTGGG GCGACACCGT GGAGATCAAC AGCTGGTTCA GCCAGAGCGG 9901 CAAGATCGGC ATGGGCCGCG AGTGGCTGAT CAGCGACTGC AACACCGGCG AGATCCTGGT 9961 GCGCGCCACC AGCGCCTGGG CCATGATGAA CCAGAAGACC CGCCGCTTCA GCAAGCTGCC 10021 CTGCGAGGTG CGCCAGGAGA TCGCCCCCCA CTTCGTGGAC GCCCCCCCCG TGATCGAGGA 10081 CAACGACCGC AAGCTGCACA AGTTCGACGT GAAGACCGGC GACAGCATCT GCAAGGGCCT 10141 GACCCCCGGC TGGAACGACT TCGACGTGAA CCAGCACGTG AGCAACGTGA AGTACATCGG 10201 CTGGATTCTG GAGAGCATGC CCACCGAGGT GCTGGAGACC CAGGAGCTGT GCAGCCTGAC 10261 CCTGGAGTAC CGCCGCGAGT GCGGCCGCGA GAGCGTGGTG GAGAGCGTGA CCAGCATGAA

10321 CCCCAGCAAG GTGGGCGACC GCAGCCAGTA CCAGCACCTG CTGCGCCTGG AGGACGGCGC

10381 CGACATCATG AAGGGCCGCA CCGAGTGGCG CCCCAAGAAC GCCGGCACCA ACCGCGCCAT

10441 CAGCACCTGA TTAATTAACT CGAGGCAGCA GCAGCTCGGA TAGTATCGAC ACACTCTGGA

10501 CGCTGGTCGT GTGATGGACT GTTGCCGCCA CACTTGCTGC CTTGACCTGT GAATATCCCT

10561 GCCGCTTTTA TCAAACAGCC TCAGTGTGTT TGATCTTGTG TGTACGCGCT TTTGCGAGTT

10621 GCTAGCTGCT TGTGCTATTT GCGAATACCA CCCCCAGCAT CCCCTTCCCT CGTTTCATAT

10681 CGCTTGCATC CCAACCGCAA CTTATCTACG CTGTCCTGCT ATCCCTCAGC GCTGCTCCTG

10741 CTCCTGCTCA CTGCCCCTCG CACAGCCTTG GTTTGGGCTC CGCCTGTATT CTCCTGGTAC

10801 TGCAACCTGT AAACCAGCAC TGCAATGCTG ATGCACGGGA AGTAGTGGGA TGGGAACACA

10861 AATGGAAAGC TTGAGCTCTT GTTTTCCAGA AGGAGTTGCT CCTTGAGCCT TTCATTCTCA

10921 GCCTCGATAA CCTCCAAAGC CGCTCTAATT GTGGAGGGGG TTCGAATTTA AAAGCTTGGA

10981 ATGTTGGTTC GTGCGTCTGG AACAAGCCCA GACTTGTTGC TCACTGGGAA AAGGACCATC

11041 AGCTCCAAAA AACTTGCCGC TCAAACCGCG TACCTCTGCT TTCGCGCAAT CTGCCCTGTT

11101 GAAATCGCCA CCACATTCAT ATTGTGACGC TTGAGCAGTC TGTAATTGCC TCAGAATGTG

11161 GAATCATCTG CCCCCTGTGC GAGCCCATGC CAGGCATGTC GCGGGCGAGG ACACCCGCCA

11221 CTCGTACAGC AGACCATTAT GCTACCTCAC AATAGTTCAT AACAGTGACC ATATTTCTCG

11281 AAGCTCCCCA ACGAGCACCT CCATGCTCTG AGTGGCCACC CCCCGGCCCT GGTGCTTGCG

11341 GAGGGCAGGT CAACCGGCAT GGGGCTACCG AAATCCCCGA CCGGATCCCA CCACCCCCGC

11401 GATGGGAAGA ATCTCTCCCC GGGATGTGGG CCCACCACCA GCACAACCTG CTGGCCCAGG

11461 CGAGCGTCAA ACCATACCAC ACAAATATCC TTGGCATCGG CCCTGAATTC CTTCTGCCGC

11521 TCTGCTACCC GGTGCTTCTG TCCGAAGCAG GGGTTGCTAG GGATCGCTCC GAGTCCGCAA

11581 ACCCTTGTCG CGTGGCGGGG CTTGTTCGAG CTTGAAGAGC CTCTAGAGTC GACCTGCAGG

11641 CATGCAAGCT TGGCGTAATC ATGGTCATAG CTGTTTCCTG TGTGAAATTG TTATCCGCTC

11701 ACAATTCCAC ACAACATACG AGCCGGAAGC ATAAAGTGTA AAGCCTGGGG TGCCTAATGA

11761 GTGAGCTAAC TCACATTAAT TGCGTTGCGC TCACTGCCCG CTTTCCAGTC GGGAAACCTG

11821 TCGTGCCAGC TGCATTAATG AATCGGCCAA CGCGCGGGGA GAGGCGGTTT GCGTATTGGG

11881 CGCTCTTCCG CTTCCTCGCT CACTGACTCG CTGCGCTCGG TCGTTCGGCT GCGGCGAGCG

11941 GTATCAGCTC ACTCAAAGGC GGTAATACGG TTATCCACAG AATCAGGGGA TAACGCAGGA

12001 AAGAACATGT GAGCAAAAGG CCAGCAAAAG GCCAGGAACC GTAAAAAGGC CGCGTTGCTG

12061 GCGTTTTTCC ATAGGCTCCG CCCCCCTGAC GAGCATCACA AAAATCGACG CTCAAGTCAG

12121 AGGTGGCGAA ACCCGACAGG ACTATAAAGA TACCAGGCGT TTCCCCCTGG AAGCTCCCTC

12181 GTGCGCTCTC CTGTTCCGAC CCTGCCGCTT ACCGGATACC TGTCCGCCTT TCTCCCTTCG

12241 GGAAGCGTGG CGCTTTCTCA TAGCTCACGC TGTAGGTATC TCAGTTCGGT GTAGGTCGTT

12301 CGCTCCAAGC TGGGCTGTGT GCACGAACCC CCCGTTCAGC CCGACCGCTG CGCCTTATCC

12361 GGTAACTATC GTCTTGAGTC CAACCCGGTA AGACACGACT TATCGC

SEQ ID NO: 233

pSZ1500

1 CACTGGCAGC AGCCACTGGT AACAGGATTA GCAGAGCGAG GTATGTAGGC GGTGCTACAG

61 AGTTCTTGAA GTGGTGGCCT AACTACGGCT ACACTAGAAG GACAGTATTT GGTATCTGCG

121 CTCTGCTGAA GCCAGTTACC TTCGGAAAAA GAGTTGGTAG CTCTTGATCC GGCAAACAAA

181 CCACCGCTGG TAGCGGTGGT TTTTTTGTTT GCAAGCAGCA GATTACGCGC AGAAAAAAAG

241 GATCTCAAGA AGATCCTTTG ATCTTTTCTA CGGGGTCTGA CGCTCAGTGG AACGAAAACT

301 CACGTTAAGG GATTTTGGTC ATGAGATTAT CAAAAAGGAT CTTCACCTAG ATCCTTTTAA

361 AT AAAAATG AAGTTTTAAA TCAATCTAAA GTATATATGA GTAAACTTGG TCTGACAGTT

421 ACCAATGCTT AATCAGTGAG GCACCTATCT CAGCGATCTG TCTATTTCGT TCATCCATAG

481 TTGCCTGACT CCCCGTCGTG TAGATAACTA CGATACGGGA GGGCTTACCA TCTGGCCCCA

541 GTGCTGCAAT GATACCGCGA GACCCACGCT CACCGGCTCC AGATT ATCA GCAATAAACC

601 AGCCAGCCGG AAGGGCCGAG CGCAGAAGTG GTCCTGCAAC TTTATCCGCC TCCATCCAGT

661 CTATTAATTG TTGCCGGGAA GCTAGAGTAA GTAGTTCGCC AGTTAATAGT TTGCGCAACG

721 TTGTTGCCAT TGCTACAGGC ATCGTGGTGT CACGCTCGTC GTTTGGTATG GCTTCATTCA

781 GCTCCGGTTC CCAACGATCA AGGCGAGTTA CATGATCCCC CATGTTGTGC AAAAAAGCGG

841 TTAGCTCCTT CGGTCCTCCG ATCGTTGTCA GAAGTAAGTT GGCCGCAGTG TTATCACTCA

901 TGGTTATGGC AGCACTGCAT AATTCTCTTA CTGTCATGCC ATCCGTAAGA TGCTTTTCTG

961 TGACTGGTGA GTACTCAACC AAGTCATTCT GAGAATAGTG TATGCGGCGA CCGAGTTGCT

1021 CTTGCCCGGC GTCAATACGG GATAATACCG CGCCACATAG CAGAACTTTA AAAGTGCTCA

1081 TCATTGGAAA ACGTTCTTCG GGGCGAAAAC TCTCAAGGAT CTTACCGCTG TTGAGATCCA

1141 GTTCGATGTA ACCCACTCGT GCACCCAACT GATCTTCAGC ATCTTTTACT TTCACCAGCG 1201 TTTCTGGGTG AGCAAAAACA GGAAGGCAAA ATGCCGCAAA AAAGGGAATA AGGGCGACAC

1261 GGAAATGTTG AATACTCATA CTCTTCCTTT TTCAATATTA TTGAAGCATT TATCAGGGTT

1321 ATTGTCTCAT GAGCGGATAC ATATTTGAAT GTATTTAGAA AAATAAACAA ATAGGGGTTC

1381 CGCGCACATT TCCCCGAAAA GTGCCACCTG ACGTCTAAGA AACCATTATT ATCATGACAT

1441 TAACCTATAA AAATAGGCGT ATCACGAGGC CCTTTCGTCT CGCGCGTTTC GGTGATGACG

1501 GTGAAAACCT CTGACACATG CAGCTCCCGG AGACGGTCAC AGCTTGTCTG TAAGCGGATG

1561 CCGGGAGCAG ACAAGCCCGT CAGGGCGCGT CAGCGGGTGT TGGCGGGTGT CGGGGCTGGC

1621 TTAACTATGC GGCATCAGAG CAGATTGTAC TGAGAGTGCA CCATATGCGG TGTGAAATAC

1681 CGCACAGATG CGTAAGGAGA AAATACCGCA TCAGGCGCCA TTCGCCATTC AGGCTGCGCA

1741 ACTGTTGGGA AGGGCGATCG GTGCGGGCCT CTTCGCTATT ACGCCAGCTG GCGAAAGGGG

1801 GATGTGCTGC AAGGCGATTA AGTTGGGTAA CGCCAGGGTT TTCCCAGTCA CGACGTTGTA

1861 AAACGACGGC CAGTGAATTG ATGCATGCTC TTCGGGCTGG TCTGAATCCT TCAGGCGGGT

1921 GTTACCCGAG AAAGAAAGGG TGCCGATTTC AAAGCAGACC CATGTGCCGG GCCCTGTGGC

1981 CTGTGTTGGC GCCTATGTAG TCACCCCCCC TCACCCAATT GTCGCCAGTT TGCGCACTCC

2041 ATAAACTCAA AACAGCAGCT TCTGAGCTGC GCTGTTCAAG AACACCTCTG GGGTTTGCTC

2101 ACCCGCGAGG TCGACGCCCA GCATGGCTAT CAAGACGAAC AGGCAGCCTG TGGAGAAGCC

2161 TCCGTTCACG ATCGGGACGC TGCGCAAGGC CATCCCCGCG CACTGTTTCG AGCGCTCGGC

2221 GCTTCGTAGC AGCATGTACC TGGCCTTTGA CATCGCGGTC ATGTCCCTGC TCTACGTCGC

2281 GTCGACGTAC ATCGACCCTG CACCGGTGCC TACGTGGGTC AAGTACGGCA TCATGTGGCC

2341 GCTCTACTGG TTCTTCCAGG TGTGTTTGAG GGTTTTGGTT GCCCGTATTG AGGTCCTGGT

2401 GGCGCGCATG GAGGAGAAGG CGCCTGTCCC GCTGACCCCC CCGGCTACCC TCCCGGCACC

2461 TTCCAGGGCG CCTTCGGCAC GGGTGTCTGG GTGTGCGCGC ACGAGTGCGG CCACCAGGCC

2521 TTTTCCTCCA GCCAGGCCAT CAACGACGGC GTGGGCCTGG TGTTCCACAG CCTGCTGCTG

2581 GTGCCCTACT ACTCCTGGAA GCACTCGCAC CGGGTACCCT TTCTTGCGCT ATGACACTTC

2641 CAGCAAAAGG TAGGGCGGGC TGCGAGACGG CTTCCCGGCG CTGCATGCAA CACCGATGAT

2701 GCTTCGACCC CCCGAAGCTC CTTCGGGGCT GCATGGGCGC TCCGATGCCG CTCCAGGGCG

2761 AGCGCTGTTT AAATAGCCAG GCCCCCGATT GCAAAGACAT TATAGCGAGC TACCAAAGCC

2821 ATATTCAAAC ACCTAGATCA CTACCACTTC TACACAGGCC ACTCGAGCTT GTGATCGCAC

2881 TCCGCTAAGG GGGCGCCTCT TCCTCTTCGT TTCAGTCACA ACCCGCAAAC TCTAGAATAT

2941 CAATGCTGCT GCAGGCCTTC CTGTTCCTGC TGGCCGGCTT CGCCGCCAAG ATCAGCGCCT

3001 CCATGACGAA CGAGACGTCC GACCGCCCCC TGGTGCACTT CACCCCCAAC AAGGGCTGGA 3061 TGAACGACCC CAACGGCCTG TGGTACGACG AGAAGGACGC CAAGTGGCAC CTGTACTTCC 3121 AGTACAACCC GAACGACACC GTCTGGGGGA CGCCCTTGTT CTGGGGCCAC GCCACGTCCG 3181 ACGACCTGAC CAACTGGGAG GACCAGCCCA TCGCCATCGC CCCGAAGCGC AACGACTCCG 3241 GCGCCTTCTC CGGCTCCATG GTGGTGGACT ACAACAACAC CTCCGGCTTC TTCAACGACA 3301 CCATCGACCC GCGCCAGCGC TGCGTGGCCA TCTGGACCTA CAACACCCCG GAGTCCGAGG 3361 AGCAGTACAT CTCCTACAGC CTGGACGGCG GCTACACCTT CACCGAGTAC CAGAAGAACC 3421 CCGTGCTGGC CGCCAACTCC ACCCAGTTCC GCGACCCGAA GGTCTTCTGG TACGAGCCCT 3481 CCCAGAAGTG GATCATGACC GCGGCCAAGT CCCAGGACTA CAAGATCGAG ATCTACTCCT 3541 CCGACGACCT GAAGTCCTGG AAGCTGGAGT CCGCGTTCGC CAACGAGGGC TTCCTCGGCT 3601 ACCAGTACGA GTGCCCCGGC CTGATCGAGG TCCCCACCGA GCAGGACCCC AGCAAGTCCT 3661 ACTGGGTGAT GTTCATCTCC ATCAACCCCG GCGCCCCGGC CGGCGGCTCC TTCAACCAGT 3721 ACTTCGTCGG CAGCTTCAAC GGCACCCACT TCGAGGCCTT CGACAACCAG TCCCGCGTGG 3781 TGGACTTCGG CAAGGACTAC TACGCCCTGC AGACCTTCTT CAACACCGAC CCGACCTACG 3841 GGAGCGCCCT GGGCATCGCG TGGGCCTCCA ACTGGGAGTA CTCCGCCTTC GTGCCCACCA 3901 ACCCCTGGCG CTCCTCCATG TCCCTCGTGC GCAAGTTCTC CCTCAACACC GAGTACCAGG

3961 CCAACCCGGA GACGGAGCTG ATCAACCTGA AGGCCGAGCC GATCCTGAAC ATCAGCAACG

4021 CCGGCCCCTG GAGCCGGTTC GCCACCAACA CCACGTTGAC GAAGGCCAAC AGCTACAACG 4081 TCGACCTGTC CAACAGCACC GGCACCCTGG AGTTCGAGCT GGTGTACGCC GTCAACACCA 4141 CCCAGACGAT CTCCAAGTCC GTGTTCGCGG ACCTCTCCCT CTGGTTCAAG GGCCTGGAGG 4201 ACCCCGAGGA GTACCTCCGC ATGGGCTTCG AGGTGTCCGC GTCCTCCTTC TTCCTGGACC 4261 GCGGGAACAG CAAGGTGAAG TTCGTGAAGG AGAACCCCTA CTTCACCAAC CGCATGAGCG 4321 TGAACAACCA GCCCTTCAAG AGCGAGAACG ACCTGTCCTA CTACAAGGTG TACGGCTTGC 4381 TGGACCAGAA CATCCTGGAG CTGTACTTCA ACGACGGCGA CGTCGTGTCC ACCAACACCT 4441 ACTTCATGAC CACCGGGAAC GCCCTGGGCT CCGTGAACAT GACGACGGGG GTGGACAACC 4501 TGTTCTACAT CGACAAGTTC CAGGTGCGCG AGGTCAAGTG ACAATTGGCA GCAGCAGCTC 4561 GGATAGTATC GACACACTCT GGACGCTGGT CGTGTGATGG ACTGTTGCCG CCACACTTGC 4621 TGCCTTGACC TGTGAATATC CCTGCCGCTT TTATCAAACA GCCTCAGTGT GTTTGATCTT 4681 GTGTGTACGC GCTTTTGCGA GTTGCTAGCT GCTTGTGCTA TTTGCGAATA CCACCCCCAG 4741 CATCCCCTTC CCTCGTTTCA TATCGCTTGC ATCCCAACCG CAACTTATCT ACGCTGTCCT 4801 GCTATCCCTC AGCGCTGCTC CTGCTCCTGC TCACTGCCCC TCGCACAGCC TTGGTTTGGG 4861 CTCCGCCTGT ATTCTCCTGG TACTGCAACC TGTAAACCAG CACTGCAATG CTGATGCACG

4921 GGAAGTAGTG GGATGGGAAC ACAAATGGAG GATCCCGCGT CTCGAACAGA GCGCGCAGAG

4981 GAACGCTGAA GGTCTCGCCT CTGTCGCACC TCAGCGCGGC ATACACCACA ATAACCACCT 5041 GACGAATGCG CTTGGTTCTT CGTCCATTAG CGAAGCGTCC GGTTCACACA CGTGCCACGT 5101 TGGCGAGGTG GCAGGTGACA ATGATCGGTG GAGCTGATGG TCGAAACGTT CACAGCCTAG 5161 GGATATCGAA TTCGGCCGAC AGGACGCGCG TCAAAGGTGC TGGTCGTGTA TGCCCTGGCC 5221 GGCAGGTCGT TGCTGCTGCT GGTTAGTGAT TCCGCAACCC TGATTTTGGC GTCTTATTTT 5281 GGCGTGGCAA ACGCTGGCGC CCGCGAGCCG GGCCGGCGGC GATGCGGTGC CCCACGGCTG 5341 CCGGAATCCA AGGGAGGCAA GAGCGCCCGG GTCAGTTGAA GGGCTTTACG CGCAAGGTAC 5401 AGCCGCTCCT GCAAGGCTGC GTGGTGGAAT TGGACGTGCA GGTCCTGCTG AAGTTCCTCC 5461 ACCGCCTCAC CAGCGGACAA AGCACCGGTG TATCAGGTCC GTGTCATCCA CTCTAAAGAA 5521 CTCGACTACG ACCTACTGAT GGCCCTAGAT TCTTCATCAA AAACGCCTGA GACACTTGCC 5581 CAGGATTGAA ACTCCCTGAA GGGACCACCA GGGGCCCTGA GTTGTTCCTT CCCCCCGTGG 5641 CGAGCTGCCA GCCAGGCTGT ACCTGTGATC GAGGCTGGCG GGAAAATAGG CTTCGTGTGC

5701 TCAGGTCATG GGAGGTGCAG GACAGCTCAT GAAACGCCAA CAATCGCACA ATTCATGTCA

5761 AGCTAATCAG CTATTTCCTC TTCACGAGCT GTAATTGTCC CAAAATTCTG GTCTACCGGG 5821 GGTGATCCTT CGTGTACGGG CCCTTCCCTC AACCCTAGGT ATGCGCGCAT GCGGTCGCCG 5881 CGCAACTCGC GCGAGGGCCG AGGGTTTGGG ACGGGCCGTC CCGAAATGCA GTTGCACCCG 5941 GATGCGTGGC ACCTTTTTTG CGATAATTTA TGCAATGGAC TGCTCTGCAA AATTCTGGCT

6001 CTGTCGCCAA CCCTAGGATC AGCGGCGTAG GATTTCGTAA TCATTCGTCC TGATGGGGAG 6061 CTACCGACTA CCCTAATATC AGCCCGACTG CCTGACGCCA GCGTCCACTT TTGTGCACAC 6121 ATTCCATTCG TGCCCAAGAC ATTTCATTGT GGTGCGAAGC GTCCCCAGTT ACGCTCACCT 6181 GTTTCCCGAC CTCCTTACTG TTCTGTCGAC AGAGCGGGCC CACAGGCCGG TCGCAGCCAC 6241 TAGTATGGCC ACCGCATCCA CTTTCTCGGC GTTCAATGCC CGCTGCGGCG ACCTGCGTCG 6301 CTCGGCGGGC TCCGGGCCCC GGCGCCCAGC GAGGCCCCTC CCCGTGCGCG GGCGCGCCGC 6361 CACCGGCGAG CAGCCCTCCG GCGTGGCCTC CCTGCGCGAG GCCGACAAGG AGAAGTCCCT 6421 GGGCAACCGC CTGCGCCTGG GCTCCCTGAC CGAGGACGGC CTGTCCTACA AGGAGAAGTT 6481 CGTGATCCGC TGCTACGAGG TGGGCATCAA CAAGACCGCC ACCATCGAGA CCATCGCCAA 6541 CCTGCTGCAG GAGGTGGGCG GCAACCACGC CCAGGGCGTG GGCTTCTCCA CCGACGGCTT 6601 CGCCACCACC ACCACCATGC GCAAGCTGCA CCTGATCTGG GTGACCGCCC GCATGCACAT 6661 CGAGATCTAC CGCTACCCCG CCTGGTCCGA CGTGATCGAG ATCGAGACCT GGGTGCAGGG 6721 CGAGGGCAAG GTGGGCACCC GCCGCGACTG GATCCTGAAG GACTACGCCA ACGGCGAGGT 6781 GATCGGCCGC GCCACCTCCA AGTGGGTGAT GATGAACGAG GACACCCGCC GCCTGCAGAA 6841 GGTGTCCGAC GACGTGCGCG AGGAGTACCT GGTGTTCTGC CCCCGCACCC TGCGCCTGGC 6901 CTTCCCCGAG GAGAACAACA ACTCCATGAA GAAGATCCCC AAGCTGGAGG ACCCCGCCGA

6961 GTACTCCCGC CTGGGCCTGG TGCCCCGCCG CTCCGACCTG GACATGAACA AGCACGTGAA 7021 CAACGTGACC TACATCGGCT GGGCCCTGGA GTCCATCCCC CCCGAGATCA TCGACACCCA 7081 CGAGCTGCAG GCCATCACCC TGGACTACCG CCGCGAGTGC CAGCGCGACG ACATCGTGGA 7141 CTCCCTGACC TCCCGCGAGC CCCTGGGCAA CGCCGCCGGC GTGAAGTTCA AGGAGATCAA 7201 CGGCTCCGTG TCCCCCAAGA AGGACGAGCA GGACCTGTCC CGCTTCATGC ACCTGCTGCG 7261 CTCCGCCGGC TCCGGCCTGG AGATCAACCG CTGCCGCACC GAGTGGCGCA AGAAGCCCGC 7321 CAAGCGCATG GACTACAAGG ACCACGACGG CGACTACAAG GACCACGACA TCGACTACAA 7381 GGACGACGAC GACAAGTGAA TCGATAGATC TCTTAAGGCA GCAGCAGCTC GGATAGTATC 7441 GACACACTCT GGACGCTGGT CGTGTGATGG ACTGTTGCCG CCACACTTGC TGCCTTGACC 7501 TGTGAATATC CCTGCCGCTT TTATCAAACA GCCTCAGTGT GTTTGATCTT GTGTGTACGC 7561 GCTTTTGCGA GTTGCTAGCT GCTTGTGCTA TTTGCGAATA CCACCCCCAG CATCCCCTTC 7621 CCTCGTTTCA TATCGCTTGC ATCCCAACCG CAACTTATCT ACGCTGTCCT GCTATCCCTC 7681 AGCGCTGCTC CTGCTCCTGC TCACTGCCCC TCGCACAGCC TTGGTTTGGG CTCCGCCTGT

7741 ATTCTCCTGG TACTGCAACC TGTAAACCAG CACTGCAATG CTGATGCACG GGAAGTAGTG 7801 GGATGGGAAC ACAAATGGAA AGCTTAATTA AGAGCTCCCG CCACCACTCC AACACGGGGT 7861 GCCTGGACAA GGACGAGGTG TTTGTGCCGC CGCACCGCGC AGTGGCGCAC GAGGGCCTGG 7921 AGTGGGAGGA GTGGCTGCCC ATCCGCATGG GCAAGGTGCT GGTCACCCTG ACCCTGGGCT 7981 GGCCGCTGTA CCTCATGTTC AACGTCGCCT CGCGGCCGTA CCCGCGCTTC GCCAACCACT

8041 TTGACCCGTG GTCGCCCATC TTCAGCAAGC GCGAGCGCAT CGAGGTGGTC ATCTCCGACC 8101 TGGCGCTGGT GGCGGTGCTC AGCGGGCTCA GCGTGCTGGG CCGCACCATG GGCTGGGCCT 8161 GGCTGGTCAA GACCTACGTG GTGCCCTACC TGATCGTGAA CATGTGGCTC GTGCTCATCA 8221 CGCTGCTCCA GCACACGCAC CCGGCGCTGC CGCACTACTT CGAGAAGGAC TGGGACTGGC 8281 TGCGCGGCGC CATGGCCACC GTGGACCGCT CCATGGGCCC GCCCTTCATG GACAACATCC 8341 TGCACCACAT CTCCGACACC CACGTGCTGC ACCACCTCTT CAGCACCATC CCGCACTACC 8401 ACGCCGAGGA GGCCTCCGCC GCCATCAGGC CCATCCTGGG CAAGTACTAC CAGTCCGACA 8461 GCCGCTGGGT CGGCCGCGCC CTGTGGGAGG ACTGGCGCGA CTGCCGCTAC GTCGTCCCGG 8521 ACGCGCCCGA GGACGACTCC GCGCTCTGGT TCCACAAGTG AG GAGTGAG AAGAGCCTCT

8581 AGAGTCGACC TGCAGGCATG CAAGCTTGGC GTAATCATGG TCATAGCTGT TTCCTGTGTG

8641 AAATTGTTAT CCGCTCACAA TTCCACACAA CATACGAGCC GGAAGCATAA AGTGTAAAGC

8701 CTGGGGTGCC TAATGAGTGA GCTAACTCAC ATTAATTGCG TTGCGCTCAC TGCCCGCTTT

8761 CCAGTCGGGA AACCTGTCGT GCCAGCTGCA TTAATGAATC GGCCAACGCG CGGGGAGAGG

8821 CGGTTTGCGT ATTGGGCGCT CTTCCGCTTC CTCGCTCACT GACTCGCTGC GCTCGGTCGT

8881 TCGGCTGCGG CGAGCGGTAT CAGCTCACTC AAAGGCGGTA ATACGGTTAT CCACAGAATC

8941 AGGGGATAAC GCAGGAAAGA ACATGTGAGC AAAAGGCCAG CAAAAGGCCA GGAACCGTAA

9001 AAAGGCCGCG TTGCTGGCGT TTTTCCATAG GCTCCGCCCC CCTGACGAGC ATCACAAAAA

9061 TCGACGCTCA AGTCAGAGGT GGCGAAACCC GACAGGACTA TAAAGATACC AGGCGTTTCC

9121 CCCTGGAAGC TCCCTCGTGC GCTCTCCTGT TCCGACCCTG CCGCTTACCG GATACCTGTC

9181 CGCCTTTCTC CCTTCGGGAA GCGTGGCGCT TTCTCATAGC TCACGCTGTA GGTATCTCAG

9241 TTCGGTGTAG GTCGTTCGCT CCAAGCTGGG CTGTGTGCAC GAACCCCCCG TTCAGCCCGA

9301 CCGCTGCGCC TTATCCGGTA ACTATCGTCT TGAGTCCAAC CCGGTAAGAC ACGACTTATC

9361 GC

SEQ ID NO: 234

pSZ1501_FADc-inv

1 CACTGGCAGC AGCCACTGGT AACAGGATTA GCAGAGCGAG GTATGTAGGC GGTGCTACAG

61 AGTTCTTGAA GTGGTGGCCT AACTACGGCT ACACTAGAAG GACAGTATTT GGTATCTGCG

121 CTCTGCTGAA GCCAGTTACC TTCGGAAAAA GAGTTGGTAG CTCTTGATCC GGCAAACAAA

181 CCACCGCTGG TAGCGGTGGT TTTTTTGTTT GCAAGCAGCA GATTACGCGC AGAAAAAAAG

241 GATCTCAAGA AGATCCTTTG ATCTTTTCTA CGGGGTCTGA CGCTCAGTGG AACGAAAACT

301 CACGTTAAGG GATTTTGGTC ATGAGATTAT CAAAAAGGAT CTTCACCTAG ATCCTTTTAA

361 ATTAAAAATG AAGTTTTAAA TCAATCTAAA GTATATATGA GTAAACTTGG TCTGACAGTT

421 ACCAATGCTT AATCAGTGAG GCACCTATCT CAGCGATCTG TCTATTTCGT TCATCCATAG

481 TTGCCTGACT CCCCGTCGTG TAGATAACTA CGATACGGGA GGGCTTACCA TCTGGCCCCA

541 GTGCTGCAAT GATACCGCGA GACCCACGCT CACCGGCTCC AGATTTATCA GCAATAAACC

601 AGCCAGCCGG AAGGGCCGAG CGCAGAAGTG GTCCTGCAAC TTTATCCGCC TCCATCCAGT

661 CTATTAATTG TTGCCGGGAA GCTAGAGTAA GTAGTTCGCC AGTTAATAGT TTGCGCAACG

721 TTGTTGCCAT TGCTACAGGC ATCGTGGTGT CACGCTCGTC GTTTGGTATG GCTTCATTCA

781 GCTCCGGTTC CCAACGATCA AGGCGAGTTA CATGATCCCC CATGTTGTGC AAAAAAGCGG

841 TTAGCTCCTT CGGTCCTCCG ATCGTTGTCA GAAGTAAGTT GGCCGCAGTG TTATCACTCA

901 TGGTTATGGC AGCACTGCAT AATTCTCTTA CTGTCATGCC ATCCGTAAGA TGCTTTTCTG

961 TGACTGGTGA GTACTCAACC AAGTCATTCT GAGAATAGTG TATGCGGCGA CCGAGTTGCT

1021 CTTGCCCGGC GTCAATACGG GATAATACCG CGCCACATAG CAGAACTTTA AAAGTGCTCA

1081 TCATTGGAAA ACGTTCTTCG GGGCGAAAAC TCTCAAGGAT CTTACCGCTG TTGAGATCCA

1141 GTTCGATGTA ACCCACTCGT GCACCCAACT GATCTTCAGC ATCTTTTACT TTCACCAGCG

1201 TTTCTGGGTG AGCAAAAACA GGAAGGCAAA ATGCCGCAAA AAAGGGAATA AGGGCGACAC

1261 GGAAATGTTG AATACTCATA CTCTTCCTTT TTCAATATTA TTGAAGCATT TATCAGGGTT

1321 ATTGTCTCAT GAGCGGATAC ATATTTGAAT GTATTTAGAA AAATAAACAA ATAGGGGTTC

1381 CGCGCACATT TCCCCGAAAA GTGCCACCTG ACGTCTAAGA AACCATTATT ATCATGACAT

1441 TAACCTATAA AAATAGGCGT ATCACGAGGC CCTTTCGTCT CGCGCGTTTC GGTGATGACG

1501 GTGAAAACCT CTGACACATG CAGCTCCCGG AGACGGTCAC AGCTTGTCTG TAAGCGGATG

1561 CCGGGAGCAG ACAAGCCCGT CAGGGCGCGT CAGCGGGTGT TGGCGGGTGT CGGGGCTGGC

1621 TTAACTATGC GGCATCAGAG CAGATTGTAC TGAGAGTGCA CCATATGCGG TGTGAAATAC

1681 CGCACAGATG CGTAAGGAGA AAATACCGCA TCAGGCGCCA TTCGCCATTC AGGCTGCGCA

1741 ACTGTTGGGA AGGGCGATCG GTGCGGGCCT CTTCGCTATT ACGCCAGCTG GCGAAAGGGG

1801 GATGTGCTGC AAGGCGATTA AGTTGGGTAA CGCCAGGGTT TTCCCAGTCA CGACGTTGTA

1861 AAACGACGGC CAGTGAATTG ATGCATGCTC TTCGGGCTGG TCTGAATCCT TCAGGCGGGT

1921 GTTACCCGAG AAAGAAAGGG TGCCGATTTC AAAGCAGACC CATGTGCCGG GCCCTGTGGC

1981 CTGTGTTGGC GCCTATGTAG TCACCCCCCC TCACCCAATT GTCGCCAGTT TGCGCACTCC

2041 ATAAACTCAA AACAGCAGCT TCTGAGCTGC GCTGTTCAAG AACACCTCTG GGGTTTGCTC

2101 ACCCGCGAGG TCGACGCCCA GCATGGCTAT CAAGACGAAC AGGCAGCCTG TGGAGAAGCC

2161 TCCGTTCACG ATCGGGACGC TGCGCAAGGC CATCCCCGCG CACTGTTTCG AGCGCTCGGC

2221 GCTTCGTAGC AGCATGTACC TGGCCTTTGA CATCGCGGTC ATGTCCCTGC TCTACGTCGC

2281 GTCGACGTAC ATCGACCCTG CACCGGTGCC TACGTGGGTC AAGTACGGCA TCATGTGGCC

2341 GCTCTACTGG TTCTTCCAGG TGTGTTTGAG GGTTTTGGTT GCCCGTATTG AGGTCCTGGT

2401 GGCGCGCATG GAGGAGAAGG CGCCTGTCCC GCTGACCCCC CCGGCTACCC TCCCGGCACC 2461 TTCCAGGGCG CCTTCGGCAC GGGTGTCTGG GTGTGCGCGC ACGAGTGCGG CCACCAGGCC

2521 TTTTCCTCCA GCCAGGCCAT CAACGACGGC GTGGGCCTGG TGTTCCACAG CCTGCTGCTG

2581 GTGCCCTACT ACTCCTGGAA GCACTCGCAC CGGGTACCCT TTCTTGCGCT ATGACACTTC

2641 CAGCAAAAGG TAGGGCGGGC TGCGAGACGG CTTCCCGGCG CTGCATGCAA CACCGATGAT

2701 GCTTCGACCC CCCGAAGCTC CTTCGGGGCT GCATGGGCGC TCCGATGCCG CTCCAGGGCG

2761 AGCGCTGTTT AAATAGCCAG GCCCCCGATT GCAAAGACAT TATAGCGAGC TACCAAAGCC

2821 ATATTCAAAC ACCTAGATCA CTACCACTTC TACACAGGCC ACTCGAGCTT GTGATCGCAC

2881 TCCGCTAAGG GGGCGCCTCT TCCTCTTCGT TTCAGTCACA ACCCGCAAAC TCTAGAATAT

2941 CAATGCTGCT GCAGGCCTTC CTGTTCCTGC TGGCCGGCTT CGCCGCCAAG ATCAGCGCCT

3001 CCATGACGAA CGAGACGTCC GACCGCCCCC TGGTGCACTT CACCCCCAAC AAGGGCTGGA 3061 TGAACGACCC CAACGGCCTG TGGTACGACG AGAAGGACGC CAAGTGGCAC CTGTACTTCC 3121 AGTACAACCC GAACGACACC GTCTGGGGGA CGCCCTTGTT CTGGGGCCAC GCCACGTCCG 3181 ACGACCTGAC CAACTGGGAG GACCAGCCCA TCGCCATCGC CCCGAAGCGC AACGACTCCG 3241 GCGCCTTCTC CGGCTCCATG GTGGTGGACT ACAACAACAC CTCCGGCTTC TTCAACGACA 3301 CCATCGACCC GCGCCAGCGC TGCGTGGCCA TCTGGACCTA CAACACCCCG GAGTCCGAGG 3361 AGCAGTACAT CTCCTACAGC CTGGACGGCG GCTACACCTT CACCGAGTAC CAGAAGAACC 3421 CCGTGCTGGC CGCCAACTCC ACCCAGTTCC GCGACCCGAA GGTCTTCTGG TACGAGCCCT 3481 CCCAGAAGTG GATCATGACC GCGGCCAAGT CCCAGGACTA CAAGATCGAG ATCTACTCCT 3541 CCGACGACCT GAAGTCCTGG AAGCTGGAGT CCGCGTTCGC CAACGAGGGC TTCCTCGGCT 3601 ACCAGTACGA GTGCCCCGGC CTGATCGAGG TCCCCACCGA GCAGGACCCC AGCAAGTCCT 3661 ACTGGGTGAT GTTCATCTCC ATCAACCCCG GCGCCCCGGC CGGCGGCTCC TTCAACCAGT 3721 ACTTCGTCGG CAGCTTCAAC GGCACCCACT TCGAGGCCTT CGACAACCAG TCCCGCGTGG 3781 TGGACTTCGG CAAGGACTAC TACGCCCTGC AGACCTTCTT CAACACCGAC CCGACCTACG 3841 GGAGCGCCCT GGGCATCGCG TGGGCCTCCA ACTGGGAGTA CTCCGCCTTC GTGCCCACCA 3901 ACCCCTGGCG CTCCTCCATG TCCCTCGTGC GCAAGTTCTC CCTCAACACC GAGTACCAGG

3961 CCAACCCGGA GACGGAGCTG ATCAACCTGA AGGCCGAGCC GATCCTGAAC ATCAGCAACG

4021 CCGGCCCCTG GAGCCGGTTC GCCACCAACA CCACGTTGAC GAAGGCCAAC AGCTACAACG 4081 TCGACCTGTC CAACAGCACC GGCACCCTGG AGTTCGAGCT GGTGTACGCC GTCAACACCA 4141 CCCAGACGAT CTCCAAGTCC GTGTTCGCGG ACCTCTCCCT CTGGTTCAAG GGCCTGGAGG 4201 ACCCCGAGGA GTACCTCCGC ATGGGCTTCG AGGTGTCCGC GTCCTCCTTC TTCCTGGACC 4261 GCGGGAACAG CAAGGTGAAG TTCGTGAAGG AGAACCCCTA CTTCACCAAC CGCATGAGCG 4321 TGAACAACCA GCCCTTCAAG AGCGAGAACG ACCTGTCCTA CTACAAGGTG TACGGCTTGC 4381 TGGACCAGAA CATCCTGGAG CTGTACTTCA ACGACGGCGA CGTCGTGTCC ACCAACACCT 4441 ACTTCATGAC CACCGGGAAC GCCCTGGGCT CCGTGAACAT GACGACGGGG GTGGACAACC 4501 TGTTCTACAT CGACAAGTTC CAGGTGCGCG AGGTCAAGTG ACAATTGGCA GCAGCAGCTC 4561 GGATAGTATC GACACACTCT GGACGCTGGT CGTGTGATGG ACTGTTGCCG CCACACTTGC 4621 TGCCTTGACC TGTGAATATC CCTGCCGCTT TTATCAAACA GCCTCAGTGT GTTTGATCTT 4681 GTGTGTACGC GCTTTTGCGA GTTGCTAGCT GCTTGTGCTA TTTGCGAATA CCACCCCCAG 4741 CATCCCCTTC CCTCGTTTCA TATCGCTTGC ATCCCAACCG CAACTTATCT ACGCTGTCCT 4801 GCTATCCCTC AGCGCTGCTC CTGCTCCTGC TCACTGCCCC TCGCACAGCC TTGGTTTGGG 4861 CTCCGCCTGT ATTCTCCTGG TACTGCAACC TGTAAACCAG CACTGCAATG CTGATGCACG 4921 GGAAGTAGTG GGATGGGAAC ACAAATGGAG GATCCCGCGT CTCGAACAGA GCGCGCAGAG

4981 GAACGCTGAA GGTCTCGCCT CTGTCGCACC TCAGCGCGGC ATACACCACA ATAACCACCT 5041 GACGAATGCG CTTGGTTCTT CGTCCATTAG CGAAGCGTCC GGTTCACACA CGTGCCACGT 5101 TGGCGAGGTG GCAGGTGACA ATGATCGGTG GAGCTGATGG TCGAAACGTT CACAGCCTAG 5161 GGATATCGAA TTCCTTTCTT GCGCTATGAC ACTTCCAGCA AAAGGTAGGG CGGGCTGCGA 5221 GACGGCTTCC CGGCGCTGCA TGCAACACCG ATGATGCTTC GACCCCCCGA AGCTCCTTCG 5281 GGGCTGCATG GGCGCTCCGA TGCCGCTCCA GGGCGAGCGC TGTTTAAATA GCCAGGCCCC 5341 CGATTGCAAA GACATTATAG CGAGCTACCA AAGCCATATT CAAACACCTA GATCACTACC 5401 ACTTCTACAC AGGCCACTCG AGCTTGTGAT CGCACTCCGC TAAGGGGGCG CCTCTTCCTC 5461 TTCGTTTCAG TCACAACCCG CAAACACTAG TATGGCCACC GCATCCACTT TCTCGGCGTT 5521 CAATGCCCGC TGCGGCGACC TGCGTCGCTC GGCGGGCTCC GGGCCCCGGC GCCCAGCGAG 5581 GCCCCTCCCC GTGCGCGGGC GCGCCGCCAC CGGCGAGCAG CCCTCCGGCG TGGCCTCCCT 5641 GCGCGAGGCC GACAAGGAGA AGTCCCTGGG CAACCGCCTG CGCCTGGGCT CCCTGACCGA

5701 GGACGGCCTG TCCTACAAGG AGAAGTTCGT GATCCGCTGC TACGAGGTGG GCATCAACAA

5761 GACCGCCACC ATCGAGACCA TCGCCAACCT GCTGCAGGAG GTGGGCGGCA ACCACGCCCA 5821 GGGCGTGGGC TTCTCCACCG ACGGCTTCGC CACCACCACC ACCATGCGCA AGCTGCACCT 5881 GATCTGGGTG ACCGCCCGCA TGCACATCGA GATCTACCGC TACCCCGCCT GGTCCGACGT 5941 GATCGAGATC GAGACCTGGG TGCAGGGCGA GGGCAAGGTG GGCACCCGCC GCGACTGGAT

6001 CCTGAAGGAC TACGCCAACG GCGAGGTGAT CGGCCGCGCC ACCTCCAAGT GGGTGATGAT 6061 GAACGAGGAC ACCCGCCGCC TGCAGAAGGT GTCCGACGAC GTGCGCGAGG AGTACCTGGT 6121 GTTCTGCCCC CGCACCCTGC GCCTGGCCTT CCCCGAGGAG AACAACAACT CCATGAAGAA

6181 GATCCCCAAG CTGGAGGACC CCGCCGAGTA CTCCCGCCTG GGCCTGGTGC CCCGCCGCTC

6241 CGACCTGGAC ATGAACAAGC ACGTGAACAA CGTGACCTAC ATCGGCTGGG CCCTGGAGTC

6301 CATCCCCCCC GAGATCATCG ACACCCACGA GCTGCAGGCC ATCACCCTGG ACTACCGCCG

6361 CGAGTGCCAG CGCGACGACA TCGTGGACTC CCTGACCTCC CGCGAGCCCC TGGGCAACGC

6421 CGCCGGCGTG AAGTTCAAGG AGATCAACGG CTCCGTGTCC CCCAAGAAGG ACGAGCAGGA

6481 CCTGTCCCGC TTCATGCACC TGCTGCGCTC CGCCGGCTCC GGCCTGGAGA TCAACCGCTG

6541 CCGCACCGAG TGGCGCAAGA AGCCCGCCAA GCGCATGGAC TACAAGGACC ACGACGGCGA

6601 CTACAAGGAC CACGACATCG ACTACAAGGA CGACGACGAC AAGTGAATCG ATAGATCTCT

6661 TAAGGCAGCA GCAGCTCGGA TAGTATCGAC ACACTCTGGA CGCTGGTCGT GTGATGGACT

6721 GTTGCCGCCA CACTTGCTGC CTTGACCTGT GAATATCCCT GCCGCTTTTA TCAAACAGCC

6781 TCAGTGTGTT TGATCTTGTG TGTACGCGCT TTTGCGAGTT GCTAGCTGCT TGTGCTATTT

6841 GCGAATACCA CCCCCAGCAT CCCCTTCCCT CGTTTCATAT CGCTTGCATC CCAACCGCAA

6901 CTTATCTACG CTGTCCTGCT ATCCCTCAGC GCTGCTCCTG CTCCTGCTCA CTGCCCCTCG

6961 CACAGCCTTG GTTTGGGCTC CGCCTGTATT CTCCTGGTAC TGCAACCTGT AAACCAGCAC 7021 TGCAATGCTG ATGCACGGGA AGTAGTGGGA TGGGAACACA AATGGAAAGC TTAATTAAGA 7081 GCTCCCGCCA CCACTCCAAC ACGGGGTGCC TGGACAAGGA CGAGGTGTTT GTGCCGCCGC 7141 ACCGCGCAGT GGCGCACGAG GGCCTGGAGT GGGAGGAGTG GCTGCCCATC CGCATGGGCA 7201 AGGTGCTGGT CACCCTGACC CTGGGCTGGC CGCTGTACCT CATGTTCAAC GTCGCCTCGC 7261 GGCCGTACCC GCGCTTCGCC AACCACTTTG ACCCGTGGTC GCCCATCTTC AGCAAGCGCG 7321 AGCGCATCGA GGTGGTCATC TCCGACCTGG CGCTGGTGGC GGTGCTCAGC GGGCTCAGCG 7381 TGCTGGGCCG CACCATGGGC TGGGCCTGGC TGGTCAAGAC CTACGTGGTG CCCTACCTGA 7441 TCGTGAACAT GTGGCTCGTG CTCATCACGC TGCTCCAGCA CACGCACCCG GCGCTGCCGC 7501 ACTACTTCGA GAAGGACTGG GACTGGCTGC GCGGCGCCAT GGCCACCGTG GACCGCTCCA 7561 TGGGCCCGCC CTTCATGGAC AACATCCTGC ACCACATCTC CGACACCCAC GTGCTGCACC 7621 ACCTCTTCAG CACCATCCCG CACTACCACG CCGAGGAGGC CTCCGCCGCC ATCAGGCCCA 7681 TCCTGGGCAA GTACTACCAG TCCGACAGCC GCTGGGTCGG CCGCGCCCTG TGGGAGGACT

7741 GGCGCGACTG CCGCTACGTC GTCCCGGACG CGCCCGAGGA CGACTCCGCG CTCTGGTTCC 7801 ACAAGTGAGT GAGTGAGAAG AGCCTCTAGA GTCGACCTGC AGGCATGCAA GCTTGGCGTA 7861 ATCATGGTCA TAGCTGTTTC CTGTGTGAAA TTGTTATCCG CTCACAATTC CACACAACAT 7921 ACGAGCCGGA AGCATAAAGT GTAAAGCCTG GGGTGCCTAA TGAGTGAGCT AACTCACATT 7981 AATTGCGTTG CGCTCACTGC CCGCTTTCCA GTCGGGAAAC CTGTCGTGCC AGCTGCATTA

8041 ATGAATCGGC CAACGCGCGG GGAGAGGCGG TTTGCGTATT GGGCGCTCTT CCGCTTCCTC 8101 GCTCACTGAC TCGCTGCGCT CGGTCGTTCG GCTGCGGCGA GCGGTATCAG CTCACTCAAA 8161 GGCGGTAATA CGGTTATCCA CAGAATCAGG GGATAACGCA GGAAAGAACA TGTGAGCAAA 8221 AGGCCAGCAA AAGGCCAGGA ACCGTAAAAA GGCCGCGTTG CTGGCGTTTT TCCATAGGCT 8281 CCGCCCCCCT GACGAGCATC ACAAAAATCG ACGCTCAAGT CAGAGGTGGC GAAACCCGAC 8341 AGGACTATAA AGATACCAGG CGTTTCCCCC TGGAAGCTCC CTCGTGCGCT CTCCTGTTCC 8401 GACCCTGCCG CTTACCGGAT ACCTGTCCGC CTTTCTCCCT TCGGGAAGCG TGGCGCTTTC 8461 TCATAGCTCA CGCTGTAGGT ATCTCAGTTC GGTGTAGGTC GTTCGCTCCA AGCTGGGCTG 8521 TGTGCACGAA CCCCCCGTTC AGCCCGACCG CTGCGCCTTA TCCGGTAACT ATCGTCTTGA 8581 GTCCAACCCG GTAAGACACG ACTTATCGC

SEQ ID NO: 235

pSZ1570

1 CACTGGCAGC AGCCACTGGT AACAGGATTA GCAGAGCGAG GTATGTAGGC GGTGCTACAG

61 AGTTCTTGAA GTGGTGGCCT AACTACGGCT ACACTAGAAG GACAGTATTT GGTATCTGCG

121 CTCTGCTGAA GCCAGTTACC TTCGGAAAAA GAGTTGGTAG CTCTTGATCC GGCAAACAAA

181 CCACCGCTGG TAGCGGTGGT TTTTTTGTTT GCAAGCAGCA GATTACGCGC AGAAAAAAAG

241 GATCTCAAGA AGATCCTTTG ATCTTTTCTA CGGGGTCTGA CGCTCAGTGG AACGAAAACT

301 CACGTTAAGG GATTTTGGTC ATGAGATTAT CAAAAAGGAT CTTCACCTAG ATCCTTTTAA

361 ATTAAAAATG AAGTTTTAAA TCAATCTAAA GTATATATGA GTAAACTTGG TCTGACAGTT

421 ACCAATGCTT AATCAGTGAG GCACCTATCT CAGCGATCTG TCTATTTCGT TCATCCATAG

481 TTGCCTGACT CCCCGTCGTG TAGATAACTA CGATACGGGA GGGCTTACCA TCTGGCCCCA

541 GTGCTGCAAT GATACCGCGA GACCCACGCT CACCGGCTCC AGATTTATCA GCAATAAACC

601 AGCCAGCCGG AAGGGCCGAG CGCAGAAGTG GTCCTGCAAC TTTATCCGCC TCCATCCAGT

661 CTATTAATTG TTGCCGGGAA GCTAGAGTAA GTAGTTCGCC AGTTAATAGT TTGCGCAACG

721 TTGTTGCCAT TGCTACAGGC ATCGTGGTGT CACGCTCGTC GTTTGGTATG GCTTCATTCA

781 GCTCCGGTTC CCAACGATCA AGGCGAGTTA CATGATCCCC CATGTTGTGC AAAAAAGCGG 841 TTAGCTCCTT CGGTCCTCCG ATCGTTGTCA GAAGTAAGTT GGCCGCAGTG TTATCACTCA

901 TGGTTATGGC AGCACTGCAT AATTCTCTTA CTGTCATGCC ATCCGTAAGA TGCTTTTCTG

961 TGACTGGTGA GTACTCAACC AAGTCATTCT GAGAATAGTG TATGCGGCGA CCGAGTTGCT

1021 CTTGCCCGGC GTCAATACGG GATAATACCG CGCCACATAG CAGAACTTTA AAAGTGCTCA

1081 TCATTGGAAA ACGTTCTTCG GGGCGAAAAC TCTCAAGGAT CTTACCGCTG TTGAGATCCA

1141 GTTCGATGTA ACCCACTCGT GCACCCAACT GATCTTCAGC ATCTTTTACT TTCACCAGCG

1201 TTTCTGGGTG AGCAAAAACA GGAAGGCAAA ATGCCGCAAA AAAGGGAATA AGGGCGACAC

1261 GGAAATGTTG AATACTCATA CTCTTCCTTT TTCAATATTA TTGAAGCATT TATCAGGGTT

1321 ATTGTCTCAT GAGCGGATAC ATATTTGAAT GTATTTAGAA AAATAAACAA ATAGGGGTTC

1381 CGCGCACATT TCCCCGAAAA GTGCCACCTG ACGTCTAAGA AACCATTATT ATCATGACAT

1441 TAACCTATAA AAATAGGCGT ATCACGAGGC CCTTTCGTCT CGCGCGTTTC GGTGATGACG

1501 GTGAAAACCT CTGACACATG CAGCTCCCGG AGACGGTCAC AGCTTGTCTG TAAGCGGATG

1561 CCGGGAGCAG ACAAGCCCGT CAGGGCGCGT CAGCGGGTGT TGGCGGGTGT CGGGGCTGGC

1621 TTAACTATGC GGCATCAGAG CAGATTGTAC TGAGAGTGCA CCATATGCGG TGTGAAATAC

1681 CGCACAGATG CGTAAGGAGA AAATACCGCA TCAGGCGCCA TTCGCCATTC AGGCTGCGCA

1741 ACTGTTGGGA AGGGCGATCG GTGCGGGCCT CTTCGCTATT ACGCCAGCTG GCGAAAGGGG

1801 GATGTGCTGC AAGGCGATTA AGTTGGGTAA CGCCAGGGTT TTCCCAGTCA CGACGTTGTA

1861 AAACGACGGC CAGTGAATTG ATGCATGCTC TTCGCCGCCG CCACTCCTGC TCGAGCGCGC

1921 CCGCGCGTGC GCCGCCAGCG CCTTGGCCTT TTCGCCGCGC TCGTGCGCGT CGCTGATGTC

1981 CATCACCAGG TCCATGAGGT CTGCCTTGCG CCGGCTGAGC CACTGCTTCG TCCGGGCGGC

2041 CAAGAGGAGC ATGAGGGAGG ACTCCTGGTC CAGGGTCCTG ACGTGGTCGC GGCTCTGGGA

2101 GCGGGCCAGC ATCATCTGGC TCTGCCGCAC CGAGGCCGCC TCCAACTGGT CCTCCAGCAG

2161 CCGCAGTCGC CGCCGACCCT GGCAGAGGAA GACAGGTGAG GGGGGTATGA ATTGTACAGA

2221 ACAACCACGA GCCTTGTCTA GGCAGAATCC CTACCAGTCA TGGCTTTACC TGGATGACGG

2281 CCTGCGAACA GCTGTCCAGC GACCCTCGCT GCCGCCGCTT CTCCCGCACG CTTCTTTCCA

2341 GCACCGTGAT GGCGCGAGCC AGCGCCGCAC GCTGGCGCTG CGCTTCGCCG ATCTGAGGAC

2401 AGTCGGGGAA CTCTGATCAG TCTAAACCCC CTTGCGCGTT AGTGTTGCCA TCCTTTGCAG

2461 ACCGGTGAGA GCCGACTTGT TGTGCGCCAC CCCCCACACC ACCTCCTCCC AGACCAATTC

2521 TGTCACCTTT TTGGCGAAGG CATCGGCCTC GGCCTGCAGA GAGGACAGCA GTGCCCAGCC

2581 GCTGGGGGTT GGCGGATGCA CGCTCAGGTA CCCTTTCTTG CGCTATGACA CTTCCAGCAA

2641 AAGGTAGGGC GGGCTGCGAG ACGGCTTCCC GGCGCTGCAT GCAACACCGA TGATGCTTCG

2701 ACCCCCCGAA GCTCCTTCGG GGCTGCATGG GCGCTCCGAT GCCGCTCCAG GGCGAGCGCT

2761 GTTTAAATAG CCAGGCCCCC GATTGCAAAG ACATTATAGC GAGCTACCAA AGCCATATTC

2821 AAACACCTAG ATCACTACCA CTTCTACACA GGCCACTCGA GCTTGTGATC GCACTCCGCT

2881 AAGGGGGCGC CTCTTCCTCT TCGTTTCAGT CACAACCCGC AAACGGCGCG CCATGCTGCT

2941 GCAGGCCTTC CTGTTCCTGC TGGCCGGCTT CGCCGCCAAG ATCAGCGCCT CCATGACGAA

3001 CGAGACGTCC GACCGCCCCC TGGTGCACTT CACCCCCAAC AAGGGCTGGA TGAACGACCC 3061 CAACGGCCTG TGGTACGACG AGAAGGACGC CAAGTGGCAC CTGTACTTCC AGTACAACCC 3121 GAACGACACC GTCTGGGGGA CGCCCTTGTT CTGGGGCCAC GCCACGTCCG ACGACCTGAC 3181 CAACTGGGAG GACCAGCCCA TCGCCATCGC CCCGAAGCGC AACGACTCCG GCGCCTTCTC 3241 CGGCTCCATG GTGGTGGACT ACAACAACAC CTCCGGCTTC TTCAACGACA CCATCGACCC 3301 GCGCCAGCGC TGCGTGGCCA TCTGGACCTA CAACACCCCG GAGTCCGAGG AGCAGTACAT 3361 CTCCTACAGC CTGGACGGCG GCTACACCTT CACCGAGTAC CAGAAGAACC CCGTGCTGGC 3421 CGCCAACTCC ACCCAGTTCC GCGACCCGAA GGTCTTCTGG TACGAGCCCT CCCAGAAGTG 3481 GATCATGACC GCGGCCAAGT CCCAGGACTA CAAGATCGAG ATCTACTCCT CCGACGACCT 3541 GAAGTCCTGG AAGCTGGAGT CCGCGTTCGC CAACGAGGGC TTCCTCGGCT ACCAGTACGA 3601 GTGCCCCGGC CTGATCGAGG TCCCCACCGA GCAGGACCCC AGCAAGTCCT ACTGGGTGAT 3661 GTTCATCTCC ATCAACCCCG GCGCCCCGGC CGGCGGCTCC TTCAACCAGT ACTTCGTCGG 3721 CAGCTTCAAC GGCACCCACT TCGAGGCCTT CGACAACCAG TCCCGCGTGG TGGACTTCGG 3781 CAAGGACTAC TACGCCCTGC AGACCTTCTT CAACACCGAC CCGACCTACG GGAGCGCCCT 3841 GGGCATCGCG TGGGCCTCCA ACTGGGAGTA CTCCGCCTTC GTGCCCACCA ACCCCTGGCG 3901 CTCCTCCATG TCCCTCGTGC GCAAGTTCTC CCTCAACACC GAGTACCAGG CCAACCCGGA

3961 GACGGAGCTG ATCAACCTGA AGGCCGAGCC GATCCTGAAC ATCAGCAACG CCGGCCCCTG

4021 GAGCCGGTTC GCCACCAACA CCACGTTGAC GAAGGCCAAC AGCTACAACG TCGACCTGTC 4081 CAACAGCACC GGCACCCTGG AGTTCGAGCT GGTGTACGCC GTCAACACCA CCCAGACGAT 4141 CTCCAAGTCC GTGTTCGCGG ACCTCTCCCT CTGGTTCAAG GGCCTGGAGG ACCCCGAGGA 4201 GTACCTCCGC ATGGGCTTCG AGGTGTCCGC GTCCTCCTTC TTCCTGGACC GCGGGAACAG 4261 CAAGGTGAAG TTCGTGAAGG AGAACCCCTA CTTCACCAAC CGCATGAGCG TGAACAACCA 4321 GCCCTTCAAG AGCGAGAACG ACCTGTCCTA CTACAAGGTG TACGGCTTGC TGGACCAGAA 4381 CATCCTGGAG CTGTACTTCA ACGACGGCGA CGTCGTGTCC ACCAACACCT ACTTCATGAC 4441 CACCGGGAAC GCCCTGGGCT CCGTGAACAT GACGACGGGG GTGGACAACC TGTTCTACAT 4501 CGACAAGTTC CAGGTGCGCG AGGTCAAGTG ACAATTGGCA GCAGCAGCTC GGATAGTATC

4561 GACACACTCT GGACGCTGGT CGTGTGATGG ACTGTTGCCG CCACACTTGC TGCCTTGACC

4621 TGTGAATATC CCTGCCGCTT TTATCAAACA GCCTCAGTGT GTTTGATCTT GTGTGTACGC

4681 GCTTTTGCGA GTTGCTAGCT GCTTGTGCTA TTTGCGAATA CCACCCCCAG CATCCCCTTC

4741 CCTCGTTTCA TATCGCTTGC ATCCCAACCG CAACTTATCT ACGCTGTCCT GCTATCCCTC

4801 AGCGCTGCTC CTGCTCCTGC TCACTGCCCC TCGCACAGCC TTGGTTTGGG CTCCGCCTGT

4861 ATTCTCCTGG TACTGCAACC TGTAAACCAG CACTGCAATG CTGATGCACG GGAAGTAGTG

4921 GGATGGGAAC ACAAATGGAG GATCCCGCGT CTCGAACAGA GCGCGCAGAG GAACGCTGAA

4981 GGTCTCGCCT CTGTCGCACC TCAGCGCGGC ATACACCACA ATAACCACCT GACGAATGCG 5041 CTTGGTTCTT CGTCCATTAG CGAAGCGTCC GGTTCACACA CGTGCCACGT TGGCGAGGTG 5101 GCAGGTGACA ATGATCGGTG GAGCTGATGG TCGAAACGTT CACAGCCTAG GGATATCGAA 5161 TTCGGCCGAC AGGACGCGCG TCAAAGGTGC TGGTCGTGTA TGCCCTGGCC GGCAGGTCGT 5221 TGCTGCTGCT GGTTAGTGAT TCCGCAACCC TGATTTTGGC GTCTTATTTT GGCGTGGCAA 5281 ACGCTGGCGC CCGCGAGCCG GGCCGGCGGC GATGCGGTGC CCCACGGCTG CCGGAATCCA 5341 AGGGAGGCAA GAGCGCCCGG GTCAGTTGAA GGGCTTTACG CGCAAGGTAC AGCCGCTCCT 5401 GCAAGGCTGC GTGGTGGAAT TGGACGTGCA GGTCCTGCTG AAGTTCCTCC ACCGCCTCAC 5461 CAGCGGACAA AGCACCGGTG TATCAGGTCC GTGTCATCCA CTCTAAAGAG CTCGACTACG 5521 ACCTACTGAT GGCCCTAGAT TCTTCATCAA AAACGCCTGA GACACTTGCC CAGGATTGAA 5581 ACTCCCTGAA GGGACCACCA GGGGCCCTGA GTTGTTCCTT CCCCCCGTGG CGAGCTGCCA 5641 GCCAGGCTGT ACCTGTGATC GAGGCTGGCG GGAAAATAGG CTTCGTGTGC TCAGGTCATG

5701 GGAGGTGCAG GACAGCTCAT GAAACGCCAA CAATCGCACA ATTCATGTCA AGCTAATCAG

5761 CTATTTCCTC TTCACGAGCT GTAATTGTCC CAAAATTCTG GTCTACCGGG GGTGATCCTT 5821 CGTGTACGGG CCCTTCCCTC AACCCTAGGT ATGCGCGCAT GCGGTCGCCG CGCAACTCGC 5881 GCGAGGGCCG AGGGTTTGGG ACGGGCCGTC CCGAAATGCA GTTGCACCCG GATGCGTGGC 5941 ACCTTTTTTG CGATAATTTA TGCAATGGAC TGCTCTGCAA AATTCTGGCT CTGTCGCCAA

6001 CCCTAGGATC AGCGGCGTAG GATTTCGTAA TCATTCGTCC TGATGGGGAG CTACCGACTA 6061 CCCTAATATC AGCCCGACTG CCTGACGCCA GCGTCCACTT TTGTGCACAC ATTCCATTCG 6121 TGCCCAAGAC ATTTCATTGT GGTGCGAAGC GTCCCCAGTT ACGCTCACCT GTTTCCCGAC 6181 CTCCTTACTG TTCTGTCGAC AGAGCGGGCC CACAGGCCGG TCGCAGCCAC TAGTATGGCT 6241 TCCGCGGCAT TCACCATGTC GGCGTGCCCC GCGATGACTG GCAGGGCCCC TGGGGCACGT 6301 CGCTCCGGAC GGCCAGTCGC CACCCGCCTG AGGGGGCGCG CCCCCAAGGC CAACGGCTCC 6361 GCCGTGTCCC TGAAGTCCGG CTCCCTGGAC ACCCAGGAGG ACACCTCCTC CTCCTCCTCC 6421 CCCCCCCGCA CCTTCATCAA CCAGCTGCCC GACTGGTCCA TGCTGCTGTC CGCCATCACC 6481 ACCGTGTTCG TGGCCGCCGA GAAGCAGTGG ACCATGCTGG ACCGCAAGTC CAAGCGCCCC 6541 GACACCCTGA TGGACCCCTT CGGCGTGGAC CGCGTGGTGC AGGACGGCGT GGTGTTCCGC 6601 CAGTCCTTCT CCATCCGCTC CTACGAGATC GGCGCCGACC GCACCGCCTC CATCGAGACC 6661 CTGATGAACA TCTTCCAGGA GACCTCCCTG AACCACTGCA AGTCCATCGG CCTGCTGAAC 6721 GACGGCTTCG GCCGCACCCC CGAGATGTGC AAGCGCGACC TGATCTGGGT GGTGACCAAG 6781 ATGCACATCG AGGTGAACCG CTACCCCACC TGGGGCGACA CCATCGAGGT GAACACCTGG 6841 GTGTCCGAGT CCGGCAAGAC CGGCATGGGC CGCGACTGGC TGATCTCCGA CTGCCACACC 6901 GGCGAGATCC TGATCCGCGC CACCTCCGTG TGCGCCATGA TGAACCAGAC CACCCGCCGC

6961 TTCTCCAAGT TCCCCTACGA GGTGCGCCAG GAGCTGGCCC CCCACTTCGT GGACTCCGCC 7021 CCCGTGATCG AGGACTACCA GAAGCTGCAC AAGCTGGACG TGAAGACCGG CGACTCCATC 7081 TGCAACGGCC TGACCCCCCG CTGGAACGAC CTGGACGTGA ACCAGCACGT GAACAACGTG 7141 AAGTACATCG GCTGGATTCT GGAGTCCGTG CCCAAGGAGG TGTTCGAGAC CCAGGAGCTG 7201 TGCGGCCTGA CCCTGGAGTA CCGCCGCGAG TGCGGCCGCG ACTCCGTGCT GAAGTCCGTG 7261 ACCGCCATGG ACCCCTCCAA GGAGGGCGAC CGCTCCCTGT ACCAGCACCT GCTGCGCCTG 7321 GAGGACGGCA CCGACATCGC CAAGGGCCGC ACCAAGTGGC GCCCCAAGAA CGCCGGCACC 7381 AACGGCGCCA TCTCCACCGG CAAGACCTCC AACGGCAACT CCATCTCCTG ATTAATTAAC 7441 TCGAGGCAGC AGCAGCTCGG ATAGTATCGA CACACTCTGG ACGCTGGTCG TGTGATGGAC 7501 TGTTGCCGCC ACACTTGCTG CCTTGACCTG TGAATATCCC TGCCGCTTTT ATCAAACAGC 7561 CTCAGTGTGT TTGATCTTGT GTGTACGCGC TTTTGCGAGT TGCTAGCTGC TTGTGCTATT 7621 TGCGAATACC ACCCCCAGCA TCCCCTTCCC TCGTTTCATA TCGCTTGCAT CCCAACCGCA 7681 ACTTATCTAC GCTGTCCTGC TATCCCTCAG CGCTGCTCCT GCTCCTGCTC ACTGCCCCTC

7741 GCACAGCCTT GGTTTGGGCT CCGCCTGTAT TCTCCTGGTA CTGCAACCTG TAAACCAGCA 7801 CTGCAATGCT GATGCACGGG AAGTAGTGGG ATGGGAACAC AAATGGAAAG CTTGAGCTCT 7861 TGTTTTCCAG AAGGAGTTGC TCCTTGAGCC TTTCATTCTC AGCCTCGATA ACCTCCAAAG 7921 CCGCTCTAAT TGTGGAGGGG GTTCGAATTT AAAAGCTTGG AATGTTGGTT CGTGCGTCTG 7981 GAACAAGCCC AGACTTGTTG CTCACTGGGA AAAGGACCAT CAGCTCCAAA AAACTTGCCG

8041 CTCAAACCGC GTACCTCTGC TTTCGCGCAA TCTGCCCTGT TGAAATCGCC ACCACATTCA 8101 TATTGTGACG CTTGAGCAGT CTGTAATTGC CTCAGAATGT GGAATCATCT GCCCCCTGTG 8161 CGAGCCCATG CCAGGCATGT CGCGGGCGAG GACACCCGCC ACTCGTACAG CAGACCATTA

8221 TGCTACCTCA CAATAGTTCA TAACAGTGAC CATATTTCTC GAAGCTCCCC AACGAGCACC

8281 TCCATGCTCT GAGTGGCCAC CCCCCGGCCC TGGTGCTTGC GGAGGGCAGG TCAACCGGCA

8341 TGGGGCTACC GAAATCCCCG ACCGGATCCC ACCACCCCCG CGATGGGAAG AATCTCTCCC

8401 CGGGATGTGG GCCCACCACC AGCACAACCT GCTGGCCCAG GCGAGCGTCA AACCATACCA

8461 CACAAATATC CTTGGCATCG GCCCTGAATT CCTTCTGCCG CTCTGCTACC CGGTGCTTCT

8521 GTCCGAAGCA GGGGTTGCTA GGGATCGCTC CGAGTCCGCA AACCCTTGTC GCGTGGCGGG

8581 GCTTGTTCGA GCTTGAAGAG CCTCTAGAGT CGACCTGCAG GCATGCAAGC TTGGCGTAAT

8641 CATGGTCATA GCTGTTTCCT GTGTGAAATT GTTATCCGCT CACAATTCCA CACAACATAC

8701 GAGCCGGAAG CATAAAGTGT AAAGCCTGGG GTGCCTAATG AGTGAGCTAA CTCACATTAA

8761 TTGCGTTGCG CTCACTGCCC GCTTTCCAGT CGGGAAACCT GTCGTGCCAG CTGCATTAAT

8821 GAATCGGCCA ACGCGCGGGG AGAGGCGGTT TGCGTATTGG GCGCTCTTCC GCTTCCTCGC

8881 TCACTGACTC GCTGCGCTCG GTCGTTCGGC TGCGGCGAGC GGTATCAGCT CACTCAAAGG

8941 CGGTAATACG GTTATCCACA GAATCAGGGG ATAACGCAGG AAAGAACATG TGAGCAAAAG

9001 GCCAGCAAAA GGCCAGGAAC CGTAAAAAGG CCGCGTTGCT GGCGTTTTTC CATAGGCTCC

9061 GCCCCCCTGA CGAGCATCAC AAAAATCGAC GCTCAAGTCA GAGGTGGCGA AACCCGACAG

9121 GACTATAAAG ATACCAGGCG TTTCCCCCTG GAAGCTCCCT CGTGCGCTCT CCTGTTCCGA

9181 CCCTGCCGCT TACCGGATAC CTGTCCGCCT TTCTCCCTTC GGGAAGCGTG GCGCTTTCTC

9241 ATAGCTCACG CTGTAGGTAT CTCAGTTCGG TGTAGGTCGT TCGCTCCAAG CTGGGCTGTG

9301 TGCACGAACC CCCCGTTCAG CCCGACCGCT GCGCCTTATC CGGTAACTAT CGTCTTGAGT

9361 CCAACCCGGT AAGACACGAC TTATCGC

SEQ ID NO: 236

pSZ941

1 CACTGGCAGC AGCCACTGGT AACAGGATTA GCAGAGCGAG GTATGTAGGC GGTGCTACAG

61 AGTTCTTGAA GTGGTGGCCT AACTACGGCT ACACTAGAAG GACAGTATTT GGTATCTGCG

121 CTCTGCTGAA GCCAGTTACC TTCGGAAAAA GAGTTGGTAG CTCTTGATCC GGCAAACAAA

181 CCACCGCTGG TAGCGGTGGT TTTTTTGTTT GCAAGCAGCA GATTACGCGC AGAAAAAAAG

241 GATCTCAAGA AGATCCTTTG ATCTTTTCTA CGGGGTCTGA CGCTCAGTGG AACGAAAACT

301 CACGTTAAGG GATTTTGGTC ATGAGATTAT CAAAAAGGAT CTTCACCTAG ATCCTTTTAA

361 AT AAAAATG AAGTTTTAAA TCAATCTAAA GTATATATGA GTAAACTTGG TCTGACAGTT

421 ACCAATGCTT AATCAGTGAG GCACCTATCT CAGCGATCTG TCTATTTCGT TCATCCATAG

481 TTGCCTGACT CCCCGTCGTG TAGATAACTA CGATACGGGA GGGCTTACCA TCTGGCCCCA

541 GTGCTGCAAT GATACCGCGA GACCCACGCT CACCGGCTCC AGATTTATCA GCAATAAACC

601 AGCCAGCCGG AAGGGCCGAG CGCAGAAGTG GTCCTGCAAC TTTATCCGCC TCCATCCAGT

661 CTATTAATTG TTGCCGGGAA GCTAGAGTAA GTAGTTCGCC AGTTAATAGT TTGCGCAACG

721 TTGTTGCCAT TGCTACAGGC ATCGTGGTGT CACGCTCGTC GTTTGGTATG GCTTCATTCA

781 GCTCCGGTTC CCAACGATCA AGGCGAGTTA CATGATCCCC CATGTTGTGC AAAAAAGCGG

841 TTAGCTCCTT CGGTCCTCCG ATCGTTGTCA GAAGTAAGTT GGCCGCAGTG TTA CACTCA

901 TGGTTATGGC AGCACTGCAT AATTCTCTTA CTGTCATGCC ATCCGTAAGA TGCTTTTCTG

961 TGACTGGTGA GTACTCAACC AAGTCATTCT GAGAATAGTG TATGCGGCGA CCGAGTTGCT

1021 CTTGCCCGGC GTCAATACGG GATAATACCG CGCCACATAG CAGAACTTTA AAAGTGCTCA

1081 TCATTGGAAA ACGTTCTTCG GGGCGAAAAC TCTCAAGGAT CTTACCGCTG TTGAGATCCA

1141 GTTCGATGTA ACCCACTCGT GCACCCAACT GATCTTCAGC ATCTTTTACT TTCACCAGCG

1201 TTTCTGGGTG AGCAAAAACA GGAAGGCAAA ATGCCGCAAA AAAGGGAATA AGGGCGACAC

1261 GGAAATGTTG AATACTCATA CTCTTCCTTT TTCAATATTA TTGAAGCATT TATCAGGGTT

1321 ATTGTCTCAT GAGCGGATAC ATATTTGAAT GTATTTAGAA AAATAAACAA ATAGGGGTTC

1381 CGCGCACATT TCCCCGAAAA GTGCCACCTG ACGTCTAAGA AACCATTATT ATCATGACAT

1441 TAACC ATAA AAATAGGCGT ATCACGAGGC CCTTTCGTCT CGCGCGTTTC GGTGATGACG

1501 GTGAAAACCT CTGACACATG CAGCTCCCGG AGACGGTCAC AGCTTGTCTG TAAGCGGATG

1561 CCGGGAGCAG ACAAGCCCGT CAGGGCGCGT CAGCGGGTGT TGGCGGGTGT CGGGGCTGGC

1621 TTAACTATGC GGCATCAGAG CAGATTGTAC TGAGAGTGCA CCATATGCGG TGTGAAATAC

1681 CGCACAGATG CGTAAGGAGA AAATACCGCA TCAGGCGCCA TTCGCCATTC AGGCTGCGCA

1741 ACTGTTGGGA AGGGCGATCG GTGCGGGCCT CTTCGCTATT ACGCCAGCTG GCGAAAGGGG

1801 GATGTGCTGC AAGGCGATTA AGTTGGGTAA CGCCAGGGTT TTCCCAGTCA CGACGTTGTA

1861 AAACGACGGC CAGTGAATTG ATGCATGCTC TTCGCCGCCG CCACTCCTGC TCGAGCGCGC

1921 CCGCGCGTGC GCCGCCAGCG CCTTGGCCTT TTCGCCGCGC TCGTGCGCGT CGCTGATGTC

1981 CATCACCAGG TCCATGAGGT CTGCCTTGCG CCGGCTGAGC CACTGCTTCG TCCGGGCGGC

2041 CAAGAGGAGC ATGAGGGAGG ACTCCTGGTC CAGGGTCCTG ACGTGGTCGC GGCTCTGGGA 2101 GCGGGCCAGC ATCATCTGGC TCTGCCGCAC CGAGGCCGCC TCCAACTGGT CCTCCAGCAG

2161 CCGCAGTCGC CGCCGACCCT GGCAGAGGAA GACAGGTGAG GGGGGTATGA ATTGTACAGA

2221 ACAACCACGA GCCTTGTCTA GGCAGAATCC CTACCAGTCA TGGCTTTACC TGGATGACGG

2281 CCTGCGAACA GCTGTCCAGC GACCCTCGCT GCCGCCGCTT CTCCCGCACG CTTCTTTCCA

2341 GCACCGTGAT GGCGCGAGCC AGCGCCGCAC GCTGGCGCTG CGCTTCGCCG ATCTGAGGAC

2401 AGTCGGGGAA CTCTGATCAG TCTAAACCCC CTTGCGCGTT AGTGTTGCCA TCCTTTGCAG

2461 ACCGGTGAGA GCCGACTTGT TGTGCGCCAC CCCCCACACC ACCTCCTCCC AGACCAATTC

2521 TGTCACCTTT TTGGCGAAGG CATCGGCCTC GGCCTGCAGA GAGGACAGCA GTGCCCAGCC

2581 GCTGGGGGTT GGCGGATGCA CGCTCAGGTA CCCTTTCTTG CGCTATGACA CTTCCAGCAA

2641 AAGGTAGGGC GGGCTGCGAG ACGGCTTCCC GGCGCTGCAT GCAACACCGA TGATGCTTCG

2701 ACCCCCCGAA GCTCCTTCGG GGCTGCATGG GCGCTCCGAT GCCGCTCCAG GGCGAGCGCT

2761 GTTTAAATAG CCAGGCCCCC GATTGCAAAG ACATTATAGC GAGCTACCAA AGCCATATTC

2821 AAACACCTAG ATCACTACCA CTTCTACACA GGCCACTCGA GCTTGTGATC GCACTCCGCT

2881 AAGGGGGCGC CTCTTCCTCT TCGTTTCAGT CACAACCCGC AAACTCTAGA ATATCAATGA

2941 TCGAGCAGGA CGGCCTCCAC GCCGGCTCCC CCGCCGCCTG GGTGGAGCGC CTGTTCGGCT

3001 ACGACTGGGC CCAGCAGACC ATCGGCTGCT CCGACGCCGC CGTGTTCCGC CTGTCCGCCC 3061 AGGGCCGCCC CGTGCTGTTC GTGAAGACCG