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Title:
MICROORGANISMS AND METHODS FOR PRODUCTION OF SPECIFIC LENGTH FATTY ALCOHOLS AND RELATED COMPOUNDS
Document Type and Number:
WIPO Patent Application WO/2014/062564
Kind Code:
A1
Abstract:
The invention provides non-naturally occurring microbial organisms containing a fatty alcohol, fatty aldehyde or fatty acid pathway, wherein the microbial organisms selectively produce a fatty alcohol, fatty aldehyde or fatty acid of a specified length. Also provided are non-naturally occurring microbial organisms having a fatty alcohol, fatty aldehyde or fatty acid pathway, wherein the microbial organisms further include an acetyl-CoA pathway. In some aspects, the microbial organisms of the invention have select gene disruptions or enzyme attenuations that increase production of fatty alcohols, fatty aldehydes or fatty acids. The invention additionally provides methods of using the above microbial organisms to produce a fatty alcohol, a fatty aldehyde or a fatty acid.

Inventors:
OSTERHOUT, Robin, E. (10520 Wateridge Circle, San Diego, CA, 92121, US)
BURGARD, Anthony, P. (10520 Wateridge Circle, San Diego, CA, 92121, US)
Application Number:
US2013/064827
Publication Date:
April 24, 2014
Filing Date:
October 14, 2013
Export Citation:
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Assignee:
GENOMATICA, INC. (10520 Wateridge Circle, San Diego, CA, 92121, US)
International Classes:
C12P7/04; C07C29/00; C07C31/02; C07C33/00; C07C47/263; C07C53/00; C07C57/00; C12N1/00; C12P7/40; C12P7/64
Foreign References:
US20110250663A12011-10-13
US20090275097A12009-11-05
Other References:
See also references of EP 2906705A4
Attorney, Agent or Firm:
HEBERT, Micheal, L. et al. (Jones Day, 222 East 41st StreetNew York, NY, 10017-6702, US)
Download PDF:
Claims:
What is claimed is:

1. A non-naturally occurring microbial organism having a malonyl-CoA independent fatty acyl-CoA elongation (MI-FAE) cycle and/or a malonyl-CoA dependent fatty acyl-CoA elongation (MD-FAE) cycle in combination with a termination pathway, wherein said MI-FAE cycle comprises one or more thiolase, one or more 3-oxoacyl- CoA reductase, one or more 3-hydroxyacyl-CoA dehydratase, and one or more enoyl-CoA reductase, wherein said MD-FAE cycle comprises one or more elongase, one or more 3- oxoacyl-CoA reductase, one or more 3-hydroxyacyl-CoA dehydratase, and one or more enoyl-CoA reductase, wherein said termination pathway comprises a pathway selected from:

(1) 1H;

(2) IK and 1L;

(3) IE and IN;

(4) IK, 1J, and IN;

(5) IE;

(6) IK and 1 J;

(?) 1H and IN;

(8) IK, 1L, and IN;

(9) IE and IF;

(10) IK, 1J, and IF;

(11) 1H, IN, and IF;

(12) IK, 1L, IN, and IF; and

(13) 1G, wherein IE is an acyl-CoA reductase (aldehyde forming), wherein IF is an alcohol dehydrogenase, wherein 1G is an acyl-CoA reductase (alcohol forming), wherein 1H is an acyl-CoA hydrolase, acyl-CoA transferase or acyl-CoA synthase, wherein 1J is an acyl-ACP reductase, wherein IK is an acyl-CoA:ACP acyltransferase, wherein 1L is a thioesterase, wherein IN is an aldehyde dehydrogenase (acid forming) or a carboxylic acid reductase, wherein an enzyme of the MI-FAE cycle, MD-FAE cycle or termination pathway is encoded by at least one exogenous nucleic acid and is expressed in a sufficient amount to produce a compound of Formula (I):

(I) wherein Rj is Ci_24 linear alkyl; R2 is CH2OH, CHO, or COOH; R3 is H, OH, or oxo

(=0); and represents a single or double bond with the proviso that the valency of the carbon atom to which R3 is attached is four, wherein the substrate of each of said enzymes of the MI-FAE cycle, MD-FAE cycle and the termination pathway are independently selected from a compound of Formula (II), malonyl-CoA, propionyl-CoA or acetyl-CoA:

wherein Ri is Ci_24 linear alkyl; R3 is H, OH, or oxo (=0); R4 is S-CoA, ACP, OH or

H; and represents a single or double bond with the proviso that the valency of the carbon atom to which R3 is attached is four; wherein said one or more enzymes of the MI-FAE cycle are each selective for a compound of Formula (II) having a number of carbon atoms at Ri that is no greater than the number of carbon atoms at Ri of said compound of Formula (I), wherein said one or more enzymes of the MD-FAE cycle are each selective for a compound of Formula (II) having a number of carbon atoms at Ri that is no greater than the number of carbon atoms at Ri of said compound of Formula (I), and wherein said one or more enzymes of the termination pathway are each selective for a compound of Formula (II) having a number of carbon atoms at Ri that is no less than the number of carbon atoms at Ri of said compound of Formula (I).

2. The non-naturally occurring microbial organism of claim 1, wherein Ri is C1-17 linear alkyl.

3. The non-naturally occurring microbial organism of claim 2, wherein Ri is C9 linear alkyl, C10 linear alkyl, Cn, linear alkyl, C12 linear alkyl or C13 linear alkyl.

4. The non-naturally occurring microbial organism of claim 1, wherein said microbial organism comprises two, three, or four exogenous nucleic acids each encoding an enzyme of said MI-FAE cycle or said MD-FAE cycle.

5. The non-naturally occurring microbial organism of claim 1, wherein said microbial organism comprises two, three, or four exogenous nucleic acids each encoding an enzyme of said termination pathway.

6. The non-naturally occurring microbial organism of claim 3, wherein said microbial organism comprises exogenous nucleic acids encoding each of the enzymes of at least one of the pathways selected from (1)-(13).

7. The non-naturally occurring microbial organism of claim 1, wherein said at least one exogenous nucleic acid is a heterologous nucleic acid.

8. The non-naturally occurring microbial organism of claim 1, wherein said non- naturally occurring microbial organism is in a substantially anaerobic culture medium.

9. The non naturally occurring microbial organism of claim 1, wherein said enzyme of the MI-FAE cycle, MD-FAE cycle or termination pathway is expressed in a sufficient amount to produce a compound selected from the Formulas (III)-(VI):

10. The non-naturally occurring microbial organism of claim 9, wherein Ri is C9 linear alkyl, C10 linear alkyl, Cn, linear alkyl, C12 linear alkyl or C13 linear alkyl. 1 1. The non-naturally occurring microbial organism of claim 1 , wherein said microbial organism further comprises an acetyl-CoA pathway and at least one exogenous nucleic acid encoding an acetyl-CoA pathway enzyme expressed in a sufficient amount to produce acetyl-CoA, wherein said acetyl-CoA pathway comprises a pathway selected from:

(1) 2 A and 2B;

(2) 2A, 2C, and 2D;

(3) 2H;

(4) 2G and 2D;

(5) 2E, 2F and 2B;

(6) 2E and 21;

(?) 2J, 2F and 2B;

(8) 2J and 21;

(9) 3A, 3B, and 3C;

(10) 3A, 3B, 3J, 3K, and 3D;

(1 1) 3A, 3B, 3G, and 3D;

(12) 3A, 3F, and 3D;

(13) 3N, 3H, 3B and 3C;

(14) 3N, 3H, 3B, 3J, 3K, and 3D;

(15) 3N, 3H, 3B, 3G, and 3D;

(16) 3N, 3H, 3F, and 3D;

(17) 3L, 3M, 3B and 3C;

(18) 3L, 3M, 3B, 3J, 3K, and 3D;

(19) 3L, 3M, 3B, 3G, and 3D; (20) 3L, 3M, 3F, and 3D;

(21) 4A, 4B, 4D, 4H, 41, and 4J;

(22) 4A, 4B, 4E, 4F, 4H, 41, and 4J;

(23) 4A, 4B, 4E, 4K, 4L, 4H, 41, and 4J;

(24) 4A, 4C, 4D, 4H, and 4J;

(25) 4A, 4C, 4E, 4F, 4H, and 4J;

(26) 4A, 4C, 4E, 4K, 4L, 4H, and 4J;

(27) 5A, 5B, 5D, and 5G;

(28) 5A, 5B, 5E, 5F, and 5G;

(29) 5A, 5B, 5E, 5K, 5L, and 5G;

(30) 5A, 5C, and 5D;

(31) 5A, 5C, 5E, and 5F; and

(32) 5A, 5C, 5E, 5K, and 5L, wherein 2A is a pyruvate oxidase (acetate-forming), wherein 2B is an acetyl-CoA synthetase, an acetyl-CoA ligase or an acetyl-CoA transferase, wherein 2C is an acetate kinase, wherein 2D is a phosphotransacetylase, wherein 2E is a pyruvate decarboxylase, wherein 2F is an acetaldehyde dehydrogenase, wherein 2G is a pyruvate oxidase (acetyl- phosphate forming), wherein 2H is a pyruvate dehydrogenase, a pyruvate :ferredoxin oxidoreductase, a pyruvate :NAD(P)H oxidoreductase or a pyruvate formate lyase, wherein 21 is an acetaldehyde dehydrogenase (acylating), wherein 2J is a threonine aldolase, wherein 3A is a phosphoenolpyruvate (PEP) carboxylase or a PEP carboxykinase, wherein 3B is an oxaloacetate decarboxylase, wherein 3C is a malonate semialdehyde dehydrogenase

(acetylating), wherein 3D is an acetyl-CoA carboxylase or a malonyl-CoA decarboxylase, wherein 3F is an oxaloacetate dehydrogenase or an oxaloacetate oxidoreductase, wherein 3G is a malonate semialdehyde dehydrogenase (acylating), wherein 3H is a pyruvate

carboxylase, wherein 3 J is a malonate semialdehyde dehydrogenase, wherein 3K is a malonyl-CoA synthetase or a malonyl-CoA transferase, wherein 3L is a malic enzyme, wherein 3M is a malate dehydrogenase or a malate oxidoreductase, wherein 3N is a pyruvate kinase or a PEP phosphatase, wherein 4A is a citrate synthase, wherein 4B is a citrate transporter, wherein 4C is a citrate/malate transporter, wherein 4D is an ATP citrate lyase, wherein 4E is a citrate lyase, wherein 4F is an acetyl-CoA synthetase or an acetyl-CoA transferase, wherein 4H is a cytosolic malate dehydrogenase, wherein 41 is a malate transporter, wherein 4J is a mitochondrial malate dehydrogenase, wherein 4K is an acetate kinase, wherein 4L is a phosphotransacetylase, wherein 5A is a citrate synthase, wherein 5B is a citrate transporter, wherein 5C is a citrate/oxaloacetate transporter, wherein 5D is an ATP citrate lyase, wherein 5E is a citrate lyase, wherein 5F is an acetyl-CoA synthetase or an acetyl-CoA transferase, wherein 5G is an oxaloacetate transporter, wherein 5K is an acetate kinase, and wherein 5L is a phosphotransacetylase.

12. The non-naturally occurring microbial organism of claim 11, wherein said microbial organism comprises two, three, four, five, six, seven or eight exogenous nucleic acids each encoding an acetyl-CoA pathway enzyme.

13. The non-naturally occurring microbial organism of claim 12, wherein said microbial organism comprises exogenous nucleic acids encoding each of the acetyl-CoA pathway enzymes of at least one of the pathways selected from (l)-(32).

14. The non-naturally occurring microbial organism of claim 1, further comprising one or more gene disruptions, said one or more gene disruptions occurring in endogenous genes encoding proteins or enzymes involved in: native production of ethanol, glycerol, acetate, formate, lactate, C02, fatty acids, or malonyl-CoA by said microbial organism; transfer of pathway intermediates to cellular compartments other than the cytosol; or native degradation of a MI-FAE cycle intermediate, MD-FAE cycle intermediate or a termination pathway intermediate by said microbial organism, wherein said one or more gene disruptions confer increased production of the compound of Formula (I) in said microbial organism. 15. The non-naturally occurring microbial organism of claim 14, wherein said protein or enzyme is selected from the group consisting of a fatty acid synthase, an acetyl- CoA carboxylase, a biotin:apoenzyme ligase, an acyl carrier protein, a thioesterase, an acyltransferase, an ACP malonyltransferase, a fatty acid elongase, an acyl-CoA synthetase, an acyl-CoA transferase, an acyl-CoA hydrolase, a pyruvate decarboxylase, a lactate dehydrogenase, an alcohol dehydrogenase, an acid-forming aldehyde dehydrogenases, an acetate kinase, a phosphotransacetylase, a pyruvate oxidase, a glycerol-3 -phosphate dehydrogenase, a glycerol-3 -phosphate phosphatase, a mitochondrial pyruvate carrier, a peroxisomal fatty acid transporter, a peroxisomal acyl-CoA transporter, a peroxisomal carnitine/acylcarnitine transferase, an acyl-CoA oxidase, and an acyl-CoA binding protein. 16. The non-naturally occurring microbial organism of claim 1, wherein one or more enzymes of the MI-FAE cycle, MD-FAE cycle or the termination pathway preferentially react with an NADH cofactor or have reduced preference for reacting with an NAD(P)H cofactor, wherein said one or more enzymes of the MI-FAE cycle or MD-FAE cycle are a 3- ketoacyl-CoA reductase or an enoyl-CoA reductase, and wherein said one or more enzymes of the termination pathway are selected from an acyl-CoA reductase (aldehyde forming), an alcohol dehydrogenase, an acyl-CoA reductase (alcohol forming), an aldehyde

decarbonylase, an acyl-ACP reductase, an aldehyde dehydrogenase (acid forming) and a carboxylic acid reductase.

17. The non-naturally occurring microbial organism of claim 1, further comprising one or more gene disruptions, said one or more gene disruptions occurring in genes encoding proteins or enzymes that result in an increased ratio of NAD(P)H to NAD(P) present in the cytosol of said microbial organism following said disruptions.

18. The non-naturally occurring microbial organism of claim 17, wherein said gene encoding a protein or enzyme that results in an increased ratio of NAD(P)H to NAD(P) present in the cytosol of said microbial organism following said disruptions is selected from the group consisting of an NADH dehydrogenase, a cytochrome oxidase, a glycerol-3- phosphate dehydrogenase, glycerol-3 -phosphate phosphatase, an alcohol dehydrogenase, a pyruvate decarboxylase, an aldehyde dehydrogenase (acid forming), a lactate dehydrogenase, a glycerol-3 -phosphate dehydrogenase, a glycerol-3 -phosphate :quinone oxidoreductase, a malic enzyme and a malate dehydrogenase.

19. The non-naturally occurring organism of claims 14 or 17, wherein said one or more gene disruptions comprises a deletion of said one or more genes.

20. The non-naturally occurring microbial organism of claim 1, wherein said microbial organism is Crabtree positive and is in culture medium comprising excess glucose, thereby increasing the ratio of NAD(P)H to NAD(P) present in the cytosol of said microbial organism.

21. The non-naturally occurring microbial organism of claim 1, further comprising at least one exogenous nucleic acid encoding an extracellular transporter or an extracellular transport system for the compound of Formula (I).

22. The non-naturally occurring microbial organism of claim 1, wherein one or more endogenous enzymes involved in: native production of ethanol, glycerol, acetate, formate, lactate, C02, fatty acids, or malonyl-CoA by said microbial organism; transfer of pathway intermediates to cellular compartments other than the cytosol; or native degradation of a MI- FAE cycle intermediate, MD-FAE cycle intermediate or a termination pathway intermediate by said microbial organism, has attenuated enzyme activity or expression levels.

23. The non-naturally occurring microbial organism of claim 22, wherein said enzyme is selected from the group consisting of a fatty acid synthase, an acetyl-CoA carboxylase, a biotin:apoenzyme ligase, a thioesterase, an acyl carrier protein, a thioesterase, an acyltransferase, an ACP malonyltransferase, a fatty acid elongase, an acyl-CoA

synthetase, an acyl-CoA transferase, an acyl-CoA hydrolase, a pyruvate decarboxylase, a lactate dehydrogenase, a short-chain alcohol dehydrogenase, an acid-forming aldehyde dehydrogenase, an acetate kinase, a phosphotransacetylase, a pyruvate oxidase, a glycerol-3 - phosphate dehydrogenase, a glycerol-3 -phosphate phosphatase, a mitochondrial pyruvate carrier, a peroxisomal fatty acid transporter, a peroxisomal acyl-CoA transporter, a peroxisomal carnitine/acylcarnitine transferase, an acyl-CoA oxidase, and an acyl-CoA binding protein.

24. The non-naturally occurring microbial organism of claim 1, wherein one or more endogenous enzymes involved in the oxidation of NAD(P)H or NADH, has attenuated enzyme activity or expression levels.

25. The non-naturally occurring microbial organism of claim 24, wherein said one or more endogenous enzymes are selected from the group consisting of an NADH

dehydrogenase, a cytochrome oxidase, a glycerol-3 -phosphate dehydrogenase, glycerol-3 - phosphate phosphatase, an alcohol dehydrogenase, a pyruvate decarboxylase, an aldehyde dehydrogenase (acid forming), a lactate dehydrogenase, a glycerol-3 -phosphate

dehydrogenase, a glycerol-3-phosphate:quinone oxidoreductase, a malic enzyme and a malate dehydrogenase.

26. A method for producing a compound of Formula (I):

(I) wherein Ri is Ci_24 linear alkyl; R2 is CH2OH, CHO, or COOH; R3 is H, OH, or oxo

(=0); and represents a single or double bond with the proviso that the valency of the carbon atom to which R3 is attached is four, comprising culturing the non-naturally occurring microbial organism of any one of claims 1-25 under conditions and for a sufficient period of time to produce said compound of Formula (I).

27. The method of claim 26, wherein said method further comprises separating the compound of Formula (I) from other components in the culture.

28. The method of claim 27, wherein the separating comprises extraction, continuous liquid-liquid extraction, pervaporation, membrane filtration, membrane separation, reverse osmosis, electrodialysis, distillation, crystallization, centrifugation, extractive filtration, ion exchange chromatography, absorption chromatography, or ultrafiltration.

29. Culture medium comprising bioderived compound of Formula (I):

(I) wherein Ri is Ci_24 linear alkyl; R2 is CH2OH, CHO, or COOH; R3 is H, OH, or oxo

(=0); and represents a single or double bond with the proviso that the valency of the carbon atom to which R3 is attached is four, wherein said bioderived compound has a carbon- 12, carbon- 13 and carbon- 14 isotope ratio that reflects an atmospheric carbon dioxide uptake source.

30. The culture medium of claim 29, wherein said culture medium is separated from a non-naturally occurring microbial organism having a malonyl-CoA independent fatty acyl- CoA elongation (MI-FAE) cycle and/or a malonyl-CoA dependent fatty acyl-CoA elongation (MD-FAE) cycle in combination with a termination pathway.

31. A bioderived compound of Formula (I) :

(I) wherein Ri is Ci_24 linear alkyl; R2 is CH2OH, CHO, or COOH; R3 is H, OH, or oxo

(=0); and represents a single or double bond with the proviso that the valency of the carbon atom to which R3 is attached is four, having a carbon- 12, carbon- 13 and carbon- 14 isotope ratio that reflects an atmospheric carbon dioxide uptake source.

32. The bioderived compound of claim 31 , wherein said bioderived compound has an Fm value of at least 80%, at least 85%, at least 90%, at least 95% or at least 98%.

33. The bioderived compound produced according to the method of any one of claims 26-28.

34. A composition comprising said bioderived compound of any one of claims 31-33 and a compound other than said bioderived compound.

35. The composition of claim 34 wherein said compound other than said bioderived compound is a trace amount of a cellular portion of a non-naturally occurring microbial organism having a malonyl-CoA independent fatty acyl-CoA elongation (MI-FAE) cycle and/or a malonyl-CoA dependent fatty acyl-CoA elongation (MD-FAE) cycle in combination with a termination pathway.

36. A composition comprising the bioderived compound of any one of claims 31-33, or a cell lysate or culture supernatant thereof.

37. A biobased product comprising said bioderived compound of any one of claims 31-33, wherein said biobased product is a biofuel, chemical, polymer, surfactant, soap, detergent, shampoo, lubricating oil additive, fragrance, flavor material or acrylate.

38. The biobased product of claim 37 comprising at least 5%>, at least 10%>, at least 20%), at least 30%>, at least 40%> or at least 50%> said bioderived compound.

39. The biobased product of claims 37 or 38, wherein said biobased product comprises a portion of said bioderived compound as a repeating unit.

40. A molded product obtained by molding a biobased product of any one of claims 37-39, biobased product is a polymer.

41. A process for producing a biobased product of any one of claims 37-39 comprising chemically reacting said bioderived compound with itself or another compound in a reaction that produces said biobased product.

Description:
MICROORGANISMS AND METHODS FOR PRODUCTION OF SPECIFIC LENGTH FATTY ALCOHOLS AND RELATED COMPOUNDS

This application claims the benefit of priority of United States Provisional application serial No. 61/714,144, filed October 15, 2012, the entire contents of which is incorporated herein by reference.

BACKGROUND OF THE INVENTION

The present invention relates generally to biosynthetic processes, and more specifically to organisms having specific length fatty alcohol, fatty aldehyde or fatty acid biosynthetic capacity.

Primary alcohols are a product class of compounds having a variety of industrial applications which include a variety of bio fuels and specialty chemicals. Primary alcohols also can be used to make a large number of additional industrial products including polymers and surfactants. For example, higher primary alcohols, also known as fatty alcohols (C 4 -C 24 ) and their ethoxylates are used as surfactants in many consumer detergents, cleaning products and personal care products worldwide such as laundry powders and liquids, dishwashing liquid and hard surface cleaners. They are also used in the manufacture of a variety of industrial chemicals and in lubricating oil additives. Specific length fatty alcohols, such as octanol and hexanol, have useful organoleptic properties and have long been employed as fragrance and flavor materials. Smaller chain length C4-C8 alcohols (e.g., butanol) are used as chemical intermediates for production of derivatives such as acrylates used in paints, coatings, and adhesives applications.

Fatty alcohols are currently produced from, for example, hydrogenation of fatty acids, hydroformylation of terminal olefins, partial oxidation of n-paraffins and the Al- catalyzed polymerization of ethylene. Unfortunately, it is not commercially viable to produce fatty alcohols directly from the oxidation of petroleum-based linear hydrocarbons (n-paraffins). This impracticality is because the oxidation of n-paraffins produces primarily secondary alcohols, tertiary alcohols or ketones, or a mixture of these compounds, but does not produce high yields of fatty alcohols. Additionally, currently known methods for producing fatty alcohols suffer from the disadvantage that they are restricted to feedstock which is relatively expensive, notably ethylene, which is produced via the thermal cracking of petroleum. In addition, current methods require several steps, and several catalyst types. Fatty alcohol production by microorganisms involves fatty acid synthesis followed by acyl-reduction steps. The universal fatty acid biosynthesis pathway found in most cells has been investigated for production of fatty alcohols and other fatty acid derivatives. There is currently a great deal of improvement that can be achieved to provide more efficient biosynthesis pathways for fatty alcohol production with significantly higher theoretical product and energy yields.

Thus, there exists a need for alternative means for effectively producing commercial quantities of fatty alcohols. The present invention satisfies this need and provides related advantages as well.

SUMMARY OF INVENTION

The invention provides non-naturally occurring microbial organisms containing fatty alcohol, fatty aldehyde or fatty acid pathways. In some embodiments, the non-naturally occurring microbial organism of the invention has a malonyl-CoA independent fatty acyl- CoA elongation (MI-FAE) cycle and/or a malonyl-CoA dependent fatty acyl-CoA elongation (MD-FAE) cycle in combination with a termination pathway as depicted in Figures 1 , 6 and 7, wherein an enzyme of the MI-FAE cycle, MD-FAE cycle or termination pathway is encoded by at least one exogenous nucleic acid and is expressed in a sufficient amount to produce a fatty alcohol, fatty aldehyde or fatty acid of Formula (I):

(I) wherein Ri is Ci_ 24 linear alkyl; R 2 is CH 2 OH, CHO, or COOH; R 3 is H, OH, or oxo

(=0); and represents a single or double bond with the proviso that the valency of the carbon atom to which R 3 is attached is four, wherein the substrate of each of said enzymes of the MI-FAE cycle, the MD-FAE cycle and the termination pathway are independently selected from a compound of Formula (II), malonyl-CoA, propionyl-CoA or acetyl-CoA:

wherein Ri is Ci_ 24 linear alkyl; R 3 is H, OH, or oxo (=0); R 4 is S-CoA, ACP, OH or

H; and represents a single or double bond with the proviso that the valency of the carbon atom to which R 3 is attached is four; wherein said one or more enzymes of the MI- FAE cycle are each selective for a compound of Formula (II) having a number of carbon atoms at Ri that is no greater than the number of carbon atoms at Ri of said compound of Formula (I), wherein said one or more enzymes of the MD-FAE cycle are each selective for a compound of Formula (II) having a number of carbon atoms at Ri that is no greater than the number of carbon atoms at Ri of said compound of Formula (I), and wherein said one or more enzymes of the termination pathway are each selective for a compound of Formula (II) having a number of carbon atoms at Ri that is no less than the number of carbon atoms at Ri of said compound of Formula (I).

In some embodiments, the invention provides a non-naturally occurring microbial organism having a fatty alcohol, fatty aldehyde or fatty acid pathway, wherein the microbial organism further includes an acetyl-CoA pathway and at least one exogenous nucleic acid encoding an acetyl-CoA pathway enzyme expressed in a sufficient amount to produce acetyl- CoA, wherein the acetyl-CoA pathway includes a pathway shown in Figures 2, 3, 4 or 5.

In some embodiments, the invention provides a non-naturally occurring microbial organism having a fatty alcohol, fatty aldehyde or fatty acid pathway, wherein the microbial organism has one or more gene disruptions, wherein the one or more gene disruptions occur in endogenous genes encoding proteins or enzymes involved in: native production of ethanol, glycerol, acetate, formate, lactate, C0 2 , fatty acids, or malonyl-CoA by said microbial organism; transfer of pathway intermediates to cellular compartments other than the cytosol; or native degradation of a MI-FAE cycle intermediate, MD-FAE cycle intermediate or a termination pathway intermediate by the microbial organism, the one or more gene disruptions confer increased production of a fatty alcohol, fatty aldehyde or fatty acid in the microbial organism. In some embodiments, the invention provides a non-naturally occurring microbial organism having a fatty alcohol, fatty aldehyde or fatty acid pathway, wherein one or more enzymes of the MI-FAE cycle, MD-FAE cycle or the termination pathway preferentially react with an NADH cofactor or have reduced preference for reacting with an NAD(P)H cofactor.

In some embodiments, the invention provides a non-naturally occurring microbial organism having a fatty alcohol, fatty aldehyde or fatty acid pathway, wherein the microbial organism has one or more gene disruptions in genes encoding proteins or enzymes that result in an increased ratio of NAD(P)H to NAD(P) present in the cytosol of the microbial organism following the disruptions.

In some embodiments, the non-naturally occurring microbial organism of the invention is Crabtree positive and is in culture medium comprising excess glucose. In such conditions, as described herein, the microbial organism can result in increasing the ratio of NAD(P)H to NAD(P) present in the cytosol of the microbial organism.

In some embodiments, the invention provides a non-naturally occurring microbial organism having a fatty alcohol, fatty aldehyde or fatty acid pathway, wherein the microbial organism has at least one exogenous nucleic acid encoding an extracellular transporter or an extracellular transport system for a fatty alcohol, fatty aldehyde or fatty acid of the invention.

In some embodiments, the invention provides a non-naturally occurring microbial organism having a fatty alcohol, fatty aldehyde or fatty acid pathway, wherein the microbial organism one or more endogenous enzymes involved in: native production of ethanol, glycerol, acetate, formate, lactate, C0 2 , fatty acids, or malonyl-CoA by said microbial organism; transfer of pathway intermediates to cellular compartments other than the cytosol; or native degradation of a MI-FAE cycle intermediate, a MD-FAE cycle intermediate or a termination pathway intermediate by said microbial organism, has attenuated enzyme activity or expression levels.

In some embodiments, the invention provides a non-naturally occurring microbial organism having a fatty alcohol, fatty aldehyde or fatty acid pathway, wherein the microbial organism has attenuated enzyme activity or expression levels for one or more endogenous enzymes involved in the oxidation of NAD(P)H or NADH. The invention additionally provides methods of using the above microbial organisms to produce a fatty alcohol, a fatty aldehyde or a fatty acid by culturing a non-naturally occurring microbial organism containing a fatty alcohol, fatty aldehyde or fatty acid pathway under conditions and for a sufficient period of time to produce a fatty alcohol, fatty aldehyde or fatty acid.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 shows an exemplary MI-FAE cycle and/or MD-FAE cycle in combination with termination pathways for production of fatty alcohols, aldehydes, or acids from the acyl- CoA intermediate of the MI-FAE cycle or MD-FAE cycle. Enzymes are: A. Thiolase; B. 3- Oxoacyl-CoA reductase; C. 3-Hydroxyacyl-CoA dehydratase; D. Enoyl-CoA reductase; E. Acyl-CoA reductase (aldehyde forming); F. Alcohol dehydrogenase; G. Acyl-CoA reductase (alcohol forming); H. acyl-CoA hydrolase, transferase or synthase; J. Acyl-ACP reductase; K. Acyl-CoA:ACP acyltransferase; L. Thioesterase; N. Aldehyde dehydrogenase (acid forming) or carboxylic acid reductase; and O. Elongase. Figure 2 shows exemplary pathways for production of cytosolic acetyl-CoA from pyruvate or threonine. Enzymes are: A. pyruvate oxidase (acetate-forming); B. acetyl-CoA synthetase, ligase or transferase; C. acetate kinase; D. phosphotransacetylase; E. pyruvate decarboxylase; F. acetaldehyde dehydrogenase; G. pyruvate oxidase (acetyl-phosphate forming); H. pyruvate dehydrogenase, pyruvate :ferredoxin oxidoreductase,

pyruvate :NAD(P)H oxidoreductase or pyruvate formate lyase; I. acetaldehyde dehydrogenase (acylating); and J. threonine aldolase.

Figure 3 shows exemplary pathways for production of acetyl-CoA from

phosphoenolpyruvate (PEP). Enzymes are: A. PEP carboxylase or PEP carboxykinase; B. oxaloacetate decarboxylase; C. malonate semialdehyde dehydrogenase (acetylating); D. acetyl-CoA carboxylase or malonyl-CoA decarboxylase; F. oxaloacetate dehydrogenase or oxaloacetate oxidoreductase; G. malonate semialdehyde dehydrogenase (acylating); H.

pyruvate carboxylase; J. malonate semialdehyde dehydrogenase; K. malonyl-CoA synthetase or transferase; L. malic enzyme; M. malate dehydrogenase or oxidoreductase; and N.

pyruvate kinase or PEP phosphatase. Figure 4 shows exemplary pathways for production of cytosolic acetyl-CoA from mitochondrial acetyl-CoA using citrate and malate transporters. Enzymes are: A. citrate synthase; B. citrate transporter; C. citrate/malate transporter; D. ATP citrate lyase; E. citrate lyase; F. acetyl-CoA synthetase or transferase; H. cytosolic malate dehydrogenase; I. malate transporter; J. mitochondrial malate dehydrogenase; K. acetate kinase; and L.

phosphotransacetylase.

Figure 5 shows exemplary pathways for production of cytosolic acetyl-CoA from mitochondrial acetyl-CoA using citrate and oxaloacetate transporters. Enzymes are: A. citrate synthase; B. citrate transporter; C. citrate/oxaloacetate transporter; D. ATP citrate lyase; E. citrate lyase; F. acetyl-CoA synthetase or transferase; G) oxaloacetate transporter; K) acetate kinase; and L) phosphotransacetylase.

Figure 6 shows an exemplary MI-FAE cycle and/or MD-FAE cycle for elongating the linear alkyl of Ri. Enzymes are: A. Thiolase; B. 3-Ketoacyl-CoA reductase; C. 3- Hydroxyacyl-CoA dehydratase; D. Enoyl-CoA reductase; and E. Elongase.

Figure 7 shows an exemplary termination cycle for generating a fatty alcohol, fatty aldehyde or fatty acid from any of the MI-FAE cycle intermediates or MD-FAE cycle intermediates of Figure 6. Enzymes are: E. MI-FAE/MD-FAE intermediate-CoA reductase (aldehyde forming); F. Alcohol dehydrogenase; G. MI-FAE/MD-FAE intermediate-CoA reductase (alcohol forming); H. MI-FAE/MD-FAE intermediate-CoA hydrolase, transferase or synthase; J. MI-FAE/MD-FAE intermediate- ACP reductase; K. MI-FAE/MD-FAE intermediate-CoA:ACP acyltransferase; L. Thioesterase; and N. Aldehyde dehydrogenase (acid forming) or carboxylic acid reductase. Rl is C I -24 linear alkyl; R 3 is H, OH, or oxo

(=0) and represents a single or double bond with the proviso that the valency of the carbon atom to which R 3 is attached is four.

Figure 8 shows exemplary compounds that can be produced from the four MI-FAE or MD-FAE cycle intermediates using the cycles depicted in Figure 6 and the termination pathways depicted in Figure 7. R is Ci_ 2 4 linear alkyl.

Figure 9 depicts the production of 1 ,3-butanediol (Figure 9 A) or ethanol (Figure 9B) in S. cerevisiae transformed with plasmids comprising genes encoding various MI-FAE cycle and termination pathway enzymes, either with or without pflAV or PDH bypass, as provided in Example X. Figure 10 depicts the production of pyruvic acid (FIG. 10A), succinic acid (FIG. 12B), acetic acid (FIG. 12C) or glucose (FIG. 12D) in S. cerevisiae transformed with plasmids comprising genes encoding various MI-FAE cycle and termination pathway enzymes, either with or without pflAV or PDH bypass, as provided in Example X. Figure 11 depicts the production of 1,3-butanediol in S. cerevisiae transformed with plasmids comprising genes encoding various MI-FAE cycle and termination pathway enzymes, either with or without pflAV or PDH bypass, as provided in Example X.

Figure 12 depicts the estimated specific activity of five thiolases for acetyl-CoA condensation activity in E. coli as provided in Example XI. Figure 13 depicts the estimated specific activity of two thiolases (1491 and 560) cloned in dual promoter yeast vectors with 1495 (a 3-hydroxybutyryl-CoA dehydrogenase) for acetyl-CoA condensation activity in E. coli as provided in Example XI.

Figure 14 depicts the time course of fluorescence detection of oxidation of NADH, which is used to measure the metabolism of acetoacetyl-CoA to 3-hydroxybutyryl-CoA by 3- hydroxybutyryl-CoA dehydrogenase, as provided in Example XI. Acetoacetyl-CoA is metabolized to 3-hydroxybutyryl-CoA by 3-hydroxybutyryl-CoA dehydrogenase. The reaction requires oxidation of NADH, which can be monitored by fluorescence at an excitation wavelength at 340 nm and an emission at 460 nm. The oxidized form, NAD+, does not fluoresce. 1495, the Hbd from Clostridium beijerinckii, was assayed in the dual promoter yeast vectors that contained either 1491 (vector id = pY3Hdl7) or 560 (vector id = pY3Hdl6).

Figure 15 depicts levels of NAD(P)H oxidation in the presence of 1 or 5 ug/ml NADH or 1 or 5 ug/ml NADPH, and shows that the Hbd prefers NADH over NADPH, as provided in Example XI. Figure 16 depicts the activity data for crude lysates of an aldehyde reductase that converts 3-hydroxybutyryl-CoA to 3-hydroxybutyraldehyde and requires NAD(P)H oxidation, which can be used to monitor enzyme activity, as provided in Example XI. The Aid from Lactobacillus brevis (Gene ID 707) was cloned in a dual vector that contained the alcohol dehydrogenase from Clostridium saccharoperbutylacetonicum (Gene ID 28). These two enzymes were cloned in another dual promoter yeast vector containing a Leu marker. A 707 lysate from E. coli was used as a standard.

Figure 17 depicts the evaluation of ADH (Gene 28) in the dual promoter vector with ALD (Gene 707) with butyraldehyde, a surrogate substrate for 3-hydroxybutyraldehyde. 1,3- BDO is formed by an alcohol dehydrogenase (Adh), which reduces 3-hydroxybutyraldehyde in the presence of NAD(P)H, and the oxidation of NAD(P)H is used to monitor the reaction.

DETAILED DESCRIPTION OF THE INVENTION

As used herein, the term "non-naturally occurring" when used in reference to a microbial organism or microorganism of the invention is intended to mean that the microbial organism has at least one genetic alteration not normally found in a naturally occurring strain of the referenced species, including wild-type strains of the referenced species. Genetic alterations include, for example, modifications introducing expressible nucleic acids encoding metabolic polypeptides, other nucleic acid additions, nucleic acid deletions and/or other functional disruption of the microbial organism's genetic material. Such modifications include, for example, coding regions and functional fragments thereof, for heterologous, homologous or both heterologous and homologous polypeptides for the referenced species. Additional modifications include, for example, non-coding regulatory regions in which the modifications alter expression of a gene or operon. Exemplary metabolic polypeptides include enzymes or proteins within a fatty alcohol, fatty aldehyde or fatty alcohol biosynthetic pathway.

A metabolic modification refers to a biochemical reaction that is altered from its naturally occurring state. Therefore, non-naturally occurring microorganisms can have genetic modifications to nucleic acids encoding metabolic polypeptides, or functional fragments thereof. Exemplary metabolic modifications are disclosed herein. As used herein, the term "isolated" when used in reference to a microbial organism is intended to mean an organism that is substantially free of at least one component as the referenced microbial organism is found in nature. The term includes a microbial organism that is removed from some or all components as it is found in its natural environment. The term also includes a microbial organism that is removed from some or all components as the microbial organism is found in non-naturally occurring environments. Therefore, an isolated microbial organism is partly or completely separated from other substances as it is found in nature or as it is grown, stored or subsisted in non-naturally occurring environments. Specific examples of isolated microbial organisms include partially pure microbes, substantially pure microbes and microbes cultured in a medium that is non-naturally occurring.

As used herein, the terms "microbial," "microbial organism" or "microorganism" are intended to mean any organism that exists as a microscopic cell that is included within the domains of archaea, bacteria or eukarya. Therefore, the term is intended to encompass prokaryotic or eukaryotic cells or organisms having a microscopic size and includes bacteria, archaea and eubacteria of all species as well as eukaryotic microorganisms such as yeast and fungi. The term also includes cell cultures of any species that can be cultured for the production of a biochemical.

As used herein, the term "CoA" or "coenzyme A" is intended to mean an organic cofactor or prosthetic group (nonprotein portion of an enzyme) whose presence is required for the activity of many enzymes (the apoenzyme) to form an active enzyme system.

Coenzyme A functions in certain condensing enzymes, acts in acetyl or other acyl group transfer and in fatty acid synthesis and oxidation, pyruvate oxidation and in other acetylation.

As used herein, the term "ACP" or "acyl carrier protein" refers to any of the relatively small acidic proteins that are associated with the fatty acid synthase system of many organisms, from bacteria to plants. ACPs can contain one 4'-phosphopantetheine prosthetic group bound covalently by a phosphate ester bond to the hydroxyl group of a serine residue. The sulfhydryl group of the 4'-phosphopantetheine moiety serves as an anchor to which acyl intermediates are (thio)esterified during fatty-acid synthesis. An example of an ACP is Escherichia coli ACP, a separate single protein, containing 77 amino-acid residues (8.85 kDa), wherein the phosphopantetheine group is linked to serine 36.

As used herein, the term "substantially anaerobic" when used in reference to a culture or growth condition is intended to mean that the amount of oxygen is less than about 10% of saturation for dissolved oxygen in liquid media. The term also is intended to include sealed chambers of liquid or solid medium maintained with an atmosphere of less than about 1% oxygen.

"Exogenous" as it is used herein is intended to mean that the referenced molecule or the referenced activity is introduced into the host microbial organism. The molecule can be introduced, for example, by introduction of an encoding nucleic acid into the host genetic material such as by integration into a host chromosome or as non-chromosomal genetic material such as a plasmid. Therefore, the term as it is used in reference to expression of an encoding nucleic acid refers to introduction of the encoding nucleic acid in an expressible form into the microbial organism. When used in reference to a biosynthetic activity, the term refers to an activity that is introduced into the host reference organism. The source can be, for example, a homologous or heterologous encoding nucleic acid that expresses the referenced activity following introduction into the host microbial organism. Therefore, the term "endogenous" refers to a referenced molecule or activity that is present in the host. Similarly, the term when used in reference to expression of an encoding nucleic acid refers to expression of an encoding nucleic acid contained within the microbial organism. The term "heterologous" refers to a molecule or activity derived from a source other than the referenced species whereas "homologous" refers to a molecule or activity derived from the host microbial organism. Accordingly, exogenous expression of an encoding nucleic acid of the invention can utilize either or both a heterologous or homologous encoding nucleic acid. It is understood that when more than one exogenous nucleic acid is included in a microbial organism that the more than one exogenous nucleic acids refers to the referenced encoding nucleic acid or biosynthetic activity, as discussed above. It is further understood, as disclosed herein, that such more than one exogenous nucleic acids can be introduced into the host microbial organism on separate nucleic acid molecules, on polycistronic nucleic acid molecules, or a combination thereof, and still be considered as more than one exogenous nucleic acid. For example, as disclosed herein a microbial organism can be engineered to express two or more exogenous nucleic acids encoding a desired pathway enzyme or protein. In the case where two exogenous nucleic acids encoding a desired activity are introduced into a host microbial organism, it is understood that the two exogenous nucleic acids can be introduced as a single nucleic acid, for example, on a single plasmid, on separate plasmids, can be integrated into the host chromosome at a single site or multiple sites, and still be considered as two exogenous nucleic acids. Similarly, it is understood that more than two exogenous nucleic acids can be introduced into a host organism in any desired combination, for example, on a single plasmid, on separate plasmids, can be integrated into the host chromosome at a single site or multiple sites, and still be considered as two or more exogenous nucleic acids, for example three exogenous nucleic acids. Thus, the number of referenced exogenous nucleic acids or biosynthetic activities refers to the number of encoding nucleic acids or the number of biosynthetic activities, not the number of separate nucleic acids introduced into the host organism.

As used herein, the term "gene disruption," or grammatical equivalents thereof, is intended to mean a genetic alteration that renders the encoded gene product inactive or attenuated. The genetic alteration can be, for example, deletion of the entire gene, deletion of a regulatory sequence required for transcription or translation, deletion of a portion of the gene which results in a truncated gene product, or by any of various mutation strategies that inactivate or attenuate the encoded gene product. One particularly useful method of gene disruption is complete gene deletion because it reduces or eliminates the occurrence of genetic reversions in the non-naturally occurring microorganisms of the invention. A gene disruption also includes a null mutation, which refers to a mutation within a gene or a region containing a gene that results in the gene not being transcribed into RNA and/or translated into a functional gene product. Such a null mutation can arise from many types of mutations including, for example, inactivating point mutations, deletion of a portion of a gene, entire gene deletions, or deletion of chromosomal segments.

As used herein, the term "growth-coupled" when used in reference to the production of a biochemical product is intended to mean that the biosynthesis of the referenced biochemical product is produced during the growth phase of a microorganism. In a particular embodiment, the growth-coupled production can be obligatory, meaning that the biosynthesis of the referenced biochemical is an obligatory product produced during the growth phase of a microorganism.

As used herein, the term "attenuate," or grammatical equivalents thereof, is intended to mean to weaken, reduce or diminish the activity or amount of an enzyme or protein.

Attenuation of the activity or amount of an enzyme or protein can mimic complete disruption if the attenuation causes the activity or amount to fall below a critical level required for a given pathway to function. However, the attenuation of the activity or amount of an enzyme or protein that mimics complete disruption for one pathway, can still be sufficient for a separate pathway to continue to function. For example, attenuation of an endogenous enzyme or protein can be sufficient to mimic the complete disruption of the same enzyme or protein for production of a fatty alcohol, fatty aldehyde or fatty acid product of the invention, but the remaining activity or amount of enzyme or protein can still be sufficient to maintain other pathways, such as a pathway that is critical for the host microbial organism to survive, reproduce or grow. Attenuation of an enzyme or protein can also be weakening, reducing or diminishing the activity or amount of the enzyme or protein in an amount that is sufficient to increase yield of a fatty alcohol, fatty aldehyde or fatty acid product of the invention, but does not necessarily mimic complete disruption of the enzyme or protein.

The term "fatty alcohol," as used herein, is intended to mean an aliphatic compound that contains one or more hydroxyl groups and contains a chain of 4 or more carbon atoms. The fatty alcohol possesses the group -CH 2 OH that can be oxidized so as to form a corresponding aldehyde or acid having the same number of carbon atoms. A fatty alcohol can also be a saturated fatty alcohol, an unsaturated fatty alcohol, a 1,3-diol, or a 3-oxo- alkan-l-ol. Exemplary fatty alcohols include a compound of Formula (III)-(VI):

(VI) wherein Ri is a Ci_ 24 linear alkyl.

The term "fatty aldehyde," as used herein, is intended to mean an aliphatic compound that contains an aldehyde (CHO) group and contains a chain of 4 or more carbon atoms. The fatty aldehyde can be reduced to form the corresponding alcohol or oxidized to form the carboxylic acid having the same number of carbon atoms. A fatty aldehyde can also be a saturated fatty aldehyde, an unsaturated fatty aldehyde, a 3 -hydroxy aldehyde or 3- oxoaldehyde. Exemplary fatty aldehydes include a compound of Formula (VII)-(X):

(X) wherein Ri is a Ci_ 24 linear alkyl.

The term "fatty acid," as used herein, is intended to mean an aliphatic compound that contains a carboxylic acid group and contains a chain of 4 or more carbon atoms. The fatty acid can be reduced to form the corresponding alcohol or aldehyde having the same number of carbon atoms. A fatty acid can also be a saturated fatty acid, an unsaturated fatty acid, a 3- hydroxyacid or a 3-oxoacids. Exemplary fatty acids include a compound of Formula (XI)- (XIV):

(XIV) wherein Ri is a Ci_ 2 4 linear alkyl.

The term "alkyl" refers to a linear saturated monovalent hydrocarbon. The alkyl can be a linear saturated monovalent hydrocarbon that has 1 to 24 (C 1-24 ), 1 to 17 (C 1-17 ), or 9 to 13 (C9-13) carbon atoms. Examples of alkyl groups include, but are not limited to, methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl, undecyl and dodecyl. For example, Cc>_i3 alkyl refers to a linear saturated monovalent hydrocarbon of 9 to 13 carbon atoms.

The invention disclosed herein is based, at least in part, on recombinant

microorganisms capable of synthesizing fatty alcohols, fatty aldehydes, or fatty acids using a malonyl-CoA-independent fatty acid elongation (MI-FAE) cycle and/or malonyl-CoA dependent fatty acid elongation cycle (MD-FAE) cycle in combination with a termination pathway. In some embodiments, the microorganisms of the invention can utilize a heterologous MI-FAE cycle and/or a MD-FAE cycle coupled with an acyl-CoA termination pathway to form fatty alcohols, fatty aldehydes, or fatty acids. The MI-FAE cycle can include a thiolase, a 3-oxoacyl-CoA reductase, a 3-hydroxyacyl-CoA dehydratase and an enoyl-CoA reductase. The MD-FAE cycle can include an elongase, a 3-oxoacyl-CoA reductase, a 3-hydroxyacyl-CoA dehydratase and an enoyl-CoA reductase. Each passage through the MI-FAE cycle and/or the MD-FAE cycle results in the formation of an acyl-CoA elongated by a single two carbon unit compared to the acyl-CoA substrate entering the elongation cycle. Products can be even or odd chain length, depending on the initial substrate entering the acyl-CoA elongation pathway, i.e. two acety-CoA substrates, malonyl-CoA or one acetyl-CoA substrate combined with a propionyl-CoA substrate. Elongation of the two acetyl-CoA substrates or malonyl-CoA produces an even chain length product, whereas elongation with the propionyl-CoA substrate produces an odd chain length product. A termination pathway catalyzes the conversion of a MI-FAE intermediate and/or a MD-FAE intermediate, such as the acyl-CoA, to its corresponding fatty alcohol, fatty aldehyde, or fatty acid product. MI-FAE cycle, MD-FAE cycle and termination pathway enzymes can be expressed in one or more compartments of the microorganism. For example, in one embodiment, all MI-FAE cycle and termination pathway enzymes are expressed in the cytosol. In another embodiment, all MD-FAE cycle and termination pathway enzymes are expressed in the cytosol. Additionally, the microorganisms of the invention can be engineered to optionally secret the desired product into the culture media or fermentation broth for further manipulation or isolation.

Products of the invention include fatty alcohols, fatty aldehydes, or fatty acids derived from intermediates of the MI-FAE cycle and/or MD-FAE cycle. For example, alcohol products can include saturated fatty alcohols, unsaturated fatty alcohols, 1,3-diols, and 3-oxo- alkan-l-ols. Aldehyde products can include saturated fatty aldehydes, unsaturated fatty aldehydes, 3 -hydroxy aldehydes and 3-oxoaldehydes. Acid products can include saturated fatty acids, unsaturated fatty acids, 3 -hydroxy acids and 3-oxoacids. These products can further be converted to derivatives such as fatty esters, either by chemical or enzymatic means. Methods for converting fatty alcohols to esters are well known in the art.

The invention also encompasses fatty alcohol, fatty aldehyde, and fatty acid chain- length control strategies in conjunction with host strain engineering strategies, such that the non-naturally occurring microorganism of the invention efficiently directs carbon and reducing equivalents toward fermentation products of a specific chain length.

Recombinant microorganisms of the invention can produce commercial quantities of a fatty alcohol, fatty aldehyde, or fatty acid ranging in chain length from four carbon atoms (C 4 ) to twenty-four carbon atoms (C 24 ) or more carbon atoms. The microorganism of the invention can produce a desired product that is at least 50%, 60%, 70%>, 75%, 85%, 90%, 95% or more selective for a particular chain length. The carbon chain-length of the product is controlled by one or more enzymes of the MI-FAE cycle (steps A/B/C/D of Figures 6) and/or one or more enzymes of the MD-FAE cycle (steps E/B/C/D of Figure 6) in

combination with one or more termination pathway enzymes (steps E-N of Figure 7). Chain length can be capped during the elongation cycle by one or more MI-FAE cycle enzymes (thiolase, 3-oxoacyl-CoA reductase, 3-hydroxyacyl-CoA dehydratase and/or enoyl-CoA reductase) exhibiting selectivity for MI-FAE cycle substrates having a number of carbon atoms that are no greater than the desired product size. Alternatively, or in addition, chain length can be capped during the elongation cycle by one or more MD-FAE cycle enzymes (elongase, 3-oxoacyl-CoA reductase, 3-hydroxyacyl-CoA dehydratase and/or enoyl-CoA reductase). Chain length can be further constrained by one or more enzymes catalyzing the conversion of the MI-FAE cycle intermediate to the fatty alcohol, fatty aldehyde or fatty acid product such that the one or more termination enzymes only reacts with substrates having a number of carbon atoms that are no less than the desired fatty alcohol, fatty aldehyde or fatty acid product.

The termination pathway enzymes catalyzing conversion of a MI-FAE-CoA intermediate or MD-FAE-CoA intermediate to a fatty alcohol can include combinations of a fatty acyl-CoA reductase (alcohol or aldehyde forming), a fatty aldehyde reductase, an acyl- ACP reductase, an acyl-CoA:ACP acyltransferase, a thioesterase, an acyl-CoA hydrolase and/or a carboxylic acid reductase (pathways G; E/F; K/J/F; H/N/F; or K/L/N/F of Figure 7). Termination pathway enzymes for converting a MI-FAE-CoA intermediate or MD-FAE-CoA intermediate to a fatty acid can include combinations of a thioesterase, a CoA hydrolase, an acyl-CoA:ACP acyltransferase, an aldehyde dehydrogenase and/or an acyl-ACP reductase (pathways H; K/L; E/N; K/J/N of Figure 7). For production of a fatty aldehyde, the termination pathway enzymes can include combinations of a fatty acyl-CoA reductase (aldehyde forming), an acyl-ACP reductase, an acyl-CoA:ACP acyltransferase, a

thioesterase, an acyl-CoA hydrolase and/or a carboxylic acid reductase (pathways E; K/J; H/N; or K/L/N of Figure 7).

The non-naturally occurring microbial organisms of the invention can also efficiently direct cellular resources, including carbon, energy and reducing equivalents, to the production of fatty alcohols, fatty aldehydes and fatty acids, thereby resulting in improved yield, productivity and/or titer relative to a naturally occurring organism. In one embodiment, the microorganism is modified to increase cytosolic acetyl-CoA levels. In another embodiment, the microorganism is modified to efficiently direct cytosolic acyl-CoA into fatty alcohols, fatty aldehydes or fatty acids rather than other byproducts or cellular processes. Enzymes or pathways that lead to the formation of byproducts can be attenuated or deleted. Exemplary byproducts include, but are not limited to, ethanol, glycerol, lactate, acetate, esters and carbon dioxide. Additional byproducts can include fatty-acyl-CoA derivatives such as alcohols, alkenes, alkanes, esters, acids and aldehydes. Accordingly, a byproduct can include any fermentation product diverting carbon and/or reducing equivalents from the product of interest.

In another embodiment, the availability of reducing equivalents or redox ratio is increased. In yet another embodiment, the cofactor requirements of the microorganism are balanced such that the same reduced cofactors generated during carbon assimilation and central metabolism are utilized by MI-FAE cycle, MD-FAE cycle and/or termination pathway enzymes. In yet another embodiment, the fatty alcohol, fatty aldehyde or fatty acid producing organism expresses a transporter which exports the fatty alcohol, fatty aldehyde or fatty acid from the cell.

Microbial organisms capable of fatty alcohol production are exemplified herein with reference to the Saccharomyces cerevisaie genetic background. However, with the complete genome sequence available now for thousands of species (with more than half of these available on public databases such as the NCBI), the identification of an alternate species homolog for one or more genes, including for example, orthologs, paralogs and

nonorthologous gene displacements, and the interchange of genetic alterations between eukaryotic organisms is routine and well known in the art. Accordingly, the metabolic alterations enabling production of fatty alcohols described herein with reference to a particular organism such as Saccharomyces cerevisiae can be readily applied to other microorganisms. Given the teachings and guidance provided herein, those skilled in the art understand that a metabolic alteration exemplified in one organism can be applied equally to other organisms. The methods of the invention are applicable to various prokaryotic and eukaryotic organisms such as bacteria, yeast and fungus. For example, the yeast can include

Saccharomyces cerevisiae and Rhizopus arrhizus. Exemplary eukaryotic organisms can also include Crabtree positive and negative yeasts, and yeasts in the genera Saccharomyces, Kluyveromyces, Candida or Pichia. Further exempalry eukaryotic species include those selected from Schizosaccharomyces pombe, Kluyveromyces lactis, Kluyveromyces marxianus, Aspergillus terreus, Aspergillus niger, Rhizopus arrhizus, Rhizopus oryzae, Candida albicans, Candida boidinii, Candida sonorensis, Candida tropicalis, Yarrowia lipolytica and Pichia pastoris. Additionally, select cells from larger eukaryotic organisms are also applicable to methods of the present invention. Exemplary bacteria include species selected from Escherichia coli, Klebsiella oxytoca, Anaerobio spirillum succiniciproducens, Actinobacillus succinogenes, Mannheimia succiniciproducens, Rhizobium etli, Bacillus subtilis, Cory neb acterium glutamicum, Gluconobacter oxydans, Zymomonas mobilis, Lactococcus lactis, Lactobacillus plantarum, Streptomyces coelicolor, Clostridium acetobutylicum, Pseudomonas fluorescens, and Pseudomonas putida.

In some aspects of the invention, production of fatty alcohols, fatty aldehydes and fatty acids through the MI-FAE cycle and termination pathways disclosed herein are particularly useful because the cycle and pathways result in higher product and ATP yields than through naturally occurring biosynthetic pathways such as the well-known malonyl-CoA dependent fatty acid synthesis pathway, or in some aspects the malonyl-ACP dependent fatty acid sysnthesis pathway. For example, using acetyl-CoA as a C 2 extension unit (e.g. step A, Figure 1) instead of malonyl-acyl carrier protein (malonyl-ACP) saves one ATP molecule per unit flux of acetyl-CoA entering the MI-FAE cycle. The MI-FAE cycle results in acyl-CoA instead of acyl-ACP, and can preclude the need of the ATP-consuming acyl-CoA synthase reactions for the production of octanol and other fatty alcohols, fatty aldehydes or fatty acids if acetyl-CoA is used as the extender unit. The fatty alcohol, fatty aldehyde and fatty acid producing organisms of the invention can additionally allow the use of biosynthetic processes to convert low cost renewable feedstock for the manufacture of chemical products.

The eukaryotic organism of the invention can be further engineered to metabolize and/or co-utilize a variety of feedstocks including glucose, xylose, fructose, syngas, methanol, and the like.

Chain length control can be achieved using a combination of highly active enzymes with suitable substrate ranges appropriate for biosynthesis of the desired fatty alcohol, fatty aldehyde, or fatty acid. Chain length of the product can be controlled using one or more enzymes of MI-FAE cycle, MD-FAE cycle or termination pathway. As described herein, chain length can be capped during the MI-FAE cycle by one or more MI-FAE cycle enzymes (thiolase, 3-oxoacyl-CoA reductase, 3-hydroxyacyl-CoA dehydratase and/or enoyl-CoA reductase) and in the case of the MD-FAE cycle, one or more MD-FAE cycle enzymes (elongase, 3-oxoacyl-CoA reductase, 3-hydroxyacyl-CoA dehydratase and/or enoyl-CoA reductase), exhibiting selectivity for MI-FAE and/or MD-FAE cycle substrates having a number of carbon atoms that are no greater than the desired product size. Since enzymes are reversible, any of the elongation pathway enzymes can serve in this capacity. Selecting enzymes with broad substrate ranges but defined chain-length boundaries enables the use of a single enzyme to catalyze multiple cycles of elongation, while conferring product specificity. To further hone specificity and prevent the accumulation of shorter byproducts, selectivity is further constrained by product-forming termination enzymes, such that one or more enzymes are selective for acyl-CoA or other termination pathway substrates having a number of carbon atoms that are no less than the desired chain length. The deletion or attenuation of endogenous pathway enzymes that produce different chain length products can further hone product specificity.

Using the approaches outlined herein, one skilled in the art can select enzymes from the literature with characterized substrate ranges that selectively produce a fatty alcohol, fatty aldehyde or fatty acid product of a specific chain length. To selectively produce fatty alcohols, fatty aldehydes or fatty acids of a desired length, one can utilize combinations of known enzymes in the literature with different selectivity ranges as described above. For example, a non-naturally occurring microbial organism that produces C 16 fatty alcohol can express enzymes such as the Rattus norvegicus Acaala thiolase and the enoyl-CoA reducatse of Mycobacterium smegmatis, which only accept substrates up to length C 16 . Coupling one or both chain elongation enzymes with a C 16 -C 18 fatty acyl-CoA reductase (alcohol or aldehyde forming) such as FAR of Simmondsia chinensis further increases product specificity by reducing the synthesis of shorter alcohol products. As another example, a non-naturally occurring microbial organism of the invention can selectively produce alcohols of length C 14 by combining the 3-hydroxyacyl-CoA dehydratase of Arabidopsis thaliana with the acyl- CoA reductase Acrl of Acinetobacter sp. Strain M-l. To produce 3-oxoacids of length C 14 , one can, for example, combine the rat thiolase with the 3-oxoacyl-CoA hydrolase of Solanum lycopersicum. As still a further example, to produce C 18 fatty acids, one can combine the Salmonella enterica fadE enoyl-CoA reductase with the tesB thioesterase of E. coli. In yet another example, selective production of C 6 alcohols are formed by combining the paaHl thiolase from Ralstonia eutropha with the Leifsonia sp. S749 alcohol dehydrogenase Isadh.

Exemplary MI-FAE cycle, MD-FAE cycle and termination pathway enzymes are described in detail in Example I. The biosynthetic enzymes described herein exhibit varying degrees of substrate specificity. Exemplary substrate ranges of enzymes characterized in the literature are shown in the table below and described in further detail in Example I.

Pathway step Chain length Gene Organism

1A C4 atoB Escherichia coli

1A C6 phaD Pseudomonas putida

1A C6-C8 bktB Ralstonia eutropha

1A C10-C16 Acaala Rattus norvegicus

IB C4 hbd Clostridium acetobutylicum

IB C4-C6 paaHl Ralstonia eutropha

IB C4-C10 HADH Sus scrofa

IB C4-C18 fadB Escherichia coli

IC C4-C6 crt Clostridium acetobutylicum

IC C4-C7 pimF Rhodopseudomonas palustris

IC C4-C14 MFP2 Arabidopsis thaliana

ID C4-C6 ECR1 Euglena gracilis

ID C6-C8 ECR3 Euglena gracilis

ID C8-10 ECR2 Euglena gracilis

ID C8-C16 ECR Rattus norvegicus

ID C10-C16 ECR Mycobacterium smegmatis

ID C2-C18 fadE Salmonella enterica

IE C2-C4 bphG Pseudomonas sp

IE C4 Bid Clostridium

saccharoperbutylacetonicum

IE C12-C20 ACR Acinetobacter calcoaceticus

IE C14-C18 Acrl Acinetobacter sp. Strain M-l

IE C16-C18 Rvl543, Rv3391 Mycobacterium tuberculosis

IF C6-C7 lsadh Leifsonia sp. S749

IF C2-C8 yqhD Escherichia coli

IF C3-C10 Adh Pseudomonas putida

IF C2-C14 alrA Acinetobacter sp. strain M-l

IF C2-C30 ADH1 Geobacillus

thermodenitrificans

1G C2 adhE Escherichia coli

1G C2-C8 adhe2 Clostridium acetobutylicum

1G C14-C16 At3gl l980 Arabidopsis thaliana

1G C16 At3g44560 Arabidopsis thaliana

1G C16-C18 FAR Simmondsia chinensis

IH C4 Cat2 Clostridium kluyveri

IH C4-C6 Acotl2 Rattus norvegicus

IH C14 MKS2 Solanum lycopersicum

Taking into account the differences in chain-length specificities of each enzyme in the MI-FAE cycle or MD-FAE cycle, one skilled in the art can select one or more enzymes for catalyzing each elongation cycle reaction step (steps A-D or steps E/B/C/D of Figure 6). For example, for the thiolase step of the MI-FAE cycle, some thiolase enzymes such as bktB of Ralstonia eutropha catalyze the elongation of short- and medium-chain acyl-CoA

intermediates (C6-C 8 ), whereas others such as Acaala of R. norvegicus are active on longer- chain substrates (Cio-Ci 6 ). Thus, an microbial organism producing a fatty alcohol, fatty aldehyde or fatty acid can comprise one, two, three, four or more variants of a thiolase, elongase, 3-oxoacyl-CoA reductase, 3-hydroxyacyl-CoA dehydratase and/or enoyl-CoA reductase.

Chain length specificity of enzymes can be assayed by methods well known in the art (eg. Wrensford et al, Anal Biochem 192:49-54 (1991)). The substrate ranges of fatty alcohol, fatty aldehyde, or fatty acid producing enzymes can be further extended or narrowed by methods well known in the art. Variants of biologically-occurring enzymes can be generated, for example, by rational and directed evolution, mutagenesis and enzyme shuffling as described herein. As one example, a rational engineering approach for altering chain length specificity was taken by Denic and Weissman (Denic and Weissman, Cell 130:663-77 (2008)). Denic and Weissman mapped the region of the yeast elongase protein ELOp responsible for chain length, and introduced mutations to vary the length of fatty acid products. In this instance, the geometry of the hydrophobic substrate pocket set an upper boundary on chain length. A similar approach can be useful for altering the chain length specificities of enzymes of the MI-FAE cycle, MD-FAE cycle and/or termination pathways. Enzyme mutagenesis, expression in a host, and screening for fatty alcohol production is another useful approach for generating enzyme variants with improved properties for the desired application. For example, US patent application 2012/0009640 lists hundreds of variants of Marinobacter algicola and Marinobacter aquaeolei FAR enzymes with improved activity over the wild type enzyme, and varying product profiles.

Enzyme mutagenesis (random or directed) in conjunction with a selection platform is another useful approach. For example, Machado and coworkers developed a selection platform aimed at increasing the activity of acyl-CoA elongation cycle enzymes on longer chain length substrates (Machado et al., Met Eng in press (2012)). Machado et al. identified the chain-length limiting step of their pathway (a 3-hydroxyacyl-CoA dehydrogenase) and evolved it for improved activity on C 6 -C8 substrates using an anaerobic growth rescue platform. Additional variants of enzymes useful for producing fatty alcohols are listed in the table below

Those skilled in the art will understand that the genetic alterations, including metabolic modifications exemplified herein, are described with reference to a suitable host organism such as E. coli or S. cerevisiae and their corresponding metabolic reactions or a suitable source organism for desired genetic material such as genes for a desired metabolic pathway. However, given the complete genome sequencing of a wide variety of organisms and the high level of skill in the area of genomics, those skilled in the art will readily be able to apply the teachings and guidance provided herein to essentially all other organisms. For example, the metabolic alterations exemplified herein can readily be applied to other species by incorporating the same or analogous encoding nucleic acid from species other than the referenced species. Such genetic alterations include, for example, genetic alterations of species homologs, in general, and in particular, orthologs, paralogs or nonorthologous gene displacements.

An ortholog is a gene or genes that are related by vertical descent and are responsible for substantially the same or identical functions in different organisms. For example, mouse epoxide hydrolase and human epoxide hydrolase can be considered orthologs for the biological function of hydrolysis of epoxides. Genes are related by vertical descent when, for example, they share sequence similarity of sufficient amount to indicate they are

homologous, or related by evolution from a common ancestor. Genes can also be considered orthologs if they share three-dimensional structure but not necessarily sequence similarity, of a sufficient amount to indicate that they have evolved from a common ancestor to the extent that the primary sequence similarity is not identifiable. Genes that are orthologous can encode proteins with sequence similarity of about 25% to 100% amino acid sequence identity. Genes encoding proteins sharing an amino acid similarity less that 25% can also be considered to have arisen by vertical descent if their three-dimensional structure also shows similarities. Members of the serine protease family of enzymes, including tissue plasminogen activator and elastase, are considered to have arisen by vertical descent from a common ancestor.

Orthologs include genes or their encoded gene products that through, for example, evolution, have diverged in structure or overall activity. For example, where one species encodes a gene product exhibiting two functions and where such functions have been separated into distinct genes in a second species, the three genes and their corresponding products are considered to be orthologs. For the production of a biochemical product, those skilled in the art will understand that the orthologous gene harboring the metabolic activity to be introduced or disrupted is to be chosen for construction of the non-naturally occurring microorganism. An example of orthologs exhibiting separable activities is where distinct activities have been separated into distinct gene products between two or more species or within a single species. A specific example is the separation of elastase proteolysis and plasminogen proteolysis, two types of serine protease activity, into distinct molecules as plasminogen activator and elastase. A second example is the separation of mycoplasma 5 '-3' exonuclease and Drosophila DNA polymerase III activity. The DNA polymerase from the first species can be considered an ortholog to either or both of the exonuclease or the polymerase from the second species and vice versa.

In contrast, paralogs are homologs related by, for example, duplication followed by evolutionary divergence and have similar or common, but not identical functions. Paralogs can originate or derive from, for example, the same species or from a different species. For example, microsomal epoxide hydrolase (epoxide hydrolase I) and soluble epoxide hydrolase (epoxide hydrolase II) can be considered paralogs because they represent two distinct enzymes, co-evolved from a common ancestor, that catalyze distinct reactions and have distinct functions in the same species. Paralogs are proteins from the same species with significant sequence similarity to each other suggesting that they are homologous, or related through co-evolution from a common ancestor. Groups of paralogous protein families include HipA homologs, luciferase genes, peptidases, and others.

A nonorthologous gene displacement is a nonorthologous gene from one species that can substitute for a referenced gene function in a different species. Substitution includes, for example, being able to perform substantially the same or a similar function in the species of origin compared to the referenced function in the different species. Although generally, a nonorthologous gene displacement will be identifiable as structurally related to a known gene encoding the referenced function, less structurally related but functionally similar genes and their corresponding gene products nevertheless will still fall within the meaning of the term as it is used herein. Functional similarity requires, for example, at least some structural similarity in the active site or binding region of a nonorthologous gene product compared to a gene encoding the function sought to be substituted. Therefore, a nonorthologous gene includes, for example, a paralog or an unrelated gene. Therefore, in identifying and constructing the non-naturally occurring microbial organisms of the invention having fatty alcohol, fatty aldehyde or fatty acid biosynthetic capability, those skilled in the art will understand with applying the teaching and guidance provided herein to a particular species that the identification of metabolic modifications can include identification and inclusion or inactivation of orthologs. To the extent that paralogs and/or nonorthologous gene displacements are present in the referenced microorganism that encode an enzyme catalyzing a similar or substantially similar metabolic reaction, those skilled in the art also can utilize these evolutionally related genes. Similarly for a gene disruption, evolutionally related genes can also be disrupted or deleted in a host microbial organism to reduce or eliminate functional redundancy of enzymatic activities targeted for disruption.

Orthologs, paralogs and nonorthologous gene displacements can be determined by methods well known to those skilled in the art. For example, inspection of nucleic acid or amino acid sequences for two polypeptides will reveal sequence identity and similarities between the compared sequences. Based on such similarities, one skilled in the art can determine if the similarity is sufficiently high to indicate the proteins are related through evolution from a common ancestor. Algorithms well known to those skilled in the art, such as Align, BLAST, Clustal W and others compare and determine a raw sequence similarity or identity, and also determine the presence or significance of gaps in the sequence which can be assigned a weight or score. Such algorithms also are known in the art and are similarly applicable for determining nucleotide sequence similarity or identity. Parameters for sufficient similarity to determine relatedness are computed based on well known methods for calculating statistical similarity, or the chance of finding a similar match in a random polypeptide, and the significance of the match determined. A computer comparison of two or more sequences can, if desired, also be optimized visually by those skilled in the art. Related gene products or proteins can be expected to have a high similarity, for example, 25% to 100% sequence identity. Proteins that are unrelated can have an identity which is essentially the same as would be expected to occur by chance, if a database of sufficient size is scanned (about 5%). Sequences between 5% and 24% may or may not represent sufficient homology to conclude that the compared sequences are related. Additional statistical analysis to determine the significance of such matches given the size of the data set can be carried out to determine the relevance of these sequences. Exemplary parameters for determining relatedness of two or more sequences using the BLAST algorithm, for example, can be as set forth below. Briefly, amino acid sequence alignments can be performed using BLASTP version 2.0.8 (Jan-05-1999) and the following parameters: Matrix: 0 BLOSUM62; gap open: 11; gap extension: 1; x dropoff: 50; expect: 10.0; wordsize: 3; filter: on. Nucleic acid sequence alignments can be performed using

BLASTN version 2.0.6 (Sept- 16- 1998) and the following parameters: Match: 1; mismatch: - 2; gap open: 5; gap extension: 2; x dropoff: 50; expect: 10.0; wordsize: 11; filter: off. Those skilled in the art will know what modifications can be made to the above parameters to either increase or decrease the stringency of the comparison, for example, and determine the relatedness of two or more sequences.

In some embodiments, the invention provides a non-naturally occurring microbial organism having a MI-FAE cycle or a MD-FAE cycle in combination with a termination pathway, wherein the MI-FAE cycle includes one or more thiolase, one or more 3-oxoacyl- CoA reductase, one or more 3-hydroxyacyl-CoA dehydratase, and one or more enoyl-CoA reductase, wherein the MD-FAE cycle includes one or more elongase, one or more 3- oxoacyl-CoA reductase, one or more 3-hydroxyacyl-CoA dehydratase, and one or more enoyl-CoA reductase, wherein the termination pathway includes a pathway shown in Figures 1, 6 or 7 selected from: (1) 1H; (2) IK and IL; (3) IE and IN; (4) IK, 1J, and IN; (5) IE; (6) IK and 1J; (7) 1H and IN; (8) IK, IL, and IN; (9) IE and IF; (10) IK, 1J, and IF; (11) 1H, IN, and IF; (12) IK, IL, IN, and IF; and (13) 1G, wherein IE is an acyl-CoA reductase (aldehyde forming), wherein IF is an alcohol dehydrogenase, wherein 1G is an acyl-CoA reductase (alcohol forming), wherein 1H is an acyl-CoA hydrolase, acyl-CoA transferase or acyl-CoA synthase, wherein 1J is an acyl-ACP reductase, wherein IK is an acyl-CoA:ACP acyltransferase, wherein IL is a thioesterase, wherein IN is an aldehyde dehydrogenase (acid forming) or a carboxylic acid reductase, wherein an enzyme of the MI-FAE cycle, MD-FAE cycle or termination pathway is encoded by at least one exogenous nucleic acid and is expressed in a sufficient amount to produce a compound of Formula (I):

(I) wherein Ri is Ci_ 24 linear alkyl; R 2 is CH 2 OH, CHO, or COOH; R 3 is H, OH, or oxo

(=0); and represents a single or double bond with the proviso that the valency of the carbon atom to which R 3 is attached is four, wherein the substrate of each of said enzymes of the MI-FAE cycle, MD-FAE cycle and the termination pathway are independently selected from a compound of Formula (II), malonyl-CoA, propionyl-CoA or acetyl-CoA:

wherein Ri is Ci_ 24 linear alkyl; R 3 is H, OH, or oxo (=0); R 4 is S-CoA, ACP, OH or

H; and represents a single or double bond with the proviso that the valency of the carbon atom to which R 3 is attached is four; wherein said one or more enzymes of the MI- FAE cycle are each selective for a compound of Formula (II) having a number of carbon atoms at Ri that is no greater than the number of carbon atoms at Ri of said compound of Formula (I), wherein said one or more enzymes of the MD-FAE cycle are each selective for a compound of Formula (II) having a number of carbon atoms at Ri that is no greater than the number of carbon atoms at Ri of said compound of Formula (I), and wherein said one or more enzymes of the termination pathway are each selective for a compound of Formula (II) having a number of carbon atoms at Ri that is no less than the number of carbon atoms at Ri of said compound of Formula (I).

In some aspects of the invention, non-naturally occurring microbial organism of the invention can produce a compound of Formula (I) wherein Ri is Ci_i 7 linear alkyl. In another aspect of the invention, the Ri of the compound of Formula (I) is Ci linear alkyl, C 2 linear alkyl, C 3 linear alkyl, C 4 linear alkyl, C 5 linear alkyl, C 6 linear alkyl, C 7 linear alkyl, Cg linear alkyl, Cg linear alkyl, Cio linear alkyl, Cn, linear alkyl, Ci 2 linear alkyl or Ci 3 linear alkyl, Ci 4 linear alkyl, C15 linear alkyl, Ci 6 linear alkyl, C 17 linear alkyl, C 18 linear alkyl, C19 linear alkyl, C 2 o linear alkyl, C 21 linear alkyl, C 22 linear alkyl, C 23 linear alkyl, or C 24 linear alkyl.

In some aspects of the invention, the microbial organism microbial organism includes two, three, or four exogenous nucleic acids each encoding an enzyme of the MI-FAE cycle or the MD-FAE cycle. In some aspects of the invention, the microbial organism includes two, three, or four exogenous nucleic acids each encoding an enzyme of the termination pathway. In some aspects of the invention, the microbial organism includes exogenous nucleic acids encoding each of the enzymes of at least one of the pathways selected from (1)-(13). In some aspects, the at least one exogenous nucleic acid is a heterologous nucleic acid. In some aspects, the non-naturally occurring microbial organism is in a substantially anaerobic culture medium.

In some embodiments, the invention provides a non naturally occurring microbial organism, wherein the one or more enzymes of the MI-FAE cycle, MD-FAE cycle or termination pathway is expressed in a sufficient amount to produce a fatty alcohol selected from the Formulas (III)-(VI):

alkyl, or alternatively Ri is Cc>_i 3 linear alkyl. In some aspects of the invention, Ri is Ci linear alkyl, C 2 linear alkyl, C 3 linear alkyl, C 4 linear alkyl, C 5 linear alkyl, C 6 linear alkyl, C 7 linear alkyl, Cg linear alkyl, C9 linear alkyl, C 10 linear alkyl, Cn, linear alkyl, C 12 linear alkyl or C 13 linear alkyl, C 14 linear alkyl, C 15 linear alkyl, C 16 linear alkyl, C 17 linear alkyl, C 18 linear alkyl, C19 linear alkyl, C 2 o linear alkyl, C 21 linear alkyl, C 22 linear alkyl, C 23 linear alkyl, or C 24 linear alkyl.

In some embodiments, the invention provides a non naturally occurring microbial organism, wherein the one or more enzymes of the MI-FAE cycle, MD-FAE cycle or termination pathway is expressed in a sufficient amount to produce a fatty aldehyde selected from the Formula (VII)-(X):

(VIII) (IX) , wherein Ri is Ci_ 24 linear alkyl, or alternatively Ri is C 1-17 linear alkyl, or alternatively Ri is C9_i 3 linear alkyl. In some aspects of the invention, Ri is Ci linear alkyl, C 2 linear alkyl, C 3 linear alkyl, C 4 linear alkyl, C 5 linear alkyl, C 6 linear alkyl, C 7 linear alkyl, Cg linear alkyl, Cg linear alkyl, C 10 linear alkyl, Cn, linear alkyl, C 12 linear alkyl or C 13 linear alkyl, C 14 linear alkyl, C 15 linear alkyl, C 16 linear alkyl, C 17 linear alkyl, C 18 linear alkyl, C19 linear alkyl, C 2 o linear alkyl, C 21 linear alkyl, C 22 linear alkyl, C 23 linear alkyl, or C 24 linear alkyl.

In some embodiments, the invention provides a non naturally occurring microbial organism, wherein the one or more enzymes of the MI-FAE cycle, MD-FAE cycle or termination pathway is expressed in a sufficient amount to produce a fatty acid selected from the Formula (XI)-(XIV):

(XIV) ^ wherein Ri is Ci_ 24 linear alkyl, or alternatively Ri is C 1-17 linear alkyl, or alternatively Ri is Cc>_i 3 linear alkyl. In some aspects of the invention, Ri is Ci linear alkyl, C 2 linear alkyl, C 3 linear alkyl, C 4 linear alkyl, C5 linear alkyl, C 6 linear alkyl, C 7 linear alkyl, Cg linear alkyl, C9 linear alkyl, C 10 linear alkyl, Cn, linear alkyl, C 12 linear alkyl or C 13 linear alkyl, C 14 linear alkyl, C 15 linear alkyl, C 16 linear alkyl, C 17 linear alkyl, C 18 linear alkyl, C19 linear alkyl, C 2 o linear alkyl, C 21 linear alkyl, C 22 linear alkyl, C 23 linear alkyl, or C 24 linear alkyl. In some embodiments, the invention provides a non-naturally occurring microbial organism as described herein, wherein the microbial organism further includes an acetyl-CoA pathway and at least one exogenous nucleic acid encoding an acetyl-CoA pathway enzyme expressed in a sufficient amount to produce acetyl-CoA, wherein the acetyl-CoA pathway includes a pathway shown in Figures 2, 3, 4 or 5 selected from: (1) 2A and 2B; (2) 2A, 2C, and 2D; (3) 2H; (4) 2G and 2D; (5) 2E, 2F and 2B; (6) 2E and 21; (7) 2J, 2F and 2B; (8) 2J and 21; (9) 3A, 3B, and 3C; (10) 3A, 3B, 3J, 3K, and 3D; (11) 3A, 3B, 3G, and 3D; (12) 3A, 3F, and 3D; (13) 3N, 3H, 3B and 3C; (14) 3N, 3H, 3B, 3J, 3K, and 3D; (15) 3N, 3H, 3B, 3G, and 3D; (16) 3N, 3H, 3F, and 3D; (17) 3L, 3M, 3B and 3C; (18) 3L, 3M, 3B, 3J, 3K, and 3D; (19) 3L, 3M, 3B, 3G, and 3D; (20) 3L, 3M, 3F, and 3D; (21) 4A, 4B, 4D, 4H, 41, and 4J; (22) 4A, 4B, 4E, 4F, 4H, 41, and 4J; (23) 4A, 4B, 4E, 4K, 4L, 4H, 41, and 4J; (24) 4A, 4C, 4D, 4H, and 4J; (25) 4A, 4C, 4E, 4F, 4H, and 4J; (26) 4A, 4C, 4E, 4K, 4L, 4H, and 4J; (27) 5A, 5B, 5D, and 5G; (28) 5A, 5B, 5E, 5F, and 5G; (29) 5A, 5B, 5E, 5K, 5L, and 5G; (30) 5A, 5C, and 5D; (31) 5A, 5C, 5E, and 5F; and (32) 5A, 5C, 5E, 5K, and 5L, wherein 2A is a pyruvate oxidase (acetate-forming), wherein 2B is an acetyl-CoA synthetase, an acetyl-CoA ligase or an acetyl-CoA transferase, wherein 2C is an acetate kinase, wherein 2D is a

phosphotransacetylase, wherein 2E is a pyruvate decarboxylase, wherein 2F is an

acetaldehyde dehydrogenase, wherein 2G is a pyruvate oxidase (acetyl-phosphate forming), wherein 2H is a pyruvate dehydrogenase, a pyruvate :ferredoxin oxidoreductase, a

pyruvate :NAD(P)H oxidoreductase or a pyruvate formate lyase, wherein 21 is an

acetaldehyde dehydrogenase (acylating), wherein 2J is a threonine aldolase, wherein 3A is a phosphoenolpyruvate (PEP) carboxylase or a PEP carboxykinase, wherein 3B is an oxaloacetate decarboxylase, wherein 3C is a malonate semialdehyde dehydrogenase

(acetylating), wherein 3D is an acetyl-CoA carboxylase or a malonyl-CoA decarboxylase, wherein 3F is an oxaloacetate dehydrogenase or an oxaloacetate oxidoreductase, wherein 3G is a malonate semialdehyde dehydrogenase (acylating), wherein 3H is a pyruvate

carboxylase, wherein 3 J is a malonate semialdehyde dehydrogenase, wherein 3K is a malonyl-CoA synthetase or a malonyl-CoA transferase, wherein 3L is a malic enzyme, wherein 3M is a malate dehydrogenase or a malate oxidoreductase, wherein 3N is a pyruvate kinase or a PEP phosphatase, wherein 4 A is a citrate synthase, wherein 4B is a citrate transporter, wherein 4C is a citrate/malate transporter, wherein 4D is an ATP citrate lyase, wherein 4E is a citrate lyase, wherein 4F is an acetyl-CoA synthetase or an acetyl-CoA transferase, wherein 4H is a cytosolic malate dehydrogenase, wherein 41 is a malate transporter, wherein 4J is a mitochondrial malate dehydrogenase, wherein 4K is an acetate kinase, wherein 4L is a phosphotransacetylase, wherein 5A is a citrate synthase, wherein 5B is a citrate transporter, wherein 5C is a citrate/oxaloacetate transporter, wherein 5D is an ATP citrate lyase, wherein 5E is a citrate lyase, wherein 5F is an acetyl-CoA synthetase or an acetyl-CoA transferase, wherein 5G is an oxaloacetate transporter, wherein 5K is an acetate kinase, and wherein 5L is a phosphotransacetylase.

In some aspects, the microbial organism of the invention can include two, three, four, five, six, seven or eight exogenous nucleic acids each encoding an acetyl-CoA pathway enzyme. In some aspects, the microbial organism includes exogenous nucleic acids encoding each of the acetyl-CoA pathway enzymes of at least one of the pathways selected from (1)- (32).

In an additional embodiment, the invention provides a non-naturally occurring microbial organism having a fatty alcohol, fatty aldehyde or fatty acid pathway, wherein the non-naturally occurring microbial organism comprises at least one exogenous nucleic acid encoding an enzyme or protein that converts a substrate to a product selected from the group consisting of two acetyl-CoA molecules to a 3-ketoacyl-CoA, acetyl-CoA plus propionyl- CoA to a ketoacyl-CoA, malonyl-CoA to 3-ketoacyl-CoA, a 3-ketoacyl-CoA to a 3- hydroxyacyl-CoA, a 3-hydroxyacyl-CoA to an enoyl-CoA, an enoyl-CoA to an acyl-CoA, an acyl-CoA plus an acetyl-CoA to a 3-ketoacyl-CoA, an acyl-CoA plus malonyl-CoA to a 3- ketoacyl-CoA, an acyl-CoA to a fatty aldehyde, a fatty aldehyde to a fatty alcohol, an acyl- CoA to a fatty alcohol, an acyl-CoA to an acyl-ACP, an acyl-ACP to a fatty acid, an acyl- CoA to a fatty acid, an acyl-ACP to a fatty aldehyde, a fatty acid to a fatty aldehyde, a fatty aldehyde to a fatty acid, pyruvate to acetate, acetate to acetyl-CoA, pyruvate to acetyl-CoA, pyruvate to acetaldehyde, threonin to acetaldehyde, acetaldehyde to acetate, acetaldehyde to acetyl-CoA, pyruvate to acetyl-phosphate, acetate to acetyl-phosphate, acetyl-phosphate to acetyl-CoA, phosphoenolpyruvate (PEP) to pyruvate, pyruvate to malate, malate to oxaloacetate, pyruvate to oxaloacetate, PEP to oxaloacetate, oxaloacetate to malonate semialdehyde, oxaloacetate to malonyl-CoA, malonate semialdehyde to malonate, malonate to malonyl-CoA, malonate semialdehyde to malonyl-CoA, malonyl-CoA to acetyl-CoA, malonate semialdehyde to acetyl-CoA, oxaloacetate plus acetyl-CoA to citrate, citrate to oxaloacetate plus acetyl-CoA, citrate to oxaloacetate plus acetate, and oxaloacetate to malate. One skilled in the art will understand that these are merely exemplary and that any of the substrate-product pairs disclosed herein suitable to produce a desired product and for which an appropriate activity is available for the conversion of the substrate to the product can be readily determined by one skilled in the art based on the teachings herein. Thus, the invention provides a non-naturally occurring microbial organism containing at least one exogenous nucleic acid encoding an enzyme or protein, where the enzyme or protein converts the substrates and products of a fatty alcohol, fatty aldehyde or fatty acid pathway, such as that shown in Figure 1-8.

While generally described herein as a microbial organism that contains a fatty alcohol, fatty aldehyde or fatty acid pathway, it is understood that the invention additionally provides a non-naturally occurring microbial organism comprising at least one exogenous nucleic acid encoding a fatty alcohol, fatty aldehyde or fatty acid pathway enzyme or protein expressed in a sufficient amount to produce an intermediate of a fatty alcohol, fatty aldehyde or fatty acid pathway. For example, as disclosed herein, a fatty alcohol, fatty aldehyde or fatty acid pathway is exemplified in Figures 1-7. Therefore, in addition to a microbial organism containing a fatty alcohol, fatty aldehyde or fatty acid pathway that produces fatty alcohol, fatty aldehyde or fatty acid, the invention additionally provides a non-naturally occurring microbial organism comprising at least one exogenous nucleic acid encoding a fatty alcohol, fatty aldehyde or fatty acid pathway enzyme, where the microbial organism produces a fatty alcohol, fatty aldehyde or fatty acid pathway intermediate, for example, a 3- ketoacyl-CoA, a 3-hydroxyacyl-CoA, an enoyl-CoA, an acyl-CoA, an acyl-ACP, acetate, acetaldehyde, acetyl-phosphate, oxaloacetate, matate, malonate semialdehyde, malonate, malonyl-CoA, acetyl-CoA, or citrate.

It is understood that any of the pathways disclosed herein, as described in the

Examples and exemplified in the Figures, including the pathways of Figures 1-7, can be utilized to generate a non-naturally occurring microbial organism that produces any pathway intermediate or product, as desired. As disclosed herein, such a microbial organism that produces an intermediate can be used in combination with another microbial organism expressing downstream pathway enzymes to produce a desired product. However, it is understood that a non-naturally occurring microbial organism that produces a fatty alcohol, fatty aldehyde or fatty acid pathway intermediate can be utilized to produce the intermediate as a desired product.

The invention is described herein with general reference to the metabolic reaction, reactant or product thereof, or with specific reference to one or more nucleic acids or genes encoding an enzyme associated with or catalyzing, or a protein associated with, the referenced metabolic reaction, reactant or product. Unless otherwise expressly stated herein, those skilled in the art will understand that reference to a reaction also constitutes reference to the reactants and products of the reaction. Similarly, unless otherwise expressly stated herein, reference to a reactant or product also references the reaction, and reference to any of these metabolic constituents also references the gene or genes encoding the enzymes that catalyze or proteins involved in the referenced reaction, reactant or product. Likewise, given the well known fields of metabolic biochemistry, enzymology and genomics, reference herein to a gene or encoding nucleic acid also constitutes a reference to the corresponding encoded enzyme and the reaction it catalyzes or a protein associated with the reaction as well as the reactants and products of the reaction.

The non-naturally occurring microbial organisms of the invention can be produced by introducing expressible nucleic acids encoding one or more of the enzymes or proteins participating in one or more fatty alcohol, fatty aldehyde or fatty acid biosynthetic pathways. Depending on the host microbial organism chosen for biosynthesis, nucleic acids for some or all of a particular fatty alcohol, fatty aldehyde or fatty acid biosynthetic pathway can be expressed. For example, if a chosen host is deficient in one or more enzymes or proteins for a desired biosynthetic pathway, then expressible nucleic acids for the deficient enzyme(s) or protein(s) are introduced into the host for subsequent exogenous expression. Alternatively, if the chosen host exhibits endogenous expression of some pathway genes, but is deficient in others, then an encoding nucleic acid is needed for the deficient enzyme(s) or protein(s) to achieve fatty alcohol, fatty aldehyde or fatty acid biosynthesis. Thus, a non-naturally occurring microbial organism of the invention can be produced by introducing exogenous enzyme or protein activities to obtain a desired biosynthetic pathway or a desired biosynthetic pathway can be obtained by introducing one or more exogenous enzyme or protein activities that, together with one or more endogenous enzymes or proteins, produces a desired product such as fatty alcohol, fatty aldehyde or fatty acid.

Host microbial organisms can be selected from, and the non-naturally occurring microbial organisms generated in, for example, bacteria, yeast, fungus or any of a variety of other microorganisms applicable or suitable to fermentation processes. Exemplary bacteria include any species selected from the order Enter obacteriales, family Enterobacteriaceae, including the genera Escherichia and Klebsiella; the order Aeromonadales, family Succinivibrionaceae, including the genus Anaerobiospirillum; the order Pasteurellales, family Pasteur ellaceae, including the genera Actinobacillus and Mannheimia; the order Rhizobiales, family Bradyrhizobiaceae, including the genus Rhizobium; the order Bacillales, family Bacillaceae, including the genus Bacillus; the order Actinomycetales, families Corynebacteriaceae and Streptomycetaceae, including the genus Corynebacterium and the genus Streptomyces, respectively; order Rhodospirillales, family Acetobacteraceae, including the genus Gluconobacter; the order Sphingomonadales, family Sphingomonadaceae, including the genus Zymomonas; the order Lactobacillales, families Lactobacillaceae and Streptococcaceae, including the genus Lactobacillus and the genus Lactococcus,

respectively; the order Clostridiales, family Clostridiaceae, genus Clostridium; and the order Pseudomonadales, family Pseudomonadaceae, including the genus Pseudomonas. Non- limiting species of host bacteria include Escherichia coli, Klebsiella oxytoca,

Anaerobiospirillum succiniciproducens, Actinobacillus succinogenes, Mannheimia succiniciproducens, Rhizobium etli, Bacillus subtilis, Corynebacterium glutamicum,

Gluconobacter oxydans, Zymomonas mobilis, Lactococcus lactis, Lactobacillus plantarum, Streptomyces coelicolor, Clostridium acetobutylicum, Pseudomonas fluorescens, and Pseudomonas putida.

Similarly, exemplary species of yeast or fungi species include any species selected from the order Saccharomycetales, family Saccaromycetaceae, including the genera

Saccharomyces, Kluyveromyces and Pichia; the order Saccharomycetales, family

Dipodascaceae, including the genus Yarrowia; the order Schizosaccharomycetales, family Schizosaccaromycetaceae, including the genus Schizosaccharomyces; the order Eurotiales, family Trichocomaceae, including the genus Aspergillus; and the order Mucorales, family Mucoraceae, including the genus Rhizopus. Non-limiting species of host yeast or fungi include Saccharomyces cerevisiae, Schizosaccharomyces pombe, Kluyveromyces lactis,

Kluyveromyces marxianus, Aspergillus terreus, Aspergillus niger, Pichia pastoris, Rhizopus arrhizus, Rhizobus oryzae, Yarrowia lipolytica, and the like. E. coli is a particularly useful host organism since it is a well characterized microbial organism suitable for genetic engineering. Other particularly useful host organisms include yeast such as Saccharomyces cerevisiae. It is understood that any suitable microbial host organism can be used to introduce metabolic and/or genetic modifications to produce a desired product. Depending on the fatty alcohol, fatty aldehyde or fatty acid biosynthetic pathway constituents of a selected host microbial organism, the non-naturally occurring microbial organisms of the invention will include at least one exogenously expressed fatty alcohol, fatty aldehyde or fatty acid pathway-encoding nucleic acid and up to all encoding nucleic acids for one or more fatty alcohol, fatty aldehyde or fatty acid biosynthetic pathways. For example, fatty alcohol, fatty aldehyde or fatty acid biosynthesis can be established in a host deficient in a pathway enzyme or protein through exogenous expression of the corresponding encoding nucleic acid. In a host deficient in all enzymes or proteins of a fatty alcohol, fatty aldehyde or fatty acid pathway, exogenous expression of all enzyme or proteins in the pathway can be included, although it is understood that all enzymes or proteins of a pathway can be expressed even if the host contains at least one of the pathway enzymes or proteins. For example, exogenous expression of all enzymes or proteins in a pathway for production of fatty alcohol, fatty aldehyde or fatty acid can be included, such as a thiolase, a 3-oxoacyl-CoA reductase, a 3-hydroxyacyl-CoA dehydratase, an enoyl-CoA redutase, an acyl-CoA reductase (aldehyde forming) and an alcohol dehydrogenase, for production of a fatty alcohol.

Given the teachings and guidance provided herein, those skilled in the art will understand that the number of encoding nucleic acids to introduce in an expressible form will, at least, parallel the fatty alcohol, fatty aldehyde or fatty acid pathway deficiencies of the selected host microbial organism. Therefore, a non-naturally occurring microbial organism of the invention can have one, two, three, four, five, six, seven or eight up to all nucleic acids encoding the enzymes or proteins constituting a fatty alcohol, fatty aldehyde or fatty acid biosynthetic pathway disclosed herein. In some embodiments, the non-naturally occurring microbial organisms also can include other genetic modifications that facilitate or optimize fatty alcohol, fatty aldehyde or fatty acid biosynthesis or that confer other useful functions onto the host microbial organism. One such other functionality can include, for example, augmentation of the synthesis of one or more of the fatty alcohol, fatty aldehyde or fatty acid pathway precursors such as acetyl-CoA, malonyl-CoA or propionyl-CoA.

Generally, a host microbial organism is selected such that it produces the precursor of a fatty alcohol, fatty aldehyde or fatty acid pathway, either as a naturally produced molecule or as an engineered product that either provides de novo production of a desired precursor or increased production of a precursor naturally produced by the host microbial organism. For example, acetyl-CoA is produced naturally in a host organism such as E. coli. A host organism can be engineered to increase production of a precursor, as disclosed herein. In addition, a microbial organism that has been engineered to produce a desired precursor can be used as a host organism and further engineered to express enzymes or proteins of a fatty alcohol, fatty aldehyde or fatty acid pathway. In some embodiments, a non-naturally occurring microbial organism of the invention is generated from a host that contains the enzymatic capability to synthesize fatty alcohol, fatty aldehyde or fatty acid. In this specific embodiment it can be useful to increase the synthesis or accumulation of a fatty alcohol, fatty aldehyde or fatty acid pathway product to, for example, drive fatty alcohol, fatty aldehyde or fatty acid pathway reactions toward fatty alcohol, fatty aldehyde or fatty acid production. Increased synthesis or accumulation can be accomplished by, for example, overexpression of nucleic acids encoding one or more of the above-described fatty alcohol, fatty aldehyde or fatty acid pathway enzymes or proteins. Overexpression of the enzyme or enzymes and/or protein or proteins of the fatty alcohol, fatty aldehyde or fatty acid pathway can occur, for example, through exogenous expression of the endogenous gene or genes, or through exogenous expression of the heterologous gene or genes. Therefore, naturally occurring organisms can be readily generated to be non-naturally occurring microbial organisms of the invention, for example, producing fatty alcohol, fatty aldehyde or fatty acid, through overexpression of one, two, three, four, five, six, seven, or eight, that is, up to all nucleic acids encoding fatty alcohol, fatty aldehyde or fatty acid biosynthetic pathway enzymes or proteins. In addition, a non-naturally occurring organism can be generated by mutagenesis of an endogenous gene that results in an increase in activity of an enzyme in the fatty alcohol, fatty aldehyde or fatty acid biosynthetic pathway.

In particularly useful embodiments, exogenous expression of the encoding nucleic acids is employed. Exogenous expression confers the ability to custom tailor the expression and/or regulatory elements to the host and application to achieve a desired expression level that is controlled by the user. However, endogenous expression also can be utilized in other embodiments such as by removing a negative regulatory effector or induction of the gene's promoter when linked to an inducible promoter or other regulatory element. Thus, an endogenous gene having a naturally occurring inducible promoter can be up-regulated by providing the appropriate inducing agent, or the regulatory region of an endogenous gene can be engineered to incorporate an inducible regulatory element, thereby allowing the regulation of increased expression of an endogenous gene at a desired time. Similarly, an inducible promoter can be included as a regulatory element for an exogenous gene introduced into a non-naturally occurring microbial organism.

It is understood that, in methods of the invention, any of the one or more exogenous nucleic acids can be introduced into a microbial organism to produce a non-naturally occurring microbial organism of the invention. The nucleic acids can be introduced so as to confer, for example, a fatty alcohol, fatty aldehyde or fatty acid biosynthetic pathway onto the microbial organism. Alternatively, encoding nucleic acids can be introduced to produce an intermediate microbial organism having the biosynthetic capability to catalyze some of the required reactions to confer fatty alcohol, fatty aldehyde or fatty acid biosynthetic capability. For example, a non-naturally occurring microbial organism having a fatty alcohol, fatty aldehyde or fatty acid biosynthetic pathway can comprise at least two exogenous nucleic acids encoding desired enzymes or proteins, such as the combination of a thiolase and an acyl-CoA reductase (alcohol forming), or alternatively a 2-oxoacyl-CoA reductase and an acyl-CoA hydrolase, or alternatively a enoyl-CoA reductase and an acyl-CoA reductase (aldehyde forming), and the like. Thus, it is understood that any combination of two or more enzymes or proteins of a biosynthetic pathway can be included in a non-naturally occurring microbial organism of the invention. Similarly, it is understood that any combination of three or more enzymes or proteins of a biosynthetic pathway can be included in a non-naturally occurring microbial organism of the invention, for example, a thiolase, an enoyl-CoA reductase and a aldehyde dehydrogenase (acid forming), or alternatively a 3 -hydroxy acyl- coA dehydratase, an acyl-CoA:ACP acyltransferase and a thioesterase, or alternatively a 3- oxoacyl-CoA reductase, an acyl-CoA hydrolase and a carboxylic acid reductase, and so forth, as desired, so long as the combination of enzymes and/or proteins of the desired biosynthetic pathway results in production of the corresponding desired product. Similarly, any combination of four, five, six, seven, eight or more enzymes or proteins of a biosynthetic pathway as disclosed herein can be included in a non-naturally occurring microbial organism of the invention, as desired, so long as the combination of enzymes and/or proteins of the desired biosynthetic pathway results in production of the corresponding desired product.

In addition to the biosynthesis of fatty alcohol, fatty aldehyde or fatty acid as described herein, the non-naturally occurring microbial organisms and methods of the invention also can be utilized in various combinations with each other and/or with other microbial organisms and methods well known in the art to achieve product biosynthesis by other routes. For example, one alternative to produce fatty alcohol, fatty aldehyde or fatty acid other than use of the fatty alcohol, fatty aldehyde or fatty acid producers is through addition of another microbial organism capable of converting a fatty alcohol, fatty aldehyde or fatty acid pathway intermediate to fatty alcohol, fatty aldehyde or fatty acid. One such procedure includes, for example, the fermentation of a microbial organism that produces a fatty alcohol, fatty aldehyde or fatty acid pathway intermediate. The fatty alcohol, fatty aldehyde or fatty acid pathway intermediate can then be used as a substrate for a second microbial organism that converts the fatty alcohol, fatty aldehyde or fatty acid pathway intermediate to fatty alcohol, fatty aldehyde or fatty acid. The fatty alcohol, fatty aldehyde or fatty acid pathway intermediate can be added directly to another culture of the second organism or the original culture of the fatty alcohol, fatty aldehyde or fatty acid pathway intermediate producers can be depleted of these microbial organisms by, for example, cell separation, and then subsequent addition of the second organism to the fermentation broth can be utilized to produce the final product without intermediate purification steps. In other embodiments, the non-naturally occurring microbial organisms and methods of the invention can be assembled in a wide variety of subpathways to achieve biosynthesis of, for example, fatty alcohol, fatty aldehyde or fatty acid. In these embodiments,

biosynthetic pathways for a desired product of the invention can be segregated into different microbial organisms, and the different microbial organisms can be co-cultured to produce the final product. In such a biosynthetic scheme, the product of one microbial organism is the substrate for a second microbial organism until the final product is synthesized. For example, the biosynthesis of fatty alcohol, fatty aldehyde or fatty acid can be accomplished by constructing a microbial organism that contains biosynthetic pathways for conversion of one pathway intermediate to another pathway intermediate or the product. Alternatively, fatty alcohol, fatty aldehyde or fatty acid also can be biosynthetically produced from microbial organisms through co-culture or co-fermentation using two organisms in the same vessel, where the first microbial organism produces a fatty alcohol, fatty aldehyde or fatty acid intermediate and the second microbial organism converts the intermediate to fatty alcohol, fatty aldehyde or fatty acid. Given the teachings and guidance provided herein, those skilled in the art will understand that a wide variety of combinations and permutations exist for the non-naturally occurring microbial organisms and methods of the invention together with other microbial organisms, with the co-culture of other non-naturally occurring microbial organisms having subpathways and with combinations of other chemical and/or biochemical procedures well known in the art to produce fatty alcohol, fatty aldehyde or fatty acid.

Similarly, it is understood by those skilled in the art that a host organism can be selected based on desired characteristics for introduction of one or more gene disruptions to increase production of fatty alcohol, fatty aldehyde or fatty acid. Thus, it is understood that, if a genetic modification is to be introduced into a host organism to disrupt a gene, any homologs, orthologs or paralogs that catalyze similar, yet non-identical metabolic reactions can similarly be disrupted to ensure that a desired metabolic reaction is sufficiently disrupted. Because certain differences exist among metabolic networks between different organisms, those skilled in the art will understand that the actual genes disrupted in a given organism may differ between organisms. However, given the teachings and guidance provided herein, those skilled in the art also will understand that the methods of the invention can be applied to any suitable host microorganism to identify the cognate metabolic alterations needed to construct an organism in a species of interest that will increase fatty alcohol, fatty aldehyde or fatty acid biosynthesis. In a particular embodiment, the increased production couples biosynthesis of fatty alcohol, fatty aldehyde or fatty acid to growth of the organism, and can obligatorily couple production of fatty alcohol, fatty aldehyde or fatty acid to growth of the organism if desired and as disclosed herein. Sources of encoding nucleic acids for a fatty alcohol, fatty aldehyde or fatty acid pathway enzyme or protein can include, for example, any species where the encoded gene product is capable of catalyzing the referenced reaction. Such species include both prokaryotic and eukaryotic organisms including, but not limited to, bacteria, including archaea and eubacteria, and eukaryotes, including yeast, plant, insect, animal, and mammal, including human. Exemplary species for such sources include, for example, Escherichia coli, 255956237 Penicillium chrysogenum Wisconsin 54-1255, Acetobacter pasteurians,

Acidaminococcus fermentans, Acinetobacter bayliyi, Acinetobacter calcoaceticus,

Acinetobacter sp. ADP1, Acinetobacter sp. Strain M-l, Actinobacillus succinogenes, Aedes aegypti, Agrobacterium tumefaciens, Alkaliphilus metalliredigens QYMF, Alkaliphilus oremlandii OhILAs, Anabaena variabilis ATCC 29413, Anaerobiospirillum

succiniciproducens, Anopheles gambiae str. PEST, Apis mellifera, Aquifex aeolicus,

Arabidopsis thaliana, Archaeoglobus fulgidus, Archaeoglobus fulgidus DSM 4304, Ascaris suum, Aspergillus fumigatus, Aspergillus nidulans, Aspergillus niger, Aspergillus niger CBS 513.88, Aspergillus terreus NIH2624, Azotobacter vinelandii DJ, Bacillus cereus, Bacillus megaterium, Bacillus methanolicus MGA3, Bacillus methanolicus PB1, Bacillus sp. SG-1, Bacillus subtilis, Bacillus weihenstephanensis KBAB4, Bacteroides fragilis, Bombyx mori, Bos taurus, Bradyrhizobium japonicum, Bradyrhizobium japonicum USDA110, Brassica napsus, Burkholderia ambifaria AMMD, Burkholderia multivorans ATCC 17616,

Burkholderia phymatum, Burkholderia stabilis, butyrate-producing bacterium L2-50, Caenorhabditis briggsae AF16, Caenorhabditis elegans, Campylobacter jejuni, Candida albicans, Candida boidinii, Candida methylica, Candida parapsilosis, Candida tropicalis, Candida tropicalis MYA-3404, Candidatus Protochlamydia amoebophila, Canis lupus familiaris (dog), Carboxydothermus hydrogenoformans, Carthamus tinctorius,

Chlamydomonas reinhardtii, Chlorobium limicola, Chlorobium tepidum, Chloroflexus aurantiacus, Citrus junos, Clostridium acetobutylicum, Clostridium aminobutyricum, Clostridium beijerinckii, Clostridium beijerinckii NCIMB 8052, Clostridium carboxidivorans P7, Clostridium kluyveri, Clostridium kluyveri DSM 555, Clostridium pasteurianum,

Clostridium saccharoperbutylacetonicum, Clostridium symbiosum, Clostridium tetani E88, Colwellia psychrerythraea 34H, Corynebacterium glutamicum, Cryptococcus neoformans var, Cryptosporidium parvum Iowa II, Cuphea hookeriana, Cuphea palustris, Cupriavidus necator, Cupriavidus taiwanensis, Cyanobium PCC7001, Cyanothece sp. PCC 7425, Danio rerio, Desulfatibacillum alkenivorans AK-01, Desulfococcus oleovorans Hxd3, Desulfovibrio africanus, Dictyostelium discoideum, Dictyostelium discoideum AX4, Drosophila

melanogaster, Erythrobacter sp. NAP I, Escherichia coli K-12 MG1655, Euglena gracilis, Flavobacteria bacterium BAL38, Fusobacterium nucleatum, Geobacillus

thermodenitrificans, Haemophilus influenza, Haloarcula marismortui, Haloarcula marismortui ATCC 43049, Halomonas sp. HTNKI, Helianthus annuus, Helicobacter pylori, Helicobacter pylori 26695, Homo sapiens, Hydrogenobacter thermophilus, Klebsiella pneumoniae, Kluyveromyces lactis, Kluyveromyces lactis NRRL Y-1140, Lactobacillus casei, Lactobacillus plantarum, Lactobacillus reuteri, Lactococcus lactis, Leifsonia sp. S749, Leuconostoc mesenteroides, Lyngbya sp. PCC 8106, Macaca mulatta, Magneto spirillum magneticum AMB-1, Mannheimia succiniciproducens, marine gamma proteobacterium

HTCC2080, Marinobacter aquaeolei, Marinobacter aquaeolei VT8, Megathyrsus maximus, Mesorhizobium loti, Metallosphaera sedula, Methanosarcina thermophila,

Methanothermobacter thermautotrophicus, Methylobacterium extorquens, Monosiga brevicollis MX1, Moorella thermoacetica, Moorella thermoacetica ATCC 39073, Mus musculus, Mycobacterium avium subsp. paratuberculosis K-10, Mycobacterium bovis BCG, Mycobacterium marinum M, Mycobacterium smegmatis, Mycobacterium smegmatis MC2 155, Mycobacterium sp. (strain JLS), Mycobacterium sp. MCS, Mycobacterium sp. strain JLS, Mycobacterium tuberculosis, Myxococcus xanthus DK 1622, Nematostella vectensis, Neurospora crassa OR74A, Nicotiana tabacum, Nocardia brasiliensis, Nocardia farcinica IFM 10152, Nocardia iowensis, Nodularia spumigena CCY9414, Nostoc azollae, Nostoc sp. PCC 7120, Opitutaceae bacterium TA V2, Paracoccus denitrificans, Penicillium

chrysogenum, Perkinsus marinus ATCC 50983, Photobacterium phosphoreum,

Photobacterium sp. SKA34, Picea sitchensis, Pichia pastoris, Pichia pastoris GS115, Plasmodium falciparum, Porphyromonas gingivalis, Porphyromonas gingivalis W83,

Prochlorococcus marinus MIT 9312, Propionigenium modestum, Pseudomonas aeruginosa, Pseudomonas aeruginosa PAOl, Pseudomonas fluorescens, Pseudomonas fluorescens PfO-1 , Pseudomonas knackmussii, Pseudomonas knackmussii (B13), Pseudomonas putida,

Pseudomonas putida GB-1, Pseudomonas sp, Pseudomonas sp. CF600, Pseudomonas stutzeri, Pseudomonas stutzeri A 1501, Pseudomonas syringae, Pyrobaculum aerophilum str. IM2, Ralstonia eutropha, Ralstonia metallidurans, Rattus norvegicus, Reinekea sp. MED297, Rhizobium etli CFN 42, Rhizobium leguminosarum, Rhodobacter sphaeroides, Rhodococcus erythropolis, Rhodococcus sp., Rhodopseudomonas palustris, Roseiflexus castenholzii, Roseovarius sp. HTCC2601, Saccharomyces cerevisiae, Saccharomyces cerevisiae s288c, Salmonella enteric, Salmonella enterica subsp. enterica serovar Typhimurium str. LT2, Salmonella typhimurium, Salmonella typhimurium LT2, Scheffersomyces stipitis,

Schizosaccharomyces pombe, Shigella dysenteriae, Shigella sonnei, Simmondsia chinensis, Solanum lycopersicum, Sordaria macrospora, Staphylococcus aureus, Stenotrophomonas maltophilia, Streptococcus mutans, Streptococcus pneumoniae, Streptococcus sanguinis, Streptomyces anulatus, Streptomyces avermitillis, Streptomyces cinnamonensis, Streptomyces coelicolor, Streptomyces griseus subsp. griseus NBRC 13350, Streptomyces luridus,

Streptomyces sp CL190, Streptomyces sp. KO-3988, Streptomyces viridochromogenes, Streptomyces wedmorensis, Strongylocentrotus purpuratus, Sulfolobus acidocaldarius, Sulfolobus solfataricus, Sulfolobus tokodaii, Sulfurihydrogenibium subterraneum,

Sulfurimonas denitrificans, Sus scrofa, Synechococcus elongatus PCC 6301, Synechococcus elongatus PCC 7942, Synechococcus sp. PCC 7002, Syntrophobacter fumaroxidans,

Syntrophus aciditrophicus, Tetraodon nigroviridis, Thermoanaerobacter ethanolicus JW 200, Thermoanaerobacter pseudethanolicus ATCC 33223, Thermococcus litoralis, Thermoproteus neutrophilus, Thermotoga maritime, Treponema denticola, Tribolium castaneum, Trichomonas vaginalis G3, Triticum aestivum, Trypanosoma brucei, Trypanosoma cruzi strain CL Brener, Tsukamurella paurometabola DSM 20162, Umbellularia California, Veillonella parvula, Vibrio cholerae V51, Xenopus tropicalis, Yarrowia lipolytica, Zea mays, Zoogloea ramiger, Zymomonas mobilis, Zymomonas mobilis subsp. mobilis ZM4, as well as other exemplary species disclosed herein or available as source organisms for corresponding genes. However, with the complete genome sequence available for now more than 550 species (with more than half of these available on public databases such as the NCBI), including 395 microorganism genomes and a variety of yeast, fungi, plant, and mammalian genomes, the identification of genes encoding the requisite fatty alcohol, fatty aldehyde or fatty acid biosynthetic activity for one or more genes in related or distant species, including for example, homologues, orthologs, paralogs and nonorthologous gene displacements of known genes, and the interchange of genetic alterations between organisms is routine and well known in the art. Accordingly, the metabolic alterations allowing biosynthesis of fatty alcohol, fatty aldehyde or fatty acid described herein with reference to a particular organism such as E. coli can be readily applied to other microorganisms, including prokaryotic and eukaryotic organisms alike. Given the teachings and guidance provided herein, those skilled in the art will know that a metabolic alteration exemplified in one organism can be applied equally to other organisms.

In some instances, such as when an alternative fatty alcohol, fatty aldehyde or fatty acid biosynthetic pathway exists in an unrelated species, fatty alcohol, fatty aldehyde or fatty acid biosynthesis can be conferred onto the host species by, for example, exogenous expression of a paralog or paralogs from the unrelated species that catalyzes a similar, yet non-identical metabolic reaction to replace the referenced reaction. Because certain differences among metabolic networks exist between different organisms, those skilled in the art will understand that the actual gene usage between different organisms may differ.

However, given the teachings and guidance provided herein, those skilled in the art also will understand that the teachings and methods of the invention can be applied to all microbial organisms using the cognate metabolic alterations to those exemplified herein to construct a microbial organism in a species of interest that will synthesize fatty alcohol, fatty aldehyde or fatty acid. A nucleic acid molecule encoding a fatty alcohol, fatty aldehyde or fatty acid pathway enzyme or protein of the invention can also include a nucleic acid molecule that hybridizes to a nucleic acid disclosed herein by SEQ ID NO, GenBank and/or GI number or a nucleic acid molecule that hybridizes to a nucleic acid molecule that encodes an amino acid sequence disclosed herein by SEQ ID NO, GenBank and/or GI number. Hybridization conditions can include highly stringent, moderately stringent, or low stringency hybridization conditions that are well known to one of skill in the art such as those described herein.

Similarly, a nucleic acid molecule that can be used in the invention can be described as having a certain percent sequence identity to a nucleic acid disclosed herein by SEQ ID NO, GenBank and/or GI number or a nucleic acid molecule that hybridizes to a nucleic acid molecule that encodes an amino acid sequence disclosed herein by SEQ ID NO, GenBank and/or GI number. For example, the nucleic acid molecule can have at least 65%, 70%>, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to a nucleic acid described herein.

Stringent hybridization refers to conditions under which hybridized polynucleotides are stable. As known to those of skill in the art, the stability of hybridized polynucleotides is reflected in the melting temperature (T m ) of the hybrids. In general, the stability of hybridized polynucleotides is a function of the salt concentration, for example, the sodium ion concentration and temperature. A hybridization reaction can be performed under conditions of lower stringency, followed by washes of varying, but higher, stringency.

Reference to hybridization stringency relates to such washing conditions. Highly stringent hybridization includes conditions that permit hybridization of only those nucleic acid sequences that form stable hybridized polynucleotides in 0.018M NaCl at 65°C, for example, if a hybrid is not stable in 0.018M NaCl at 65°C, it will not be stable under high stringency conditions, as contemplated herein. High stringency conditions can be provided, for example, by hybridization in 50% formamide, 5X Denhart's solution, 5X SSPE, 0.2% SDS at 42°C, followed by washing in 0.1X SSPE, and 0.1% SDS at 65°C. Hybridization conditions other than highly stringent hybridization conditions can also be used to describe the nucleic acid sequences disclosed herein. For example, the phrase moderately stringent hybridization refers to conditions equivalent to hybridization in 50% formamide, 5X Denhart's solution, 5X SSPE, 0.2% SDS at 42°C, followed by washing in 0.2X SSPE, 0.2% SDS, at 42°C. The phrase low stringency hybridization refers to conditions equivalent to hybridization in 10% formamide, 5X Denhart's solution, 6X SSPE, 0.2% SDS at 22°C, followed by washing in IX SSPE, 0.2% SDS, at 37°C. Denhart's solution contains 1% Ficoll, 1% polyvinylpyrolidone, and 1%) bovine serum albumin (BSA). 20X SSPE (sodium chloride, sodium phosphate, ethylene diamide tetraacetic acid (EDTA)) contains 3M sodium chloride, 0.2M sodium phosphate, and 0.025 M (EDTA). Other suitable low, moderate and high stringency hybridization buffers and conditions are well known to those of skill in the art and are described, for example, in Sambrook et al, Molecular Cloning: A Laboratory Manual, Third Ed., Cold Spring Harbor Laboratory, New York (2001); and Ausubel et al, Current

Protocols in Molecular Biology, John Wiley and Sons, Baltimore, MD (1999). A nucleic acid molecule encoding a fatty alcohol, fatty aldehyde or fatty acid pathway enzyme or protein of the invention can have at least a certain sequence identity to a nucleotide sequence disclosed herein. According, in some aspects of the invention, a nucleic acid molecule encoding a fatty alcohol, fatty aldehyde or fatty acid pathway enzyme or protein has a nucleotide sequence of at least 65% identity, at least 70%> identity, at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, at least 91% identity, at least 92% identity, at least 93% identity, at least 94% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, or at least 99% identity to a nucleic acid disclosed herein by SEQ ID NO, GenBank and/or GI number or a nucleic acid molecule that hybridizes to a nucleic acid molecule that encodes an amino acid sequence disclosed herein by SEQ ID NO, GenBank and/or GI number.

Sequence identity (also known as homology or similarity) refers to sequence similarity between two nucleic acid molecules or between two polypeptides. Identity can be determined by comparing a position in each sequence, which may be aligned for purposes of comparison. When a position in the compared sequence is occupied by the same base or amino acid, then the molecules are identical at that position. A degree of identity between sequences is a function of the number of matching or homologous positions shared by the sequences. The alignment of two sequences to determine their percent sequence identity can be done using software programs known in the art, such as, for example, those described in Ausubel et al, Current Protocols in Molecular Biology, John Wiley and Sons, Baltimore, MD (1999). Preferably, default parameters are used for the alignment. One alignment program well known in the art that can be used is BLAST set to default parameters. In particular, programs are BLASTN and BLASTP, using the following default parameters: Genetic code = standard; filter = none; strand = both; cutoff = 60; expect = 10; Matrix = BLOSUM62; Descriptions = 50 sequences; sort by = HIGH SCORE; Databases = non- redundant, GenBank + EMBL + DDBJ + PDB + GenBank CDS translations + SwissProtein + SPupdate + PIR. Details of these programs can be found at the National Center for Biotechnology Information. Methods for constructing and testing the expression levels of a non-naturally occurring fatty alcohol, fatty aldehyde or fatty acid-producing host can be performed, for example, by recombinant and detection methods well known in the art. Such methods can be found described in, for example, Sambrook et al, Molecular Cloning: A Laboratory Manual, Third Ed., Cold Spring Harbor Laboratory, New York (2001); and Ausubel et al, Current Protocols in Molecular Biology, John Wiley and Sons, Baltimore, MD (1999).

Exogenous nucleic acid sequences involved in a pathway for production of fatty alcohol, fatty aldehyde or fatty acid can be introduced stably or transiently into a host cell using techniques well known in the art including, but not limited to, conjugation,

electroporation, chemical transformation, transduction, transfection, and ultrasound transformation. For exogenous expression in E. or other prokaryotic cells, some nucleic acid sequences in the genes or cDNAs of eukaryotic nucleic acids can encode targeting signals such as an N-terminal mitochondrial or other targeting signal, which can be removed before transformation into prokaryotic host cells, if desired. For example, removal of a mitochondrial leader sequence led to increased expression in E. coli (Hoffmeister et al, J. Biol. Chem. 280:4329-4338 (2005)). For exogenous expression in yeast or other eukaryotic cells, genes can be expressed in the cytosol without the addition of leader sequence, or can be targeted to mitochondrion or other organelles, or targeted for secretion, by the addition of a suitable targeting sequence such as a mitochondrial targeting or secretion signal suitable for the host cells. Thus, it is understood that appropriate modifications to a nucleic acid sequence to remove or include a targeting sequence can be incorporated into an exogenous nucleic acid sequence to impart desirable properties. Furthermore, genes can be subjected to codon optimization with techniques well known in the art to achieve optimized expression of the proteins. An expression vector or vectors can be constructed to include one or more fatty alcohol, fatty aldehyde or fatty acid biosynthetic pathway encoding nucleic acids as exemplified herein operably linked to expression control sequences functional in the host organism. Expression vectors applicable for use in the microbial host organisms of the invention include, for example, plasmids, phage vectors, viral vectors, episomes and artificial chromosomes, including vectors and selection sequences or markers operable for stable integration into a host chromosome. Additionally, the expression vectors can include one or more selectable marker genes and appropriate expression control sequences. Selectable marker genes also can be included that, for example, provide resistance to antibiotics or toxins, complement auxotrophic deficiencies, or supply critical nutrients not in the culture media. Expression control sequences can include constitutive and inducible promoters, transcription enhancers, transcription terminators, and the like which are well known in the art. When two or more exogenous encoding nucleic acids are to be co-expressed, both nucleic acids can be inserted, for example, into a single expression vector or in separate expression vectors. For single vector expression, the encoding nucleic acids can be operationally linked to one common expression control sequence or linked to different expression control sequences, such as one inducible promoter and one constitutive promoter. The transformation of exogenous nucleic acid sequences involved in a metabolic or synthetic pathway can be confirmed using methods well known in the art. Such methods include, for example, nucleic acid analysis such as Northern blots or polymerase chain reaction (PCR) amplification of mRNA, or immunoblotting for expression of gene products, or other suitable analytical methods to test the expression of an introduced nucleic acid sequence or its corresponding gene product. It is understood by those skilled in the art that the exogenous nucleic acid is expressed in a sufficient amount to produce the desired product, and it is further understood that expression levels can be optimized to obtain sufficient expression using methods well known in the art and as disclosed herein.

In some embodiments, the invention provides a method for producing a compound of Formula (I):

(I) wherein Ri is Ci_ 24 linear alkyl; R 2 is CH 2 OH, CHO, or COOH; R 3 is H, OH, or oxo

(=0); and represents a single or double bond with the proviso that the valency of the carbon atom to which R 3 is attached is four, comprising culturing a non-naturally occurring microbial organism of under conditions and for a sufficient period of time to produce the compound of Formula (I), wherein the non-naturally occurring microbial organism has a MI FAE cycle and/or a MD-FAE cycle in combination with a termination pathway, wherein the MI-FAE cycle includes one or more thiolase, one or more 3-oxoacyl-CoA reductase, one or more 3-hydroxyacyl-CoA dehydratase, and one or more enoyl-CoA reductase, wherein the MD-FAE cycle includes one or more elongase, one or more 3-oxoacyl-CoA reductase, one or more 3-hydroxyacyl-CoA dehydratase, and one or more enoyl-CoA reductase, wherein the termination pathway includes a pathway shown in Figures 1 , 6 or 7 selected from: (1) 1H; (2) IK and 1L; (3) IE and IN; (4) IK, 1 J, and IN; (5) IE; (6) IK and 1 J; (7) 1H and IN; (8) IK, 1L, and IN; (9) IE and IF; (10) IK, IJ, and IF; (1 1) 1H, IN, and IF; (12) IK, 1L, IN, and IF; and (13) 1G, wherein IE is an acyl-CoA reductase (aldehyde forming), wherein IF is an alcohol dehydrogenase, wherein 1G is an acyl-CoA reductase (alcohol forming), wherein 1H is an acyl-CoA hydrolase, acyl-CoA transferase or acyl-CoA synthase, wherein IJ is an acyl- ACP reductase, wherein IK is an acyl-CoA:ACP acyltransferase, wherein 1L is a

thioesterase, wherein IN is an aldehyde dehydrogenase (acid forming) or a carboxylic acid reductase, wherein an enzyme of the MI-FAE cycle, MD-FAE cycle or termination pathway is encoded by at least one exogenous nucleic acid and is expressed in a sufficient amount to produce the compound of Formula (I), wherein the substrate of each of said enzymes of the MI-FAE cycle, MD-FAE cycle and the termination pathway are independently selected from a compound of Formula (II), malonyl-CoA, propionyl-CoA or acetyl-CoA:

wherein Ri is Ci_ 2 4 linear alkyl; R 3 is H, OH, or oxo (=0); R 4 is S-CoA, ACP, OH or H; and represents a single or double bond with the proviso that the valency of the carbon atom to which R 3 is attached is four; wherein said one or more enzymes of the MI- FAE cycle are each selective for a compound of Formula (II) having a number of carbon atoms at Ri that is no greater than the number of carbon atoms at Ri of said compound of Formula (I), wherein said one or more enzymes of the MD-FAE cycle are each selective for a compound of Formula (II) having a number of carbon atoms at Ri that is no greater than the number of carbon atoms at Ri of said compound of Formula (I), and wherein said one or more enzymes of the termination pathway are each selective for a compound of Formula (II) having a number of carbon atoms at Ri that is no less than the number of carbon atoms at Ri of said compound of Formula (I). In some embodiments, the invention provides a method for producing a compound of Formula (I) wherein Ri is C 1-17 linear alkyl. In another aspect of the invention, the Ri of the compound of Formula (I) is Ci linear alkyl, C 2 linear alkyl, C 3 linear alkyl, C 4 linear alkyl, C 5 linear alkyl, C 6 linear alkyl, C 7 linear alkyl, Cg linear alkyl, C9 linear alkyl, C 10 linear alkyl, C11, linear alkyl, C 12 linear alkyl or C 13 linear alkyl, C 14 linear alkyl, C 15 linear alkyl, C 16 linear alkyl, C 17 linear alkyl, C 18 linear alkyl, C19 linear alkyl, C 2 o linear alkyl, C 21 linear alkyl, C 22 linear alkyl, C 23 linear alkyl, or C 24 linear alkyl.

In some aspects of the invention, the microbial organism microbial organism used in the method of the invention includes two, three, or four exogenous nucleic acids each encoding an enzyme of the MI-FAE cycle or the MD-FAE cycle. In some aspects of the invention, the microbial organism used in the method of the invention includes two, three, or four exogenous nucleic acids each encoding an enzyme of the termination pathway. In some aspects of the invention, the microbial organism used in the method of the invention includes exogenous nucleic acids encoding each of the enzymes of at least one of the pathways selected from (1)-(13). In some aspects, the at least one exogenous nucleic acid is a heterologous nucleic acid. In some aspects, the non-naturally occurring microbial organism used in the method of the invention is in a substantially anaerobic culture medium.

In some embodiments, the invention provides a method for producing a fatty alcohol selected from the Formulas (III)-(VI):

R ^ "OH

, wherein Ri is Ci_ 24 linear alkyl, or alternatively Ri is C 1-17 linear alkyl, or alternatively Ri is Cc > _i 3 linear alkyl. In some aspects of the invention, Ri is Ci linear alkyl, C 2 linear alkyl, C 3 linear alkyl, C 4 linear alkyl, C 5 linear alkyl, C 6 linear alkyl, C 7 linear alkyl, Cg linear alkyl, C9 linear alkyl, C 10 linear alkyl, Cn, linear alkyl, C 12 linear alkyl or C 13 linear alkyl, C 14 linear alkyl, C 15 linear alkyl, C 16 linear alkyl, C 17 linear alkyl, C 18 linear alkyl, C19 linear alkyl, C 2 o linear alkyl, C 21 linear alkyl, C 22 linear alkyl, C 23 linear alkyl, or C 24 linear alkyl. In some embodiments, the invention provides a method for producing a fatty aldehyde selected from the Formulas (VII)-(X):

(VIII) (IX) , wherein Ri is Ci_ 24 linear alkyl, or alternatively Ri is C 1-17 linear alkyl, or alternatively Ri is Cg_i 3 linear alkyl. In some aspects of the invention, Ri is Ci linear alkyl, C 2 linear alkyl, C 3 linear alkyl, C 4 linear alkyl, C 5 linear alkyl, C 6 linear alkyl, C 7 linear alkyl, Cg linear alkyl, C 9 linear alkyl, C 10 linear alkyl, Cn, linear alkyl, C 12 linear alkyl or C 13 linear alkyl, C 14 linear alkyl, C 15 linear alkyl, C 16 linear alkyl, C 17 linear alkyl, C 18 linear alkyl, C 19 linear alkyl, C 2 o linear alkyl, C 21 linear alkyl, C 22 linear alkyl, C 23 linear alkyl, or C 24 linear alkyl.

In some embodiments, the invention provides a method for producing a fatty acid selected from the Formulas (XI)-(XIV):

(XIV) ^ wherein Ri is Ci_ 24 linear alkyl, or alternatively Ri is C 1-17 linear alkyl, or alternatively Ri is C 9 _i 3 linear alkyl. In some aspects of the invention, Ri is Ci linear alkyl, C 2 linear alkyl, C 3 linear alkyl, C 4 linear alkyl, C5 linear alkyl, C 6 linear alkyl, C 7 linear alkyl, Cg linear alkyl, C 9 linear alkyl, C 10 linear alkyl, Cn, linear alkyl, C 12 linear alkyl or C 13 linear alkyl, C 14 linear alkyl, C 15 linear alkyl, C 16 linear alkyl, C 17 linear alkyl, C 18 linear alkyl, C 19 linear alkyl, C 2 o linear alkyl, C 21 linear alkyl, C 22 linear alkyl, C 23 linear alkyl, or C 24 linear alkyl. In some embodiments, the method for producing a fatty alcohol, fatty aldehyde or fatty acid described herein includes using a non-naturally occurring microbial organism that has an acetyl-CoA pathway and at least one exogenous nucleic acid encoding an acetyl-CoA pathway enzyme expressed in a sufficient amount to produce acetyl-CoA, wherein the acetyl- CoA pathway includes a pathway shown in Figures 2, 3, 4 or 5 selected from: (1) 2A and 2B; (2) 2A, 2C, and 2D; (3) 2H; (4) 2G and 2D; (5) 2E, 2F and 2B; (6) 2E and 21; (7) 2J, 2F and 2B; (8) 2J and 21; (9) 3A, 3B, and 3C; (10) 3A, 3B, 3J, 3K, and 3D; (11) 3A, 3B, 3G, and 3D; (12) 3A, 3F, and 3D; (13) 3N, 3H, 3B and 3C; (14) 3N, 3H, 3B, 3J, 3K, and 3D; (15) 3N, 3H, 3B, 3G, and 3D; (16) 3N, 3H, 3F, and 3D; (17) 3L, 3M, 3B and 3C; (18) 3L, 3M, 3B, 3J, 3K, and 3D; (19) 3L, 3M, 3B, 3G, and 3D; (20) 3L, 3M, 3F, and 3D; (21) 4A, 4B, 4D, 4H, 41, and 4J; (22) 4A, 4B, 4E, 4F, 4H, 41, and 4J; (23) 4A, 4B, 4E, 4K, 4L, 4H, 41, and 4J; (24) 4A, 4C, 4D, 4H, and 4J; (25) 4A, 4C, 4E, 4F, 4H, and 4J; (26) 4A, 4C, 4E, 4K, 4L, 4H, and 4J; (27) 5A, 5B, 5D, and 5G; (28) 5A, 5B, 5E, 5F, and 5G; (29) 5A, 5B, 5E, 5K, 5L, and 5G; (30) 5A, 5C, and 5D; (31) 5A, 5C, 5E, and 5F; and (32) 5A, 5C, 5E, 5K, and 5L, wherein 2A is a pyruvate oxidase (acetate-forming), wherein 2B is an acetyl-CoA synthetase, an acetyl- CoA ligase or an acetyl-CoA transferase, wherein 2C is an acetate kinase, wherein 2D is a phosphotransacetylase, wherein 2E is a pyruvate decarboxylase, wherein 2F is an

acetaldehyde dehydrogenase, wherein 2G is a pyruvate oxidase (acetyl-phosphate forming), wherein 2H is a pyruvate dehydrogenase, a pyruvate :ferredoxin oxidoreductase, a

pyruvate :NAD(P)H oxidoreductase or a pyruvate formate lyase, wherein 21 is an

acetaldehyde dehydrogenase (acylating), wherein 2J is a threonine aldolase, wherein 3A is a phosphoenolpyruvate (PEP) carboxylase or a PEP carboxykinase, wherein 3B is an oxaloacetate decarboxylase, wherein 3C is a malonate semialdehyde dehydrogenase

(acetylating), wherein 3D is an acetyl-CoA carboxylase or a malonyl-CoA decarboxylase, wherein 3F is an oxaloacetate dehydrogenase or an oxaloacetate oxidoreductase, wherein 3G is a malonate semialdehyde dehydrogenase (acylating), wherein 3H is a pyruvate

carboxylase, wherein 3 J is a malonate semialdehyde dehydrogenase, wherein 3K is a malonyl-CoA synthetase or a malonyl-CoA transferase, wherein 3L is a malic enzyme, wherein 3M is a malate dehydrogenase or a malate oxidoreductase, wherein 3N is a pyruvate kinase or a PEP phosphatase, wherein 4 A is a citrate synthase, wherein 4B is a citrate transporter, wherein 4C is a citrate/malate transporter, wherein 4D is an ATP citrate lyase, wherein 4E is a citrate lyase, wherein 4F is an acetyl-CoA synthetase or an acetyl-CoA transferase, wherein 4H is a cytosolic malate dehydrogenase, wherein 41 is a malate transporter, wherein 4J is a mitochondrial malate dehydrogenase, wherein 4K is an acetate kinase, wherein 4L is a phosphotransacetylase, wherein 5A is a citrate synthase, wherein 5B is a citrate transporter, wherein 5C is a citrate/oxaloacetate transporter, wherein 5D is an ATP citrate lyase, wherein 5E is a citrate lyase, wherein 5F is an acetyl-CoA synthetase or an acetyl-CoA transferase, wherein 5G is an oxaloacetate transporter, wherein 5K is an acetate kinase, and wherein 5L is a phosphotransacetylase.

In some aspects, the microbial organism used in the method of the invention includes two, three, four, five, six, seven or eight exogenous nucleic acids each encoding an acetyl- CoA pathway enzyme. In some aspects, the microbial organism used in the method of the invention includes exogenous nucleic acids encoding each of the acetyl-CoA pathway enzymes of at least one of the pathways selected from (l)-(32).

Suitable purification and/or assays to test for the production of fatty alcohol, fatty aldehyde or fatty acid can be performed using well known methods. Suitable replicates such as triplicate cultures can be grown for each engineered strain to be tested. For example, product and byproduct formation in the engineered production host can be monitored. The final product and intermediates, and other organic compounds, can be analyzed by methods such as HPLC (High Performance Liquid Chromatography), GC-MS (Gas Chromatography- Mass Spectroscopy) and LC-MS (Liquid Chromatography-Mass Spectroscopy) or other suitable analytical methods using routine procedures well known in the art. The release of product in the fermentation broth can also be tested with the culture supernatant. Byproducts and residual glucose can be quantified by HPLC using, for example, a refractive index detector for glucose and alcohols, and a UV detector for organic acids (Lin et al, Biotechnol. Bioeng. 90:775-779 (2005)), or other suitable assay and detection methods well known in the art. The individual enzyme or protein activities from the exogenous DNA sequences can also be assayed using methods well known in the art. The fatty alcohol, fatty aldehyde or fatty acid can be separated from other components in the culture using a variety of methods well known in the art. Such separation methods include, for example, extraction procedures as well as methods that include continuous liquid-liquid extraction, pervaporation, membrane filtration, membrane separation, reverse osmosis, electrodialysis, distillation, crystallization, centrifugation, extractive filtration, ion exchange chromatography, size exclusion chromatography, adsorption chromatography, and ultrafiltration. All of the above methods are well known in the art. Any of the non-naturally occurring microbial organisms described herein can be cultured to produce and/or secrete the biosynthetic products of the invention. For example, the fatty alcohol, fatty aldehyde or fatty acid producers can be cultured for the biosynthetic production of fatty alcohol, fatty aldehyde or fatty acid. Accordingly, in some embodiments, the invention provides culture medium having the fatty alcohol, fatty aldehyde or fatty acid pathway intermediate described herein. In some aspects, the culture medium can also be separated from the non-naturally occurring microbial organisms of the invention that produced the fatty alcohol, fatty aldehyde or fatty acid pathway intermediate. Methods for separating a microbial organism from culture medium are well known in the art. Exemplary methods include filtration, flocculation, precipitation, centrifugation, sedimentation, and the like.

For the production of fatty alcohol, fatty aldehyde or fatty acid, the recombinant strains are cultured in a medium with carbon source and other essential nutrients. It is sometimes desirable and can be highly desirable to maintain anaerobic conditions in the fermenter to reduce the cost of the overall process. Such conditions can be obtained, for example, by first sparging the medium with nitrogen and then sealing the flasks with a septum and crimp-cap. For strains where growth is not observed anaerobically, microaerobic or substantially anaerobic conditions can be applied by perforating the septum with a small hole for limited aeration. Exemplary anaerobic conditions have been described previously and are well-known in the art. Exemplary aerobic and anaerobic conditions are described, for example, in United State publication 2009/0047719, filed August 10, 2007. Fermentations can be performed in a batch, fed-batch or continuous manner, as disclosed herein.

Fermentations can also be conducted in two phases, if desired. The first phase can be aerobic to allow for high growth and therefore high productivity, followed by an anaerobic phase of high fatty alcohol, fatty aldehyde or fatty acid yields.

If desired, the pH of the medium can be maintained at a desired pH, in particular neutral pH, such as a pH of around 7 by addition of a base, such as NaOH or other bases, or acid, as needed to maintain the culture medium at a desirable pH. The growth rate can be determined by measuring optical density using a spectrophotometer (600 nm), and the glucose uptake rate by monitoring carbon source depletion over time.

The growth medium can include, for example, any carbohydrate source which can supply a source of carbon to the non-naturally occurring microorganism. Such sources include, for example: sugars such as glucose, xylose, arabinose, galactose, mannose, fructose, sucrose and starch; or glycerol, and it is understood that a carbon source can be used alone as the sole source of carbon or in combination with other carbon sources described herein or known in the art. Other sources of carbohydrate include, for example, renewable feedstocks and biomass. Exemplary types of biomasses that can be used as feedstocks in the methods of the invention include cellulosic biomass, hemicellulosic biomass and lignin feedstocks or portions of feedstocks. Such biomass feedstocks contain, for example, carbohydrate substrates useful as carbon sources such as glucose, xylose, arabinose, galactose, mannose, fructose and starch. Given the teachings and guidance provided herein, those skilled in the art will understand that renewable feedstocks and biomass other than those exemplified above also can be used for culturing the microbial organisms of the invention for the production of fatty alcohol, fatty aldehyde or fatty acid.

In addition to renewable feedstocks such as those exemplified above, the fatty alcohol, fatty aldehyde or fatty acid microbial organisms of the invention also can be modified for growth on syngas as its source of carbon. In this specific embodiment, one or more proteins or enzymes are expressed in the fatty alcohol, fatty aldehyde or fatty acid producing organisms to provide a metabolic pathway for utilization of syngas or other gaseous carbon source.

Synthesis gas, also known as syngas or producer gas, is the major product of gasification of coal and of carbonaceous materials such as biomass materials, including agricultural crops and residues. Syngas is a mixture primarily of H 2 and CO and can be obtained from the gasification of any organic feedstock, including but not limited to coal, coal oil, natural gas, biomass, and waste organic matter. Gasification is generally carried out under a high fuel to oxygen ratio. Although largely H 2 and CO, syngas can also include C0 2 and other gases in smaller quantities. Thus, synthesis gas provides a cost effective source of gaseous carbon such as CO and, additionally, C0 2 .

The Wood-Ljungdahl pathway catalyzes the conversion of CO and H 2 to acetyl-CoA and other products such as acetate. Organisms capable of utilizing CO and syngas also generally have the capability of utilizing C0 2 and C0 2 /H 2 mixtures through the same basic set of enzymes and transformations encompassed by the Wood-Ljungdahl pathway. Independent conversion of C0 2 to acetate by microorganisms was recognized long before it was revealed that CO also could be used by the same organisms and that the same pathways were involved. Many acetogens have been shown to grow in the presence of C0 2 and produce compounds such as acetate as long as hydrogen is present to supply the necessary reducing equivalents (see for example, Drake, Acetogenesis, pp. 3-60 Chapman and Hall, New York, (1994)). This can be summarized by the following equation:

2 C0 2 + 4 H 2 + n ADP + n Pi→ CH 3 COOH + 2 H 2 0 + n ATP

Hence, non-naturally occurring microorganisms possessing the Wood-Ljungdahl pathway can utilize C0 2 and H 2 mixtures as well for the production of acetyl-CoA and other desired products.

The Wood-Ljungdahl pathway is well known in the art and consists of 12 reactions which can be separated into two branches: (1) methyl branch and (2) carbonyl branch. The methyl branch converts syngas to methyl-tetrahydrofolate (methyl-THF) whereas the carbonyl branch converts methyl-THF to acetyl-CoA. The reactions in the methyl branch are catalyzed in order by the following enzymes or proteins: ferredoxin oxidoreductase, formate dehydrogenase, formyltetrahydrofolate synthetase, methenyltetrahydrofolate

cyclodehydratase, methylenetetrahydrofolate dehydrogenase and methylenetetrahydrofolate reductase. The reactions in the carbonyl branch are catalyzed in order by the following enzymes or proteins: methyltetrahydrofolatexorrinoid protein methyltransferase (for example, AcsE), corrinoid iron-sulfur protein, nickel-protein assembly protein (for example, AcsF), ferredoxin, acetyl-CoA synthase, carbon monoxide dehydrogenase and nickel-protein assembly protein (for example, CooC). Following the teachings and guidance provided herein for introducing a sufficient number of encoding nucleic acids to generate a fatty alcohol, fatty aldehyde or fatty acid pathway, those skilled in the art will understand that the same engineering design also can be performed with respect to introducing at least the nucleic acids encoding the Wood-Ljungdahl enzymes or proteins absent in the host organism. Therefore, introduction of one or more encoding nucleic acids into the microbial organisms of the invention such that the modified organism contains the complete Wood-Ljungdahl pathway will confer syngas utilization ability.

Additionally, the reductive (reverse) tricarboxylic acid cycle coupled with carbon monoxide dehydrogenase and/or hydrogenase activities can also be used for the conversion of CO, C0 2 and/or H 2 to acetyl-CoA and other products such as acetate. Organisms capable of fixing carbon via the reductive TCA pathway can utilize one or more of the following enzymes: ATP citrate-lyase, citrate lyase, aconitase, isocitrate dehydrogenase, alpha- ketoglutarate:ferredoxin oxidoreductase, succinyl-CoA synthetase, succinyl-CoA transferase, fumarate reductase, fumarase, malate dehydrogenase, NAD(P)H:ferredoxin oxidoreductase, carbon monoxide dehydrogenase, and hydrogenase. Specifically, the reducing equivalents extracted from CO and/or H 2 by carbon monoxide dehydrogenase and hydrogenase are utilized to fix C0 2 via the reductive TCA cycle into acetyl-CoA or acetate. Acetate can be converted to acetyl-CoA by enzymes such as acetyl-CoA transferase, acetate

kinase/phosphotransacetylase, and acetyl-CoA synthetase. Acetyl-CoA can be converted to the fatty alcohol, fatty aldehyde or fatty acid precursors, glyceraldehyde-3 -phosphate, phosphoenolpyruvate, and pyruvate, by pyruvate :ferredoxin oxidoreductase and the enzymes of gluconeogenesis. Following the teachings and guidance provided herein for introducing a sufficient number of encoding nucleic acids to generate a fatty alcohol, fatty aldehyde or fatty acid pathway, those skilled in the art will understand that the same engineering design also can be performed with respect to introducing at least the nucleic acids encoding the reductive TCA pathway enzymes or proteins absent in the host organism. Therefore, introduction of one or more encoding nucleic acids into the microbial organisms of the invention such that the modified organism contains a reductive TCA pathway can confer syngas utilization ability.

Accordingly, given the teachings and guidance provided herein, those skilled in the art will understand that a non-naturally occurring microbial organism can be produced that secretes the biosynthesized compounds of the invention when grown on a carbon source such as a carbohydrate. Such compounds include, for example, fatty alcohol, fatty aldehyde or fatty acid and any of the intermediate metabolites in the fatty alcohol, fatty aldehyde or fatty acid pathway. All that is required is to engineer in one or more of the required enzyme or protein activities to achieve biosynthesis of the desired compound or intermediate including, for example, inclusion of some or all of the fatty alcohol, fatty aldehyde or fatty acid biosynthetic pathways. Accordingly, the invention provides a non-naturally occurring microbial organism that produces and/or secretes fatty alcohol, fatty aldehyde or fatty acid when grown on a carbohydrate or other carbon source and produces and/or secretes any of the intermediate metabolites shown in the fatty alcohol, fatty aldehyde or fatty acid pathway when grown on a carbohydrate or other carbon source. The fatty alcohol, fatty aldehyde or fatty acid producing microbial organisms of the invention can initiate synthesis from an intermediate, for example, a 3-ketoacyl-CoA, a 3-hydroxyacyl-CoA, an enoyl-CoA, an acyl-CoA, an acyl-ACP, acetate, acetaldehyde, acetyl-phosphate, oxaloacetate, matate, malonate semialdehyde, malonate, malonyl-CoA, acetyl-CoA, or citrate.

The non-naturally occurring microbial organisms of the invention are constructed using methods well known in the art as exemplified herein to exogenously express at least one nucleic acid encoding a fatty alcohol, fatty aldehyde or fatty acid pathway enzyme or protein in sufficient amounts to produce fatty alcohol, fatty aldehyde or fatty acid. It is understood that the microbial organisms of the invention are cultured under conditions sufficient to produce fatty alcohol, fatty aldehyde or fatty acid. Following the teachings and guidance provided herein, the non-naturally occurring microbial organisms of the invention can achieve biosynthesis of fatty alcohol, fatty aldehyde or fatty acid resulting in intracellular concentrations between about 0.1-200 mM or more. Generally, the intracellular

concentration of fatty alcohol, fatty aldehyde or fatty acid is between about 3-150 mM, particularly between about 5-125 mM and more particularly between about 8-100 mM, including about 10 mM, 20 mM, 50 mM, 80 mM, or more. Intracellular concentrations between and above each of these exemplary ranges also can be achieved from the non- naturally occurring microbial organisms of the invention.

In some embodiments, culture conditions include anaerobic or substantially anaerobic growth or maintenance conditions. Exemplary anaerobic conditions have been described previously and are well known in the art. Exemplary anaerobic conditions for fermentation processes are described herein and are described, for example, in U.S. publication

2009/0047719, filed August 10, 2007. Any of these conditions can be employed with the non-naturally occurring microbial organisms as well as other anaerobic conditions well known in the art. Under such anaerobic or substantially anaerobic conditions, the fatty alcohol, fatty aldehyde or fatty acid producers can synthesize fatty alcohol, fatty aldehyde or fatty acid at intracellular concentrations of 5-10 mM or more as well as all other

concentrations exemplified herein. It is understood that, even though the above description refers to intracellular concentrations, fatty alcohol, fatty aldehyde or fatty acid producing microbial organisms can produce fatty alcohol, fatty aldehyde or fatty acid intracellularly and/or secrete the product into the culture medium. Exemplary fermentation processes include, but are not limited to, fed-batch fermentation and batch separation; fed-batch fermentation and continuous separation; and continuous fermentation and continuous separation. In an exemplary batch fermentation protocol, the production organism is grown in a suitably sized bioreactor sparged with an appropriate gas. Under anaerobic conditions, the culture is sparged with an inert gas or combination of gases, for example, nitrogen, N 2 /C0 2 mixture, argon, helium, and the like. As the cells grow and utilize the carbon source, additional carbon source(s) and/or other nutrients are fed into the bioreactor at a rate approximately balancing consumption of the carbon source and/or nutrients. The temperature of the bioreactor is maintained at a desired temperature, generally in the range of 22-37 degrees C, but the temperature can be maintained at a higher or lower temperature depending on the the growth characteristics of the production organism and/or desired conditions for the fermentation process. Growth continues for a desired period of time to achieve desired characteristics of the culture in the fermenter, for example, cell density, product concentration, and the like. In a batch fermentation process, the time period for the fermentation is generally in the range of several hours to several days, for example, 8 to 24 hours, or 1 , 2, 3, 4 or 5 days, or up to a week, depending on the desired culture conditions. The pH can be controlled or not, as desired, in which case a culture in which pH is not controlled will typically decrease to pH 3-6 by the end of the run. Upon completion of the cultivation period, the fermenter contents can be passed through a cell separation unit, for example, a centrifuge, filtration unit, and the like, to remove cells and cell debris. In the case where the desired product is expressed

intracellularly, the cells can be lysed or disrupted enzymatically or chemically prior to or after separation of cells from the fermentation broth, as desired, in order to release additional product. The fermentation broth can be transferred to a product separations unit. Isolation of product occurs by standard separations procedures employed in the art to separate a desired product from dilute aqueous solutions. Such methods include, but are not limited to, liquid- liquid extraction using a water immiscible organic solvent (e.g. , toluene or other suitable solvents, including but not limited to diethyl ether, ethyl acetate, tetrahydrofuran (THF), methylene chloride, chloroform, benzene, pentane, hexane, heptane, petroleum ether, methyl tertiary butyl ether (MTBE), dioxane, dimethylformamide (DMF), dimethyl sulfoxide (DMSO), and the like) to provide an organic solution of the product, if appropriate, standard distillation methods, and the like, depending on the chemical characteristics of the product of the fermentation process.

In an exemplary fully continuous fermentation protocol, the production organism is generally first grown up in batch mode in order to achieve a desired cell density. When the carbon source and/or other nutrients are exhausted, feed medium of the same composition is supplied continuously at a desired rate, and fermentation liquid is withdrawn at the same rate. Under such conditions, the product concentration in the bioreactor generally remains constant, as well as the cell density. The temperature of the fermenter is maintained at a desired temperature, as discussed above. During the continuous fermentation phase, it is generally desirable to maintain a suitable pH range for optimized production. The pH can be monitored and maintained using routine methods, including the addition of suitable acids or bases to maintain a desired pH range. The bioreactor is operated continuously for extended periods of time, generally at least one week to several weeks and up to one month, or longer, as appropriate and desired. The fermentation liquid and/or culture is monitored periodically, including sampling up to every day, as desired, to assure consistency of product

concentration and/or cell density. In continuous mode, fermenter contents are constantly removed as new feed medium is supplied. The exit stream, containing cells, medium, and product, are generally subjected to a continuous product separations procedure, with or without removing cells and cell debris, as desired. Continuous separations methods employed in the art can be used to separate the product from dilute aqueous solutions, including but not limited to continuous liquid-liquid extraction using a water immiscible organic solvent (e.g., toluene or other suitable solvents, including but not limited to diethyl ether, ethyl acetate, tetrahydrofuran (THF), methylene chloride, chloroform, benzene, pentane, hexane, heptane, petroleum ether, methyl tertiary butyl ether (MTBE), dioxane, dimethylformamide (DMF), dimethyl sulfoxide (DMSO), and the like), standard continuous distillation methods, and the like, or other methods well known in the art.

In addition to the culturing and fermentation conditions disclosed herein, growth condition for achieving biosynthesis of fatty alcohol, fatty aldehyde or fatty acid can include the addition of an osmoprotectant to the culturing conditions. In certain embodiments, the non-naturally occurring microbial organisms of the invention can be sustained, cultured or fermented as described herein in the presence of an osmoprotectant. Briefly, an

osmoprotectant refers to a compound that acts as an osmolyte and helps a microbial organism as described herein survive osmotic stress. Osmoprotectants include, but are not limited to, betaines, amino acids, and the sugar trehalose. Non-limiting examples of such are glycine betaine, praline betaine, dimethylthetin, dimethylslfonioproprionate, 3-dimethylsulfonio-2- methylproprionate, pipecolic acid, dimethylsulfonioacetate, choline, L-carnitine and ectoine. In one aspect, the osmoprotectant is glycine betaine. It is understood to one of ordinary skill in the art that the amount and type of osmoprotectant suitable for protecting a microbial organism described herein from osmotic stress will depend on the microbial organism used. The amount of osmoprotectant in the culturing conditions can be, for example, no more than about 0.1 mM, no more than about 0.5 mM, no more than about 1.0 mM, no more than about 1.5 mM, no more than about 2.0 mM, no more than about 2.5 mM, no more than about 3.0 mM, no more than about 5.0 mM, no more than about 7.0 mM, no more than about lOmM, no more than about 50mM, no more than about lOOmM or no more than about 500mM.

In some embodiments, the carbon feedstock and other cellular uptake sources such as phosphate, ammonia, sulfate, chloride and other halogens can be chosen to alter the isotopic distribution of the atoms present in fatty alcohol, fatty aldehyde or fatty acid or any fatty alcohol, fatty aldehyde or fatty acid pathway intermediate. The various carbon feedstock and other uptake sources enumerated above will be referred to herein, collectively, as "uptake sources." Uptake sources can provide isotopic enrichment for any atom present in the product fatty alcohol, fatty aldehyde or fatty acid or fatty alcohol, fatty aldehyde or fatty acid pathway intermediate, or for side products generated in reactions diverging away from a fatty alcohol, fatty aldehyde or fatty acid pathway. Isotopic enrichment can be achieved for any target atom including, for example, carbon, hydrogen, oxygen, nitrogen, sulfur, phosphorus, chloride or other halogens.

In some embodiments, the uptake sources can be selected to alter the carbon- 12, carbon- 13, and carbon- 14 ratios. In some embodiments, the uptake sources can be selected to alter the oxygen- 16, oxygen- 17, and oxygen- 18 ratios. In some embodiments, the uptake sources can be selected to alter the hydrogen, deuterium, and tritium ratios. In some embodiments, the uptake sources can be selected to alter the nitrogen- 14 and nitrogen- 15 ratios. In some embodiments, the uptake sources can be selected to alter the sulfur-32, sulfur- 33, sulfur-34, and sulfur-35 ratios. In some embodiments, the uptake sources can be selected to alter the phosphorus-31 , phosphorus-32, and phosphorus-33 ratios. In some embodiments, the uptake sources can be selected to alter the chlorine-35, chlorine-36, and chlorine-37 ratios.

In some embodiments, the isotopic ratio of a target atom can be varied to a desired ratio by selecting one or more uptake sources. An uptake source can be derived from a natural source, as found in nature, or from a man-made source, and one skilled in the art can select a natural source, a man-made source, or a combination thereof, to achieve a desired isotopic ratio of a target atom. An example of a man-made uptake source includes, for example, an uptake source that is at least partially derived from a chemical synthetic reaction. Such isotopically enriched uptake sources can be purchased commercially or prepared in the laboratory and/or optionally mixed with a natural source of the uptake source to achieve a desired isotopic ratio. In some embodiments, a target atom isotopic ratio of an uptake source can be achieved by selecting a desired origin of the uptake source as found in nature. For example, as discussed herein, a natural source can be a biobased derived from or synthesized by a biological organism or a source such as petroleum-based products or the atmosphere. In some such embodiments, a source of carbon, for example, can be selected from a fossil fuel- derived carbon source, which can be relatively depleted of carbon- 14, or an environmental or atmospheric carbon source, such as C0 2 , which can possess a larger amount of carbon- 14 than its petroleum-derived counterpart.

The unstable carbon isotope carbon-14 or radiocarbon makes up for roughly 1 in 10 12 carbon atoms in the earth's atmosphere and has a half-life of about 5700 years. The stock of carbon is replenished in the upper atmosphere by a nuclear reaction involving cosmic rays and ordinary nitrogen ( 14 N). Fossil fuels contain no carbon-14, as it decayed long ago.

Burning of fossil fuels lowers the atmospheric carbon-14 fraction, the so-called "Suess effect".

Methods of determining the isotopic ratios of atoms in a compound are well known to those skilled in the art. Isotopic enrichment is readily assessed by mass spectrometry using techniques known in the art such as accelerated mass spectrometry (AMS), Stable Isotope Ratio Mass Spectrometry (SIRMS) and Site-Specific Natural Isotopic Fractionation by Nuclear Magnetic Resonance (SNIF-NMR). Such mass spectral techniques can be integrated with separation techniques such as liquid chromatography (LC), high performance liquid chromatography (HPLC) and/or gas chromatography, and the like. In the case of carbon, ASTM D6866 was developed in the United States as a standardized analytical method for determining the biobased content of solid, liquid, and gaseous samples using radiocarbon dating by the American Society for Testing and Materials (ASTM) International. The standard is based on the use of radiocarbon dating for the determination of a product's biobased content. ASTM D6866 was first published in 2004, and the current active version of the standard is ASTM D6866-11 (effective April 1, 2011).

Radiocarbon dating techniques are well known to those skilled in the art, including those described herein. The biobased content of a compound is estimated by the ratio of carbon- 14 ( C) to carbon- 12 ( 12 C). Specifically, the Fraction Modern (Fm) is computed from the expression: Fm = (S-B)/(M-B), where B, S and M represent the 14 C/ 12 C ratios of the blank, the sample and the modern reference, respectively. Fraction Modern is a measurement of the deviation of the 14 C/ 12 C ratio of a sample from "Modern." Modern is defined as 95% of the radiocarbon concentration (in AD 1950) of National Bureau of Standards (NBS) Oxalic Acid I (i.e., standard reference materials (SRM) 4990b) normalized to per mil (Olsson, The use of Oxalic acid as a Standard, in, Radiocarbon Variations and Absolute Chronology, Nobel Symposium, 12th Proc, John Wiley & Sons, New York (1970)). Mass spectrometry results, for example, measured by ASM, are calculated using the internationally agreed upon definition of 0.95 times the specific activity of NBS Oxalic Acid I (SRM 4990b) normalized to 5 13 C VPDB =-19 per mil. This is equivalent to an absolute (AD 1950) 14 C/ 12 C ratio of 1.176 ± 0.010 x 10 "12 (Karlen et al, Arkiv Geofysik, 4:465-471 (1968)). The standard calculations take into account the differential uptake of one isotope with respect to another, for example, the preferential uptake in biological systems of C 12 over C 13 over C 14 , and these corrections are reflected as a Fm corrected for δ 13 .

An oxalic acid standard (SRM 4990b or HOx 1) was made from a crop of 1955 sugar beet. Although there were 1000 lbs made, this oxalic acid standard is no longer

commercially available. The Oxalic Acid II standard (HOx 2; N.I.S.T designation SRM 4990 C) was made from a crop of 1977 French beet molasses. In the early 1980's, a group of 12 laboratories measured the ratios of the two standards. The ratio of the activity of Oxalic acid II to 1 is 1.2933±0.001 (the weighted mean). The isotopic ratio of HOx II is -17.8 per mil. ASTM D6866-11 suggests use of the available Oxalic Acid II standard SRM 4990 C (Hox2) for the modern standard (see discussion of original vs. currently available oxalic acid standards in Mann, Radiocarbon, 25(2):519-527 (1983)). A Fm = 0% represents the entire lack of carbon- 14 atoms in a material, thus indicating a fossil (for example, petroleum based) carbon source. A Fm = 100%, after correction for the post-1950 injection of carbon-14 into the atmosphere from nuclear bomb testing, indicates an entirely modern carbon source. As described herein, such a "modern" source includes biobased sources. As described in ASTM D6866, the percent modern carbon (pMC) can be greater than

100%) because of the continuing but diminishing effects of the 1950s nuclear testing programs, which resulted in a considerable enrichment of carbon-14 in the atmosphere as described in ASTM D6866-11. Because all sample carbon- 14 activities are referenced to a "pre-bomb" standard, and because nearly all new biobased products are produced in a post- bomb environment, all pMC values (after correction for isotopic fraction) must be multiplied by 0.95 (as of 2010) to better reflect the true biobased content of the sample. A biobased content that is greater than 103% suggests that either an analytical error has occurred, or that the source of biobased carbon is more than several years old.

ASTM D6866 quantifies the biobased content relative to the material's total organic content and does not consider the inorganic carbon and other non-carbon containing substances present. For example, a product that is 50% starch-based material and 50%> water would be considered to have a Biobased Content = 100% (50%> organic content that is 100% biobased) based on ASTM D6866. In another example, a product that is 50% starch-based material, 25% petroleum-based, and 25% water would have a Biobased Content = 66.7% (75%) organic content but only 50%> of the product is biobased). In another example, a product that is 50%> organic carbon and is a petroleum-based product would be considered to have a Biobased Content = 0%> (50%> organic carbon but from fossil sources). Thus, based on the well known methods and known standards for determining the biobased content of a compound or material, one skilled in the art can readily determine the biobased content and/or prepared downstream products that utilize of the invention having a desired biobased content. Applications of carbon- 14 dating techniques to quantify bio-based content of materials are known in the art (Currie et al., Nuclear Instruments and Methods in Physics Research B, 172:281-287 (2000)). For example, carbon- 14 dating has been used to quantify bio-based content in terephthalate-containing materials (Colonna et al., Green Chemistry, 13:2543-2548 (2011)). Notably, polypropylene terephthalate (PPT) polymers derived from renewable 1 ,3-propanediol and petroleum-derived terephthalic acid resulted in Fm values near 30% (i.e., since 3/11 of the polymeric carbon derives from renewable 1,3-propanediol and 8/11 from the fossil end member terephthalic acid) (Currie et al., supra, 2000). In contrast, polybutylene terephthalate polymer derived from both renewable 1 ,4-butanediol and renewable terephthalic acid resulted in bio-based content exceeding 90% (Colonna et al., supra, 2011).

Accordingly, in some embodiments, the present invention provides fatty alcohol, fatty aldehyde or fatty acid or a fatty alcohol, fatty aldehyde or fatty acid pathway intermediate that has a carbon- 12, carbon- 13, and carbon- 14 ratio that reflects an atmospheric carbon, also referred to as environmental carbon, uptake source. For example, in some aspects the fatty alcohol, fatty aldehyde or fatty acid or a fatty alcohol, fatty aldehyde or fatty acid pathway intermediate can have an Fm value of at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%o or as much as 100%. In some such embodiments, the uptake source is C0 2 . In some embodiments, the present invention provides fatty alcohol, fatty aldehyde or fatty acid or a fatty alcohol, fatty aldehyde or fatty acid pathway intermediate that has a carbon- 12, carbon- 13, and carbon- 14 ratio that reflects petroleum-based carbon uptake source. In this aspect, the fatty alcohol, fatty aldehyde or fatty acid or a fatty alcohol, fatty aldehyde or fatty acid pathway intermediate can have an Fm value of less than 95%, less than 90%, less than 85%o, less than 80%>, less than 75%, less than 70%>, less than 65%, less than 60%>, less than 55%, less than 50%, less than 45%, less than 40%, less than 35%, less than 30%, less than 25%o, less than 20%>, less than 15%, less than 10%, less than 5%, less than 2% or less than 1%. In some embodiments, the present invention provides fatty alcohol, fatty aldehyde or fatty acid or a fatty alcohol, fatty aldehyde or fatty acid pathway intermediate that has a carbon- 12, carbon- 13, and carbon- 14 ratio that is obtained by a combination of an

atmospheric carbon uptake source with a petroleum-based uptake source. Using such a combination of uptake sources is one way by which the carbon- 12, carbon-13, and carbon- 14 ratio can be varied, and the respective ratios would reflect the proportions of the uptake sources.

Further, the present invention relates to the biologically produced fatty alcohol, fatty aldehyde or fatty acid or fatty alcohol, fatty aldehyde or fatty acid pathway intermediate as disclosed herein, and to the products derived therefrom, wherein the fatty alcohol, fatty aldehyde or fatty acid or a fatty alcohol, fatty aldehyde or fatty acid pathway intermediate has a carbon- 12, carbon-13, and carbon- 14 isotope ratio of about the same value as the C0 2 that occurs in the environment. For example, in some aspects the invention provides bioderived fatty alcohol, fatty aldehyde or fatty acid or a bioderived fatty alcohol, fatty aldehyde or fatty acid intermediate having a carbon- 12 versus carbon-13 versus carbon- 14 isotope ratio of about the same value as the C0 2 that occurs in the environment, or any of the other ratios disclosed herein. It is understood, as disclosed herein, that a product can have a carbon- 12 versus carbon-13 versus carbon- 14 isotope ratio of about the same value as the C0 2 that occurs in the environment, or any of the ratios disclosed herein, wherein the product is generated from bioderived fatty alcohol, fatty aldehyde or fatty acid or a bioderived fatty alcohol, fatty aldehyde or fatty acid pathway intermediate as disclosed herein, wherein the bioderived product is chemically modified to generate a final product. Methods of chemically modifying a bioderived product of fatty alcohol, fatty aldehyde or fatty acid, or an intermediate thereof, to generate a desired product are well known to those skilled in the art, as described herein. The invention further provides biofuels, chemicals, polymers, surfactants, soaps, detergents, shampoos, lubricating oil additives, fragrances, flavor materials or acrylates having a carbon- 12 versus carbon- 13 versus carbon- 14 isotope ratio of about the same value as the C0 2 that occurs in the environment, wherein the biofuels, chemicals, polymers, surfactants, soaps, detergents, shampoos, lubricating oil additives, fragrances, flavor materials or acrylates are generated directly from or in combination with bioderived fatty alcohol, fatty aldehyde or fatty acid or a bioderived fatty alcohol, fatty aldehyde or fatty acid pathway intermediate as disclosed herein. Fatty alcohol, fatty aldehyde or fatty acid is a chemical used in commercial and industrial applications. Non-limiting examples of such applications include production of biofuels, chemicals, polymers, surfactants, soaps, detergents, shampoos, lubricating oil additives, fragrances, flavor materials and acrylates. Accordingly, in some embodiments, the invention provides biobased biofuels, chemicals, polymers, surfactants, soaps, detergents, shampoos, lubricating oil additives, fragrances, flavor materials and acrylates comprising one or more bioderived fatty alcohol, fatty aldehyde or fatty acid or bioderived fatty alcohol, fatty aldehyde or fatty acid pathway intermediate produced by a non-naturally occurring microorganism of the invention or produced using a method disclosed herein.

As used herein, the term "bioderived" means derived from or synthesized by a biological organism and can be considered a renewable resource since it can be generated by a biological organism. Such a biological organism, in particular the microbial organisms of the invention disclosed herein, can utilize feedstock or biomass, such as, sugars or carbohydrates obtained from an agricultural, plant, bacterial, or animal source. Alternatively, the biological organism can utilize atmospheric carbon. As used herein, the term "biobased" means a product as described above that is composed, in whole or in part, of a bioderived compound of the invention. A biobased or bioderived product is in contrast to a petroleum derived product, wherein such a product is derived from or synthesized from petroleum or a petrochemical feedstock.

In some embodiments, the invention provides a biofuel, chemical, polymer, surfactant, soap, detergent, shampoo, lubricating oil additive, fragrance, flavor material or acrylate comprising bioderived fatty alcohol, fatty aldehyde or fatty acid or bioderived fatty alcohol, fatty aldehyde or fatty acid pathway intermediate, wherein the bioderived fatty alcohol, fatty aldehyde or fatty acid or bioderived fatty alcohol, fatty aldehyde or fatty acid pathway intermediate includes all or part of the fatty alcohol, fatty aldehyde or fatty acid or fatty alcohol, fatty aldehyde or fatty acid pathway intermediate used in the production of a biofuel, chemical, polymer, surfactant, soap, detergent, shampoo, lubricating oil additive, fragrance, flavor material or acrylate. For example, the final biofuel, chemical, polymer, surfactant, soap, detergent, shampoo, lubricating oil additive, fragrance, flavor material or acrylate can contain the bioderived fatty alcohol, fatty aldehyde or fatty acid, fatty alcohol, fatty aldehyde or fatty acid pathway intermediate, or a portion thereof that is the result of the manufacturing of the biofuel, chemical, polymer, surfactant, soap, detergent, shampoo, lubricating oil additive, fragrance, flavor material or acrylate. Such manufacturing can include chemically reacting the bioderived fatty alcohol, fatty aldehyde or fatty acid, or bioderived fatty alcohol, fatty aldehyde or fatty acid pathway intermediate (e.g. chemical conversion, chemical functionalization, chemical coupling, oxidation, reduction,

polymerization, copolymerization and the like) with itself or another compound in a reaction that produces the final biofuel, chemical, polymer, surfactant, soap, detergent, shampoo, lubricating oil additive, fragrance, flavor material or acrylate. Thus, in some aspects, the invention provides a biobased biofuel, chemical, polymer, surfactant, soap, detergent, shampoo, lubricating oil additive, fragrance, flavor material or acrylate comprising at least 2%, at least 3%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least

30%, at least 35%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98% or 100% bioderived fatty alcohol, fatty aldehyde or fatty acid or bioderived fatty alcohol, fatty aldehyde or fatty acid pathway intermediate as disclosed herein. In some aspects, when the product is a biobased polymer that includes or is obtained from a bioderived fatty alcohol, fatty aldehyde or fatty acid, or or fatty alcohol, fatty aldehyde or fatty acid pathway intermediate described herein, the biobased polymer can be molded using methods well known in the art. Accordingly, in some embodiments, provided herein is a molded product comprising the biobased polymer described herein. Additionally, in some embodiments, the invention provides a composition having a bioderived fatty alcohol, fatty aldehyde or fatty acid, or fatty alcohol, fatty aldehyde or fatty acid pathway intermediate disclosed herein and a compound other than the bioderived fatty alcohol, fatty aldehyde or fatty acid or fatty alcohol, fatty aldehyde or fatty acid pathway intermediate. For example, in some aspects, the invention provides a biobased biofuel, chemical, polymer, surfactant, soap, detergent, shampoo, lubricating oil additive, fragrance, flavor material or acrylate wherein the fatty alcohol, fatty aldehyde or fatty acid or fatty alcohol, fatty aldehyde or fatty acid pathway intermediate used in its production is a combination of bioderived and petroleum derived fatty alcohol, fatty aldehyde or fatty acid or fatty alcohol, fatty aldehyde or fatty acid pathway intermediate. For example, a biobased a biofuel, chemical, polymer, surfactant, soap, detergent, shampoo, lubricating oil additive, fragrance, flavor material or acrylate can be produced using 50% bioderived fatty alcohol, fatty aldehyde or fatty acid and 50% petroleum derived fatty alcohol, fatty aldehyde or fatty acid or other desired ratios such as 60%/40%, 70%/30%, 80%/20%, 90%/10%, 95%/5%, 100%/0%, 40%/60%, 30%/70%, 20%/80%, 10%/90% of bioderived/petroleum derived precursors, so long as at least a portion of the product comprises a bioderived product produced by the microbial organisms disclosed herein. It is understood that methods for producing a biofuel, chemical, polymer, surfactant, soap, detergent, shampoo, lubricating oil additive, fragrance, flavor material or acrylate using the bioderived fatty alcohol, fatty aldehyde or fatty acid or bioderived fatty alcohol, fatty aldehyde or fatty acid pathway intermediate of the invention are well known in the art.

The invention further provides a composition comprising bioderived fatty alcohol, fatty aldehyde or fatty acid, and a compound other than the bioderived fatty alcohol, fatty aldehyde or fatty acid. The compound other than the bioderived product can be a cellular portion, for example, a trace amount of a cellular portion of, or can be fermentation broth or culture medium, or a purified or partially purified fraction thereof produced in the presence of, a non-naturally occurring microbial organism of the invention having a fatty alcohol, fatty aldehyde or fatty acid pathway. The composition can comprise, for example, a reduced level of a byproduct when produced by an organism having reduced byproduct formation, as disclosed herein. The composition can comprise, for example, bioderived fatty alcohol, fatty aldehyde or fatty acid, or a cell lysate or culture supernatant of a microbial organism of the invention. In certain embodiments, provided herein is a composition comprising a bioderived fatty alcohol, fatty aldehyde or fatty acid provided herein, for example, a bioderived fatty alcohol, fatty aldehyde or fatty acid produced by culturing a non-naturally occurring microbial organism having a MI-FAE cycle and/or a MD-FAE cycle in combination with a termination pathway, as provided herein. In some embodiments, the composition further comprises a compound other than said bioderived bioderived fatty alcohol, fatty aldehyde or fatty acid. In certain embodiments, the compound other than said bioderived fatty alcohol, fatty aldehyde or fatty acid is a trace amount of a cellular portion of a non-naturally occurring microbial organism having a MI-FAE cycle and/or a MD-FAE cycle in combination with a termination pathway.

In some embodiments, provided herein is a biobased product comprising a bioderived fatty alcohol, fatty aldehyde or fatty acid provided herein. In certain embodiments, the biobased product is a biofuel, chemical, polymer, surfactant, soap, detergent, shampoo, lubricating oil additive, fragrance, flavor material or acrylate. In certain embodiments, the biobased product comprises at least 5% bioderived fatty alcohol, fatty aldehyde or fatty acid. In certain embodiments, the biobased product comprises at least 10% bioderived fatty alcohol, fatty aldehyde or fatty acid. In some embodiments, the biobased product comprises at least 20% bioderived fatty alcohol, fatty aldehyde or fatty acid. In other embodiments, the biobased product comprises at least 30%> bioderived fatty alcohol, fatty aldehyde or fatty acid. In some embodiments, the biobased product comprises at least 40%> bioderived fatty alcohol, fatty aldehyde or fatty acid. In other embodiments, the biobased product comprises at least 50% bioderived fatty alcohol, fatty aldehyde or fatty acid. In one embodiment, the biobased product comprises a portion of said bioderived fatty alcohol, fatty aldehyde or fatty acid as a repeating unit. In another embodiment, provided herein is a molded product obtained by molding the biobased product provided herein. In other embodiments, provided herein is a process for producing a biobased product provided herein, comprising chemically reacting said bioderived fatty alcohol, fatty aldehyde or fatty acid with itself or another compound in a reaction that produces said biobased product. In certain embodiments, provided herein is a polymer comprising or obtained by converting the bioderived fatty alcohol, fatty aldehyde or fatty acid. In other embodiments, provided herein is a method for producing a polymer, comprising chemically of enzymatically converting the bioderived fatty alcohol, fatty aldehyde or fatty acid to the polymer. In yet other embodiments, provided herein is a composition comprising the bioderived fatty alcohol, fatty aldehyde or fatty acid, or a cell lysate or culture supernatant thereof.

The culture conditions can include, for example, liquid culture procedures as well as fermentation and other large scale culture procedures. As described herein, particularly useful yields of the biosynthetic products of the invention can be obtained under anaerobic or substantially anaerobic culture conditions.

As described herein, one exemplary growth condition for achieving biosynthesis of fatty alcohol, fatty aldehyde or fatty acid includes anaerobic culture or fermentation conditions. In certain embodiments, the non-naturally occurring microbial organisms of the invention can be sustained, cultured or fermented under anaerobic or substantially anaerobic conditions. Briefly, an anaerobic condition refers to an environment devoid of oxygen.

Substantially anaerobic conditions include, for example, a culture, batch fermentation or continuous fermentation such that the dissolved oxygen concentration in the medium remains between 0 and 10% of saturation. Substantially anaerobic conditions also includes growing or resting cells in liquid medium or on solid agar inside a sealed chamber maintained with an atmosphere of less than 1% oxygen. The percent of oxygen can be maintained by, for example, sparging the culture with an N 2 /C0 2 mixture or other suitable non-oxygen gas or gases.

The culture conditions described herein can be scaled up and grown continuously for manufacturing of fatty alcohol, fatty aldehyde or fatty acid. Exemplary growth procedures include, for example, fed-batch fermentation and batch separation; fed-batch fermentation and continuous separation, or continuous fermentation and continuous separation. All of these processes are well known in the art. Fermentation procedures are particularly useful for the biosynthetic production of commercial quantities of fatty alcohol, fatty aldehyde or fatty acid. Generally, and as with non-continuous culture procedures, the continuous and/or near- continuous production of fatty alcohol, fatty aldehyde or fatty acid will include culturing a non-naturally occurring fatty alcohol, fatty aldehyde or fatty acid producing organism of the invention in sufficient nutrients and medium to sustain and/or nearly sustain growth in an exponential phase. Continuous culture under such conditions can include, for example, growth or culturing for 1 day, 2, 3, 4, 5, 6 or 7 days or more. Additionally, continuous culture can include longer time periods of 1 week, 2, 3, 4 or 5 or more weeks and up to several months. Alternatively, organisms of the invention can be cultured for hours, if suitable for a particular application. It is to be understood that the continuous and/or near- continuous culture conditions also can include all time intervals in between these exemplary periods. It is further understood that the time of culturing the microbial organism of the invention is for a sufficient period of time to produce a sufficient amount of product for a desired purpose.

Fermentation procedures are well known in the art. Briefly, fermentation for the biosynthetic production of fatty alcohol, fatty aldehyde or fatty acid can be utilized in, for example, fed-batch fermentation and batch separation; fed-batch fermentation and continuous separation, or continuous fermentation and continuous separation. Examples of batch and continuous fermentation procedures are well known in the art.

In addition to the above fermentation procedures using the fatty alcohol, fatty aldehyde or fatty acid producers of the invention for continuous production of substantial quantities of fatty alcohol, fatty aldehyde or fatty acid, the fatty alcohol, fatty aldehyde or fatty acid producers also can be, for example, simultaneously subjected to chemical synthesis and/or enzymatic procedures to convert the product to other compounds or the product can be separated from the fermentation culture and sequentially subjected to chemical and/or enzymatic conversion to convert the product to other compounds, if desired.

To generate better producers, metabolic modeling can be utilized to optimize growth conditions. Modeling can also be used to design gene knockouts that additionally optimize utilization of the pathway (see, for example, U.S. patent publications US 2002/0012939, US 2003/0224363, US 2004/0029149, US 2004/0072723, US 2003/0059792, US 2002/0168654 and US 2004/0009466, and U.S. Patent No. 7,127,379). Modeling analysis allows reliable predictions of the effects on cell growth of shifting the metabolism towards more efficient production of fatty alcohol, fatty aldehyde or fatty acid. In addition to active and selective enzymes producing fatty alcohols, fatty aldehydes, or fatty acids at high yield, titer and productivity, a robust host organism that can efficiently direct carbon and reducing equivalents to fatty alcohol, fatty aldehyde and fatty acid biosynthesis can be beneficial. Host modifications described herein are particularly useful in combination with selective enzymes described herein that favor formation of the desired fatty alcohol, fatty aldehyde, or fatty acid product. Several host modifications described herein entail introducing heterologous enzyme activities into the host organism. Other modifications involve overexpressing or elevating enzyme activity relative to wild type levels. Yet other modifications include disrupting endogenous genes or attenuating endogenous enzyme activities.

In one embodiment of the invention, the microbial organisms efficiently directs carbon and energy sources into production of acetyl-CoA, which is used as both a primer and extension unit in the MI-FAE cycle. In one embodiment of the invention, the microbial organisms efficiently directs carbon and energy sources into production of malonyl-CoA, which is used as both a primer and extension unit in the MD-FAE cycle. In unmodified microbial organism, fatty alcohol, fatty aldehyde and fatty acid production in the cytosol relies on the native cell machinery to provide the necessary precursors. Thus, high concentrations of cytosolic acetyl-CoA and/or malonyl-CoA are desirable for facilitating deployment of a cytosolic fatty alcohol, fatty aldehyde or fatty acid production pathway that originates from acetyl-CoA or malonyl-CoA. Metabolic engineering strategies for increasing cytosolic acetyl-CoA and malonyl-CoA are disclosed herein. Since many eukaryotic organisms synthesize most of their acetyl-CoA in the mitochondria during growth on glucose, increasing the availability of acetyl-CoA in the cytosol can be obtained by introduction of a cytosolic acetyl-CoA biosynthesis pathway. Accordingly, acetyl-CoA biosynthesis pathways are described herein. In one embodiment, utilizing the pathways shown in Figure 2, acetyl-CoA can be synthesized in the cytosol from a pyruvate or threonine precursor. In other embodiment, acetyl-CoA can be synthesized in the cytosol from phosphoenolpyruvate (PEP) or pyruvate (Figure 3). In yet another embodiment acetyl-CoA can be synthesized in cellular compartments and transported to the cytosol. For example, one mechanism involves converting mitochondrial acetyl-CoA to a metabolic intermediate such as citrate or citramalate, transporting those intermediates to the cytosol, and then regenerating the acetyl-CoA (see Figures 4 and 5). Exemplary acetyl-CoA pathways and corresponding enzymes are further described in Examples II-IV.

In another embodiment, increasing cytosolic acetyl-CoA availability for fatty alcohol, fatty aldehyde, or fatty acid biosynthesis is to disrupt or attenuate competing enzymes and pathways that utilize acetyl-CoA or its precursors. Exemplary competing enzyme activities include, but are not limited to, pyruvate decarboxylase, lactate dehydrogenase, short-chain aldehyde and alcohol dehydrogenases, acetate kinase, phosphotransacetylase,

glyceraldehyde-3 -phosphate dehydrogenases, pyruvate oxidase and acetyl-CoA carboxylase. Exemplary acetyl-CoA consuming pathways whose disruption or attenuation can improve fatty alcohol, fatty aldehyde, or fatty acid production include the mitochondrial TCA cycle, fatty acid biosynthesis, ethanol production and amino acid biosynthesis. These enzymes and pathways are further described herein. Yet another strategy for increasing cytosolic acetyl-CoA production is to increase the pool of CoA available in the cytoplasm. This can be accomplished by overexpression of CoA biosynthetic enzymes in the cytosol. In particular, expression of pantothenate kinase (EC 2.7.1.33) can be used. This enzyme catalyzes the first step and rate-limiting enzyme of CoA biosynthesis. Exemplary pantothenate kinase variants resistant to feedback inhibition by CoA are well known in the art (Rock et al, J Bacteriol 185: 3410-5 (2003)) and are described in the below table.

Competing enzymes and pathways that divert acyl-CoA substrates from production of fatty alcohols, fatty aldehydes or fatty acids of the invention can also be attenuated or disrupted. Exemplary enzymes for attenuation include acyltransferases, carnitine shuttle enzymes and negative regulators of MI-FAE cycle, MD-FAE cycle or termination pathway enzymes.

Disruption or attenuation of acyltransferases that transfer acyl moieties from CoA to other acceptors such as ACP, glycerol, ethanol and others, can increase the availability of acyl-CoA for fatty alcohol, fatty aldehyde or fatty acid production. For example, Acyl- CoA:ACP transacylase (EC 2.3.1.38; 2.3.1.39) enzymes such asfabH (KASIII) of E. coli transfer acyl moieties from CoA to ACP. FabH is active on acetyl-CoA and butyryl-CoA (Prescott et al, Adv. Enzymol. Relat. Areas Mol, 36:269-311 (1972)). Acetyl-CoA:ACP transacylase enzymes from Plasmodium falciparum and Streptomyces avermitillis have been heterologously expressed in E. coli (Lobo et al, Biochem 40: 11955-64 (2001)). A synthetic KASIII (FabH) from P. falciparum expressed in a fabH-deficient Lactococcus lactis host was able to complement the native fadH activity (Du et al, AEM 76:3959-66 (2010)). The acetyl- CoA:ACP transacylase enzyme from Spinacia oleracea accepts other acyl-ACP molecules as substrates, including butyryl-ACP (Shimakata et al, Methods Enzym 122:53-9 (1986)). Malonyl-CoA:ACP transacylase enzymes include FabD of E. coli and Brassica napsus (Verwoert et al, J Bacteriol, 174:2851-7 (1992); Simon et al, FEBS Lett 435:204-6 (1998)). FabD of B. napsus was able to complement ^a^D-deficient E. coli. The multifunctional eukaryotic fatty acid synthase enzyme complexes (described herein) also catalyze this activity. Other exemplary acyltransferases include diacylglycerol acyltransferases such as LROl and DGA1 of S. cerevisiae and DGA1 and DGA2 of Yarrowia lipolytica, glycerolipid acyltransferase enzymes such as plsB of E. coli (GenBank: AAC77011.2, GL87082362; Heath and Rock, J Bacteriol 180: 1425-30 (1998)), sterol acyltransferases such as AREl and ARE2 of S. cerevisiae, ethanol acyltransferases (EEB1, EHT1), putative acyltransferases (YMR210W) and others.

Increasing production of fatty alcohols, fatty aldehydes or fatty acids may necessitate disruption or attenuation of enzymes involved in the trafficking of acetyl-CoA and acyl-CoA molecules from the cytosol to other compartments of the organism such as mitochondria, endoplasmic reticulum, proteoliposomes and peroxisomes. In these compartments, the acyl- CoA intermediate can be degraded or used as building blocks to synthesize fatty acids, cofactors and other byproducts. Acetyl-CoA and acyl-CoA molecules localized in the cytosol can be transported into other cellular compartments with the aid of the carrier molecule carnitine via carnitine shuttles (van Roermund et al., EMBO J 14:3480-86 (1995)). Acyl-carnitine shuttles between cellular compartments have been characterized in yeasts such as Candida albicans (Strijbis et al, J Biol Chem 285:24335-46 (2010)). In these shuttles, the acyl moiety of acyl-CoA is reversibly transferred to carnitine by acylcarnitine transferase enzymes. Acetylcarnitine can then be transported across the membrane by organelle-specific acylcarnitine/carnitine translocase enzymes. After translocation, the acyl-CoA is regenerated by acetylcarnitine transferase. Enzymes suitable for disruption or attenuation include carnitine acyltransferase enzymes, acylcarnitine translocases, acylcarnitine carrier proteins and enzymes involved in carnitine biosynthesis.

Carnitine acetyltransferase (CAT, EC 2.3.1.7) reversibly links acetyl units from acetyl-CoA to the carrier molecule, carnitine. Candida albicans encodes three CAT isozymes: Cat2, Yatl and Yat2 (Strijbis et al, J Biol Chem 285:24335-46 (2010)). Cat2 is expressed in both the mitochondrion and the peroxisomes, whereas Yatl and Yat2 are cytosolic. The Cat2 transcript contains two start codons that are regulated under different carbon source conditions. The longer transcript contains a mitochondrial targeting sequence whereas the shorter transcript is targeted to peroxisomes. Cat2 of Saccharomyces cerevisiae and AcuJ of Aspergillus nidulans employ similar mechanisms of dual localization (Elgersma et al, EMBO J 14:3472-9 (1995); Hynes et al, Euk Cell 10:547-55 (2011)). The cytosolic CAT of A. nidulans is encoded by facC. Other exemplary CAT enzymes are found in Rattus norvegicus and Homo sapiens (Cordente et al, Biochem 45:6133-41 (2006)). Exemplary carnitine acyltransferase enzymes (EC 2.3.1.21) are the Cptl and Cpt2 gene products of Rattus norvegicus (de Vries et al, Biochem 36:5285-92 (1997)).

Protein Accession # GI number Organism

Cat2 AAN31660.1 23394954 Candida albicans

Yatl AAN31659.1 23394952 Candida albicans

Yat2 XP 711005.1 68490355 Candida albicans

Cat2 CAA88327.1 683665 Saccharomyces cerevisiae

Yatl AAC09495.1 456138 Saccharomyces cerevisiae

Yat2 NP 010941.1 6320862 Saccharomyces cerevisiae

AcuJ CBF69795.1 259479509 Aspergillus nidulans

FacC AAC82487.1 2511761 Aspergillus nidulans

Crat AAH83616.1 53733439 Rattus norvegicus Crat P43155.5 215274265 Homo sapiens

Cptl AAB48046.1 1850590 Rattus norvegicus

Cpt2 AAB02339.1 1374784 Rattus norvegicus

Carnitine-acylcarnitine translocases can catalyze the bidirectional transport of carnitine and carnitine-fatty acid complexes. The Cact gene product provides a mechanism for transporting acyl-carnitine substrates across the mitochondrial membrane (Ramsay et al Biochim Biophys Acta 1546:21-42 (2001)). A similar protein has been studied in humans (Sekoguchi et al, J Biol Chem 278:38796-38802 (2003)). The Saccharomyces cerevisiae mitochondrial carnitine carrier is Crcl (van Roermund et al., supra; Palmieri et al.,

Biochimica et Biophys Acta 1757: 1249-62 (2006)). The human carnitine translocase was able to complement a Crcl -deficient strain of S. cerevisiae (van Roermund et al, supra). Two additional carnitine translocases found in Drosophila melanogaster and Caenorhabditis elegans were also able to complement Crcl -deficient yeast (Oey et al., Mol Genet Metab 85: 121-24 (2005)). Four mitochondrial carnitine/acetylcarnitine carriers were identified in Trypanosoma brucei based on sequence homology to the yeast and human transporters (Colasante et al., Mol Biochem Parasit 167: 104-117 (2009)). The carnitine transporter of Candida albicans was also identified by sequence homology. An additional mitochondrial carnitine transporter is the acuH gene product of Aspergillus nidulans, which is exclusively localized to the mitochondrial membrane (Lucas et al., FEMS Microbiol Lett 201 : 193-8 (2006)).

Transport of carnitine and acylcarnitine across the peroxisomal membrane has not been well-characterized. Specific peroxisomal acylcarnitine carrier proteins in yeasts have not been identified to date. However, mitochonidrial carnitine translocases can also function in the peroxisomal transport of carnitine and acetylcarnitine. Experimental evidence suggests that the OCTN3 protein of Mus musculus is a peroxisomal carnitine/acylcarnitine translocase.

Yet another possibility is that acyl-CoA or acyl-carnitine are transported across the peroxisomal or mitochondrial membranes by an acyl-CoA transporter such as the Pxal and Pxa2 ABC transporter of Saccharomyces cerevisiae or the ALDP ABC transporter of Homo sapiens (van Roermund et al, FASEB J 22:4201-8 (2008)). Pxal and Pxa2 (Patl and Pat2) form a heterodimeric complex in the peroxisomal membrane and catalyze the ATP-dependent transport of fatty acyl-CoA esters into the peroxisome (Verleur et al., Eur J Biochem 249: 657-61 (1997)). The mutant phenotype of a pxal/pxa2 deficient yeast can be rescued by heterologous expression of ALDP, which was shown to transport a range of acyl-CoA substrates (van Roermund et al., FASEB J 22:4201-8 (2008)). Deletion of the Pxal2 transport system, in tandem with deletion of the peroxisomal fatty acyl-CoA synthetase (Faa2) abolished peroxisomal beta-oxidation in S. cerevisiae. Yet another strategy for reducing transport of pathway intermediates or products into the peroxisome is to attenuate or eliminate peroxisomal function, by interfering with systems involved in peroxisomal biogenesis. An exemplary target is PexlO of Yarrowia lipolytica and homo logs.

Carnitine biosynthetic pathway enzymes are also suitable candidates for disruption or attenuation. In Candida albicans, for example, carnitine is synthesized from trimethyl-L- lysine in four enzymatic steps (Strijbis et al., FASEB J 23:2349-59 (2009)). The carnitine pathway precursor, trimethyllysine (TML), is produced during protein degradation. TML dioxygenase (Ca013.4316) hydroxylates TML to form 3-hydroxy-6-N-trimethyllysine. A pyridoxal-5'-phoshpate dependent aldolase (CaO19.6305) then cleaves HTML into 4- trimethylaminobutyraldehyde. The 4-trimethylaminobutyraldehyde is subsequently oxidized to 4-trimethylaminobutyrate by a dehydrogenase (CaO 19.6306). In the final step, 4- trimethylaminobutyrate is hydroxylated to form carnitine by the gene product of Ca019.7131. Flux through the carnitine biosynthesis pathway is limited by the availability of the pathway substrate and very low levels of carnitine seem to be sufficient for normal carnitine shuttle activity (Strejbis et al, IUBMB Life 62:357-62 (2010)).

Carbon flux towards production of fatty alcohols, fatty aldehydes or fatty acids can be improved by deleting or attenuating competing pathways. Typical fermentation products of yeast include ethanol, glycerol and C0 2 . The elimination or reduction of these byproducts can be accomplished by approaches described herein. For example, carbon loss due to respiration can be reduced. Other potential byproducts include lactate, acetate, formate, fatty acids and amino acids.

The conversion of acetyl-CoA into ethanol can be detrimental to the production of fatty alcohols, fatty aldehyes or fatty acids because the conversion process can draw away both carbon and reducing equivalents from the MI-FAE cycle, MD-FAE cycle and termination pathway. Ethanol can be formed from pyruvate in two enzymatic steps catalyzed by pyruvate decarboxylase and ethanol dehydrogenase. Saccharomyces cerevisiae has three pyruvate decarboxylases (PDC1, PDC5 and PDC6). PDC1 is the major isozyme and is strongly expressed in actively fermenting cells. PDC5 also functions during glycolytic fermentation, but is expressed only in the absence of PDC1 or under thiamine limitating conditions. PDC6 functions during growth on nonfermentable carbon sources. Deleting PDC1 and PDC5 can reduce ethanol production significantly; however these deletions can lead to mutants with increased PDC6 expression. Deletion of all three eliminates ethanol formation completely but also can cause a growth defect because of inability of the cells to form sufficient acetyl-CoA for biomass formation. This, however, can be overcome by evolving cells in the presence of reducing amounts of C2 carbon source (ethanol or acetate) (van Maris et al, AEM 69:2094-9 (2003)). It has also been reported that deletion of the positive regulator PDC2 of pyruvate decarboxylases PDC1 and PDC5, reduced ethanol formation to -10% of that made by wild-type (Hohmann et al, Mol Gen Genet 241 : 657-66 (1993)). Protein sequences and identifiers of PDC enzymes are listed in Example II. Alternatively, alcohol dehydrogenases that convert acetaldehyde into ethanol and/or other short chain alcohol dehydrogenases can be disrupted or attenuated to provide carbon and reducing equivalents for the MI-FAE cycle, MD-FAE or termination pathway. To date, seven alcohol dehydrogenases, ADHI-ADHVII, have been reported in S. cerevisiae (de Smidt et al, FEMS Yeast Res 8:967-78 (2008)). ADHl (GI: 1419926) is the key enzyme responsible for reducing acetaldehyde to ethanol in the cytosol under anaerobic conditions. It has been reported that a yeast strain deficient in ADHl cannot grow anaerobically because an active respiratory chain is the only alternative path to regenerate NADH and lead to a net gain of ATP (Drewke et al, J Bacteriol 172:3909-17 (1990)). This enzyme is an ideal candidate for downregulation to limit ethanol production. ADHl is severely repressed in the presence of glucose. In K. lactis, two NAD-dependent cytosolic alcohol dehydrogenases have been identified and characterized. These genes also show activity for other aliphatic alcohols. The genes ADHl (GI: 113358) and ADHII (GI:51704293) are preferentially expressed in glucose- grown cells (Bozzi et al, Biochim Biophys Acta 1339: 133-142 (1997)). Cytosolic alcohol dehydrogenases are encoded by ADHl (GL608690) in C albicans, ADHl (GL3810864) in S pombe, ADHl (GL5802617) in Y. lipolytics ADHl (GI:2114038) and ADHII

(GI:2143328)in Pichia stipitis or Scheffersomyces stipitis (Passoth et al, Yeast 14: 1311-23 (1998)). Candidate alcohol dehydrogenases are shown the table below.

Protein ( ,cn Bank ID GI number Organism

SADH BAA24528.1 2815409 Candida parapsilosis

ADHl NP 014555.1 6324486 Saccharomyces cerevisiae s288c

ADH2 NP 014032.1 6323961 Saccharomyces cerevisiae s288c

ADH3 NP 013800.1 6323729 Saccharomyces cerevisiae s288c

ADH4 NP 011258.2 269970305 Saccharomyces cerevisiae s288c

ADH5 (SFA1) NP 010113.1 6320033 Saccharomyces cerevisiae s288c

ADH6 NP 014051.1 6323980 Saccharomyces cerevisiae s288c

ADH7 NP 010030.1 6319949 Saccharomyces cerevisiae s288c adhP CAA44614.1 2810 Kluyveromyces lactis

ADHl P20369.1 113358 Kluyveromyces lactis

ADH2 CAA45739.1 2833 Kluyveromyces lactis

ADH3 P49384.2 51704294 Kluyveromyces lactis

ADHl CAA57342.1 608690 Candida albicans

ADH2 CAA21988.1 3859714 Candida albicans

SAD XP 712899.1 68486457 Candida albicans

ADHl CAA21782.1 3810864 Schizosaccharomyces pombe

ADHl AAD51737.1 5802617 Yarrowia lipolytica

ADH2 AAD51738.1 5802619 Yarrowia lipolytica

ADH3 AAD51739.1 5802621 Yarrowia lipolytica

AlcB AAX53105.1 61696864 Aspergillus niger ANI 1 282024 XP 001399347.1 145231748 Aspergillus niger

ANI 1 126164 XP 001398574.2 317037131 Aspergillus niger

ANI 1 1756104 XP 001395505.2 317033815 Aspergillus niger

ADH2 CAA73827.1 2143328 Scheffersomyces stipitis

Attenuation or disruption of one or more glycerol-3-phosphatase or glycerol-3- phosphate (G3P) dehydrogenase enzymes can eliminate or reduce the formation of glycerol, and thereby conserving carbon and reducing equivalents for production of fatty alcohols, fatty aldehydes or fatty acids.

G3P phosphatase catalyzes the hydrolysis of G3P to glycerol. Enzymes with this activity include the glycerol- 1 -phosphatase (EC 3.1.3.21) enzymes of Saccharomyces cerevisiae (GPP1 and GPP2), Candida albicans and Dunaleilla parva (Popp et al, Biotechnol Bioeng 100:497-505 (2008); Fan et al, FEMS Microbiol Lett 245 : 107- 16 (2005)). The D. parva gene has not been identified to date. These and additional G3P phosphatase enzymes are shown in the table below.

S. cerevisiae has three G3P dehydrogenase enzymes encoded by GPDl and GDP2 in the cytosol and GUT2 in the mitochondrion. GPD2 is known to encode the enzyme responsible for the majority of the glycerol formation and is responsible for maintaining the redox balance under anaerobic conditions. GPDl is primarily responsible for adaptation of S. cerevisiae to osmotic stress (Bakker et al, FEMS Microbiol Rev 24: 15-37 (2001)).

Attenuation of GPDl, GPD2 and/or GUT2 will reduce glycerol formation. GPDl and GUT2 encode G3P dehydrogenases in Yarrowia lipolytica (Beopoulos et al, AEM 4:7779-89 (2008)). GPDl and GPD2 encode for G3P dehydrogenases in S pombe. Similarly, G3P dehydrogenase is encoded by CTRG 02011 in Candida tropicalis and a gene represented by

GL20522022 in Candida albicans. Protein ( ,cn Bank ID GI number Organism

GPD1 CAA98582.1 1430995 Saccharomyces cerevisiae

GPD2 NP 014582.1 6324513 Saccharomyces cerevisiae

GUT2 NP 012111.1 6322036 Saccharomyces cerevisiae

GPD1 CAA22119.1 6066826 Yarrowia lipolytica

GUT2 CAG83113.1 49646728 Yarrowia lipolytica

GPD1 CAA22119.1 3873542 Schizosaccharomyces pombe

GPD2 CAA91239.1 1039342 Schizosaccharomyces pombe

ANI 1 786014 XP 001389035.2 317025419 Aspergillus niger

ANI 1 1768134 XP 001397265.1 145251503 Aspergillus niger

KLLA0C04004g XP 452375.1 50304839 Kluyveromyces lactis

CTRG 02011 XP 002547704.1 255725550 Candida tropicalis

GPD1 XP 714362.1 68483412 Candida albicans

GPD2 XP 713824.1 68484586 Candida albicans

Enzymes that form acid byproducts such as acetate, formate and lactate can also be attenuated or disrupted. Such enzymes include acetate kinase, phosphotransacetylase and pyruvate oxidase. Disruption or attenuation of pyruvate formate lyase and formate dehydrogenase could limit formation of formate and carbon dioxide. These enzymes are described in further detail in Example II.

Alcohol dehydrogenases that convert pyruvate to lactate are also candidates for disruption or attenuation. Lactate dehydrogenase enzymes include IdhA of E. coli and Idh from Ralstonia eutropha (Steinbuchel and Schlegel, Eur. J. Biochem. 130:329-334 (1983)). Other alcohol dehydrogenases listed above may also exhibit LDH activity.

Tuning down activity of the mitochondrial pyruvate dehydrogenase complex will limit flux into the mitochondrial TCA cycle. Under anaerobic conditions and in conditions where glucose concentrations are high in the medium, the capacity of this mitochondrial enzyme is very limited and there is no significant flux through it. However, in some embodiments, this enzyme can be disrupted or attenuated to increase fatty alcohol, fatty aldehyde or fatty acid production. Exemplary pyruvate dehydrogenase genes include PDB1, PDA1, LAT1 and LPD1. Accession numbers and homo logs are listed in Example II. Another strategy for reducing flux into the TCA cycle is to limit transport of pyruvate into the mitochondria by tuning down or deleting the mitochondrial pyruvate carrier.

Transport of pyruvate into the mitochondria in S. cerevisiae is catalyzed by a heterocomplex encoded by MPCl and MPC2 (Herzig et al, Science 337:93-6 (2012); Bricker et al, Science 337:96-100 (2012)). S. cerevisiae encodes five other putative monocarboxylate transporters (MCHl-5), several of which may be localized to the mitochondrial membrane (Makuc et al, Yeast 18: 1131-43 (2001)). NDTl is another putative pyruvate transporter, although the role of this protein is disputed in the literature (Todisco et al, J Biol Chem 20: 1524-31 (2006)). Exemplary pyruvate and monocarboxylate transporters are shown in the table below:

Disruption or attenuation of enzymes that synthesize malonyl-CoA and fatty acids can increase the supply of carbon available for fatty alcohol, fatty aldehyde or fatty acid biosynthesis from acetyl-CoA. Exemplary enzymes for disruption or attenuation include fatty acid synthase, acetyl-CoA carboxylase, biotin:apoenzyme ligase, acyl carrier protein, thioesterase, acyltransferases, ACP malonyltransferase, fatty acid elongase, acyl-CoA synthetase, acyl-CoA transferase and acyl-CoA hydrolase.

Another strategy to reduce fatty acid biosynthesis is expression or overexpression of regulatory proteins which repress fatty acid forming genes. Acetyl-CoA carboxylase (EC 6.4.1.2) catalyzes the first step of fatty acid biosynthesis in many organisms: the ATP- dependent carboxylation of acetyl-CoA to malonyl-CoA. This enzyme utilizes biotin as a cofactor. Exemplary ACC enzymes are encoded by accABCD of E. coli (Davis et al, J Biol Chem 275:28593-8 (2000)), ACC1 of Saccharomyces cerevisiae and homologs (Sumper et al, Methods Enzym 71 :34-7 (1981)). The mitochondrial acetyl-CoA carboxylase of S. cerevisiae is encoded by HFA1. Acetyl-CoA carboxylase holoenzyme formation requires attachment of biotin by a biotin: apoprotein ligase such as BPL1 of S. cerevisiae.

Proteins participating in the synthesis of fatty acids are shown below. The fatty acid synthase enzyme complex of yeast is composed of two multifunctional subunits, FAS1 and FAS2, which together catalyze the net conversion of acetyl-CoA and malonyl-CoA to fatty acids (Lomakin et al, Cell 129: 319-32 (2007)). Additional proteins associated with mitochondrial fatty acid synthesis include OAR1, Mctl, ETR1, ACPI and PPT2. ACPI is the mitochondrial acyl carrier protein and PPT2 encodes a phosphopantetheine transferase, which pantetheinylates mitochondrial ACP and is required for fatty acid biosynthesis in the mitochondria (Stuible et al, J Biol Chem: 273: 22334-9 (1998)). A non-genetic strategy for reducing activity of fatty acid synthases is to add an inhibitor such as cerulenin. Global regulators of lipid biosynthesis can also be altered to tune down endogenous fatty acid biosynthesis pathways during production of long chain alcohols or related products. An exemplary global regulator is SNF1 of Yarrowia lipolytica and Saccharomyces cerevisiae.

Protein ( ,cn Bank ID GI Number Organism

FAS1 NP 012739.1 6322666 Saccharomyces cerevisiae

FAS2 NP 015093.1 6325025 Saccharomyces cerevisiae

FAS1 XP 451653.1 50303423 Kluyveromyces lactis

FAS2 XP 452914.1 50305907 Kluyveromyces lactis

FAS1 XP 716817.1 68478392 Candida albicans

FAS2 XP 723014.1 68465892 Candida albicans

FAS1 XP 500912.1 50546885 Yarrowia lipolytica

FAS2 XP 501096.1 50547253 Yarrowia lipolytica FAS1 XP 001393490.2 317031809 Aspergillus niger

FAS2 XP 001388458.1 145228299 Aspergillus niger

OAR1 NP 012868.1 6322795 Saccharomyces cerevisiae

MCT1 NP 014864.4 398365823 Saccharomyces cerevisiae

ETR1 NP 009582.1 6319500 Saccharomyces cerevisiae

ACPI NP 012729.1 6322656 Saccharomyces cerevisiae

PPT2 NP 015177.2 37362701 Saccharomyces cerevisiae

SNF1 CAG80498.1 49648180 Yarrowia lipolytica

SNF1 P06782.1 134588 Saccharomyces cerevisiae

Disruption or attenuation of elongase enzymes which convert acyl-CoA substrates to longer-chain length fatty acids can also be used to increase fatty alcohol, fatty aldehyde or fatty acid production. Elongase enzymes are found in compartments such as the

mitochondria, endoplasmic reticulum, proteoliposomes and peroxisomes. For example, some yeast such as S. cerevisiae are able to synthesize long-chain fatty acids of chain length C16 and higher via a mitochondrial elongase which accepts exogenous or endogenous acyl-CoA substrates (Bessoule et al, FEBS Lett 214: 158-162 (1987)). This system requires ATP for activity. The endoplasmic reticulum also has an elongase system for synthesizing very long chain fatty acids (CI 8+) from acyl-CoA substrates of varying lengths (Kohlwein et al, Mol Cell Biol 21 : 109-25 (2001)). Genes involved in this system include TSC13, EL02 and EL03. ELOl catalyzes the elongation of C12 acyl-CoAs to C16-C18 fatty acids.

Native enzymes converting acyl-CoA pathway intermediates to acid byproducts can also reduce fatty alcohol, fatty aldehyde or fatty acid yield. For example, CoA hydrolases, transferases and synthetases can act on acyl-CoA intermediates to form short-, medium- or long chain acids. Disruption or attenuation of endogenous CoA hydrolases, CoA transerases and/or reversible CoA synthetases can be used to increase fatty alcohol, fatty aldehyde or fatty acid yield. Exempalry enzymes are shown in the table below. Protein ( ,cn Bank ID GI number Organism

Tesl NP 012553.1 6322480 Saccharomyces cerevisiae s288c

ACH1 NP 009538.1 6319456 Saccharomyces cerevisiae s288c

EHD3 NP 010321.1 6320241 Saccharomyces cerevisiae s288c

YALI0F14729p XP 505426.1 50556036 Yarrowia lipolytica

YALI0E30965p XP 504613.1 50554409 Yarrowia lipolytica

KLLA0E16523g XP 454694.1 50309373 Kluyveromyces lactis

KLLA0E10561g XP 454427.1 50308845 Kluyveromyces lactis

ACH1 P83773.2 229462795 Candida albicans

CaO19.10681 XP 714720.1 68482646 Candida albicans

ANI 1 318184 XP 001401512.1 145256774 Aspergillus niger

ANI 1 1594124 XP 001401252.2 317035188 Aspergillus niger

tesB NP 414986.1 16128437 Escherichia coli

tesB NP 355686.2 159185364 Agrobacterium tumefaciens atoA 2492994 P76459.1 Escherichia coli

atoD 2492990 P76458.1 Escherichia coli

Enzymes that favor the degradation of products, MI-FAE cycle intermediates, MD- FAE cycle intermeidates or termination pathway intermediates can also be disrupted or attenuated. Examples include aldehyde dehydrogenases, aldehyde decarbonylases, oxidative alcohol dehydrogenases, and irreversible fatty acyl-CoA degrading enzymes.

For production of fatty alcohols, fatty aldehydes or fatty acids of the invention, deletion or attenuation of non-specific aldehyde dehydrogenases can improve yield. For production of fatty acids, expression of such an enzyme may improve product formation. Such enzymes can, for example, convert acetyl-CoA into acetaldehyde, fatty aldehydes to fatty acids, or fatty alcohols to fatty acids. Acylating aldehyde dehydrogenase enzymes are described in Example I. Acid-forming aldehyde dehydrogenase are described in Examples III and IX.

The pathway enzymes that favor the reverse direction can also be disrupted or attenuated, if they are detrimental to fatty alcohol, fatty aldehyde or fatty acid production. An example is long chain alcohol dehydrogenases (EC 1.1.1.192) that favor the oxidative direction. Exemplary long chain alcohol dehydrogenases are ADH1 and ADH2 of

Geobacillus thermodenitrificans, which oxidize alcohols up to a chain length of C30 (Liu et al, Physiol Biochem 155:2078-85 (2009)). These and other exemplary fatty alcohol dehydrogenase enzymes are listed in Examples I and II. If an alcohol-forming acyl-CoA reductase is utilized for fatty alcohol, fatty aldehyde or fatty acid biosynthesis, deletion of endogenous fatty alcohol dehydrogenases will substantially reduce backflux. Beta-oxidation enzymes may be reversible and operate in the direction of acyl-CoA synthesis. However, if they are irreversible or strongly favored in the degradation direction they are candidates for disruption or attenuation. An enzyme that fall into this category includes FOX2 of S. cerevisiae, a multifunctional enzyme with 3-hydroxyacyl-CoA dehydrogenase and enoyl-CoA hydratase activity (Hiltunen et al, J Biol Chem 267: 6646- 6653 (1992)). Additional genes include degradative thiolases such as POTl and acyl-CoA dehydrogenases that utilize cofactors other than NAD(P)H (EG. EC 1.3.8.-) such as fadE of E. coli.

Fatty acyl-CoA oxidase enzymes such as POXl of S. cerevisiae catalyze the oxygen- dependent oxidation of fatty acyl-CoA substrates. Enzymes with this activity can be disrupted or attenuated, if they are expressed under fatty alcohol, fatty aldehyde or fatty acid producing conditions. POXl (EC 1.3.3.6) genes and homologs are shown in the table below. POXl is subject to regulation by OAF1, which also activates genes involved in peroxisomal beta-oxidation, organization and biogenesis (Luo et al, J Biol Chem 271 : 12068-75 (1996)). Regulators with functions similar to OAF1, and peroxisomal fatty acid transporters PXAl and PXA2 are also candidates for deletion.

Protein ( ,cn Bank ID GI Number Organism

POXl NP 011310.1 6321233 Saccharomyces cerevisiae

OAF1 NP 009349.3 330443370 Saccharomyces cerevisiae

PXAl NP 015178.1 6325110 Saccharomyces cerevisiae

PXA2 NP 012733.1 6322660 Saccharomyces cerevisiae

YALI0F10857g XP 505264.1 50555712 Yarrowia lipolytica

YALI0D24750p XP 503244.1 50551539 Yarrowia lipolytica

YALI0E32835p XP 504703.1 50554589 Yarrowia lipolytica

YALI0E06567p XP 503632.1 50552444 Yarrowia lipolytica

YALI0E27654p XP 504475.1 50554133 Yarrowia lipolytica

YALI0C23859p XP 502199.1 50549457 Yarrowia lipolytica

POX XP 455532.1 50311017 Kluyveromyces lactis

POXl 04 XP 721610.1 68468582 Candida albicans

POXl 05 XP 717995.1 68475844 Candida albicans

POXl 02 XP 721613.1 68468588 Candida albicans Another candidate for disruption or attenuation is an acyl-CoA binding protein. The acyl binding protein ACB 1 of S. cerevisiae, for example, binds acyl-CoA esters and shuttles them to acyl-CoA utilizing processes (Schjerling et al, J Biol Chem 271 : 22514-21 (1996)). Deletion of this protein did not impact growth rate and lead to increased accumulation of longer-chain acyl-CoA molecules. Acyl-CoA esters are involved in diverse cellular processes including lipid biosynthesis and homeostatis, signal transduction, growth regulation and cell differentiation (Rose et al, PNAS USA 89: 11287-11291 (1992)).

To achieve high yields of fatty alcohols, fatty aldehydes or fatty acids, it is desirable that the host organism can supply the co factors required by the MI-FAE cycle, MD-FAE and/or the termination pathway in sufficient quantities. In several organisms, in particular eukaryotic organisms, such as several Saccharomyces, Kluyveromyces, Candida, Aspergillus, and Yarrowia species, NADH is more abundant than NADPH in the cytosol as it is produced in large quantities by glycolysis. NADH can be made even more abundant by converting pyruvate to acetyl-CoA by means of heterologous or native NAD-dependant enzymes such as NAD-dependant pyruvate dehydrogenase, NAD-dependant formate dehydrogenase,

NADH:ferredoxin oxidoreductase, or NAD-dependant acylating acetylaldehyde

dehydrogenase in the cytosol. Given the abundance of NADH in the cytosol of most organisms, it can be beneficial for all reduction steps of the MI-FAE cycle, MD-FAE cycle and/or terminatio pathway to accept NADH as the reducing agent preferentially over other reducing agents such as NADPH. High yields of fatty alcohols, fatty aldehydes or fatty acids can thus be accomplished by, for example: 1) identifying and implementing endogenous or exogenous MI-FAE cycle, MD-FAE cycle and/or termination pathway enzymes with a stronger preference for NADH than other reducing equivalents such as NADPH; 2) attenuating one or more endogenous MI-FAE cycle, MD-FAE cycle or termination pathway enzymes that contribute NADPH-dependant reduction activity; 3) altering the cofactor specificity of endogenous or exogenous MI-FAE cycle, MD-FAE cycle or termination pathway enzymes so that they have a stronger preference for NADH than their natural versions; or 4) altering the cofactor specificity of endogenous or exogenous MI-FAE cycle, MD-FAE cycle or termination pathway enzymes so that they have a weaker preference for NADPH than their natural versions.

Strategies for engineering NADH-favoring MI-FAE cycle, MD-FAE cycle and/or termination pathways are described in further detail in Example V. Methods for changing the cofactor specificity of an enzyme are well known in the art, and an example is described in Example VI.

If one or more of the MI-FAE cycle, MD-FAE cycle and/or termination pathway enzymes utilizes NADPH as the cofactor, it can be beneficial to increase the production of NADPH in the host organism. In particular, if the MI-FAE cycle, MD-FAE cycle and/or termination pathway is present in the cytosol of the host organism, methods for increasing NADPH production in the cytosol can be beneficial. Several approaches for increasing cytosolic production of NADPH can be implemented including channeling an increased amount of flux through the oxidative branch of the pentose phosphate pathway relative to wild-type, channeling an increased amount of flux through the Entner Doudoroff pathway relative to wild-type, introducing a soluble or membrane -bound transhydrogenase to convert NADH to NADPH, or employing NADP-dependant versions of the following enzymes:

phosphorylating or non-phosphorylating glyceraldehyde-3 -phosphate dehydrogenase, pyruvate dehydrogenase, formate dehydrogenase, or acylating acetylaldehyde dehydrogenase. These activities can be augmented by disrupting or attenuating native NAD-dependant enzymes including glyceraldehyde-3 -phosphate dehydrogenase, pyruvate dehydrogenase, formate dehydrogenase, or acylating acetylaldehyde dehydrogenase. Strategies for

engineering increased NADPH availability are described in Example VII.

Synthesis of fatty alcohols, fatty aldehyes or fattyc acids in the cytosol can be dependent upon the availability of sufficient carbon and reducing equivalents. Therefore, without being bound to any particular theory of operation, increasing the redox ratio of

NAD(P)H to NAD(P) can help drive the MI-FAE cycle, MD-FAE cycle and/or termination pathway in the forward direction. Methods for increasing the redox ratio of NAD(P)H to NAD(P) include limiting respiration, attenuating or disrupting competing pathways that produce reduced byproducts such as ethanol and glycerol, attenuating or eliminating the use of NADH by NADH dehydrogenases, and attenuating or eliminating redox shuttles between compartments. One exemplary method to provide an increased number of reducing equivalents, such as NAD(P)H, for enabling the formation of fatty alcohols, fatty aldehydes or fatty acids is to constrain the use of such reducing equivalents during respiration. Respiration can be limited by: reducing the availability of oxygen, attenuating NADH dehydrogenases and/or cytochrome oxidase activity, attenuating G3P dehydrogenase, and/or providing excess glucose to Crabtree positive organisms.

Restricting oxygen availability by culturing the non-naturally occurring eukaryotic organisms in a fermenter is one exmaple for limiting respiration and thereby increasing the ratio of NAD(P)H to NAD(P). The ratio of NAD(P)H/NAD(P) increases as culture conditions become more anaerobic, with completely anaerobic conditions providing the highest ratios of the reduced cofactors to the oxidized ones. For example, it has been reported that the ratio of NADH/NAD = 0.02 in aerobic conditions and 0.75 in anaerobic conditions in E. coli (de Graes et al, J Bacteriol 181 :2351-57 (1999)).

Respiration can also be limited by reducing expression or activity of NADH dehydrogenases and/or cytochrome oxidases in the cell under aerobic conditions. In this case, respiration can be limited by the capacity of the electron transport chain. Such an approach has been used to enable anaerobic metabolism of E. coli under completely aerobic conditions (Portnoy et al, AEM 74:7561-9 (2008)). S. cerevisiae can oxidize cytosolic NADH directly using external NADH dehydrogenases, encoded by NDE1 and NDE2. One such NADH dehydrogenase in Yarrowia lipolytica is encoded by NDH2 (Kerscher et al, J Cell Sci 112:2347-54 (1999)). These and other NADH dehydrogenase enzymes are listed in the table below.

Protein ( ,cn Bank ID GI number Organism

NDE1 NP 013865.1 6323794 Saccharomyces cerevisiae s288c

NDE2 NP 010198.1 6320118 Saccharomyces cerevisiae s288c

NDH2 AJ006852.1 3718004 Yarrowia lipolytica

ANI 1 610074 XP 001392541.2 317030427 Aspergillus niger

ANI 1 2462094 XP 001394893.2 317033119 Aspergillus niger

KLLA0E21891g XP 454942.1 50309857 Kluyveromyces lactis

KLLA0C06336g XP 452480.1 50305045 Kluyveromyces lactis

NDE1 XP 720034.1 68471982 Candida albicans

NDE2 XP 717986.1 68475826 Candida albicans Cytochrome oxidases of Saccharomyces cerevisiae include the COX gene products. COX 1-3 are the three core subunits encoded by the mitochondrial genome, whereas COX4- 13 are encoded by nuclear genes. Attenuation or disruption of any of the cytochrome genes results in a decrease or block in respiratory growth (Hermann and Funes, Gene 354:43-52 (2005)). Cytochrome oxidase genes in other organisms can be inferred by sequence homology.

Cytosolic NADH can also be oxidized by the respiratory chain via the G3P dehydrogenase shuttle, consisting of cytosolic NADH-linked G3P dehydrogenase and a membrane-bound G3P:ubiquinone oxidoreductase. The deletion or attenuation of G3P dehydrogenase enzymes will also prevent the oxidation of NADH for respiration. Enzyme candidates encoding these enzymes are described herein.

Additionally, in Crabtree positive organisms, fermentative metabolism can be achieved in the presence of excess of glucose. For example, S. cerevisiae makes ethanol even under aerobic conditions. The formation of ethanol and glycerol can be reduced/eliminated and replaced by the production of fatty alcohol, fatty aldehyde or fatty acid in a Crabtree positive organism by feeding excess glucose to the Crabtree positive organism. In another aspect, provided herein is a method for producing fatty alcohols, fatty aldehydes or fatty acids, comprising culturing a non-naturally occurring eukaryotic organism under conditions and for a sufficient period of time to produce fatty alcohol, fatty aldehyde or fatty acid, wherein the eukaryotic organism is a Crabtree positive organism that comprises at least one exogenous nucleic acid encoding a MI-FAE cycle, MD-FAE cycle and/or termination pathway enzyme and wherein eukaryotic organism is in a culture medium comprising excess glucose.

Preventing formation of reduced fermentation byproducts will increase the availability of both carbon and reducing equivalents for fatty alcohol, fatty aldehyde or fatty acid production. The two key reduced byproducts under anaerobic and microaerobic conditions are ethanol and glycerol. Ethanol is typically formed from pyruvate in two enzymatic steps catalyzed by pyruvate decarboxylase and ethanol dehydrogenase. Glycerol is formed from the glycolytic intermediate dihydroxyacetone phosphate by the enzymes glycerol-3-phsophate dehydrogenase and glycero 1-3 -phosphate phosphatase. Attenuation of one or more of these enzyme activities will increase the yield of fatty alcohols, fatty aldehydes or fatty acids. Strain engineering strategies for reducing or eliminating ethanol and glycerol formation are described herein.

Yeast such as S. cerevisiae can produce glycerol to allow for regeneration of NAD(P) under anaerobic conditions. Another way to reduce or eliminate glycerol production is by oxygen- limited cultivation (Bakker et al, supra). Glycerol formation only sets in when the specific oxygen uptake rates of the cells decrease below the rate that is required to reoxidize the NADH formed in biosynthesis.

In addition to the redox sinks listed above, malate dehydrogenase can potentially draw away reducing equivalents when it functions in the reductive direction. Several redox shuttles believed to be functional in S. cerevisiae utilize this enzyme to transfer reducing equivalents between the cytosol and the mitochondria. This transfer of redox can be prevented by attenuating malate dehydrogenase and/or malic enzyme activity. The redox shuttles that can be blocked by the attenuation of mdh include (i) malate-asparate shuttle, (ii) malate-oxaloacetate shuttle, and (iii) malate-pyruvate shuttle. Genes encoding malate dehydrogenase and malic enzymes are listed in the table below.

Protein ( ,cn Bank ID GI Number Organism

MDH1 NP 012838.1 6322765 Saccharomyces cerevisiae

MDH2 NP 014515.2 116006499 Saccharomyces cerevisiae

MDH3 NP 010205.1 6320125 Saccharomyces cerevisiae

MAE1 NP 012896.1 6322823 Saccharomyces cerevisiae

MDH1 XP 722674.1 68466384 Candida albicans

MDH2 XP 718638.1 68474530 Candida albicans

MAE1 XP 716669.1 68478574 Candida albicans

KLLA0F25960g XP 456236.1 50312405 Kluyveromyces lactis KLLA0E18635g XP 454793.1 50309563 Kluyveromyces lactis

KLLA0E07525g XP 454288.1 50308571 Kluyveromyces lactis

YALI0D16753p XP 502909.1 50550873 Yarrowia lipolytica

YALI0E18634p XP 504112.1 50553402 Yarrowia lipolytica

ANI 1 268064 XP 001391302.1 145237310 Aspergillus niger

ANI 1 12134 XP 001396546.1 145250065 Aspergillus niger

ANI 1 22104 XP 001395105.2 317033225 Aspergillus niger

Overall, disruption or attenuation of the aforementioned sinks for redox either individually or in combination with the other redox sinks can eliminate or lower the use of reducing power for respiration or byproduct formation. It has been reported that the deletion of the external NADH dehydrogenases (NDE1 and NDE2) and the mitochondrial G3P dehydrogenase (GUT2) almost completely eliminates cytosolic NAD+ regeneration in S. cerevisiae (Overkamp et al, J Bacteriol 182:2823-30 (2000)).

Microorganisms of the invention produce fatty alcohols, fatty aldehydes or fatty acids and optionally secrete the fatty alcohols, fatty aldehydes or fatty acis into the culture medium. S. cerevisiae, Yarrowia lipolytica and E. coli harboring heterologous fatty alcohol forming activities accululated fatty alcohols intracellularly; however fatty alcohols were not detected in the culture medium (Behrouzian et al, United States Patent Application 20100298612). The introduction of fatty acyl-CoA reductase enzymes with improved activity resulted in higher levels of fatty alcohol secreted into the culture media. Alternately, introduction of a fatty alcohol, fatty aldehyde or fatty acid transporter or transport system can improve extracellular accumulation of fatty alcohols, fatty aldehydes or fatty acids. Exemplary transporters are listed in the table below.

Protein ( ,cn Bank ID GI Organism

Number

Fatp NP 524723.2 24583463 Drosophila melanogaster

AY161280.1 :45..1757 AAN73268.1 34776949 Rhodococcus erythropolis acrA CAF23274.1 46399825 Candidatus Protochlamydia

amoebophila

acrB CAF23275.1 46399826 Candidatus Protochlamydia

amoebophila

CER5 AY734542.1 52354013 Arabidopsis thaliana

AmiS2 JC5491 7449112 Rhodococcus sp.

ANI 1 1160064 XP 001391993.1 145238692 Aspergillus niger

YALI0E16016g XP 504004.1 50553188 Yarrowia lipolytica Thus, in some embodiments, the invention provides a non-naturally occurring microbial organism as disclosed herein having one or more gene disruptions, wherein the one or more gene disruptions occurr in endogenous genes encoding proteins or enzymes involved in: native production of ethanol, glycerol, acetate, formate, lactate, C0 2 , fatty acids, or malonyl-CoA by said microbial organism; transfer of pathway intermediates to cellular compartments other than the cytosol; or native degradation of a MI-FAE cycle intermediate, a MD-FAE cycle intermediate or a termination pathway intermediate by the microbial organism, the one or more gene disruptions confer increased production of a fatty alcohol, fatty aldehyde or fatty acid in the microbial organism. Accordingly, the protein or enzyme can be a fatty acid synthase, an acetyl-CoA carboxylase, a biotin:apoenzyme ligase, an acyl carrier protein, a thioesterase, an acyltransferase, an ACP malonyltransferase, a fatty acid elongase, an acyl-CoA synthetase, an acyl-CoA transferase, an acyl-CoA hydrolase, a pyruvate decarboxylase, a lactate dehydrogenase, an alcohol dehydrogenase, an acid-forming aldehyde dehydrogenases, an acetate kinase, a phosphotransacetylase, a pyruvate oxidase, a glycerol-3 -phosphate dehydrogenase, a glycerol-3 -phosphate phosphatase, a mitochondrial pyruvate carrier, a peroxisomal fatty acid transporter, a peroxisomal acyl-CoA transporter, a peroxisomal carnitine/acylcarnitine transferase, an acyl-CoA oxidase, or an acyl-CoA binding protein. In some aspects, the one or more gene disruptions include a deletion of the one or more genes. In some embodiments, the invention provides a non-naturally occurring microbial organism as described herein, wherein one or more enzymes of the MI-FAE cycle, the MD- FAE cycle or the termination pathway preferentially react with an NADH cofactor or have reduced preference for reacting with an NAD(P)H cofactor. For example, the one or more enzymes of the MI-FAE cycle can be a 3-ketoacyl-CoA reductase or an enoyl-CoA reductase. For the termination pathway, the one or more enzymes can be an acyl-CoA reductase

(aldehyde forming), an alcohol dehydrogenase, an acyl-CoA reductase (alcohol forming), an aldehyde decarbonylase, an acyl- ACP reductase, an aldehyde dehydrogenase (acid forming) or a carboxylic acid reductase.

In some embodiments, the invention provides a non-naturally occurring microbial organism as described herein having one or more gene disruptions in genes encoding proteins or enzymes that result in an increased ratio of NAD(P)H to NAD(P) present in the cytosol of the microbial organism following the disruptions. Accordingly, the gene encoding a protein or enzyme that results in an increased ratio of NAD(P)H to NAD(P) present in the cytosol of the microbial organism following the disruptions can be an NADH dehydrogenase, a cytochrome oxidase, a G3P dehydrogenase, G3P phosphatase, an alcohol dehydrogenase, a pyruvate decarboxylase, an aldehyde dehydrogenase (acid forming), a lactate dehydrogenase, a glycerol-3 -phosphate dehydrogenase, a glycerol-3 -phosphate :quinone oxidoreductase, a malic enzyme and a malate dehydrogenase. In some aspects, the one or more gene disruptions include a deletion of the one or more genes.

In some embodiments, the non-naturally occurring microbial organism of the invention is Crabtree positive and is in culture medium comprising excess glucose. In such conditions, as described herein, the microbial organism can result in increasing the ratio of NAD(P)H to NAD(P) present in the cytosol of the microbial organism.

In some embodiments, the invention provides a non-naturally occurring microbial organism as described herein having at least one exogenous nucleic acid encoding an extracellular transporter or an extracellular transport system for a fatty alcohol, fatty aldehyde or fatty acid of the invention.

In some embodiments, the invention provides a non-naturally occurring microbial organism as described herein, wherein one or more endogenous enzymes involved in: native production of ethanol, glycerol, acetate, formate, lactate, C0 2 , fatty acids, or malonyl-CoA by said microbial organism; transfer of pathway intermediates to cellular compartments other than the cytosol; or native degradation of a MI-FAE cycle intermediate, a MD-FAE cycle intermediate or a termination pathway intermediate by said microbial organism, has attenuated enzyme activity or expression levels. Accordingly, the endogenous enzyme can be a fatty acid synthase, an acetyl-CoA carboxylase, a biotin:apoenzyme ligase, an acyl carrier protein, a thioesterase, an acyltransferase, an ACP malonyltransferase, a fatty acid elongase, an acyl-CoA synthetase, an acyl-CoA transferase, an acyl-CoA hydrolase, a pyruvate decarboxylase, a lactate dehydrogenase, an alcohol dehydrogenase, an acid-forming aldehyde dehydrogenases, an acetate kinase, a phosphotransacetylase, a pyruvate oxidase, a glycerol-3 - phosphate dehydrogenase, a glycerol-3 -phosphate phosphatase, a mitochondrial pyruvate carrier, a peroxisomal fatty acid transporter, a peroxisomal acyl-CoA transporter, a peroxisomal carnitine/acylcarnitine transferase, an acyl-CoA oxidase, or an acyl-CoA binding protein. In some embodiments, the invention provides a non-naturally occurring microbial organism as described herein, wherein one or more endogenous enzymes involved in the oxidation of NAD(P)H or NADH, has attenuated enzyme activity or expression levels.

Accordingly, the one or more endogenous enzymes can be a NADH dehydrogenase, a cytochrome oxidase, a G3P dehydrogenase, G3P phosphatase, an alcohol dehydrogenase, a pyruvate decarboxylase, an aldehyde dehydrogenase (acid forming), a lactate dehydrogenase, a glycerol-3 -phosphate dehydrogenase, a glycerol-3 -phosphate :quinone oxidoreductase, a malic enzyme and a malate dehydrogenase.

The non-naturally occurring microbal organisms of the invention can contain stable genetic alterations, which refers to microorganisms that can be cultured for greater than five generations without loss of the alteration. Generally, stable genetic alterations include modifications that persist greater than 10 generations, particularly stable modifications will persist more than about 25 generations, and more particularly, stable genetic modifications will be greater than 50 generations, including indefinitely. In the case of gene disruptions, a particularly useful stable genetic alteration is a gene deletion. The use of a gene deletion to introduce a stable genetic alteration is particularly useful to reduce the likelihood of a reversion to a phenotype prior to the genetic alteration. For example, stable growth-coupled production of a biochemical can be achieved, for example, by deletion of a gene encoding an enzyme catalyzing one or more reactions within a set of metabolic modifications. The stability of growth-coupled production of a biochemical can be further enhanced through multiple deletions, significantly reducing the likelihood of multiple compensatory reversions occurring for each disrupted activity.

Also provided is a method of producing a non-naturally occurring microbial organisms having stable growth-coupled production of fatty alcohol, fatty aldehyde or fatty acid. The method can include identifying in silico a set of metabolic modifications that increase production of fatty alcohol, fatty aldehyde or fatty acid, for example, increase production during exponential growth; genetically modifying an organism to contain the set of metabolic modifications that increase production of fatty alcohol, fatty aldehyde or fatty acid, and culturing the genetically modified organism. If desired, culturing can include adaptively evolving the genetically modified organism under conditions requiring production of fatty alcohol, fatty aldehyde or fatty acid. The methods of the invention are applicable to bacterium, yeast and fungus as well as a variety of other cells and microorganism, as disclosed herein.

Thus, the invention provides a non-naturally occurring microbial organism

comprising one or more gene disruptions that confer increased production of fatty alcohol, fatty aldehyde or fatty acid. In one embodiment, the one or more gene disruptions confer growth-coupled production of fatty alcohol, fatty aldehyde or fatty acid, and can, for example, confer stable growth-coupled production of fatty alcohol, fatty aldehyde or fatty acid. In another embodiment, the one or more gene disruptions can confer obligatory coupling of fatty alcohol, fatty aldehyde or fatty acid production to growth of the microbial organism. Such one or more gene disruptions reduce the activity of the respective one or more encoded enzymes.

The non-naturally occurring microbial organism can have one or more gene disruptions included in a gene encoding a enzyme or protein disclosed herein. As disclosed herein, the one or more gene disruptions can be a deletion. Such non-naturally occurring microbial organisms of the invention include bacteria, yeast, fungus, or any of a variety of other microorganisms applicable to fermentation processes, as disclosed herein.

Thus, the invention provides a non-naturally occurring microbial organism, comprising one or more gene disruptions, where the one or more gene disruptions occur in genes encoding proteins or enzymes where the one or more gene disruptions confer increased production of fatty alcohol, fatty aldehyde or fatty acid in the organism. The production of fatty alcohol, fatty aldehyde or fatty acid can be growth-coupled or not growth-coupled. In a particular embodiment, the production of fatty alcohol, fatty aldehyde or fatty acid can be obligatorily coupled to growth of the organism, as disclosed herein.

The invention provides non naturally occurring microbial organisms having genetic alterations such as gene disruptions that increase production of fatty alcohol, fatty aldehyde or fatty acid, for example, growth-coupled production of fatty alcohol, fatty aldehyde or fatty acid. Product production can be, for example, obligatorily linked to the exponential growth phase of the microorganism by genetically altering the metabolic pathways of the cell, as disclosed herein. The genetic alterations can increase the production of the desired product or even make the desired product an obligatory product during the growth phase. Metabolic alterations or transformations that result in increased production and elevated levels of fatty alcohol, fatty aldehyde or fatty acid biosynthesis are exemplified herein. Each alteration corresponds to the requisite metabolic reaction that should be functionally disrupted.

Functional disruption of all reactions within one or more of the pathwyas can result in the increased production of fatty alcohol, fatty aldehyde or fatty acid by the engineered strain during the growth phase.

Each of these non-naturally occurring alterations result in increased production and an enhanced level of fatty alcohol, fatty aldehyde or fatty acid production, for example, during the exponential growth phase of the microbial organism, compared to a strain that does not contain such metabolic alterations, under appropriate culture conditions. Appropriate conditions include, for example, those disclosed herein, including conditions such as particular carbon sources or reactant availabilities and/or adaptive evolution.

Given the teachings and guidance provided herein, those skilled in the art will understand that to introduce a metabolic alteration such as attenuation of an enzyme, it can be necessary to disrupt the catalytic activity of the one or more enzymes involved in the reaction. Alternatively, a metabolic alteration can include disrupting expression of a regulatory protein or cofactor necessary for enzyme activity or maximal activity.

Furthermore, genetic loss of a cofactor necessary for an enzymatic reaction can also have the same effect as a disruption of the gene encoding the enzyme. Disruption can occur by a variety of methods including, for example, deletion of an encoding gene or incorporation of a genetic alteration in one or more of the encoding gene sequences. The encoding genes targeted for disruption can be one, some, or all of the genes encoding enzymes involved in the catalytic activity. For example, where a single enzyme is involved in a targeted catalytic activity, disruption can occur by a genetic alteration that reduces or eliminates the catalytic activity of the encoded gene product. Similarly, where the single enzyme is multimeric, including heteromeric, disruption can occur by a genetic alteration that reduces or destroys the function of one or all subunits of the encoded gene products. Destruction of activity can be accomplished by loss of the binding activity of one or more subunits required to form an active complex, by destruction of the catalytic subunit of the multimeric complex or by both. Other functions of multimeric protein association and activity also can be targeted in order to disrupt a metabolic reaction of the invention. Such other functions are well known to those skilled in the art. Similarly, a target enzyme activity can be reduced or eliminated by disrupting expression of a protein or enzyme that modifies and/or activates the target enzyme, for example, a molecule required to convert an apoenzyme to a holoenzyme. Further, some or all of the functions of a single polypeptide or multimeric complex can be disrupted according to the invention in order to reduce or abolish the catalytic activity of one or more enzymes involved in a reaction or metabolic modification of the invention. Similarly, some or all of enzymes involved in a reaction or metabolic modification of the invention can be disrupted so long as the targeted reaction is reduced or eliminated.

Given the teachings and guidance provided herein, those skilled in the art also will understand that an enzymatic reaction can be disrupted by reducing or eliminating reactions encoded by a common gene and/or by one or more orthologs of that gene exhibiting similar or substantially the same activity. Reduction of both the common gene and all orthologs can lead to complete abolishment of any catalytic activity of a targeted reaction. However, disruption of either the common gene or one or more orthologs can lead to a reduction in the catalytic activity of the targeted reaction sufficient to promote coupling of growth to product biosynthesis. Exemplified herein are both the common genes encoding catalytic activities for a variety of metabolic modifications as well as their orthologs. Those skilled in the art will understand that disruption of some or all of the genes encoding a enzyme of a targeted metabolic reaction can be practiced in the methods of the invention and incorporated into the non-naturally occurring microbial organisms of the invention in order to achieve the increased production of fatty alcohol, fatty aldehyde or fatty acid or growth-coupled product production.

Given the teachings and guidance provided herein, those skilled in the art also will understand that enzymatic activity or expression can be attenuated using well known methods. Reduction of the activity or amount of an enzyme can mimic complete disruption of a gene if the reduction causes activity of the enzyme to fall below a critical level that is normally required for a pathway to function. Reduction of enzymatic activity by various techniques rather than use of a gene disruption can be important for an organism's viability. Methods of reducing enzymatic activity that result in similar or identical effects of a gene disruption include, but are not limited to: reducing gene transcription or translation;

destabilizing mRNA, protein or catalytic RNA; and mutating a gene that affects enzyme activity or kinetics (See, Sambrook et al, Molecular Cloning: A Laboratory Manual, Third Ed., Cold Spring Harbor Laboratory, New York (2001); and Ausubel et al, Current

Protocols in Molecular Biology, John Wiley and Sons, Baltimore, MD (1999). Natural or imposed regulatory controls can also accomplish enzyme attenuation including: promoter replacement (See, Wang et al, Mol. Biotechnol. 52(2):300-308 (2012)); loss or alteration of transcription factors (Dietrick et al, Annu. Rev. Biochem. 79:563-590 (2010); and Simicevic et al, Mol. Biosyst. 6(3):462-468 (2010)); introduction of inhibitory R As or peptides such as siRNA, antisense RNA, RNA or peptide/small-molecule binding aptamers, ribozymes, aptazymes and riboswitches (Wieland et al, Methods 56(3):351-357 (2012); O'Sullivan, Anal. Bioanal. Chem. 372(l):44-48 (2002); and Lee et al, Curr. Opin. Biotechnol. 14(5):505- 511 (2003)); and addition of drugs or other chemicals that reduce or disrupt enzymatic activity such as an enzyme inhibitor, an antibiotic or a target-specific drug. One skilled in the art will also understand and recognize that attenuation of an enzyme can be done at various levels. For example, at the gene level, a mutation causing a partial or complete null phenotype, such as a gene disruption, or a mutation causing epistatic genetic effects that mask the activity of a gene product (Miko, Nature Education 1(1) (2008)), can be used to attenuate an enzyme. At the gene expression level, methods for attenuation include: coupling transcription to an endogenous or exogenous inducer, such as isopropylthio-β- galactoside (IPTG), then adding low amounts of inducer or no inducer during the production phase (Donovan et al, J. Ind. Microbiol. 16(3): 145-154 (1996); and Hansen et al, Curr. Microbiol. 36(6):341-347 (1998)); introducing or modifying a positive or a negative regulator of a gene; modify histone acetylation/deacetylation in a eukaryotic chromosomal region where a gene is integrated (Yang et al, Curr. Opin. Genet. Dev. 13(2): 143-153 (2003) and Kurdistani et al, Nat. Rev. Mol. Cell Biol. 4(4):276-284 (2003)); introducing a transposition to disrupt a promoter or a regulatory gene (Bleykasten-Brosshans et al, C. R. Biol. 33(8- 9):679-686 (2011); and McCue et al, PLoS Genet. 8(2):el002474 (2012)); flipping the orientation of a transposable element or promoter region so as to modulate gene expression of an adjacent gene (Wang et al, Genetics 120(4):875-885 (1988); Hayes, Annu. Rev. Genet. 37:3-29 (2003); in a diploid organism, deleting one allele resulting in loss of heterozygosity (Daigaku et al., Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis 600(1-2)177-183 (2006)); introducing nucleic acids that increase RNA degradation

(Houseley et al, Cell, 136(4):763-776 (2009); or in bacteria, for example, introduction of a transfer-messenger RNA (tmRNA) tag, which can lead to RNA degradation and ribosomal stalling (Sunohara et al, RNA 10(3):378-386 (2004); and Sunohara et al, J. Biol. Chem. 279: 15368-15375 (2004)). At the translational level, attenuation can include: introducing rare codons to limit translation (Angov, Biotechnol. J. 6(6):650-659 (2011)); introducing R A interference molecules that block translation (Castel et al., Nat. Rev. Genet. 14(2): 100- 112 (2013); and Kawasaki et al, Curr. Opin. Mol. Ther. 7(2): 125-131 (2005); modifying regions outside the coding sequence, such as introducing secondary structure into an untranslated region (UTR) to block translation or reduce efficiency of translation (Ringner et al, PLoS Comput. Biol. I(7):e72 (2005)); adding RNAase sites for rapid transcript degradation (Pasquinelli, Nat. Rev. Genet. 13(4):271-282 (2012); and Arraiano et al., FEMS Microbiol. Rev. 34(5):883-932 (2010); introducing antisense RNA oligomers or antisense transcripts (Nashizawa et al, Front. Biosci. 17:938-958 (2012)); introducing RNA or peptide aptamers, ribozymes, aptazymes, riboswitches (Wieland et al, Methods 56(3):351-357 (2012); O'Sullivan, Anal. Bioanal. Chem. 372(l):44-48 (2002); and Lee et al, Curr. Opin.

Biotechnol. 14(5):505-511 (2003)); or introducing translational regulatory elements involving RNA structure that can prevent or reduce translation that can be controlled by the presence or absence of small molecules (Araujo et al., Comparative and Functional Genomics, Article ID 475731, 8 pages (2012)). At the level of enzyme localization and/or longevity, enzyme attenuation can include: adding a degradation tag for faster protein turnover (Hochstrasser, Annual Rev. Genet. 30:405-439 (1996); and Yuan et al, PLoS One 8(4):e62529 (2013)); or adding a localization tag that results in the enzyme being secreted or localized to a subcellular compartment in a eukaryotic cell, where the enzyme would not be able to react with its normal substrate (Nakai et al. Genomics 14(4):897-911 (1992); and Russell et al, J. Bact. 189(21)7581-7585 (2007)). At the level of post-translational regulation, enzyme attenuation can include: increasing intracellular concentration of known inhibitors; or modifying post- translational modified sites (Mann et al., Nature Biotech. 21 :255-261 (2003)). At the level of enzyme activity, enzyme attenuation can include: adding an endogenous or an exogenous inhibitor, such as an enzyme inhibitor, an antibiotic or a target-specific drug, to reduce enzyme activity; limiting availability of essential cofactors, such as vitamin B 12, for an enzyme that requires the cofactor; chelating a metal ion that is required for enzyme activity; or introducing a dominant negative mutation. The applicability of a technique for attenuation described above can depend upon whether a given host microbial organism is prokaryotic or eukaryotic, and it is understand that a determination of what is the appropriate technique for a given host can be readily made by one skilled in the art.

In some embodiments, microaerobic designs can be used based on the growth- coupled formation of the desired product. To examine this, production cones can be constructed for each strategy by first maximizing and, subsequently minimizing the product yields at different rates of biomass formation feasible in the network. If the rightmost boundary of all possible phenotypes of the mutant network is a single point, it implies that there is a unique optimum yield of the product at the maximum biomass formation rate possible in the network. In other cases, the rightmost boundary of the feasible phenotypes is a vertical line, indicating that at the point of maximum biomass the network can make any amount of the product in the calculated range, including the lowest amount at the bottommost point of the vertical line. Such designs are given a low priority.

The fatty alcohol, fatty aldehyde or fatty acid-production strategies identified in the various tables disclosed herein can be disrupted to increase production of fatty alcohol, fatty aldehyde or fatty acid. Accordingly, the invention also provides a non-naturally occurring microbial organism having metabolic modifications coupling fatty alcohol, fatty aldehyde or fatty acid production to growth of the organism, where the metabolic modifications includes disruption of one or more genes selected from the genes encoding proteins and/or enzymes shown in the various tables disclosed herein.

Each of the strains can be supplemented with additional deletions if it is determined that the strain designs do not sufficiently increase the production of fatty alcohol, fatty aldehyde or fatty acid and/or couple the formation of the product with biomass formation. Alternatively, some other enzymes not known to possess significant activity under the growth conditions can become active due to adaptive evolution or random mutagenesis. Such activities can also be knocked out. However, the list of gene deletion disclosed herein allows the construction of strains exhibiting high-yield production of fatty alcohol, fatty aldehyde or fatty acid, including growth-coupled production of fatty alcohol, fatty aldehyde or fatty acid.

In some embodiments, the invention provides a method for producing a compound of Formula (I):

(I) wherein Ri is Ci_ 24 linear alkyl; R 2 is CH 2 OH, CHO, or COOH; R 3 is H, OH, or oxo (=0); and represents a single or double bond with the proviso that the valency of the carbon atom to which R 3 is attached is four, comprising culturing a non-naturally occurring microbial organism described herein under conditions and for a sufficient period of time to produce the compound of Formula (I), wherein the non-naturally occurring microbial organism has one or more gene disruptions, wherein the one or more gene disruptions occurr in endogenous genes encoding proteins or enzymes involved in: native production of ethanol, glycerol, acetate, formate, lactate, C0 2 , fatty acids, or malonyl-CoA by said microbial organism; transfer of pathway intermediates to cellular compartments other than the cytosol; or native degradation of a MI-FAE cycle intermediate, a MD-FAE cycle intermediate or a termination pathway intermediate by the microbial organism, the one or more gene disruptions confer increased production of a fatty alcohol, fatty aldehyde or fatty acid in the microbial organism. Accordingly, the protein or enzyme can be a fatty acid synthase, an acetyl-CoA carboxylase, a biotin:apoenzyme ligase, an acyl carrier protein, a thioesterase, an acyltransferases, an ACP malonyltransferase, a fatty acid elongase, an acyl-CoA synthetase, an acyl-CoA transferase, an acyl-CoA hydrolase, a pyruvate decarboxylase, a lactate dehydrogenase, an alcohol dehydrogenase, an acid- forming aldehyde dehydrogenases, an acetate kinase, a phosphotransacetylase, a pyruvate oxidase, a glycerol-3 -phosphate dehydrogenase, a glycerol-3 -phosphate phosphatase, a mitochondrial pyruvate carrier, a peroxisomal fatty acid transporters, a peroxisomal acyl-CoA transporters, a peroxisomal carnitine/acylcarnitine transferases, an acyl-CoA oxidase, or an acyl-CoA binding protein. In some aspects, the one or more gene disruptions include a deletion of the one or more genes.

In some embodiments, the invention provides a method for producing a fatty alcohol, fatty aldehyde or fatty acid using a non-naturally occurring microbial organism as described herein, wherein one or more enzymes of the MI-FAE cycle, MD-FAE cycle or the termination pathway preferentially react with an NADH cofactor or have reduced preference for reacting with an NAD(P)H cofactor. For example, the one or more enzymes of the MI- FAE cycle or MD-FAE cycle can be a 3-ketoacyl-CoA reductase or an enoyl-CoA reductase. For the termination pathway, the one or more enzymes can be an acyl-CoA reductase (aldehyde forming), an alcohol dehydrogenase, an acyl-CoA reductase (alcohol forming), an aldehyde decarbonylase, an acyl- ACP reductase, an aldehyde dehydrogenase (acid forming) or a carboxylic acid reductase.

In some embodiments, the invention provides a method for producing a fatty alcohol, fatty aldehyde or fatty acid using a non-naturally occurring microbial organism as described herein having one or more gene disruptions in genes encoding proteins or enzymes that result in an increased ratio of NAD(P)H to NAD(P) present in the cytosol of the microbial organism following the disruptions. Accordingly, the gene encoding a protein or enzyme that results in an increased ratio of NAD(P)H to NAD(P) present in the cytosol of the microbial organism following the disruptions can be an NADH dehydrogenase, a cytochrome oxidase, a glycerol- 3-phosphate (G3P) dehydrogenase, a glycerol-3 -phosphate (G3P) phosphatase, an alcohol dehydrogenase, a pyruvate decarboxylase, an aldehyde dehydrogenase (acid forming), a lactate dehydrogenase, a glycerol-3 -phosphate dehydrogenase, a glycerol-3 - phosphate :quinone oxidoreductase, a malic enzyme and a malate dehydrogenase. In some aspects, the one or more gene disruptions include a deletion of the one or more genes.

In some embodiments, the invention provides a method for producing a fatty alcohol, fatty aldehyde or fatty acid using a non-naturally occurring microbial organism of the invention that is Crabtree positive and is in culture medium comprising excess glucose. In such conditions, as described herein, the microbial organism can result in increasing the ratio of NAD(P)H to NAD(P) present in the cytosol of the microbial organism.

In some embodiments, the invention provides a method for producing a fatty alcohol, fatty aldehyde or fatty acid using a non-naturally occurring microbial organism as described herein having at least one exogenous nucleic acid encoding an extracellular transporter or an extracellular transport system for a fatty alcohol, fatty aldehyde or fatty acid of the invention. In some embodiments, the invention provides a method for producing a fatty alcohol, fatty aldehyde or fatty acid using a non-naturally occurring microbial organism as described herein, wherein one or more endogenous enzymes involved in: native production of ethanol, glycerol, acetate, formate, lactate, C0 2 , fatty acids, or malonyl-CoA by said microbial organism; transfer of pathway intermediates to cellular compartments other than the cytosol; or native degradation of a MI-FAE cycle intermediate, a MD-FAE cycle intermediate or a termination pathway intermediate by said microbial organism, has attenuated enzyme activity or expression levels. Accordingly, the endogenous enzyme can be a fatty acid synthase, an acetyl-CoA carboxylase, a biotin:apoenzyme ligase, an acyl carrier protein, a thioesterase, an acyltransferase, an ACP malonyltransferase, a fatty acid elongase, an acyl-CoA synthetase, an acyl-CoA transferase, an acyl-CoA hydrolase, a pyruvate decarboxylase, a lactate dehydrogenase, an alcohol dehydrogenase, an acid-forming aldehyde dehydrogenases, an acetate kinase, a phosphotransacetylase, a pyruvate oxidase, a glycerol-3 -phosphate dehydrogenase, a glycero 1-3 -phosphate phosphatase, a mitochondrial pyruvate carrier, a peroxisomal fatty acid transporter, a peroxisomal acyl-CoA transporter, a peroxisomal carnitine/acylcarnitine transferase, an acyl-CoA oxidase, and an acyl-CoA binding protein.

In some embodiments, the invention provides a method for producing a fatty alcohol, fatty aldehyde or fatty acid using a non-naturally occurring microbial organism as described herein, wherein one or more endogenous enzymes involved in the oxidation of NAD(P)H or NADH, has attenuated enzyme activity or expression levels. Accordingly, the one or more endogenous enzymes can be NADH dehydrogenase, a cytochrome oxidase, a glycerol-3 - phosphate dehydrogenase, glycerol-3 -phosphate phosphatase, an alcohol dehydrogenase, a pyruvate decarboxylase, an aldehyde dehydrogenase (acid forming), a lactate dehydrogenase, a glycerol-3 -phosphate dehydrogenase, a glycerol-3 -phosphate :quinone oxidoreductase, a malic enzyme and a malate dehydrogenase.

A fatty alcohol, fatty aldehyde or fatty acid can be harvested or isolated at any time point during the culturing of the microbial organism, for example, in a continuous and/or near-continuous culture period, as disclosed herein. Generally, the longer the

microorganisms are maintained in a continuous and/or near-continuous growth phase, the proportionally greater amount of fatty alcohol, fatty aldehyde or fatty acid can be produced.

Therefore, the invention additionally provides a method for producing fatty alcohol, fatty aldehyde or fatty acid that includes culturing a non-naturally occurring microbial organism having one or more gene disruptions, as disclosed herein. The disruptions can occur in one or more genes encoding an enzyme that increases production of fatty alcohol, fatty aldehyde or fatty acid, including optionally coupling fatty alcohol, fatty aldehyde or fatty acid production to growth of the microorganism when the gene disruption reduces or eliminates an activity of the enzyme. For example, the disruptions can confer stable growth- coupled production of fatty alcohol, fatty aldehyde or fatty acid onto the non-naturally microbial organism.

In some embodiments, the gene disruption can include a complete gene deletion. In some embodiments other methods to disrupt a gene include, for example, frameshifting by omission or addition of oligonucleotides or by mutations that render the gene inoperable. One skilled in the art will recognize the advantages of gene deletions, however, because of the stability it confers to the non-naturally occurring organism from reverting to a parental phenotype in which the gene disruption has not occurred. In particular, the gene disruptions are selected from the gene sets as disclosed herein.

Once computational predictions are made of gene sets for disruption to increase production of fatty alcohol, fatty aldehyde or fatty acid, the strains can be constructed, evolved, and tested. Gene disruptions, including gene deletions, are introduced into host organism by methods well known in the art. A particularly useful method for gene disruption is by homologous recombination, as disclosed herein.

The engineered strains can be characterized by measuring the growth rate, the substrate uptake rate, and/or the product/byproduct secretion rate. Cultures can be grown and used as inoculum for a fresh batch culture for which measurements are taken during exponential growth. The growth rate can be determined by measuring optical density using a spectrophotometer (A600). Concentrations of glucose and other organic acid byproducts in the culture supernatant can be determined by well known methods such as HPLC, GC-MS or other well known analytical methods suitable for the analysis of the desired product, as disclosed herein, and used to calculate uptake and secretion rates.

Strains containing gene disruptions can exhibit suboptimal growth rates until their metabolic networks have adjusted to their missing functionalities. To assist in this adjustment, the strains can be adaptively evolved. By subjecting the strains to adaptive evolution, cellular growth rate becomes the primary selection pressure and the mutant cells are compelled to reallocate their metabolic fluxes in order to enhance their rates of growth. This reprogramming of metabolism has been recently demonstrated for several E. coli mutants that had been adaptively evolved on various substrates to reach the growth rates predicted a priori by an in silico model (Fong and Palsson, Nat. Genet. 36: 1056-1058 (2004)). The growth improvements brought about by adaptive evolution can be accompanied by enhanced rates of fatty alcohol, fatty aldehyde or fatty acid production. The strains are generally adaptively evolved in replicate, running in parallel, to account for differences in the evolutionary patterns that can be exhibited by a host organism (Fong and Palsson, Nat. Genet. 36: 1056-1058 (2004); Fong et al, J. Bacteriol. 185:6400-6408 (2003); Ibarra et al, Nature 420: 186-189 (2002)) that could potentially result in one strain having superior production qualities over the others. Evolutions can be run for a period of time, typically 2-6 weeks, depending upon the rate of growth improvement attained. In general, evolutions are stopped once a stable phenotype is obtained. Following the adaptive evolution process, the new strains are characterized again by measuring the growth rate, the substrate uptake rate, and the product/byproduct secretion rate. These results are compared to the theoretical predictions by plotting actual growth and production yields along side the production envelopes from metabolic modeling. The most successful design/evolution combinations are chosen to pursue further, and are characterized in lab-scale batch and continuous fermentations. The growth-coupled biochemical production concept behind the methods disclosed herein such as OptKnock approach should also result in the generation of genetically stable overproduces. Thus, the cultures are maintained in continuous mode for an extended period of time, for example, one month or more, to evaluate long-term stability. Periodic samples can be taken to ensure that yield and productivity are maintained.

Adaptive evolution is a powerful technique that can be used to increase growth rates of mutant or engineered microbial strains, or of wild-type strains growing under unnatural environmental conditions. It is especially useful for strains designed via methods such as OptKnock, which results in growth-coupled product formation. Therefore, evolution toward optimal growing strains will indirectly optimize production as well. Unique strains of E. coli K-12 MG1655 were created through gene knockouts and adaptive evolution. (Fong and Palsson, Nat. Genet. 36: 1056-1058 (2004)). In this work, all adaptive evolutionary cultures were maintained in prolonged exponential growth by serial passage of batch cultures into fresh medium before the stationary phase was reached, thus rendering growth rate as the primary selection pressure. Knockout strains were constructed and evolved on minimal medium supplemented with different carbon substrates (four for each knockout strain).

Evolution cultures were carried out in duplicate or triplicate, giving a total of 50 evolution knockout strains. The evolution cultures were maintained in exponential growth until a stable growth rate was reached. The computational predictions were accurate (within 10%) at predicting the post-evolution growth rate of the knockout strains in 38 out of the 50 cases examined. Furthermore, a combination of OptKnock design with adaptive evolution has led to improved lactic acid production strains. (Fong et al, Biotechnol. Bioeng. 91 :643-648 (2005)). Similar methods can be applied to the strains disclosed herein and applied to various host strains.

There are a number of developed technologies for carrying out adaptive evolution. Exemplary methods are disclosed herein. In some embodiments, optimization of a non- naturally occurring organism of the present invention includes utilizing adaptive evolution techniques to increase fatty alcohol, fatty aldehyde or fatty acid production and/or stability of the producing strain.

Serial culture involves repetitive transfer of a small volume of grown culture to a much larger vessel containing fresh growth medium. When the cultured organisms have grown to saturation in the new vessel, the process is repeated. This method has been used to achieve the longest demonstrations of sustained culture in the literature (Lenski and

Travisano, Proc. Natl. Acad. Sci. USA 91 :6808-6814 (1994)) in experiments which clearly demonstrated consistent improvement in reproductive rate over a period of years. Typically, transfer of cultures is usually performed during exponential phase, so each day the transfer volume is precisely calculated to maintain exponential growth through the next 24 hour period. Manual serial dilution is inexpensive and easy to parallelize.

In continuous culture the growth of cells in a chemostat represents an extreme case of dilution in which a very high fraction of the cell population remains. As a culture grows and becomes saturated, a small proportion of the grown culture is replaced with fresh media, allowing the culture to continually grow at close to its maximum population size.

Chemostats have been used to demonstrate short periods of rapid improvement in

reproductive rate (Dykhuizen, Methods Enzymol. 613-631 (1993)). The potential usefulness of these devices was recognized, but traditional chemostats were unable to sustain long periods of selection for increased reproduction rate, due to the unintended selection of dilution-resistant (static) variants. These variants are able to resist dilution by adhering to the surface of the chemostat, and by doing so, outcompete less adherent individuals, including those that have higher reproductive rates, thus obviating the intended purpose of the device (Chao and Ramsdell, J. Gen. Microbiol 20: 132-138 (1985)). One possible way to overcome this drawback is the implementation of a device with two growth chambers, which periodically undergo transient phases of sterilization, as described previously (Marliere and Mutzel, U.S. Patent No. 6,686,194).

Evolugator™ is a continuous culture device developed by Evolugate, LLC

(Gainesville, FL) and exhibits significant time and effort savings over traditional evolution techniques (de Crecy et al.,. Appl. Microbiol. Biotechnol. 77:489-496 (2007)). The cells are maintained in prolonged exponential growth by the serial passage of batch cultures into fresh medium before the stationary phase is attained. By automating optical density measurement and liquid handling, the Evolugator can perform serial transfer at high rates using large culture volumes, thus approaching the efficiency of a chemostat in evolution of cell fitness. For example, a mutant of Acinetobacter sp ADP1 deficient in a component of the translation apparatus, and having severely hampered growth, was evolved in 200 generations to 80% of the wild-type growth rate. However, in contrast to the chemostat which maintains cells in a single vessel, the machine operates by moving from one "reactor" to the next in subdivided regions of a spool of tubing, thus eliminating any selection for wall-growth. The transfer volume is adjustable, and normally set to about 50%. A drawback to this device is that it is large and costly, thus running large numbers of evolutions in parallel is not practical.

Furthermore, gas addition is not well regulated, and strict anaerobic conditions are not maintained with the current device configuration. Nevertheless, this is an alternative method to adaptively evolve a production strain.

As disclosed herein, a nucleic acid encoding a desired activity of a fatty alcohol, fatty aldehyde or fatty acid pathway can be introduced into a host organism. In some cases, it can be desirable to modify an activity of a fatty alcohol, fatty aldehyde or fatty acid pathway enzyme or protein to increase production of fatty alcohol, fatty aldehyde or fatty acid. For example, known mutations that increase the activity of a protein or enzyme can be introduced into an encoding nucleic acid molecule. Additionally, optimization methods can be applied to increase the activity of an enzyme or protein and/or decrease an inhibitory activity, for example, decrease the activity of a negative regulator.

One such optimization method is directed evolution. Directed evolution is a powerful approach that involves the introduction of mutations targeted to a specific gene in order to improve and/or alter the properties of an enzyme. Improved and/or altered enzymes can be identified through the development and implementation of sensitive high-throughput screening assays that allow the automated screening of many enzyme variants (for example, >10 4 ). Iterative rounds of mutagenesis and screening typically are performed to afford an enzyme with optimized properties. Computational algorithms that can help to identify areas of the gene for mutagenesis also have been developed and can significantly reduce the number of enzyme variants that need to be generated and screened. Numerous directed evolution technologies have been developed (for reviews, see Hibbert et al, Biomol.Eng 22: 11-19 (2005); Huisman and Lalonde, In Biocatalysis in the pharmaceutical and biotechnology industries pgs. 717-742 (2007), Patel (ed.), CRC Press; Otten and Quax. Biomol.Eng 22: 1-9 (2005).; and Sen et al, Appl Biochem.Biotechnol 143:212-223 (2007)) to be effective at creating diverse variant libraries, and these methods have been successfully applied to the improvement of a wide range of properties across many enzyme classes.

Enzyme characteristics that have been improved and/or altered by directed evolution technologies include, for example: selectivity/specificity, for conversion of non-natural substrates; temperature stability, for robust high temperature processing; pH stability, for bioprocessing under lower or higher pH conditions; substrate or product tolerance, so that high product titers can be achieved; binding (K m ), including broadening substrate binding to include non-natural substrates; inhibition (K;), to remove inhibition by products, substrates, or key intermediates; activity (kcat), to increases enzymatic reaction rates to achieve desired flux; expression levels, to increase protein yields and overall pathway flux; oxygen stability, for operation of air sensitive enzymes under aerobic conditions; and anaerobic activity, for operation of an aerobic enzyme in the absence of oxygen.

Described below in more detail are exemplary methods that have been developed for the mutagenesis and diversification of genes to target desired properties of specific enzymes. Such methods are well known to those skilled in the art. Any of these can be used to alter and/or optimize the activity of a fatty alcohol, fatty aldehyde or fatty acid pathway enzyme or protein.

EpPCR (Pritchard et al, J Theor.Biol. 234:497-509 (2005)) introduces random point mutations by reducing the fidelity of DNA polymerase in PCR reactions by the addition of Mn 2+ ions, by biasing dNTP concentrations, or by other conditional variations. The five step cloning process to confine the mutagenesis to the target gene of interest involves: 1) error- prone PCR amplification of the gene of interest; 2) restriction enzyme digestion; 3) gel purification of the desired DNA fragment; 4) ligation into a vector; 5) transformation of the gene variants into a suitable host and screening of the library for improved performance.

This method can generate multiple mutations in a single gene simultaneously, which can be useful to screen a larger number of potential variants having a desired activity. A high number of mutants can be generated by EpPCR, so a high-throughput screening assay or a selection method, for example, using robotics, is useful to identify those with desirable characteristics.

Error-prone Rolling Circle Amplification (epRCA) (Fujii et al., Nucleic Acids Res. 32:el45 (2004); and Fujii et al, Nat. Protoc. 1 :2493-2497 (2006)) has many of the same elements as epPCR except a whole circular plasmid is used as the template and random 6- mers with exonuclease resistant thiophosphate linkages on the last 2 nucleotides are used to amplify the plasmid followed by transformation into cells in which the plasmid is re- circularized at tandem repeats. Adjusting the Mn 2+ concentration can vary the mutation rate somewhat. This technique uses a simple error-prone, single-step method to create a full copy of the plasmid with 3 - 4 mutations/kbp. No restriction enzyme digestion or specific primers are required. Additionally, this method is typically available as a commercially available kit.

DNA or Family Shuffling (Stemmer, Proc Natl Acad Sci USA 91 : 10747- 10751 (1994)); and Stemmer, Nature 370:389-391 (1994)) typically involves digestion of two or more variant genes with nucleases such as Dnase I or EndoV to generate a pool of random fragments that are reassembled by cycles of annealing and extension in the presence of DNA polymerase to create a library of chimeric genes. Fragments prime each other and recombination occurs when one copy primes another copy (template switch). This method can be used with >lkbp DNA sequences. In addition to mutational recombinants created by fragment reassembly, this method introduces point mutations in the extension steps at a rate similar to error-prone PCR. The method can be used to remove deleterious, random and neutral mutations.

Staggered Extension (StEP) (Zhao et al, Nat. Biotechnol. 16:258-261 (1998)) entails template priming followed by repeated cycles of 2 step PCR with denaturation and very short duration of annealing/extension (as short as 5 sec). Growing fragments anneal to different templates and extend further, which is repeated until full-length sequences are made.

Template switching means most resulting fragments have multiple parents. Combinations of low-fidelity polymerases (Taq and Mutazyme) reduce error-prone biases because of opposite mutational spectra. In Random Priming Recombination (RPR) random sequence primers are used to generate many short DNA fragments complementary to different segments of the template (Shao et al, Nucleic Acids Res 26:681-683 (1998)). Base misincorporation and mispriming via epPCR give point mutations. Short DNA fragments prime one another based on homology and are recombined and reassembled into full-length by repeated thermocycling. Removal of templates prior to this step assures low parental recombinants. This method, like most others, can be performed over multiple iterations to evolve distinct properties. This technology avoids sequence bias, is independent of gene length, and requires very little parent DNA for the application.

In Heteroduplex Recombination linearized plasmid DNA is used to form

heteroduplexes that are repaired by mismatch repair (Volkov et al, Nucleic Acids Res. 27:el8 (1999); and Volkov et al, Methods Enzymol. 328:456-463 (2000)). The mismatch repair step is at least somewhat mutagenic. Heteroduplexes transform more efficiently than linear homoduplexes. This method is suitable for large genes and whole operons.

Random Chimeragenesis on Transient Templates (RACHITT) (Coco et al., Nat. Biotechnol. 19:354-359 (2001)) employs Dnase I fragmentation and size fractionation of single stranded DNA (ssDNA). Homologous fragments are hybridized in the absence of polymerase to a complementary ssDNA scaffold. Any overlapping unhybridized fragment ends are trimmed down by an exonuclease. Gaps between fragments are filled in and then ligated to give a pool of full-length diverse strands hybridized to the scaffold, which contains U to preclude amplification. The scaffold then is destroyed and is replaced by a new strand complementary to the diverse strand by PCR amplification. The method involves one strand (scaffold) that is from only one parent while the priming fragments derive from other genes, and the parent scaffold is selected against. Thus, no reannealing with parental fragments occurs. Overlapping fragments are trimmed with an exonuclease. Otherwise, this is conceptually similar to DNA shuffling and StEP. Therefore, there should be no siblings, few inactives, and no unshuffled parentals. This technique has advantages in that few or no parental genes are created and many more crossovers can result relative to standard DNA shuffling.

Recombined Extension on Truncated templates (RETT) entails template switching of unidirectionally growing strands from primers in the presence of unidirectional ssDNA fragments used as a pool of templates (Lee et al, J. Molec. Catalysis 26: 119-129 (2003)). No DNA endonucleases are used. Unidirectional ssDNA is made by DNA polymerase with random primers or serial deletion with exonuclease. Unidirectional ssDNA are only templates and not primers. Random priming and exonucleases do not introduce sequence bias as true of enzymatic cleavage of DNA shuffling/RACHITT. RETT can be easier to optimize than StEP because it uses normal PCR conditions instead of very short extensions. Recombination occurs as a component of the PCR steps, that is, no direct shuffling. This method can also be more random than StEP due to the absence of pauses. In Degenerate Oligonucleotide Gene Shuffling (DOGS) degenerate primers are used to control recombination between molecules; (Bergquist and Gibbs, Methods Mol.Biol 352: 191-204 (2007); Bergquist et al, Biomol.Eng 22:63-72 (2005); Gibbs et al, Gene 271 : 13-20 (2001)) this can be used to control the tendency of other methods such as DNA shuffling to regenerate parental genes. This method can be combined with random mutagenesis (epPCR) of selected gene segments. This can be a good method to block the reformation of parental sequences. No endonucleases are needed. By adjusting input concentrations of segments made, one can bias towards a desired backbone. This method allows DNA shuffling from unrelated parents without restriction enzyme digests and allows a choice of random mutagenesis methods.

Incremental Truncation for the Creation of Hybrid Enzymes (ITCHY) creates a combinatorial library with 1 base pair deletions of a gene or gene fragment of interest (Ostermeier et al, Proc. Natl. Acad. Sci. USA 96:3562-3567 (1999); and Ostermeier et al, Nat. Biotechnol. 17: 1205-1209 (1999)). Truncations are introduced in opposite direction on pieces of 2 different genes. These are ligated together and the fusions are cloned. This technique does not require homology between the 2 parental genes. When ITCHY is combined with DNA shuffling, the system is called SCRATCHY (see below). A major advantage of both is no need for homology between parental genes; for example, functional fusions between an E. coli and a human gene were created via ITCHY. When ITCHY libraries are made, all possible crossovers are captured.

Thio-Incremental Truncation for the Creation of Hybrid Enzymes (THIO-ITCHY) is similar to ITCHY except that phosphothioate dNTPs are used to generate truncations (Lutz et al, Nucleic Acids Res 29:E16 (2001)). Relative to ITCHY, THIO-ITCHY can be easier to optimize, provide more reproducibility, and adjustability. SCRATCHY combines two methods for recombining genes, ITCHY and DNA shuffling (Lutz et al, Proc. Natl. Acad. Sci. USA 98: 11248-11253 (2001)). SCRATCHY combines the best features of ITCHY and DNA shuffling. First, ITCHY is used to create a comprehensive set of fusions between fragments of genes in a DNA homology-independent fashion. This artificial family is then subjected to a DNA- shuffling step to augment the number of crossovers. Computational predictions can be used in optimization. SCRATCHY is more effective than DNA shuffling when sequence identity is below 80%. In Random Drift Mutagenesis (RNDM) mutations are made via epPCR followed by screening/selection for those retaining usable activity (Bergquist et al, Biomol. Eng. 22:63-72 (2005)). Then, these are used in DOGS to generate recombinants with fusions between multiple active mutants or between active mutants and some other desirable parent. Designed to promote isolation of neutral mutations; its purpose is to screen for retained catalytic activity whether or not this activity is higher or lower than in the original gene. RNDM is usable in high throughput assays when screening is capable of detecting activity above background. RNDM has been used as a front end to DOGS in generating diversity. The technique imposes a requirement for activity prior to shuffling or other subsequent steps; neutral drift libraries are indicated to result in higher/quicker improvements in activity from smaller libraries. Though published using epPCR, this could be applied to other large-scale mutagenesis methods.

Sequence Saturation Mutagenesis (SeSaM) is a random mutagenesis method that: 1) generates a pool of random length fragments using random incorporation of a phosphothioate nucleotide and cleavage; this pool is used as a template to 2) extend in the presence of

"universal" bases such as inosine; 3) replication of an inosine-containing complement gives random base incorporation and, consequently, mutagenesis (Wong et al, Biotechnol. J. 3:74- 82 (2008); Wong et al, Nucleic Acids Res. 32:e26 (2004); and Wong et al, Anal. Biochem. 341 : 187- 189 (2005)). Using this technique it can be possible to generate a large library of mutants within 2 to 3 days using simple methods. This technique is non-directed in comparison to the mutational bias of DNA polymerases. Differences in this approach makes this technique complementary (or an alternative) to epPCR.

In Synthetic Shuffling, overlapping oligonucleotides are designed to encode "all genetic diversity in targets" and allow a very high diversity for the shuffled progeny (Ness et al., Nat. Biotechnol. 20: 1251-1255 (2002)). In this technique, one can design the fragments to be shuffled. This aids in increasing the resulting diversity of the progeny. One can design sequence/codon biases to make more distantly related sequences recombine at rates approaching those observed with more closely related sequences. Additionally, the technique does not require physically possessing the template genes. Nucleotide Exchange and Excision Technology NexT exploits a combination of dUTP incorporation followed by treatment with uracil DNA glycosylase and then piperidine to perform endpoint DNA fragmentation (Muller et al., Nucleic Acids Res. 33:el 17 (2005)). The gene is reassembled using internal PCR primer extension with proofreading polymerase. The sizes for shuffling are directly controllable using varying dUPT::dTTP ratios. This is an end point reaction using simple methods for uracil incorporation and cleavage. Other nucleotide analogs, such as 8-oxo-guanine, can be used with this method. Additionally, the technique works well with very short fragments (86 bp) and has a low error rate. The chemical cleavage of DNA used in this technique results in very few unshuffled clones.

In Sequence Homology-Independent Protein Recombination (SHIPREC), a linker is used to facilitate fusion between two distantly related or unrelated genes. Nuclease treatment is used to generate a range of chimeras between the two genes. These fusions result in libraries of single-crossover hybrids (Sieber et al, Nat. Biotechnol. 19:456-460 (2001)). This produces a limited type of shuffling and a separate process is required for mutagenesis. In addition, since no homology is needed, this technique can create a library of chimeras with varying fractions of each of the two unrelated parent genes. SHIPREC was tested with a heme-binding domain of a bacterial CP450 fused to N-terminal regions of a mammalian CP450; this produced mammalian activity in a more soluble enzyme.

In Gene Site Saturation Mutagenesis™ (GSSM™) the starting materials are a supercoiled dsDNA plasmid containing an insert and two primers which are degenerate at the desired site of mutations (Kretz et al., Methods Enzymol. 388:3-11 (2004)). Primers carrying the mutation of interest, anneal to the same sequence on opposite strands of DNA. The mutation is typically in the middle of the primer and flanked on each side by approximately 20 nucleotides of correct sequence. The sequence in the primer is NNN or NNK (coding) and MNN (noncoding) (N = all 4, K = G, T, M = A, C). After extension, Dpnl is used to digest dam-methylated DNA to eliminate the wild-type template. This technique explores all possible amino acid substitutions at a given locus (that is, one codon). The technique facilitates the generation of all possible replacements at a single-site with no nonsense codons and results in equal to near-equal representation of most possible alleles. This technique does not require prior knowledge of the structure, mechanism, or domains of the target enzyme. If followed by shuffling or Gene Reassembly, this technology creates a diverse library of recombinants containing all possible combinations of single-site up-mutations. The usefulness of this technology combination has been demonstrated for the successful evolution of over 50 different enzymes, and also for more than one property in a given enzyme. Combinatorial Cassette Mutagenesis (CCM) involves the use of short oligonucleotide cassettes to replace limited regions with a large number of possible amino acid sequence alterations (Reidhaar-Olson et al. Methods Enzymol. 208:564-586 (1991); and Reidhaar- Olson et al. Science 241 :53-57 (1988)). Simultaneous substitutions at two or three sites are possible using this technique. Additionally, the method tests a large multiplicity of possible sequence changes at a limited range of sites. This technique has been used to explore the information content of the lambda repressor DNA-binding domain.

Combinatorial Multiple Cassette Mutagenesis (CMCM) is essentially similar to CCM except it is employed as part of a larger program: 1) use of epPCR at high mutation rate to 2) identify hot spots and hot regions and then 3) extension by CMCM to cover a defined region of protein sequence space (Reetz et al, Angew. Chem. Int. Ed Engl. 40:3589-3591 (2001)). As with CCM, this method can test virtually all possible alterations over a target region. If used along with methods to create random mutations and shuffled genes, it provides an excellent means of generating diverse, shuffled proteins. This approach was successful in increasing, by 51 -fold, the enantioselectivity of an enzyme.

In the Mutator Strains technique, conditional ts mutator plasmids allow increases of 20 to 4000-X in random and natural mutation frequency during selection and block accumulation of deleterious mutations when selection is not required (Selifonova et al, Appl. Environ. Microbiol. 67:3645-3649 (2001)). This technology is based on a plasmid-derived mutD5 gene, which encodes a mutant subunit of DNA polymerase III. This subunit binds to endogenous DNA polymerase III and compromises the proofreading ability of polymerase III in any strain that harbors the plasmid. A broad-spectrum of base substitutions and frameshift mutations occur. In order for effective use, the mutator plasmid should be removed once the desired phenotype is achieved; this is accomplished through a temperature sensitive (ts) origin of replication, which allows for plasmid curing at 41°C. It should be noted that mutator strains have been explored for quite some time (see Low et al, J. Mol. Biol. 260:359- 3680 (1996)). In this technique, very high spontaneous mutation rates are observed. The conditional property minimizes non-desired background mutations. This technology could be combined with adaptive evolution to enhance mutagenesis rates and more rapidly achieve desired phenotypes.

Look-Through Mutagenesis (LTM) is a multidimensional mutagenesis method that assesses and optimizes combinatorial mutations of selected amino acids (Rajpal et al., Proc. Natl. Acad. Sci. USA 102:8466-8471 (2005)). Rather than saturating each site with all possible amino acid changes, a set of nine is chosen to cover the range of amino acid R-group chemistry. Fewer changes per site allows multiple sites to be subjected to this type of mutagenesis. A >800-fold increase in binding affinity for an antibody from low nanomolar to picomolar has been achieved through this method. This is a rational approach to minimize the number of random combinations and can increase the ability to find improved traits by greatly decreasing the numbers of clones to be screened. This has been applied to antibody engineering, specifically to increase the binding affinity and/or reduce dissociation. The technique can be combined with either screens or selections. Gene Reassembly is a DNA shuffling method that can be applied to multiple genes at one time or to create a large library of chimeras (multiple mutations) of a single gene

(Tunable GeneReassembly™ (TGR™) Technology supplied by Verenium Corporation). Typically this technology is used in combination with ultra-high-throughput screening to query the represented sequence space for desired improvements. This technique allows multiple gene recombination independent of homology. The exact number and position of cross-over events can be pre-determined using fragments designed via bioinformatic analysis. This technology leads to a very high level of diversity with virtually no parental gene reformation and a low level of inactive genes. Combined with GSSM™, a large range of mutations can be tested for improved activity. The method allows "blending" and "fine tuning" of DNA shuffling, for example, codon usage can be optimized.

In Silico Protein Design Automation (PDA) is an optimization algorithm that anchors the structurally defined protein backbone possessing a particular fold, and searches sequence space for amino acid substitutions that can stabilize the fold and overall protein energetics (Hayes et al, Proc. Natl. Acad. Sci. USA 99: 15926-15931 (2002)). This technology uses in silico structure-based entropy predictions in order to search for structural tolerance toward protein amino acid variations. Statistical mechanics is applied to calculate coupling interactions at each position. Structural tolerance toward amino acid substitution is a measure of coupling. Ultimately, this technology is designed to yield desired modifications of protein properties while maintaining the integrity of structural characteristics. The method computationally assesses and allows filtering of a very large number of possible sequence variants (10 50 ). The choice of sequence variants to test is related to predictions based on the most favorable thermodynamics. Ostensibly only stability or properties that are linked to stability can be effectively addressed with this technology. The method has been successfully used in some therapeutic proteins, especially in engineering immunoglobulins. In silico predictions avoid testing extraordinarily large numbers of potential variants. Predictions based on existing three-dimensional structures are more likely to succeed than predictions based on hypothetical structures. This technology can readily predict and allow targeted screening of multiple simultaneous mutations, something not possible with purely

experimental technologies due to exponential increases in numbers.

Iterative Saturation Mutagenesis (ISM) involves: 1) using knowledge of

structure/function to choose a likely site for enzyme improvement; 2) performing saturation mutagenesis at chosen site using a mutagenesis method such as Stratagene QuikChange (Stratagene; San Diego CA); 3) screening/selecting for desired properties; and 4) using improved clone(s), start over at another site and continue repeating until a desired activity is achieved (Reetz et al, Nat. Protoc. 2:891-903 (2007); and Reetz et al, Angew. Chem. Int. Ed Engl. 45:7745-7751 (2006)). This is a proven methodology, which assures all possible replacements at a given position are made for screening/selection.

Any of the aforementioned methods for mutagenesis can be used alone or in any combination. Additionally, any one or combination of the directed evolution methods can be used in conjunction with adaptive evolution techniques, as described herein.

It is understood that modifications which do not substantially affect the activity of the various embodiments of this invention are also provided within the definition of the invention provided herein. Accordingly, the following examples are intended to illustrate but not limit the present invention.

EXAMPLE I

Production of Fatty Alcohols and Fatty Aldehydes by MI-FAE Cycle, MD-FAE Cycle and Acyl-CoA Termination Pathways

Encoding nucleic acids and species that can be used as sources for conferring fatty alcohol and fatty aldehyde production capability onto a host microbial organism are exemplified further below. Multienzyme complexes

In one exemplary embodiment, the genes fadA and fadB encode a multienzyme complex that exhibits three constituent activities of the malonyl-CoA independent FAS pathway, namely, ketoacyl-CoA thiolase, 3-hydroxyacyl-CoA dehydrogenase, and enoyl- CoA hydratase activities (Nakahigashi, K. and H. Inokuchi, Nucleic Acids Research 18:4937 (1990); Yang et al, Journal of Bacteriology 173:7405-7406 (1991); Yang et al, Journal of Biological Chemistry 265: 10424-10429 (1990); Yang et al, Biochemistry 30:6788-6795 (1990)). The fadl and fadJ genes encode similar activities which can substitute for the above malonyl-CoA independent FAS conferring genes fadA and fadB. The acyl-Coa

dehydrogenase of E. coli is encoded by fadE (Campbell et al, J Bacteriol 184: 3759-64)). This enzyme catalyzes the rate-limiting step of beta-oxidation (O'Brien et al, J Bacteriol 132:532-40 (1977)). The nucleic acid sequences for each of the above fad genes are well known in the art and can be accessed in the public databases such as Genbank using the following accession numbers.

Step A. Thiolase

Thiolase enzymes, also know as beta-keto thiolase, acyl-CoA C-acetyltransferase, acyl-CoA:acetyl-CoA C-acyltransferase, 3-oxoacyl-CoA thiolase, 3-ketoacyl-CoA thiolase, beta-ketoacyl-CoA thiolase, and acyl-CoA thiolase, that are suitable for fatty alcohol, fatty aldehyde or fatty acid production are described herein (Figures 1 A and 6A). Exemplary acetoacetyl-CoA thiolase enzymes include the gene products of atoB and homolog yqeF from E. coli (Martin et al, Nat.Biotechnol 21 :796-802 (2003)), thlA and MB from C.

acetobutylicum (Hanai et al, Appl Environ Microbiol 73:7814-7818 (2007); Winzer et al, J. Mol. Microbiol Biotechnol 2:531-541 (2000)), and ERG10 from S. cerevisiae (Hiser et al, J.Biol. Chem. 269:31383-31389 (1994)). A degradative thiolase of S. cerevisiae is encoded by POT1. Another candidate thiolase is the phaA gene product of R. eutropha (Jenkins et al, Journal of Bacteriology 169:42-52 (1987)). The acetoacetyl-CoA thiolase from Zoogloea ramigera is irreversible in the biosynthetic direction and a crystal structure is available (Merilainen et al, Biochem 48: 11011-25 (2009)). Accession numbers for these thiolases and homologs are included in the table below.

Many thiolase enzymes catalyze the formation of longer-chain acyl-CoA products. Exemplary thiolases include, for example, 3-oxoadipyl-CoA thiolase (EC 2.3.1.174) and acyl-CoA thiolase (EC 2.3.1.16). 3-Oxoadipyl-CoA thiolase converts succinyl-CoA and acetyl-CoA to 3-oxoadipyl-CoA, and is a key enzyme of the beta-ketoadipate pathway for aromatic compound degradation. The enzyme is widespread in soil bacteria and fungi including Pseudomonas putida (Harwood et al, J Bacteriol. 176:6479-6488 (1994)) and Acinetobacter calcoaceticus (Doten et al, J Bacteriol. 169:3168-3174 (1987)). The gene products encoded by pcaF in Pseudomonas strain B 13 (Kaschabek et al, J Bacteriol.

184:207-215 (2002)), phaD in Pseudomonas putida U (Olivera et al, Proc.Natl.Acad.Sci

U.S.A 95:6419-6424 (1998)), paaE in Pseudomonas fluorescens ST (Oi et al, Arch. Microbiol 188: 117-125 (2007)), and paaJ from E. coli (Nogales et al, Microbiology 153:357-365 (2007)) also catalyze this transformation. Several beta-ketothiolases exhibit significant and selective activities in the oxoadipyl-CoA forming direction including bkt from Pseudomonas putida, pcaF and bkt from Pseudomonas aeruginosa PAOl, bkt from Burkholderia ambifaria AMMD, paaJ from E. coli, and phaD from P. putida. Two gene products of Ralstonia eutropha (formerly known as Alcaligenes eutrophus), encoded by genes bktB and bktC, catalyze the formation of 3-oxopimeloyl-CoA (Slater et al., J.Bacteriol. 180: 1979-1987 (1998); Haywood et al, FEMS Microbiology Letters 52:91-96 (1988)). The sequence of the BktB protein is known; however, the sequence of the BktC protein has not been reported. BktB is also active on substrates of length C6 and C8 (Machado et al, Met Eng in press

(2012)). The pirn, operon of Rhodopseudomonas palustris also encodes a beta-ketothiolase, encoded by pimB, predicted to catalyze this transformation in the degradative direction during benzoyl-CoA degradation (Harrison et al, Microbiology 151 :727-736 (2005)). A beta- ketothiolase enzyme candidate in S. aciditrophicus was identified by sequence homology to bktB (43% identity, evalue = 1 e-93).

Acyl-CoA thiolase (EC 2.3.1.16) enzymes involved in the beta-oxidation cycle of fatty acid degradation exhibit activity on a broad range of acyl-CoA substrates of varying chain length. Exemplary acyl-CoA thiolases are found in Arabidopsis thaliana (Cruz et al, Plant Physiol 135:85-94 (2004)), Homo sapiens (Mannaerts et al, Cell Biochem Biphys 32:73-87 (2000)), Helianthus annuus (Schiedel et al, Prot Expr Purif 33:25-33 (2004)). The chain length specificity of thiolase enzymes can be assayed by methods well known in the art (Wrensford et al, Anal Biochem 192:49-54 (1991)). A peroxisomal thiolase found in rat liver catalyze the acetyl-CoA dependent formation of longer chain acyl-CoA products from octanoyl-CoA (Horie et al, Arch Biochem Biophys 214: 64-73 (1989); Hijikata et al, J Biol Chem 265, 4600-4606 (1990)).

Acetoacetyl-CoA can also be synthesized from acetyl-CoA and malonyl-CoA by acetoacetyl-CoA synthase (EC 2.3.1.194). This enzyme (FhsA) has been characterized in the soil bacterium Streptomyces sp. CL190 where it participates in mevalonate biosynthesis (Okamura et al, PNAS USA 107: 11265-70 (2010)). As this enzyme catalyzes an essentially irreversible reaction, it is particularly useful for metabolic engineering applications for overproducing metabolites, fuels or chemicals derived from acetoacetyl-CoA such as long chain alcohols. Other acetoacetyl-CoA synthase genes can be identified by sequence homology to flisA. Acyl-CoA synthase enzymes such as fhsA and homo logs can be engineered or evolved to accept longer acyl-CoA substrates by methods known in the art.

Chain length selectivity of selected thiolase enzymes described above is summarized the table below.

Chain length Gene Organism

C4 atoB Escherichia coli

C6 phaD Pseudomonas putida

C6-C8 bktB Ralstonia eutropha

C10-C16 Acaala Rattus norvegicus Step B. 3-Oxoacyl-CoA reductase

3-Oxoacyl-CoA reductases (also known as 3-hydroxyacyl-CoA dehydrogenases, 3- ketoacyl-CoA reductases, beta-ketoacyl-CoA reductases, beta-hydroxyacyl-CoA

dehydrogenases, hydroxyacyl-CoA dehydrogenases, and ketoacyl-CoA reductases) catalyze the reduction of 3-oxoacyl-CoA substrates to 3-hydroxyacyl-CoA products (Figure IB and Figure 6B). These enzymes are often involved in fatty acid beta-oxidation and aromatic degradation pathways. For example, subunits of two fatty acid oxidation complexes in E. coli, encoded by fadB and fadJ, function as 3-hydroxyacyl-CoA dehydrogenases (Binstock et al, Methods Enzymol. 71 Pt C:403-411 (1981)). Knocking out a negative regulator encoded by fadR can be utilized to activate the fadB gene product (Sato et al, J Biosci.Bioeng 103:38- 44 (2007)). Another 3-hydroxyacyl-CoA dehydrogenase from E. coli is paaH (Ismail et al, European Journal of Biochemistry 270:3047-3054 (2003)). Additional 3-oxoacyl-CoA enzymes include the gene products of phaC in Pseudomonas putida (Olivera et al,

Proc.Natl.Acad.Sci U.S.A 95:6419-6424 (1998)) and paaC in Pseudomonas fluorescens (Di et al., 188: 117-125 (2007)). These enzymes catalyze the reversible oxidation of 3- hydroxyadipyl-CoA to 3-oxoadipyl-CoA during the catabolism of phenylacetate or styrene. Other suitable enzyme candidates include AA072312.1 from E. gracilis (Winkler et al., Plant Physiology 131 :753-762 (2003)) and paaC from Pseudomonas putida (Olivera et al., PNAS USA 95:6419-6424 (1998)). Enzymes catalyzing the reduction of acetoacetyl-CoA to 3-hydroxybutyryl-CoA include hbd of Clostridium acetobutylicum (Youngleson et al, J Bacteriol. 171 :6800-6807 (1989)), phbB from Zoogloea ramigera (Ploux et al, EurJ Biochem. 174: 177-182 (1988)), phaB from Rhodobacter sphaeroides (Alber et al,

Mol.Microbiol 61 :297-309 (2006)) and paaHl of Ralstonia eutropha (Machado et al, Met Eng, In Press (2012)). The Z. ramigera enzyme is NADPH-dependent and also accepts 3- oxopropionyl-CoA as a substrate (Ploux et al, Eur. J Biochem. 174: 177-182 (1988)).

Additional genes include phaB in Paracoccus denitrificans, Hbdl (C -terminal domain) and Hbd2 (N-terminal domain) in Clostridium kluyveri (Hillmer and Gottschalk, Biochim.

Biophys. Acta 3334: 12-23 (1974)) and HSD17B10 in Bos taurus (Wakil et al, J Biol.Chem. 207:631-638 (1954)). The enzyme from Paracoccus denitrificans has been functionally expressed and characterized in E. coli (Yabutani et al., FEMS Microbiol Lett. 133:85-90

(1995)). A number of similar enzymes have been found in other species of Clostridia and in Metallosphaera sedula (Berg et al, Science. 318: 1782-1786 (2007)). The enzyme from Candida tropicalis is a component of the peroxisomal fatty acid beta-oxidation multifunctional enzyme type 2 (MFE-2). The dehydrogenase B domain of this protein is catalytically active on acetoacetyl-CoA. The domain has been functionally expressed in E. coli, a crystal structure is available, and the catalytic mechanism is well-understood

(Ylianttila et al., Biochem Biophys Res Commun 324:25-30 (2004); Ylianttila et al., J Mol Biol 358: 1286-1295 (2006)). 3-Hydroxyacyl-CoA dehydrogenases that accept longer acyl- CoA substrates (eg. EC 1.1.1.35) are typically involved in beta-oxidation. An example is HSD17B10 in Bos taurus (Wakil et al, J Biol.Chem. 207:631-638 (1954)). The pig liver enzyme is preferentially active on short and medium chain acyl-CoA substrates whereas the heart enzyme is less selective (He et al, Biochim Biophys Acta 1392: 119-26 (1998)). The S. cerevisiae enzyme FOX2 is active in beta-degradation pathways and also has enoyl-CoA hydratase activity (Hiltunen et al, J Biol Chem 267: 6646-6653 (1992)).

Chain length specificity of selected hydroxyacyl-CoA dehydrogenase enzymes is shown below. Directed evolution can enhance selectivity of enzymes for longer-chain substrates. For example, Machado and coworkers developed a selection platform for directed evolution of chain elongation enzymes that favor longer acyl-CoA substrates. This group evolved paaHl of Ralstonia eutropha for improved activity on 3-oxo-hexanoyl-CoA

(Machado et al, Met Eng, In Press (2012)).

Step C. 3-Hydroxyacyl-CoA dehydratase

3-Hydroxyacyl-CoA dehydratases (eg. EC 4.2.1.17, also known as enoyl-CoA hydratases) catalyze the dehydration of a range of 3-hydroxyacyl-CoA substrates (Roberts et al, ArchMicrobiol 117:99-108 (1978); Agnihotri et al, Bioorg.Med.Chem. 11 :9-20 (2003); Conrad et al, J Bacteriol. 118:103-111 (1974)) and can be used in the conversion of 3- hydroxyacyl-CoA to enoyl-CoA (Figures 1C and 6C). The ech gene product of Pseudomonas putida catalyzes the conversion of 3-hydroxybutyryl-CoA to crotonyl-CoA (Roberts et al, ArchMicrobiol 117:99-108 (1978)). This transformation is also catalyzed by the crt gene product of Clostridium acetobutylicum, the crtl gene product of C. kluyveri, and other clostridial organisms Atsumi et al, Metab Eng 10:305-311 (2008); Boynton et al, J

Bacteriol. 178:3015-3024 (1996); Hillmer et al, FEBS Lett. 21 :351-354 (1972)). Additional enoyl-CoA hydratase candidates are phaA and phaB, of P. putida, and paaA and paaB from P.fluorescens (Olivera et al, Proc.Natl.Acad.Sci U.S.A 95:6419-6424 (1998)). The gene product of pimF in Rhodopseudomonas palustris is predicted to encode an enoyl-CoA hydratase that participates in pimeloyl-CoA degradation (Harrison et al., Microbiology 151 :727-736 (2005)). Lastly, a number of Escherichia coli genes have been shown to demonstrate enoyl-CoA hydratase functionality including maoC (Park et al., J Bacteriol. 185:5391-5397 (2003)), paaF (Ismail et al, Eur.J Biochem. 270:3047-3054 (2003); Park et al, Appl.Biochem.Biotechnol 113-116:335-346 (2004); Park et al, Biotechnol Bioeng 86:681-686 (2004)) and paaG (Ismail et al, Eur.J Biochem. 270:3047-3054 (2003); Park and Lee, Appl.Biochem.Biotechnol 113-116:335-346 (2004); Park and Yup, Biotechnol Bioeng 86:681-686 (2004)). Enzymes with 3-hydroxyacyl-CoA dehydratase activity in S. cerevisiae include PHS1 and FOX2. Gene GenBank Accession No. GI No. Organism

ech NP 745498.1 26990073 Pseudomonas putida

crt NP 349318.1 15895969 Clostridium acetobutylicum crtl YP 001393856 153953091 Clostridium kluyveri

phaA ABF82233.1 26990002 Pseudomonas putida

phaB ABF82234.1 26990001 Pseudomonas putida

paaA NP 745427.1 106636093 Pseudomonas fluorescens paaB NP 745426.1 106636094 Pseudomonas fluorescens pimF CAE29158.1 39650635 Rhodopseudomonas palustris maoC NP 415905.1 16129348 Escherichia coli

paaF NP 415911.1 16129354 Escherichia coli

paaG NP 415912.1 16129355 Escherichia coli

FOX2 NP 012934.1 6322861 Saccharomyces cerevisiae

PHS1 NP 012438.1 6322364 Saccharomyces cerevisiae

Enoyl-CoA hydratases involved in beta-oxidation can also be used in an fatty alcohol, fatty aldehyde and fatty acid biosynthetic pathway. For example, the multifunctional MFP2 gene product of Arabidopsis thaliana exhibits an enoyl-CoA reductase activity selective for chain lengths less than or equal to C14 (Arent et al, J Biol Chem 285:24066-77 (2010)). Alternatively, the E. coli gene products of fadA and fadB encode a multienzyme complex involved in fatty acid oxidation that exhibits enoyl-CoA hydratase activity (Yang et al., Biochemistry 30:6788-6795 (1991); Yang, J Bacteriol. 173:7405-7406 (1991); Nakahigashi et al, Nucleic Acids Res. 18:4937 (1990)). The fadl and fadJ genes encode similar functions and are naturally expressed under anaerobic conditions (Campbell et al., Mol.Microbiol 47:793-805 (2003)).

Chain length specificity of selected 3-hydroxyacyl-CoA dehydratase enzymes is shown below. Chain length Gene Organism

C4-C6 crt Clostridium acetobutylicum

C4-C7 pimF Rhodopseudomonas

palustris

C4-C14 MFP2 Arabidopsis thaliana

Step D. Enoyl-CoA reductase

Enoyl-CoA reductases (also known as acyl-CoA dehydrogenases, trans-2-enoyl-CoA reductases, or acyl-CoA oxidoreductases) catalyze the conversion of an enoyl-CoA to an acyl-CoA (step D of Figures 1 and 6). Exemplary acyl-CoA dehydrogenase or enoyl-CoA reductase (ECR) enzymes are the gene products of fadE of E.coli and Salmonella enterica (Iram et al, J Bacteriol 188:599-608 (2006)). The bed gene product from Clostridium acetobutylicum (Atsumi et al, 10:305-311 (2008); Boynton et al, J Bacteriol. 178:3015- 3024 (1996)) catalyzes the reduction of crotonyl-CoA to butyryl-CoA (EC 1.3.99.2). This enzyme participates in the acetyl-CoA fermentation pathway to butyrate in Clostridial species (Jones et al, Microbiol Rev. 50:484-524 (1986)). Activity of butyryl-CoA reductase can be enhanced by expressing bed in conjunction with expression of the C. acetobutylicum etfAB genes, which encode an electron transfer flavoprotein. An additional candidate for the enoyl-CoA reductase step is the enoyl-CoA reductase (EC 1.3.1.44) TER from E. gracilis (Hoffmeister et al, J Biol.Chem 280:4329-4338 (2005)). A construct derived from this sequence following the removal of its mitochondrial targeting leader sequence was cloned in E. coli resulting in an active enzyme. A close homolog of the ECR protein from the prokaryote Treponema denticola, encoded by TDE0597, has also been cloned and expressed in E. coli (Tucci et al, FEBS Lett, 581 :1561-1566 (2007)). Six genes in Syntrophus aciditrophicus were identified by sequence homology to the C. acetobutylicum bed gene product. The S. aciditrophicus genes syn_02637 and syn_02636 bear high sequence homology to the etfAB genes of C. acetobutylicum, and are predicted to encode the alpha and beta subunits of an electron transfer flavoprotein.

Protein ( ,cn Bank ID GI Number Organism

fadE AAC73325.2 87081702 Escherichia coli

fadE YP 005241256.1 379699528 Salmonella enterica

bed NP 349317.1 15895968 Clostridium acetobutylicum etfA NP 349315.1 15895966 Clostridium acetobutylicum etfB NP 349316.1 15895967 Clostridium acetobutylicum

TER Q5EU90.1 62287512 Euglena gracilis TER NP 612558.1 19924091 Rattus norvegicus

TDE0597 NP 971211.1 42526113 Treponema denticola

syn 02587 ABC76101 85721158 Syntrophus aciditrophicus syn 02586 ABC76100 85721157 Syntrophus aciditrophicus syn 01146 ABC76260 85721317 Syntrophus aciditrophicus syn 00480 ABC77899 85722956 Syntrophus aciditrophicus syn 02128 ABC76949 85722006 Syntrophus aciditrophicus syn 01699 ABC78863 85723920 Syntrophus aciditrophicus syn 02637 ABC78522.1 85723579 Syntrophus aciditrophicus syn 02636 ABC78523.1 85723580 Syntrophus aciditrophicus

Additional enoyl-CoA reductase enzyme candidates are found in organisms that degrade aromatic compounds. Rhodopseudomonas palustris, a model organism for benzoate degradation, has the enzymatic capability to degrade pimelate via beta-oxidation of pimeloyl- CoA. Adjacent genes in the pim operon, pimC and pimD, bear sequence homology to C. acetobutylicum bed and are predicted to encode a flavin-containing pimeloyl-CoA

dehydrogenase (Harrison et al, 151 :727-736 (2005)). The genome of nitrogen-fixing soybean symbiont Bradyrhizobium japonicum also contains a pim operon composed of genes with high sequence similarity to pimC and pimD of R. palustris (Harrison and Harwood, Microbiology 151 :727-736 (2005)).

An additional candidate is 2-methyl-branched chain enoyl-CoA reductase (EC 1.3.1.52 and EC 1.3.99.12), an enzyme catalyzing the reduction of sterically hindered trans- enoyl-CoA substrates. This enzyme participates in branched-chain fatty acid synthesis in the nematode Ascaris suum and is capable of reducing a variety of linear and branched chain substrates including 2-methylvaleryl-CoA, 2-methylbutanoyl-CoA, 2-methylpentanoyl-CoA, octanoyl-CoA and pentanoyl-CoA (Duran et al., 268:22391-22396 (1993)). Two isoforms of the enzyme, encoded by genes acadl and acad, have been characterized.

Protein ( ,cn Bank ID GI Number Organism

acadl AAC48316.1 2407655 Ascaris suum acad AAA16096.1 347404 Ascaris suum At least three mitochondrial enoyl-CoA reductase enzymes exist in E. gracilis and are applicable for use in the invention. Three mitochondrial enoyl-CoA reductase enzymes of E. gracilis (ECRl-3) exhibit different chain length preferences (Inui et al, European Journal of Biochemistry 142: 121-126 (1984)), which is particularly useful for dictating the chain length of the desired fatty alcohol, fatty aldehyde or fatty acid products. EST's ELL00002199, ELL00002335, and ELL00002648, which are all annotated as mitochondrial trans-2-enoyl- CoA reductases, can be used to isolate these additional enoyl-CoA reductase genes by methods known in the art. Two ECR enzymes from rat liver microsomes also exhibit different substrate specificities (Nagi et al, Arc/z Biochem Biophys 226:50-64 (1983)). The sequences of these enzymes have not been identified to date. The Mycobacterium smegmatis enoyl-CoA reductase accepts acyl-CoA substrates of chain lengths between C10-C16 (Shimakata et al, J Biochem 89: 1075-80 (1981)).

Enoyl-CoA reductases and their chain length specificities are shown in the table below.

Step E. Acyl-CoA reductase (aldehyde forming)

Reduction of an acyl-CoA to a fatty alcohol is catalyzed by either a single enzyme or pair of enzymes that exhibit acyl-CoA reductase and alcohol dehydrogenase activities. Acyl- CoA dehydrogenases that reduce an acyl-CoA to its corresponding aldehyde include fatty acyl-CoA reductase (EC 1.2.1.42, 1.2.1.50), succinyl-CoA reductase (EC 1.2.1.76), acetyl- CoA reductase, butyryl-CoA reductase and propionyl-CoA reductase (EC 1.2.1.3). Aldehyde forming acyl-CoA reductase enzymes with demonstrated activity on acyl-CoA, 3- hydroxyacyl-CoA and 3-oxoacyl-CoA substrates are known in the literature. Several acyl- CoA reductase enzymes are active on 3-hydroxyacyl-CoA substrates. For example, some butyryl-CoA reductases from Clostridial organisms, are active on 3-hydroxybutyryl-CoA and propionyl-CoA reductase of L. reuteri is active on 3-hydroxypropionyl-CoA. An enzyme for converting 3-oxoacyl-CoA substrates to their corresponding aldehydes is malonyl-CoA reductase. Enzymes in this class that demonstrate activity on enoyl-CoA substrates have not been identified to date. Specificity for a particular substrate can be refined using evolution or enzyme engineering methods known in the art. Exemplary fatty acyl-CoA reductases enzymes are encoded by acrl of Acinetobacter calcoaceticus (Reiser, Journal of Bacteriology 179:2969-2975 (1997)) and Acinetobacter sp. M-1 (Ishige et al, Appl. Environ. Microbiol. 68: 1192-1195 (2002)). Two gene products from Mycobacterium tuberculosis accept longer chain fatty acyl-CoA substrates of length C16-C18 (Harminder Singh, U. Central Florida (2007)). Yet another fatty acyl-CoA reductase is LuxC of Photobacterium phosphoreum (Lee et al, Biochim Biohys Acta 1388:215-22 (1997)).

Enzymes with succinyl-CoA reductase activity are encoded by sucD of Clostridium kluyveri (Sohling, J. Bacteriol. 178:871-880 (1996)) and sucD of P. gingivalis (Takahashi, J.

Bacteriol 182:4704-4710 (2000)). Additional succinyl-CoA reductase enzymes participate in the 3-hydroxypropionate/4-hydroxybutyrate cycle of thermophilic archaea including

Metallosphaera sedula (Berg et al., Science 318: 1782-1786 (2007)) and Thermoproteus neutrophilus (Ramos-Vera et al, J Bacteriol, 191 :4286-4297 (2009)). The sedula enzyme, encoded by Msed_0709, is strictly NADPH-dependent and also has malonyl-CoA reductase activity. The T. neutrophilus enzyme is active with both NADPH and NADH. The enzyme acylating acetaldehyde dehydrogenase in Pseudomonas sp, encoded by bphG, is yet another as it has been demonstrated to oxidize and acylate acetaldehyde, propionaldehyde, butyraldehyde, isobutyraldehyde and formaldehyde (Powlowski, J. Bacteriol. 175:377-385 (1993)). In addition to reducing acetyl-CoA to ethanol, the enzyme encoded by adhE in Leuconostoc mesenteroides has been shown to oxidize the branched chain compound isobutyraldehyde to isobutyryl-CoA (Kazahaya, J. Gen. Appl. Microbiol. 18:43-55 (1972); and Koo et al, Biotechnol Lett. 27:505-510 (2005)). Butyraldehyde dehydrogenase catalyzes a similar reaction, conversion of butyryl-CoA to butyraldehyde, in solventogenic organisms such as Clostridium saccharoperbutylacetonicum (Kosaka et al., Biosci Biotechnol Biochem., 71 :58-68 (2007)). Exemplary propionyl-CoA reductase enzymes include pduP of Salmonella typhimurium LT2 ( Leal, Arch. Microbiol. 180:353-361 (2003)) and eutE from E. coli (Skraly, WO Patent No. 2004/024876). The propionyl-CoA reductase of Salmonella typhimurium LT2, which naturally converts propionyl-CoA to propionaldehyde, also catalyzes the reduction of 5-hydroxyvaleryl-CoA to 5-hydroxypentanal (WO 2010/068953 A2). The propionaldehyde dehydrogenase of Lactobacillus reuten, PduP, has a broad substrate range that includes butyraldehyde, valeraldehyde and 3-hydroxypropionaldehyde (Luo et al, Appl Microbiol Biotech, 89: 697-703 (2011). Additionally, some acyl-ACP reductase enzymes such as the orfl594 gene product of Synechococcus elongatus PCC7942 also exhibit aldehyde-forming acyl-CoA reductase activity (Schirmer et al, Science, 329: 559-62 (2010)). Acyl-ACP reductase enzymes and homologs are described in further detail in Example IX.

An additional enzyme type that converts an acyl-CoA to its corresponding aldehyde is malonyl-CoA reductase which transforms malonyl-CoA to malonic semialdehyde. Malonyl- CoA reductase is a key enzyme in autotrophic carbon fixation via the 3-hydroxypropionate cycle in thermoacidophilic archaeal bacteria (Berg, Science 318:1782- 1786 (2007); and

Thauer, Science 318: 1732-1733 (2007)). The enzyme utilizes NADPH as a cofactor and has been characterized in Metallosphaera and Sulfolobus sp. (Alber et al, J. Bacteriol. 188:8551- 8559 (2006); and Hugler, J. Bacteriol. 184:2404-2410 (2002)). The enzyme is encoded by Msed_0709 in Metallosphaera sedula (Alber et al, J. Bacteriol. 188:8551-8559 (2006); and Berg, Science 318: 1782- 1786 (2007)). A gene encoding a malonyl-CoA reductase from Sulfolobus tokodaii was cloned and heterologously expressed in E. coli (Alber et al, J.

Bacteriol 188:8551-8559 (2006). This enzyme has also been shown to catalyze the conversion of methylmalonyl-CoA to its corresponding aldehyde (WO2007141208 (2007)). Although the aldehyde dehydrogenase functionality of these enzymes is similar to the bifunctional dehydrogenase from Chloroflexus aurantiacus, there is little sequence similarity. Both malonyl-CoA reductase enzyme candidates have high sequence similarity to aspartate- semialdehyde dehydrogenase, an enzyme catalyzing the reduction and concurrent

dephosphorylation of aspartyl-4-phosphate to aspartate semialdehyde. Additional gene candidates can be found by sequence homology to proteins in other organisms including Sulfolobus solfataricus and Sulfolobus acidocaldarius and have been listed below. Yet another candidate for CoA-acylating aldehyde dehydrogenase is the aid gene from

Clostridium beijerinckii (Toth, Appl. Environ. Microbiol. 65:4973-4980 (1999). This enzyme has been reported to reduce acetyl-CoA and butyryl-CoA to their corresponding aldehydes. This gene is very similar to eutE that encodes acetaldehyde dehydrogenase of Salmonella typhimurium and E. coli (Toth, Appl. Environ. Microbiol. 65:4973-4980 (1999).

Chain length specificity ranges of selected aldehyde-forming acyl-CoA reductase enzymes are show in the table below.

Step G. Acyl-CoA reductase (alcohol forming)

Bifunctional alcohol-forming acyl-CoA reductase enzymes catalyze step G (i.e. step E and F) of Figures 1 and 6. Enzymes with this activity include adhE of E. coli (Kessler et al, FEBS.Lett. 281 :59-63 (1991))) and adhE2 of Clostridium acetobutylicum (Fontaine et al, J.Bacteriol. 184:821-830 (2002))). The E. coli enzyme is active on C2 substrates, whereas the C. acetobutylicum enzyme has a broad substrate range that spans C2-C8 (Dekishima et al, J Am Chem Soc 133: 11399-11401 (2011)). The C. acetobutylicum enzymes encoded by bdh I and bdh II (Walter, et al, J. Bacteriol. 174:7149-7158 (1992)), reduce acetyl-CoA and butyryl-CoA to ethanol and butanol, respectively. The adhE gene produce from Leuconostoc mesenteroides is active on acetyl-CoA and isobutyryl-CoA (Kazahaya et al.,

J. Gen.Appl.Microbiol. 18:43-55 (1972); Koo et al, Biotechnol Lett, 27:505-510 (2005)). Enzyme candidates in other organisms including Roseiflexus castenholzii, Erythrobacter sp. NAP1 and marine gamma proteobacterium HTCC2080 can be inferred by sequence similarity. Longer chain acyl-CoA molecules can be reduced to their corresponding alcohols by enzymes such as the jojoba (Simmondsia chinensis) FAR which encodes an alcohol- forming fatty acyl-CoA reductase. Its overexpression in E. coli resulted in FAR activity and the accumulation of C16-C18 fatty alcohols (Metz et al, Plant Physiol, 122:635-644 (2000)). FAR enzymes in Arabidopsis thaliana include the gene products of At3gll980 and

At3g44560 (Doan et al, J Plant Physiol 166 (2006)). Bifunctional prokaryotic FAR enzymes are found in Marinobacter aquaeolei VT8 (Hofvander et al, FEBS Lett 3538-43 (2011)), Marinobacter algicola and Oceanobacter strain RED65 (US Pat Appl 20110000125). Other suitable enzymes include bfar from Bombyx mori, mfarl and mfar2 from Mus musculus; mfar2 from Mus musculus; acrMl from Acinetobacter sp. Ml; and hfar from H. sapiens.

Chain length specificity ranges of selected alcohol-forming acyl-CoA reductase enzymes are show in the table below. Chain length Gene Organism

C2 adhE Escherichia coli

C2-C8 adhe2 Clostridium acetobutylicum

C14-C16 At3gl l980 Arabidopsis thaliana

C16 At3g44560 Arabidopsis thaliana

C16-C18 FAR Simmondsia chinensis

C14-C18 FAR Marinobacter aquaeolei

Step F. Fatty aldehyde reductase

Exemplary genes encoding enzymes that catalyze the conversion of an aldehyde to alcohol (i.e., alcohol dehydrogenase or equivalently aldehyde reductase) include alrA encoding a medium-chain alcohol dehydrogenase for C2-C14 (Tani et al.,

Appl.Environ.Microbiol. 66:5231-5235 (2000)), yqhD and fucO from E. coli (Sulzenbacher et al, 342:489-502 (2004)), and bdh I and bdh II from C. acetobutylicum which converts butyryaldehyde into butanol (Walter et al, J Bacteriol 174:7149-7158 (1992)). The alrA gene product showed no activity on aldehydes longer than C14, and favored the reductive direction (Tani et al, supra). YqhD catalyzes the reduction of a wide range of aldehydes using NADPH as the cofactor, with a preference for chain lengths longer than C(3) (Sulzenbacher et al, J Mol Biol 342:489-502 (2004);Perez et al, J Biol.Chem. 283:7346-7353 (2008)). The adhA gene product from Zymomonas mobilis has been demonstrated to have activity on a number of aldehydes including formaldehyde, acetaldehyde, propionaldehyde, butyraldehyde, and acrolein (Kinoshita et al., Appl Microbiol Biotechnol 22:249-254 (1985)). Additional aldehyde reductase candidates are encoded by bdh in C. saccharoperbutylacetonicum and Cbei_1722, Cbei_2181 and Cbei_2421 in C. beijerinckii. The alcohol dehydrogenase from Leifsonia sp. S749 shows maximal activity on medium chain-length substrates of length C6- C7 (Inoue et al, AEM 71 : 3633-3641 (2005). The adh gene product of Pseudomonas putida is active on substrates of length C3-C10 (Nagashima et al, J Ferment Bioeng 82:328-33(1996)). The alcohol dehydrogenase enzymes ADH1 and ADH2 of Geobacillus thermodenitrificans oxidize alcohols up to a chain length of C30 (Liu et al, Physiol Biochem 155:2078-85 (2009)).

Protein ( ,cn Bank ID GI Number Organism

alrA BAB 12273.1 9967138 Acinetobacter sp. strain M-l

ADH2 NP 014032.1 6323961 Saccharomyces cerevisiae yqhD NP 417484.1 16130909 Escherichia coli

fucO NP 417279.1 16130706 Escherichia coli bdh l NP 349892.1 15896543 Clostridium acetobutylicum bdh II NP 349891.1 15896542 Clostridium acetobutylicum adhA YP 162971.1 56552132 Zymomonas mobilis

bdh BAF45463.1 124221917 Clostridium

saccharoperbutylacetonicum

Cbei 1722 YP 001308850 150016596 Clostridium beijerinckii

Cbei 2181 YP 001309304 150017050 Clostridium beijerinckii

Cbei 2421 YP 001309535 150017281 Clostridium beijerinckii lsadh BAD99642.1 67625613 Leifsonia sp. S749

adh Pseudomonas putida

Native alcohol dehydrogenases also convert aldehyde substrates to alcohol products. To date, seven alcohol dehydrogenases, ADHI-ADHVII, have been reported in S. cerevisiae (de Smidt et al, FEMS Yeast Res 8:967-78 (2008)). ADHl (GI: 1419926) is the key enzyme responsible for reducing acetaldehyde to ethanol in the cytosol under anaerobic conditions. In K. lactis, two NAD-dependent cytosolic alcohol dehydrogenases have been identified and characterized. These genes also show activity for other aliphatic alcohols. The genes ADHl (GI: 113358) and ADHII (GI:51704293) are preferentially expressed in glucose-grown cells (Bozzi et al, Biochim Biophys Acta 1339:133-142 (1997)). Cytosolic alcohol dehydrogenases are encoded by ADHl (GL608690) in C albicans, ADHl (GL3810864) in S pombe, ADHl (GL5802617) in Y. lipolytica, ADHl (GI:2114038) and ADHII (GI:2143328)in Pichia stipitis or Scheffersomyces stipitis (Passoth et al, Yeast 14: 1311-23 (1998)). Candidate alcohol dehydrogenases are shown the table below.

Protein ( ,cn Bank ID GI number Organism

SADH BAA24528.1 2815409 Candida parapsilosis

ADHl NP 014555.1 6324486 Saccharomyces cerevisiae s288c

ADH2 NP 014032.1 6323961 Saccharomyces cerevisiae s288c

ADH3 NP 013800.1 6323729 Saccharomyces cerevisiae s288c

ADH4 NP 011258.2 269970305 Saccharomyces cerevisiae s288c

ADH5 (SFA1) NP 010113.1 6320033 Saccharomyces cerevisiae s288c

ADH6 NP 014051.1 6323980 Saccharomyces cerevisiae s288c

ADH7 NP 010030.1 6319949 Saccharomyces cerevisiae s288c adhP CAA44614.1 2810 Kluyveromyces lactis

ADHl P20369.1 113358 Kluyveromyces lactis

ADH2 CAA45739.1 2833 Kluyveromyces lactis

ADH3 P49384.2 51704294 Kluyveromyces lactis

ADHl YP 001126968.1 138896515 Geobacillus thermodenitrificans

ADH2 YP 001125863.1 138895410 Geobacillus thermodenitrificans Substrate specificity ranges of selected alcohol dehydrogenase enzymes are show in the table below.

Step O. Elongase Elongase (ELO) enzymes utilize malonyl-CoA to add a C2 unit to a growing acyl-

CoA chain. This process also involves decarboxylation and is thus largely irreversible.

Trypanosoma brucei, a eukaryotic human parasite, is known to produce long chain fatty acids using an elongase system. The process is initiated by butyryl-CoA. In particular, the ELO system esterifies the growing fatty acid chain to CoA intermediates rather than ACP intermediates like the bacterial and other microbial counterparts (Lee et al, Cell 126, 691-699, 2006; Cronan, Cell, 126, 2006). This is in contrast to typical bacterial fatty acid elongation which is initiated following the formation of acetoacetyl acyl-ACP from malonyl-ACP. So far, four ELOs (encoded by ELO 1-4) that are homologous to their animal counterparts have been found in T. brucei (Lee et al, Nature Reviews Microbiology, Vol 5, 287-297, 2007). ELO 1-3 together account for synthesis of saturated fatty acids up to a chain length of CI 8. ELOl converts C4 to CIO, EL02 extends the chain length from CIO to myristate (CI 4), and EL03 extends myristate to CI 8. There is some overlap in ELO specificity; for example, ELOl can extend a CIO primer to CI 2, albeit with low activity. EL04 is an example of an ELO that is specific for poly unsaturated fatty acids (PUFAs). It extends arachidonate (C20:4) by two carbon atoms. Several additional ELO enzymes can be found by sequence homology (see Lee et al, Nature Reviews Microbiology, Vol 5, 287-297, 2007).

Elongase enzymes are found in several compartments including the mitochondria, endoplasmic reticulum, proteoliposomes and peroxisomes. For example, some yeast such as S. cerevisiae are able to synthesize long-chain fatty acids of chain length C16 and higher via a mitochondrial elongase which accepts exogenous or endogenous acyl-CoA substrates

(Bessoule et al, FEBS Lett 214: 158-162 (1987)). This system requires ATP for activity. The endoplasmic reticulum also has an elongase system for synthesizing very long chain fatty acids (CI 8+) from acyl-CoA substrates of varying lengths (Kohlwein et al, Mol Cell Biol 21 : 109-25 (2001)). Genes involved in this system include TSC13, EL02 and EL03. ELOl catalyzes the elongation of C12 acyl-CoAs to C16-C18 fatty acids.

Those skilled in the art also can obtain nucleic acids encoding any or all of the malonyl-CoA independent FAS pathway or acyl-reduction pathway enzymes by cloning using known sequences from available sources. For example, any or all of the encoding nucleic acids for the malonyl-CoA independent FAS pathway can be readily obtained using methods well known in the art from E. gracilis as this pathway has been well characterized in this organism. E. gracilis encoding nucleic acids can be isolated from, for example, an E. gracilis cDNA library using probes of known sequence. The probes can be designed with whole or partial DNA sequences from the following EST sequences from the publically available sequence database TBestDB (http://tbestdb.bcm.umontreal.ca). The nucleic acids generated from this process can be inserted into an appropriate expression vector and transformed into E. coli or other microorganisms to generate fatty alcohols, fatty aldehydes or fatty acids production organisms of the invention.

Thiolase (Figure 1A): ELL00002550, ELL00002493,ELL00000789

3-Hydroxyacyl-CoA dehydrogenase (Figure IB): ELL00000206, ELL00002419, ELL00006286, ELL00006656

Enoyl-CoA hydratase (Figure 1C): ELL00005926, ELL00001952, ELL00002235, ELL00006206

Enoyl-CoA reductase (Figure ID): ELL00002199, ELL00002335, ELL00002648 Acyl-CoA reductase (Figure IE; 1E/F): ELL00002572, ELL00002581, ELL00000108

Alternatively, the above EST sequences can be used to identify homologue polypeptides in GenBank through BLAST search. The resulting homologue polypeptides and their corresponding gene sequences provide additional encoding nucleic acids for

transformation into E. coli or other microorganisms to generate the fatty alcohols, fatty aldehydes or fatty acids producing organisms of the invention. Listed below are exemplary homologue polypeptide and their gene accession numbers in GenBank which are applicable for use in the non-naturally occurring organisms of the invention.

Ketoacyl-CoA acyltransferase (or ketoacyl-CoA thiolase)

3-Hydroxyacyl-CoA dehydrogenase

Enoyl-CoA hydratase

Protein GenBank ID GI Organism

number

Tb927.3.4850 XP 844077 72387305 Trypanosoma brucei

TcOO.1047053509701.10 XP_802711 71399112 Trypanosoma cruzi strain CL

Brener

PputGBl_3629 YP 0016698 167034625 Pseudomonas putida GB-1

56 Enoyl-CoA reductase

In addition to the above exemplary encoding nucleic acids, nucleic acids other than those within the MI-FAE cycle, MD-FAE and/or termination pathways of the invention also can be introduced into a host organism for further production of fatty alcohols, fatty aldehydes or fatty acids. For example, the Ralstonia eutropha BktB and PhbB genes catalyze the condensation of butyryl-CoA and acetyl-CoA to form β-keto-hexanoyl-CoA and the reduction of β-keto-hexanoyl-CoA to 3-hydroxy-hexanoyl-CoA (Fukui et al.,

Biomacromolecules 3:618-624 (2002)). To improve the production of fatty alcohols, exogenous DNA sequences encoding for these specific enzymes can be expressed in the production host of interest. Furthermore, the above described enzymes can be subjected to directed evolution to generate improved versions of these enzymes with high activity and high substrate specificity. A similar approach also can be utilized with any or all other enzymatic steps in the fatty alcohol, fatty aldehyde or fatty acid producing pathways of the invention to, for example, improve enzymatic activity and/or specificity and/or to generate a fatty alcohol, a fatty aldehyde or a fatty acid of a predetermined chain length or lengths.

EXAMPLE II

Pathways For Producing Cytosolic Acetyl-CoA from Cytosolic Pyruvate

The following example describes exemplary pathways for the conversion of cytosolic pyruvate and threonine to cytosolic acetyl-CoA, as shown in Figure 2.

Pathways for the conversion of cytosolic pyruvate and threonine to cytosolic acetyl- CoA could enable deployment of a cytosolic fatty alcohol, fatty aldehyde or fatty acid production pathway that originates from acetyl-CoA. Several pathways for converting cytosolic pyruvate to cytosolic acetyl-CoA are shown in Figure 2. Direct conversion of pyruvate to acetyl-CoA can be catalyzed by pyruvate dehydrogenase, pyruvate formate lyase, pyruvate :NAD(P) oxidoreductase or pyruvate :ferredoxin oxidoreductase. If a pyruvate formate lyase is utilized, the formate byproduct can be further converted to C02 by formate dehydrogenase or formate hydrogen lyase.

Indirect conversion of pyruvate to acetyl-CoA can proceed through several alternate routes. Pyruvate can be converted to acetaldehyde by a pyruvate decarboxylase.

Acetaldehyde can then converted to acetyl-CoA by an acylating (CoA-dependent) acetaldehyde dehydrogenase. Alternately, acetaldehyde generated by pyruvate decarboxylase can be converted to acetyl-CoA by the "PDH bypass" pathway. In this pathway,

acetaldehyde is oxidized by acetaldehyde dehydrogenase to acetate, which is then converted to acetyl-CoA by a CoA ligase, synthetase or transferase. In another embodiment, the acetate intermediate is converted by an acetate kinase to acetyl-phosphate that is then converted to acetyl-CoA by a phosphotransacetylase. In yet another embodiment, pyruvate is directly converted to acetyl-phosphate by a pyruvate oxidase (acetyl-phosphate forming). Conversion of pyruvate to acetate is also catalyzed by acetate-forming pyruvate oxidase. Cytosolic acetyl-CoA can also be synthesized from threonine by expressing a native or heterologous threonine aldolase (Figure 5 J) (van Maris et al, AEM 69:2094-9 (2003)). Threonine aldolase converts threonine into acetaldehyde and glycine. The acetaldehyde product is subsequently converted to acetyl-CoA by various pathways described above.

Gene candidates for the acetyl-CoA forming enzymes shown in Figure 2 are described below.

Pyruvate oxidase (acetate-forming) (Figure 2 A) or pyruvate :quinone oxidoreductase (PQO) can catalyze the oxidative decarboxylation of pyruvate into acetate, using ubiquione (EC 1.2.5.1) or quinone (EC 1.2.2.1) as an electron acceptor. The E. coli enzyme, PoxB, is localized on the inner membrane (Abdel-Hamid et al., Microbiol 147: 1483-98 (2001)). The enzyme has thiamin pyrophosphate and flavin adenine dinucleotide (FAD) cofactors (Koland and Gennis, Biochemistry 21 :4438-4442 (1982)); O'Brien et al, Biochemistry 16:3105-3109 (1977); O'Brien and Gennis, J. Biol. Chem. 255:3302-3307 (1980)). PoxB has similarity to pyruvate decarboxylase of S. cerevisiae and Zymomonas mobilis. The pqo transcript of Cory neb acterium glutamicum encodes a quinone-dependent and acetate-forming pyruvate oxidoreductase (Schreiner et al, J Bacteriol 188:1341-50 (2006)). Similar enzymes can be inferred by sequence homology. Protein ( ,cn Bank ID GI Number Organism

poxB NP 415392.1 16128839 Escherichia coli

pqo YP 226851.1 62391449 Corymb acterium glutamicum poxB YP 309835.1 74311416 Shigella sonnei

poxB ZP 03065403.1 194433121 Shigella dysenteriae

The acylation of acetate to acetyl-CoA (Figure 2B) can be catalyzed by enzymes with acetyl-CoA synthetase, ligase or transferase activity. Two enzymes that can catalyze this reaction are AMP-forming acetyl-CoA synthetase or ligase (EC 6.2.1.1) and ADP-forming acetyl-CoA synthetase (EC 6.2.1.13). AMP-forming acetyl-CoA synthetase (ACS) is the predominant enzyme for activation of acetate to acetyl-CoA. Exemplary ACS enzymes are found in E. coli (Brown et al, J. Gen. Microbiol. 102:327-336 (1977)), Ralstonia eutropha (Priefert and Steinbuchel, J. Bacteriol. 174:6590-6599 (1992)), Methanothermobacter thermautotrophicus (Ingram-Smith and Smith, Archaea 2:95-107 (2007)), Salmonella enterica (Gulick et al, Biochemistry 42:2866-2873 (2003)) and Saccharomyces cerevisiae (Jogl and Tong, Biochemistry 43: 1425-1431 (2004)). ADP-forming acetyl-CoA synthetases are reversible enzymes with a generally broad substrate range (Musfeldt and Schonheit, J. Bacteriol. 184:636-644 (2002)). Two isozymes of ADP-forming acetyl-CoA synthetases are encoded in the Archaeoglobus fulgidus genome by are encoded by AF1211 and AF1983 (Musfeldt and Schonheit, supra (2002)). The enzyme from Haloarcula marismortui

(annotated as a succinyl-CoA synthetase) also accepts acetate as a substrate and reversibility of the enzyme was demonstrated (Brasen and Schonheit, Arch. Microbiol. 182:277-287 (2004)). The ACD encoded by PAE3250 from hyperthermophilic crenarchaeon

Pyrobaculum aerophilum showed the broadest substrate range of all characterized ACDs, reacting with acetate, isobutyryl-CoA (preferred substrate) and phenylacetyl-CoA (Brasen and Schonheit, supra (2004)). Directed evolution or engineering can be used to modify this enzyme to operate at the physiological temperature of the host organism. The enzymes from A.fulgidus, H. marismortui and P. aerophilum have all been cloned, functionally expressed, and characterized in E. coli (Brasen and Schonheit, supra (2004); Musfeldt and Schonheit, supra (2002)). Additional candidates include the succinyl-CoA synthetase encoded by sucCD in E. coli (Buck et al, Biochemistry 24:6245-6252 (1985)) and the acyl-CoA ligase from Pseudomonas putida (Fernandez- Valverde et al, Appl. Environ. Microbiol. 59: 1149- 1154 (1993)). The aforementioned proteins are shown below. Protein ( ,cn Bank ID GI Number Organism

acs AAC77039.1 1790505 Escherichia coli

acoE AAA21945.1 141890 Ralstonia eutropha

acsl ABC87079.1 86169671 Methanothermobacter

thermautotrophicus acsl AAL23099.1 16422835 Salmonella enterica

ACS1 Q01574.2 257050994 Saccharomyces cerevisiae

AF1211 NP 070039.1 11498810 Archaeoglobus fulgidus

AF1983 NP 070807.1 11499565 Archaeoglobus fulgidus scs YP 135572.1 55377722 Haloarcula marismortui

PAE3250 NP 560604.1 18313937 Pyrobaculum aerophilum str.

IM2

sucC NP 415256.1 16128703 Escherichia coli

sucD AAC73823.1 1786949 Escherichia coli

paaF AAC24333.2 22711873 Pseudomonas putida

The acylation of acetate to acetyl-CoA can also be catalyzed by CoA transferase enzymes (Figure 2B). Numerous enzymes employ acetate as the CoA acceptor, resulting in the formation of acetyl-CoA. An exemplary CoA transferase is acetoacetyl-CoA transferase, encoded by the E. coli atoA (alpha subunit) and atoD (beta subunit) genes (Korolev et al, Acta Crystallogr.D.Biol.Crystallogr. 58:2116-2121 (2002); Vanderwinkel et al, 33:902-908 (1968)). This enzyme has a broad substrate range (Sramek et al., Arch Biochem Biophys 171 : 14-26 (1975)) and has been shown to transfer the CoA moiety to acetate from a variety of branched and linear acyl-CoA substrates, including isobutyrate (Matthies et al, Appl Environ.Microbiol 58: 1435-1439 (1992)), valerate (Vanderwinkel et al,

Biochem.Biophys.Res.Commun. 33:902-908 (1968)) and butanoate (Vanderwinkel et al, Biochem.Biophys.Res.Commun. 33:902-908 (1968)). Similar enzymes exist in

Cory neb acterium glutamicum ATCC 13032 (Duncan et al, 68:5186-5190 (2002)),

Clostridium acetobutylicum (Cary et al., Appl Environ Microbiol 56: 1576-1583 (1990); Wiesenborn et al, Appl Environ Microbiol 55:323-329 (1989)), and Clostridium

saccharoperbutylacetonicum (Kosaka et al., Biosci.Biotechnol Biochem. 71 :58-68 (2007)).

Gene GI # Accession No. Organism

atoA 2492994 P76459.1 Escherichia coli

atoD 2492990 P76458.1 Escherichia coli

actA 62391407 YP 226809.1 Coryneb acterium glutamicum

cg0592 62389399 YP 224801.1 Coryneb acterium glutamicum

ctfA 15004866 NP 149326.1 Clostridium acetobutylicum

ctfB 15004867 NP 149327.1 Clostridium acetobutylicum ctfA 31075384 AAP42564.1 Clostridium saccharoperbutylacetonicum ctfB 31075385 AAP42565.1 Clostridium saccharoperbutylacetonicum

Acetate kinase (EC 2.7.2.1) can catalyzes the reversible ATP-dependent

phosphorylation of acetate to acetylphosphate (Figure 2C). Exemplary acetate kinase enzymes have been characterized in many organisms including E. coli, Clostridium acetobutylicum and Methanosarcina thermophila (Ingram-Smith et al., J. Bacteriol.

187:2386-2394 (2005); Fox and Roseman, J. Biol. Chem. 261 : 13487-13497 (1986); Winzer et al, Microbioloy 143 (Pt 10):3279-3286 (1997)). Acetate kinase activity has also been demonstrated in the gene product of E. coli purT (Marolewski et al, Biochemistry 33:2531- 2537 (1994). Some butyrate kinase enzymes (EC 2.7.2.7), for example bukl and buk2 from Clostridium acetobutylicum, also accept acetate as a substrate (Hartmanis, M.G., J. Biol. Chem. 262:617-621 (1987)). Homologs exist in several other organisms including

Salmonella enterica and Chlamydomonas reinhardtii.

The formation of acetyl-CoA from acetyl-phosphate can be catalyzed by

phosphotransacetylase (EC 2.3.1.8) (Figure 2D). The pta gene from E. coli encodes an enzyme that reversibly converts acetyl-CoA into acetyl-phosphate (Suzuki, T., Biochim. Biophys. Acta 191 :559-569 (969)). Additional acetyltransferase enzymes have been characterized in Bacillus subtilis (Rado and Hoch, Biochim. Biophys. Acta 321 : 114-125 (1973), Clostridium kluyveri (Stadtman, E., Methods Enzymol. 1 :5896-599 (1955), and Thermotoga maritima (Bock et al. , J. Bacteriol. 181 : 1861-1867 (1999)). This reaction can also be catalyzed by some phosphotranbutyrylase enzymes (EC 2.3.1.19), including the ptb gene products from Clostridium acetobutylicum (Wiesenborn et al, App. Environ. Microbiol. 55:317-322 (1989); Walter et al, Gene 134:107-111 (1993)). Additional ptb genes are found in butyrate-producing bacterium L2-50 (Louis et al, J. Bacteriol. 186:2099-2106 (2004) and Bacillus megaterium (Vazquez et al, Curr. Microbiol. 42:345-349 (2001). Homologs to the E. colipta gene exist in several other organisms including Salmonella enterica and

Chlamydomonas reinhardtii.

Pyruvate decarboxylase (PDC) is a key enzyme in alcoholic fermentation, catalyzing the decarboxylation of pyruvate to acetaldehyde (Figure 2E). The PDC1 enzyme from Saccharomyces cerevisiae has been extensively studied (Killenberg-Jabs et al,

EurJ.Biochem. 268: 1698-1704 (2001); Li et al, Biochemistry. 38: 10004-10012 (1999); ter Schure et al, Appl.Environ.Microbiol. 64: 1303-1307 (1998)). Other well-characterized PDC enzymes are found in Zymomonas mobilus (Siegert et al, Protein Eng Des Sel 18:345-357 (2005)), Acetobacter pasteurians (Chandra et al, 176:443-451 (2001)) and Kluyveromyces lactis (Krieger et al, 269:3256-3263 (2002)). The PDC1 and PDC5 enzymes of

Saccharomyces cerevisiae are subject to positive transcriptional regulation by PDC2 (Hohmann et al, Mol Gen Genet 241 :657-66 (1993)). Pyruvate decarboxylase activity is also possessed by a protein encoded by CTRG 03826 (GL255729208) in Candida tropicalis, PDC1 (GI number: 1226007) in Kluyveromyces lactis, YALI0D10131g (GL50550349) in Yarrowia lipolytica, PAS_chr3_0188 (GL254570575) in Pichia pastoris, pyruvate decarboxylase (GI: GI: 159883897) in Schizosaccharomyces pombe, ANI_1_1024084 (GI: 145241548), ANI_1_796114 (GI:317034487), ANI_1_936024 (GI:317026934) and ANI_1_2276014 (GI:317025935) in Aspergillus niger. Protein ( ,cn Bank ID GI Organism

Number

pdc P06672.1 118391 Zymomonas mobilis pdcl P06169 30923172 Saccharomyces cerevisiae

Pdc2 NP 010366.1 6320286 Saccharomyces cerevisiae

Pdc5 NP 013235.1 6323163 Saccharomyces cerevisiae

CTRG 03826 XP 002549529 255729208 Candida tropicalis,

CU329670.1 :585597.587312 CAA90807 159883897 Schizosaccharomyces

pombe

YALI0D10131g XP 502647 50550349 Yarrowia lipolytica

PAS chr3 0188 XP 002492397 254570575 Pichia pastoris

pdc Q8L388 20385191 Acetobacter pasteurians pdcl Q 12629 52788279 Kluyveromyces lactis

ANI 1 1024084 XP 001393420 145241548 Aspergillus niger

ANI 1 796114 XP 001399817 317026934 Aspergillus niger

ANI 1 936024 XP 001396467 317034487 Aspergillus niger

ANI 1 2276014 XP 001388598 317025935 Aspergillus niger

Aldehyde dehydrogenase enzymes in EC class 1.2.1 catalyze the oxidation of acetaldehyde to acetate (Figure 2F). Exemplary genes encoding this activity were described above. The oxidation of acetaldehyde to acetate can also be catalyzed by an aldehyde oxidase with acetaldehyde oxidase activity. Such enzymes can convert acetaldehyde, water and C"2 to acetate and hydrogen peroxide. Exemplary aldehyde oxidase enzymes that have been shown to catalyze this transformation can be found in Bos taurus and Mus musculus (Garattini et al, Cell Mol Life Sci 65: 1019-48 (2008); Cabre et al, Biochem Soc Trans 15:882-3 (1987)). Additional aldehyde oxidase gene candidates include the two flavin- and molybdenum- containing aldehyde oxidases of Zea mays, encoded by zmAO-1 and zmAO-2 (Sekimoto et al, J Biol Chem 272: 15280-85 (1997)).

Pyruvate oxidase (acetyl-phosphate forming) can catalyze the conversion of pyruvate, oxygen and phosphate to acetyl-phosphate and hydrogen peroxide (Figure 2G). This type of pyruvate oxidase is soluble and requires the cofactors thiamin diphosphate and flavin adenine dinucleotide (FAD). Acetyl-phosphate forming pyruvate oxidase enzymes can be found in lactic acid bacteria Lactobacillus delbrueckii and Lactobacillus plantarum (Lorquet et al., J Bacteriol 186:3749-3759 (2004); Hager et al, FedProc 13:734-38 (1954)). A crystal structure of the L. plantarum enzyme has been solved (Muller et al, (1994)). In

Streptococcus sanguinis and Streptococcus pneumonia, acetyl-phosphate forming pyruvate oxidase enzymes are encoded by the spxB gene (Spellerberg et al, Mol Micro 19:803-13 (1996); Ramos-Montanez et al, Mol Micro 67:729-46 (2008)). The SpxR was shown to positively regulate the transcription of spxB in S. pneumoniae (Ramos-Montanez et al., supra). A similar regulator in S. sanguinis was identified by sequence homology.

Introduction or modification of catalase activity can reduce accumulation of the hydrogen peroxide product.

The pyruvate dehydrogenase (PDH) complex catalyzes the conversion of pyruvate to acetyl-CoA (Figure 2H). The E. coli PDH complex is encoded by the genes aceEF and IpdA. Enzyme engineering efforts have improved the E. coli PDH enzyme activity under anaerobic conditions (Kim et al, J.Bacteriol. 190:3851-3858 (2008); Kim et al,

Appl.Environ.Microbiol. 73:1766-1771 (2007); Zhou et al, Biotechnol.Lett. 30:335-342 (2008)). In contrast to the E. coli PDH, the B. subtilis complex is active and required for growth under anaerobic conditions (Nakano et al, 179:6749-6755 (1997)). The Klebsiella pneumoniae PDH, characterized during growth on glycerol, is also active under anaerobic conditions (Menzel et al, 56: 135-142 (1997)). Crystal structures of the enzyme complex from bovine kidney (Zhou et al. , 98 : 14802- 14807 (2001 )) and the E2 catalytic domain from Azotobacter vinelandii are available (Mattevi et al., Science. 255: 1544-1550 (1992)). Some mammalian PDH enzymes complexes can react on alternate substrates such as 2- oxobutanoate. Comparative kinetics of Rattus norvegicus PDH and BCKAD indicate that BCKAD has higher activity on 2-oxobutanoate as a substrate (Paxton et al., Biochem.J. 234:295-303 (1986)). The S. cerevisiae PDH complex canconsist of an E2 (LAT1) core that binds El (PDAl, PDBI), E3 (LPDI), and Protein X (PDXI) components (Pronk et al, Yeast 12: 1607-1633 (1996)). The PDH complex of S. cerevisiae is regulated by phosphorylation of El involving PKP1 (PDH kinase I), PTC5 (PDH phosphatase I), PKP2 and PTC6. Modification of these regulators may also enhance PDH activity. Coexpression of lipoyl ligase {LplA of E. coli and AIM22 in S. cerevisiae) with PDH in the cytosol may be necessary for activating the PDH enzyme complex. Increasing the supply of cytosolic lipoate, either by modifying a metabolic pathway or media supplementation with lipoate, may also improve PDH activity.

As an alternative to the large multienzyme PDH complexes described above, some organisms utilize enzymes in the 2-ketoacid oxidoreductase family (OFOR) to catalyze acylating oxidative decarboxylation of 2-keto-acids. Unlike the PDH complexes, PFOR enzymes contain iron-sulfur clusters, utilize different cofactors and use ferredoxin or flavodixin as electron acceptors in lieu of NAD(P)H. Pyruvate ferredoxin oxidoreductase (PFOR) can catalyze the oxidation of pyruvate to form acetyl-CoA (Figure 2H). The PFOR from Desulfovibrio africanus has been cloned and expressed in E. coli resulting in an active recombinant enzyme that was stable for several days in the presence of oxygen (Pieulle et al., J Bacteriol. 179:5684-5692 (1997)). Oxygen stability is relatively uncommon in PFORs and is believed to be conferred by a 60 residue extension in the polypeptide chain of the D.

africanus enzyme. The M. thermoacetica PFOR is also well characterized (Menon et al., Biochemistry 36:8484-8494 (1997)) and was even shown to have high activity in the direction of pyruvate synthesis during autotrophic growth (Furdui et al, J Biol Chem.

275:28494-28499 (2000)). Further, E. coli possesses an uncharacterized open reading frame, ydbK, that encodes a protein that is 51% identical to the M. thermoacetica PFOR. Evidence for pyruvate oxidoreductase activity in E. coli has been described (Blaschkowski et al, Eur. J Biochem. 123:563-569 (1982)). Several additional PFOR enzymes are described in Ragsdale, Chem.Rev. 103:2333-2346 (2003). Finally, flavodoxin reductases (e.g., fqrB from

Helicobacter pylori or Campylobacter jejuni (St Maurice et al., J.Bacteriol. 189:4764-4773 (2007))) or Rnf-type proteins (Seedorf et al, Proc.NatlAcad.Sci. USA. 105:2128-2133 (2008); Herrmann et al, J.Bacteriol. 190:784-791 (2008)) provide a means to generate NADH or NADPH from the reduced ferredoxin generated by PFOR. These proteins are identified below.

Pyruvate formate-lyase (PFL, EC 2.3.1.54) (Figure 2H), encoded by pflB in E. coli, can convert pyruvate into acetyl-CoA and formate. The activity of PFL can be enhanced by an activating enzyme encoded by pflA (Knappe et al, Proc.NatlAcad.Sci U.S.A 81 :1332- 1335 (1984); Wong et al, Biochemistry 32:14102-14110 (1993)). Keto-acid formate-lyase (EC 2.3.1.-), also known as 2-ketobutyrate formate-lyase (KFL) and pyruvate formate-lyase 4, is the gene product of tdcE in E. coli. This enzyme catalyzes the conversion of 2- ketobutyrate to propionyl-CoA and formate during anaerobic threonine degradation, and can also substitute for pyruvate formate-lyase in anaerobic catabolism (Simanshu et al., JBiosci. 32: 1195-1206 (2007)). The enzyme is oxygen-sensitive and, like PflB, can require post- translational modification by PFL-AE to activate a glycyl radical in the active site

(Hesslinger et al., Mol.Microbiol 2Ί ΊΊ-492 (1998)). A pyruvate formate-lyase from Archaeoglobus fulgidus encoded by pflD has been cloned, expressed in E. coli and characterized (Lehtio et al, Protein Eng Des Sel 17:545-552 (2004)). The crystal structures of the A.fulgidus and E. coli enzymes have been resolved (Lehtio et al, J Mol.Biol. 357:221- 235 (2006); Leppanen et al, Structure. 7:733-744 (1999)). Additional PFL and PFL-AE candidates are found in Lactococcus lactis (Melchiorsen et al, Appl Microbiol Biotechnol 58:338-344 (2002)), and Streptococcus mutans (Takahashi-Abbe et al, Oral. Microbiol Immunol. 18:293-297 (2003)), Chlamydomonas reinhardtii (Hemschemeier et al.,

Eukaryot.Cell 7:518-526 (2008b); Atteia et al, J.Biol.Chem. 281 :9909-9918 (2006)) and Clostridium pasteurianum (Weidner et al, J Bacteriol. 178:2440-2444 (1996)).

If a pyruvate formate lyase is utilized to convert pyruvate to acetyl-CoA, coexpression of a formate dehydrogenase or formate hydrogen lyase enzyme will converte formate to carbon dioxide. Formate dehydrogenase (FDH) catalyzes the reversible transfer of electrons from formate to an acceptor. Enzymes with FDH activity utilize various electron carriers such as, for example, NADH (EC 1.2.1.2), NADPH (EC 1.2.1.43), quinols (EC 1.1.5.6), cytochromes (EC 1.2.2.3) and hydrogenases (EC 1.1.99.33). FDH enzymes have been characterized from Moorella thermoacetica (Andreesen and Ljungdahl, J Bacteriol 116:867- 873 (1973); Li et al, J Bacteriol 92:405-412 (1966); Yamamoto et al, J Biol Chem.

258: 1826-1832 (1983). The loci, Moth_2312 is responsible for encoding the alpha subunit of formate dehydrogenase while the beta subunit is encoded by Moth_2314 (Pierce et al.,

Environ Microbiol (2008)). Another set of genes encoding formate dehydrogenase activity with a propensity for C0 2 reduction is encoded by Sfum_2703 through Sfum_2706 in Syntrophobacter fumaroxidans (de Bok et al., Eur J Biochem. 270:2476-2485 (2003)); Reda et al, PNAS 105: 10654-10658 (2008)). A similar set of genes presumed to carry out the same function are encoded by CHY 0731 , CHY 0732, and CHY 0733 in C. hydrogenoformans (Wu et al, PLoS Genet 1 :e65 (2005)). Formate dehydrogenases are also found many additional organisms including C. carboxidivorans P7, Bacillus methanolicus, Burkholderia stabilis, Moorella thermoacetica ATCC 39073, Candida boidinii, Candida methylica, and Saccharomyces cerevisiae S288c.

Alternately, a formate hydrogen lyase enzyme can be employed to convert formate to carbon dioxide and hydrogen. An exemplary formate hydrogen lyase enzyme can be found in Escherichia coli. The E. coli formate hydrogen lyase consists of hydrogenase 3 and formate dehydrogenase-H (Maeda et al, Appl Microbiol Biotechnol 77:879-890 (2007)). It is activated by the gene product of fhlA . (Maeda et al. , Appl Microbiol Biotechnol 77 : 879-890 (2007)). The addition of the trace elements, selenium, nickel and molybdenum, to a fermentation broth has been shown to enhance formate hydrogen lyase activity (Soini et al., Microb. Cell Fact. 7:26 (2008)). Various hydrogenase 3, formate dehydrogenase and transcriptional activator genes are shown below. A formate hydrogen lyase enzyme also exists in the hyperthermophilic archaeon, Thermococcus litoralis (Takacs et al., BMC.Microbiol 8:88 (2008)). Additional formate hydrogen lyase systems have been found in Salmonella typhimurium, Klebsiella pneumoniae, Rhodospirillum rubrum,

Methanobacterium formicicum (Vardar-Schara et al., Microbial Biotechnology 1 : 107-125 (2008)).

Pyruvate :NADP oxidoreductase (PNO) catalyzes the conversion of pyruvate to acetyl-CoA. This enzyme is encoded by a single gene and the active enzyme is a homodimer, in contrast to the multi-subunit PDH enzyme complexes described above. The enzyme from Euglena gracilis is stabilized by its cofactor, thiamin pyrophosphate (Nakazawa et al, Arch Biochem Biophys 411 : 183-8 (2003)). The mitochondrial targeting sequence of this enzyme should be removed for expression in the cytosol. The PNO protein of E. gracilis protein and other NADP-dependant pyruvate :NADP+ oxidoreductase enzymes are listed in the table below.

Protein ( ,cn Bank ID GI Number Organism

PNO Q94IN5.1 33112418 Euglena gracilis

cgd4 690 XP 625673.1 66356990 Cryptosporidium parvum Iowa II

TPP PFOR PNO XP 002765111.11 294867463 Perkinsus marinus ATCC 50983 The NAD(P) dependent oxidation of acetaldehyde to acetyl-CoA (Figure 21) can be catalyzed by an acylating acetaldehyde dehydrogenase (EC 1.2.1.10). Acylating

acetaldehyde dehydrogenase enzymes of E. coli are encoded by adhE, eutE, and mhpF (Ferrandez et al, J Bacteriol 179:2573-81 (1997)). The Pseudomonas sp. CF600 enzyme, encoded by dmpF, participates in meta-cleavage pathways and forms a complex with 4- hydroxy-2-oxo valerate aldolase (Shingler et al, J Bacteriol 11 '4:711-24 (1992)).

Solventogenic organisms such as Clostridium acetobutylicum encode bifunctional enzymes with alcohol dehydrogenase and acetaldehyde dehydrogenase activities. The bifunctional C. acetobutylicum enzymes are encoded by bdh I and adhE2 (Walter, et al, J. Bacteriol.

174:7149-7158 (1992); Fontaine et al, J.Bacteriol. 184:821-830 (2002)). Yet another candidate for acylating acetaldehyde dehydrogenase is the aid gene from Clostridium beijerinckii (Toth, Appl. Environ. Microbiol. 65:4973-4980 (1999). This gene is very similar to the eutE acetaldehyde dehydrogenase genes of Salmonella typhimurium and E. coli (Toth, Appl. Environ. Microbiol. 65:4973-4980 (1999).

Threonine aldolase (EC 4.1.2.5) catalyzes the cleavage of threonine to glycine and acetaldehyde (Figure 2J). The Saccharomyces cerevisiae and Candida albicans enzymes are encoded by GLY1 (Liu et al, Eur J Biochem 245:289-93 (1997); McNeil et al, Yeast 16: 167- 75 (2000)). The ItaE and glyA gene products of E. coli also encode enzymes with this activity (Liu et al, Eur J Biochem 255:220-6 (1998)).

Protein ( ,cn Bank ID GI Number Organism

GLY1 NP 010868.1 6320789 Saccharomyces cerevisiae

GLY1 AAB64198.1 2282060 Candida albicans

ItaE AAC73957.1 1787095 Escherichia coli

glyA AAC75604.1 1788902 Escherichia coli EXAMPLE III

Pathways for Producing Acetyl-CoA from PEP and Pyruvate

Pathways for the conversion of cytosolic phosphoenolpyruvate (PEP) and pyruvate to cytosolic acetyl-CoA can also enable deployment of a cytosolic fatty alcohol, fatty aldehyde or fatty acid production pathway from acetyl-CoA. Figure 3 shows numerous pathways for converting PEP and pyruvate to acetyl-CoA.

The conversion of PEP to oxaloacetate is catalyzed in one, two or three enzymatic steps. Oxaloacetate is further converted to acetyl-CoA via malonate semialdehyde or malonyl-CoA intermediates. In one pathway, PEP carboxylase or PEP carboxykinase converts PEP to oxaloacetate (step A); oxaloacetate decarboxylase converts the oxaloacetate to malonate (step B); and malonate semialdehyde dehydrogenase (acetylating) converts the malonate semialdehyde to acetyl-CoA (step C). In another pathway pyruvate kinase or PEP phosphatase converts PEP to pyruvate (step N); pyruvate carboxylase converts the pyruvate to (step H); oxaloacetate decarboxylase converts the oxaloacetate to malonate (step B); and malonate semialdehyde dehydrogenase (acetylating) converts the malonate semialdehyde to acetyl-CoA (step C). In another pathway pyruvate kinase or PEP phosphatase converts PEP to pyruvate (step N); malic enzyme converts the pyruvate to malate (step L); malate dehydrogenase or oxidoreductase converts the malate to oxaloacetate (step M); oxaloacetate decarboxylase converts the oxaloacetate to malonate (step B); and malonate semialdehyde dehydrogenase (acetylating) converts the malonate semialdehyde to acetyl-CoA (step C). In another pathway, PEP carboxylase or PEP carboxykinase converts PEP to oxaloacetate (step A); oxaloacetate decarboxylase converts the oxaloacetate to malonate semialdehyde (step B); malonyl-CoA reductase converts the malonate semialdehyde to malonyl-CoA (step G); and malonyl-CoA decarboxylase converts the malonyl-CoA to acetyl-CoA (step (D). In another pathway, pyruvate kinase or PEP phosphatase converts PEP to pyruvate (step N); pyruvate carboxylase converts the pyruvate to oxaloacetate (step H); (oxaloacetate decarboxylase converts the oxaloacetate to malonate semialdehyde (step B); malonyl-CoA reductase converts the malonate semialdehyde to malonyl-CoA (step G); and malonyl-CoA

decarboxylase converts the malonyl-CoA to acetyl-CoA (step (D). In another pathway, pyruvate kinase or PEP phosphatase converts PEP to pyruvate (step N); malic enzyme converts the pyruvate to malate (step L); malate dehydrogenase or oxidoreductase converts the malate to oxaloacetate (step M); oxaloacetate decarboxylase converts the oxaloacetate to malonate semialdehyde (step B); malonyl-CoA reductase converts the malonate semialdehyde to malonyl-CoA (step G); and malonyl-CoA decarboxylase converts the malonyl-CoA to acetyl-CoA (step (D). In another pathway, PEP carboxylase or PEP carboxykinase converts PEP to oxaloacetate (step A); oxaloacetate decarboxylase converts the oxaloacetate to malonate semialdehyde (step B); malonate semialdehyde dehydrogenase converts the malonate semialdehyde to malonate (step J); malonyl-CoA synthetase or transferase converts the malonate to malonyl-CoA (step K); and malonyl-CoA decarboxylase converts the malonyl-CoA to acetyl-CoA (step D). In another pathway, pyruvate kinase or PEP phosphatase converts PEP to pyruvate (step N); pyruvate carboxylase converts the pyruvate to oxaloacetate (step H); oxaloacetate decarboxylase converts the oxaloacetate to malonate semialdehyde (step B); malonate semialdehyde dehydrogenase converts the malonate semialdehyde to malonate (step J); malonyl-CoA synthetase or transferase converts the malonate to malonyl-CoA (step K); and malonyl-CoA decarboxylase converts the malonyl-CoA to acetyl-CoA (step D). In another pathway, pyruvate kinase or PEP phosphatase converts PEP to pyruvate (step N); malic enzyme converts the pyruvate to malate (step L); malate dehydrogenase or oxidoreductase converts the malate to oxaloacetate (step M); oxaloacetate decarboxylase converts the oxaloacetate to malonate semialdehyde (step B); malonate semialdehyde dehydrogenase converts the malonate semialdehyde to malonate (step J); malonyl-CoA synthetase or transferase converts the malonate to malonyl- CoA (step K); and malonyl-CoA decarboxylase converts the malonyl-CoA to acetyl-CoA (step D). In another pathway, PEP carboxylase or PEP carboxykinase converts PEP to oxaloacetate (step A); oxaloacetate dehydrogenase or oxaloacetate oxidoreductase converts the oxaloacetate to malonyl-CoA (step F); and malonyl-CoA decarboxylase converts the malonyl-CoA to acetyl-CoA (step D). In another pathway, pyruvate kinase or PEP phosphatase converts PEP to pyruvate (step N); pyruvate carboxylase converts the pyruvate to oxaloacetate (step H); oxaloacetate dehydrogenase or oxaloacetate oxidoreductase converts the oxaloacetate to malonyl-CoA (step F); and malonyl-CoA decarboxylase converts the malonyl-CoA to acetyl-CoA (step D). In another pathway, pyruvate kinase or PEP phosphatase converts PEP to pyruvate (step N); malic enzyme converts the pyruvate to malate (step L); malate dehydrogenase or oxidoreductase converts the malate to oxaloacetate (step M); oxaloacetate dehydrogenase or oxaloacetate oxidoreductase converts the oxaloacetate to malonyl-CoA (step F); and malonyl-CoA decarboxylase converts the malonyl-CoA to acetyl-CoA (step D). Enzymes candidates for the reactions shown in Figure 3 are described below.

Enzyme candidates for several enzymes in Figure 3 have been described elsewhere herein. These include acetyl-CoA carboxylase, acetoacetyl-CoA synthase, acetoacetyl-CoA thiolase, malonyl-CoA reductase (also called malonate semialdehyde dehydrogenase

(acylating) , malate dehydrogenase.

1.1. n.a Oxidoreductase (alcohol to oxo)

Malate dehydrogenase or oxidoreductase catalyzes the oxidation of malate to oxaloacetate. Different carriers can act as electron acceptors for enzymes in this class. Malate dehydrogenase enzymes utilize NADP or NAD as electron acceptors. Malate dehydrogenase (Step M) enzyme candidates are described above in example 1 (Table 7, 23). Malate :quinone oxidoreductase enzymes (EC 1.1.5.4) are membrane-associated and utilize quinones, flavoproteins or vitamin K as electron acceptors. Malate :quinone oxidoreductase enzymes of E. coli, Helicobacter pylori and Pseudomonas syringae are encoded by mqo (Kather et al, J Bacteriol 182:3204-9 (2000); Mellgren et al, J Bacteriol 191 :3132-42 (2009)). The Cgl2001 gene of C. gluamicum also encodes an MQO enzyme (Mitsuhashi et al, Biosci Biotechnol Biochem 70:2803-6 (2006)).

Protein ( ,cn Bank ID GI Number Organism

mqo NP 416714.1 16130147 Escherichia coli

mqo NP 206886.1 15644716 Helicobacter pylori

mqo NP 790970.1 28868351 Pseudomonas syringae

Cgl2001 NP 601207.1 19553205 Corynebacterium glutamicum 1.1. l.d Malic enzyme

Malic enzyme (malate dehydrogenase) catalyzes the reversible oxidative

carboxylation of pyruvate to malate. E. coli encodes two malic enzymes, MaeA and MaeB (Takeo, J. Biochem. 66:379-387 (1969)). Although malic enzyme is typically assumed to operate in the direction of pyruvate formation from malate, the NAD-dependent enzyme, encoded by maeA, has been demonstrated to operate in the carbon- fixing direction (Stols and Donnelly, ^/?/?/. Environ. Microbiol. 63(7) 2695-2701 (1997)). A similar observation was made upon overexpressing the malic enzyme from Ascaris suum in E. coli (Stols et al, Appl. Biochem. Biotechnol. 63-65(1), 153-158 (1997)). The second E. coli malic enzyme, encoded by maeB, is NADP-dependent and also decarboxylates oxaloacetate and other alpha-keto acids (Iwakura et al, J. Biochem. 85(5): 1355-65 (1979)). Another suitable enzyme candidate is mel from Zea mays (Furumoto et al, Plant Cell Physiol 41 : 1200-1209 (2000)).

1.2.1. a Oxidoreductase (aldehyde to acid) The oxidation of malonate semialdehyde to malonate is catalyzed by malonate semialdehyde dehydrogenase (EC 1.2.1.15). This enzyme was characterized in Pseudomonas aeruginosa (Nakamura et al, Biochim Biophys Acta 50:147-52 (1961)). The NADP and NAD-dependent succinate semialdehyde dehydrogenase enzymes of Euglena gracilas accept malonate semialdehyde as substrates (Tokunaga et al, Biochem Biophys Act 429:55-62 (1976)). Genes encoding these enzymes has not been identified to date. Aldehyde

dehydrogenase enzymes from eukoryotic organisms such as S. cerevisiae, C. albicans, Y. lipolytica and A. niger typically have broad substrate specificity and are suitable candidates. These enzymes and other acid forming aldehyde dehydrogenase and aldehyde oxidase enzymes are described earlier and listed in Tables 9 and 30. Additional MSA dehydrogenase enzyme candidates include NAD(P)+-dependent aldehyde dehydrogenase enzymes (EC

1.2.1.3). Two aldehyde dehydrogenases found in human liver, ALDH-1 and ALDH-2, have broad substrate ranges for a variety of aliphatic, aromatic and polycyclic aldehydes (Klyosov, Biochemistry 35:4457-4467 (1996a)). Active ALDH-2 has been efficiently expressed in E. coli using the GroEL proteins as chaperonins (Lee et al, Biochem.Biophys.Res.Commun. 298:216-224 (2002)). The rat mitochondrial aldehyde dehydrogenase also has a broad substrate range (Siew et al, Arch.Biochem.Biophys. 176:638-649 (1976)). The E. coli genes astD and aldH encode NAD+-dependent aldehyde dehydrogenases. AstD is active on succinic semialdehyde (Kuznetsova et al, FEMS Microbiol Rev 29:263-279 (2005)) and aldH is active on a broad range of aromatic and aliphatic substrates (Jo et al, Appl Microbiol Biotechnol 81 :51-60 (2008)).

1.2.1. f Oxidoreductase (decarboxylating acyl-CoA to aldehyde) Malonate semialdehyde dehydrogenase (acetylating) (EC 1.2.1.18) catalyzes the oxidative decarboxylation of malonate semialdehyde to acetyl-CoA. Exemplary enzymes are encoded by ddcC of Halomonas sp. HTNK1 (Todd et al, Environ Microbiol 12:237-43 (2010)) and IolA of Lactobacillus casei (Yebra et al, AEM 73:3850-8 (2007)). The DdcC enzyme has homologs in A. niger and C. albicans, shown in the table below. The malonate semialdehyde dehydrogenase enzyme in Rattus norvegicus, Mmsdh, also converts malonate semialdehyde to acetyl-CoA (US 8048624). A malonate semialdehyde dehydrogenase (acetylating) enzyme has also been characterized in Pseudomonas fluorescens, although the gene has not been identified to date (Hayaishi et al, J Biol Chem 236:781-90 (1961)).

Methylmalonate semialdehyde dehydrogenase (acetylating) enzymes (EC 1.2.1.27) are also suitable candidates, as several enzymes in this class accept malonate semialdehyde as a substrate including Msdh of Bacillus subtilis (Stines-Chaumeil et al, Biochem J 395: 107- 15 (2006)) and the methylmalonate semialdehyde dehydrogenase of R. norvegicus (Kedishvii et al, Methods Enzymol 324:207-18 (2000)).

Protein GenBank ID GI Number Organism

ddcC ACV84070.1 258618587 Halomonas sp. HTNK1

ANI 1 1120014 XP 001389265.1 145229913 Aspergillus niger

ALD6 XP 710976.1 68490403 Candida albicans

YALI0C01859g XP 501343.1 50547747 Yarrowia lipolytica

mmsA 1 YP_257876.1 70734236 Pseudomonas fluorescens mmsA 2 YP_257884.1 70734244 Pseudomonas fluorescens

PA0130 NP_248820.1 15595328 Pseudomonas aeruginosa

Mmsdh Q02253.1 400269 Rattus norvegicus

msdh NP_391855.1 16081027 Bacillus subtilis

IolA ABP57762.1 145309085 Lactobacillus casei

2.7.2.a Kinase

Pyruvate kinase (Step ION), also known as phosphoenolpyruvate synthase (EC 2 .9.2), converts pyruvate and ATP to PEP and AMP. This enzyme is encoded by the PYKl (Burke et al, J. Biol. Chem. 258:2193-2201 (1983)) and PYK2 (Boles et al, J. Bacteriol.

179:2987-2993 (1997)) genes in S. cerevisiae. In E. coli, this activity is catalyzed by the gene products of pykF and pykA. Selected homo logs of the S. cerevisiae enzymes are also shown in the table below.

2.8.3. a CoA transferase

Activation of malonate to malonyl-CoA is catalyzed by a CoA transferase in EC class 2.8.3. a. Malonyl-CoA:acetate CoA transferase (EC 2.8.3.3) enzymes have been characterized in Pseudomonas species including Pseudomonas fluorescens and Pseudomonas putida (Takamura et al, Biochem Int 3:483-91 (1981); Hayaishi et al, J Biol Chem 215: 125-36 (1955)). Genes associated with these enzymes have not been identified to date. A

mitochondrial CoA transferase found in Rattus norvegicus liver also catalyzes this reaction and is able to utilize a range of CoA donors and acceptors (Deana et al, Biochem Int 26:767- 73 (1992)). Several CoA transferase enzymes described above can also be applied to catalyze step K of Figure 10. These enzymes include acetyl-CoA transferase (Table 26), 3-HB CoA transferase (Table 8), acetoacetyl-CoA transferase (table 55), SCOT (table 56) and other CoA transferases (table 57).

3.1.3. a Phosphatase

Phosphoenolpyruvate phosphatase (EC 3.1.3.60, Step ION) catalyzes the hydrolysis of PEP to pyruvate and phosphate. Numerous phosphatase enzymes catalyze this activity, including alkaline phosphatase (EC 3.1.3.1), acid phosphatase (EC 3.1.3.2), phosphoglycerate phosphatase (EC 3.1.3.20) and PEP phosphatase (EC 3.1.3.60). PEP phosphatase enzymes have been characterized in plants such as Vignia radiate, Bruguiera sexangula and Brassica nigra. The phytase from Aspergillus fumigates, the acid phosphatase from Homo sapiens and the alkaline phosphatase of E. coli also catalyze the hydrolysis of PEP to pyruvate (Brugger et al, Appl Microbiol Biotech 63:383-9 (2004); Hayman et al, Biochem J 261 :601-9 (1989); et al, The Enzymes 3 rd Ed. 4:373-415 (1971))). Similar enzymes have been characterized in Campylobacter jejuni (van Mourik et al., Microbiol. 154:584-92 (2008)), Saccharomyces cerevisiae (Oshima et al., Gene 179: 171-7 (1996)) and Staphylococcus aureus (Shah and Blobel, J. Bacteriol. 94:780-1 (1967)). Enzyme engineering and/or removal of targeting sequences may be required for alkaline phosphatase enzymes to function in the cytoplasm.

4.1.1. a Decarboxylase

Several reactions in Figure 10 are catalyzed by decarboxylase enzymes in EC class 4.1.1, including oxaloacetate decarboxylase (Step B), malonyl-CoA decarboxylase (step D) and pyruvate carboxylase or carboxykinase (step A).

Carboxylation of phosphoenolpyruvate to oxaloacetate is catalyzed by

phosphoenolpyruvate carboxylase (EC 4.1.1.31). Exemplary PEP carboxylase enzymes are encoded by ppc in E. coli (Kai et al, Arch. Biochem. Biophys. 414:170-179 (2003), ppcA in Methylobacterium extorquens AMI (Arps et al, J. Bacteriol. 175:3776-3783 (1993), and ppc in Corymb acterium glutamicum (Eikmanns et al, Mol. Gen. Genet. 218:330-339 (1989).

An alternative enzyme for carboxylating phosphoenolpyruvate to oxaloacetate is PEP carboxykinase (EC 4.1.1.32, 4.1.1.49), which simultaneously forms an ATP or GTP. In most organisms PEP carboxykinase serves a gluconeogenic function and converts oxaloacetate to PEP at the expense of one ATP. S. cerevisiae is one such organism whose native PEP carboxykinase, PCK1, serves a gluconeogenic role (Valdes-Hevia et al, FEBS Lett. 258:313- 316 (1989). E. coli is another such organism, as the role of PEP carboxykinase in producing oxaloacetate is believed to be minor when compared to PEP carboxylase (Kim et al., Appl. Environ. Microbiol. 70: 1238-1241 (2004)). Nevertheless, activity of the native E. coli PEP carboxykinase from PEP towards oxaloacetate has been recently demonstrated in ppc mutants of E. coli K-12 (Kwon et al, J. Microbiol. Biotechnol. 16: 1448-1452 (2006)). These strains exhibited no growth defects and had increased succinate production at high NaHC0 3 concentrations. Mutant strains of E. coli can adopt Pck as the dominant C0 2 -fixing enzyme following adaptive evolution (Zhang et al. 2009). In some organisms, particularly rumen bacteria, PEP carboxykinase is quite efficient in producing oxaloacetate from PEP and generating ATP. Examples of PEP carboxykinase genes that have been cloned into E. coli include those from Mannheimia succiniciproducens (Lee et al., Biotechnol. Bioprocess Eng. 7:95-99 (2002)), Anaerobio spirillum succiniciproducens (Laivenieks et al., Appl. Environ. Microbiol. 63:2273-2280 (1997), and Actinobacillus succinogenes (Kim et al. supra). The PEP carboxykinase enzyme encoded by Haemophilus influenza is effective at forming oxaloacetate from PEP. Another suitable candidate is the PEPCK enzyme from Megathyrsus maximus, which has a low Km for C0 2 , a substrate thought to be rate-limiting in the E. coli enzyme (Chen et al, Plant Physiol 128: 160-164 (2002); Cotelesage et al, Int.J Biochem.Cell Biol. 39: 1204-1210 (2007)). The kinetics of the GTP-dependent pepck gene product from Cupriavidus necator favor oxaloacetate formation (US8048624 and Lea et al, Amino Acids 20:225-41 (2001)). Protein ( ,cn Bank ID GI Number Organism

PCK1 NP 013023 6322950 Saccharomyces cerevisiae pck NP 417862.1 16131280 Escherichia coli

pckA YP 089485.1 52426348 Mannheimia

succiniciproducens

pckA 009460.1 3122621 Anaerobio spirillum

succiniciproducens

pckA Q6W6X5 75440571 Actinobacillus succinogenes pckA P43923.1 1172573 Haemophilus influenza

AF532733.1 : 1..1929 AAQ 10076.1 33329363 Megathyrsus maximus pepck YP 728135.1 113869646 Cupriavidus necator

Oxaloacetate decarboxylase catalyzes the decarboxylation of oxaloacetate to malonate semialdehyde. Enzymes catalyzing this reaction include kgd of Mycobacterium tuberculosis (GenBank ID: O50463.4, GI: 160395583). Enzymes evolved from kgd with improved activity and/or substrate specificity for oxaloacetate have also been described (US patent 8048624). Additional enzymes useful for catalyzing this reaction include keto-acid decarboxylases shown in the table below.

The decarboxylation of keto-acids is catalyzed by a variety of enzymes with varied substrate specificities, including pyruvate decarboxylase (EC 4.1.1.1), benzoylformate decarboxylase (EC 4.1.1.7), alpha-ketoglutarate decarboxylase and branched-chain alpha- ketoacid decarboxylase. Pyruvate decarboxylase (PDC), also termed keto-acid decarboxylase, is a key enzyme in alcoholic fermentation, catalyzing the decarboxylation of pyruvate to acetaldehyde. The PDCl enzyme from Saccharomyces cerevisiae has a broad substrate range for aliphatic 2-keto acids including 2-ketobutyrate, 2-keto valerate, 3-hydroxypyruvate and 2- phenylpyruvate (22). This enzyme has been extensively studied, engineered for altered activity, and functionally expressed in E. coli (Killenberg-Jabs et al., EurJ.Biochem.

268: 1698-1704 (2001); Li et al, Biochemistry. 38: 10004-10012 (1999); ter Schure et al, Appl.Environ.Microbiol. 64:1303-1307 (1998)). The PDC from Zymomonas mobilus, encoded by pdc, also has a broad substrate range and has been a subject of directed engineering studies to alter the affinity for different substrates (Siegert et al, Protein Eng Des Sel 18:345-357 (2005)). The crystal structure of this enzyme is available (Killenberg-Jabs et al., EurJ.Biochem. 268: 1698-1704 (2001)). Other well-characterized PDC candidates include the enzymes from Acetobacter pasteurians (Chandra et al, 176:443-451 (2001)) and Kluyveromyces lactis (Krieger et al., 269:3256-3263 (2002)).

Like PDC, benzoylformate decarboxylase (EC 4.1.1.7) has a broad substrate range and has been the target of enzyme engineering studies. The enzyme from Pseudomonas putida has been extensively studied and crystal structures of this enzyme are available (Polovnikova et al, 42: 1820-1830 (2003); Hasson et al, 37:9918-9930 (1998)). Site- directed mutagenesis of two residues in the active site of the Pseudomonas putida enzyme altered the affinity (Km) of naturally and non-naturally occurring substrates (Siegert et al., Protein Eng Des Sel 18:345-357 (2005)). The properties of this enzyme have been further modified by directed engineering (Lingen et al, Chembiochem. 4:721-726 (2003); Lingen et al, Protein Eng 15:585-593 (2002)). The enzyme from Pseudomonas aeruginosa, encoded by mdlC, has also been characterized experimentally (Barrowman et al., 34:57-60 (1986)). Additional gene candidates from Pseudomonas stutzeri, Pseudomonas fluorescens and other organisms can be inferred by sequence homology or identified using a growth selection system developed in Pseudomonas putida (Henning et al, Appl.Environ.Microbiol. 72:7510- 7517 (2006)).

Protein ( ,cn Bank ID GI Number Organism

mdlC P20906.2 3915757 Pseudomonas putida

mdlC Q9HUR2.1 81539678 Pseudomonas aeruginosa dpgB ABN80423.1 126202187 Pseudomonas stutzeri

ilvB-1 YP 260581.1 70730840 Pseudomonas fluorescens A third enzyme capable of decarboxylating 2-oxoacids is alpha-ketoglutarate decarboxylase (KGD, EC 4.1.1.71). The substrate range of this class of enzymes has not been studied to date. An exemplarly KDC is encoded by kad in Mycobacterium tuberculosis (Tian et al, PNAS 102: 10670-10675 (2005)). KDC enzyme activity has also been detected in several species of rhizobia including Bradyrhizobium japonicum and Mesorhizobium loti (Green et al, J Bacteriol 182:2838-2844 (2000)). Although the KDC-encoding gene(s) have not been isolated in these organisms, the genome sequences are available and several genes in each genome are annotated as putative KDCs. A KDC from Euglena gracilis has also been characterized but the gene associated with this activity has not been identified to date

(Shigeoka et al., Arch.Biochem.Biophys. 288:22-28 (1991)). The first twenty amino acids starting from the N-terminus were sequenced MTYKAPVKDVKFLLDKVFKV (Shigeoka and Nakano, Arch.Biochem.Biophys. 288:22-28 (1991)). The gene could be identified by testing candidate genes containing this N-terminal sequence for KDC activity. A novel class of AKG decarboxylase enzymes has recently been identified in cyanobacteria such as Synechococcus sp. PCC 7002 and homologs (Zhang and Bryant, Science 334: 1551-3 (2011)).

A fourth candidate enzyme for catalyzing this reaction is branched chain alpha- ketoacid decarboxylase (BCKA). This class of enzyme has been shown to act on a variety of compounds varying in chain length from 3 to 6 carbons (Oku et al., J Biol Chem. 263: 18386- 18396 (1988); S it et al, Appl Environ Microbiol 71 :303-311 (2005)). The enzyme in Lactococcus lactis has been characterized on a variety of branched and linear substrates including 2-oxobutanoate, 2-oxohexanoate, 2-oxopentanoate, 3-methyl-2-oxobutanoate, 4- methyl-2-oxobutanoate and isocaproate (Smit et al., Appl Environ Microbiol 71 :303-311 (2005)). The enzyme has been structurally characterized (Berg et al., Science. 318: 1782-1786 (2007)). Sequence alignments between the Lactococcus lactis enzyme and the pyruvate decarboxylase of Zymomonas mobilus indicate that the catalytic and substrate recognition residues are nearly identical (Siegert et al, Protein Eng Des Sel 18:345-357 (2005)), so this enzyme would be a promising candidate for directed engineering. Several ketoacid decarboxylases of Saccharomyces cerevisiae catalyze the decarboxylation of branched substrates, including ARO10, PDC6, PDC5, PDC1 and THI3 (Dickenson et al, J Biol Chem 275: 10937-42 (2000)). Yet another BCKAD enzyme is encoded by rv0853c of

Mycobacterium tuberculosis (Werther et al, J Biol Chem 283:5344-54 (2008)). This enzyme is subject to allosteric activation by alpha-ketoacid substrates. Decarboxylation of alpha- ketoglutarate by a BCKA was detected in Bacillus subtilis; however, this activity was low (5%) relative to activity on other branched-chain substrates (Oku and Kaneda, J Biol Chem. 263: 18386-18396 (1988)) and the gene encoding this enzyme has not been identified to date. Additional BCKA gene candidates can be identified by homology to the Lactococcus lactis protein sequence. Many of the high-scoring BLASTp hits to this enzyme are annotated as indolepyruvate decarboxylases (EC 4.1.1.74). Indolepyruvate decarboxylase (IPDA) is an enzyme that catalyzes the decarboxylation of indolepyruvate to indoleacetaldehyde in plants and plant bacteria. Recombinant branched chain alpha-keto acid decarboxylase enzymes derived from the El subunits of the mitochondrial branched-chain keto acid dehydrogenase complex from Homo sapiens and Bos taurus have been cloned and functionally expressed in E. coli (Davie et al, J.Biol.Chem. 267: 16601-16606 (1992); Wynn et al, J.Biol.Chem.

267: 12400-12403 (1992); Wynn et al, J.Biol.Chem. 267: 1881-1887 (1992)). In these studies, the authors found that co-expression of chaperonins GroEL and GroES enhanced the specific activity of the decarboxylase by 500-fold (Wynn et al, J.Biol.Chem. 267: 12400-12403 (1992)). These enzymes are composed of two alpha and two beta subunits.

3-Phosphonopyruvate decarboxylase (EC 4.1.1.82) catalyzes the decarboxylation of 3-phosphonopyruvate to 2-phosphonoacetaldehyde. Exemplary phosphonopyruvate decarboxylase enzymes are encoded by dhpF of Streptomyces luridus, ppd of Streptomyces viridochromogenes,fom2 of Streptomyces wedmorensis and bcpC of Streptomyces hygroscopius (Circello et al, Chem Biol 17:402-11 (2010); Blodgett et al, FEMS Microbiol Lett 163: 149-57 (2005); Hidaka et al, Mol Gen Genet 249:274-80 (1995); Nakashita et al, Biochim Biophys Acta 1490: 159-62 (2000)). The Bacteroides fragilis enzyme, encoded by aepY, also decarboxylates pyruvate and sulfopyruvate (Zhang et al, J Biol Chem 278:41302-8 (2003)).

Many oxaloacetate decarboxylase enzymes such as the eda gene product in E. coli (EC 4.1.1.3), act on the terminal acid of oxaloacetate to form pyruvate. Because

decarboxylation at the 3-keto acid position competes with the malonate semialdehyde forming decarboxylation at the 2-keto-acid position, this enzyme activity can be knocked out in a host strain with a pathway proceeding through a malonate semilaldehyde intermediate.

Malonyl-CoA decarboxylase (EC 4.1.1.9) catalyzes the decarboxylation of malonyl- CoA to acetyl-CoA. Enzymes have been characterized in Rhizobium leguminosarum and Acinetobacter calcoaceticus (An et al, Eur J Biochem 257: 395-402 (1998); Koo et al, Eur J Biochem 266:683-90 (1999)). Similar enzymes have been characterized in Streptomyces erythreus (Hunaiti et al, Arch Biochem Biophys 229:426-39 (1984)). A recombinant human malonyl-CoA decarboxylase was overexpressed in E. coli (Zhou et al, Prot Expr Pur 34:261- 9 (2004)). Methylmalonyl-CoA decarboxylase enzymes that decarboxylate malonyl-CoA are also suitable candidates. For example, the Veillonella parvula enzyme accepts malonyl-CoA as a substrate (Hilpert et al, Nature 296:584-5 (1982)). The E. coli enzyme is encoded by ygfG (Benning et al, Biochemistry. 39:4630-4639 (2000); Haller et al, Biochemistry.

39:4622-4629 (2000)). The stereo specificity of the E. coli enzyme was not reported, but the enzyme in Propionigenium modestum (Bott et al, Eur. J. Biochem. 250:590-599 (1997)) and Veillonella parvula (Huder et al, J.Biol.Chem. 268:24564-24571 (1993)) catalyzes the decarboxylation of the (S)-stereoisomer of methylmalonyl-CoA (Hoffmann et al., FEBS.Lett. 220: 121-125 (1987)). The enzymes from P. modestum and V. parvula are comprised of multiple subunits that not only decarboxylate (S)-methylmalonyl-CoA, but also create a pump that transports sodium ions across the cell membrane as a means to generate energy. Protein ( ,cn Bank ID GI Number Organism

YgfG NP 417394 90111512 Escherichia coli

matA Q9ZIP6 75424899 Rhizobium leguminosarum mdcD AAB97628.1 2804622 Acinetobacter calcoaceticus mdcE AAF20287.1 6642782 Acinetobacter calcoaceticus mdcA AAB97627.1 2804621 Acinetobacter calcoaceticus mdcC AAB97630.1 2804624 Acinetobacter calcoaceticus mcd NP 036345.2 110349750 Homo sapiens

mmdA CAA05137 2706398 Propionigenium modestum mmdD CAA05138 2706399 Propionigenium modestum mmdC CAA05139 2706400 Propionigenium modestum mmdB CAA05140 2706401 Propionigenium modestum mmdA CAA80872 415915 Veillonella parvula

mmdC CAA80873 415916 Veillonella parvula

mmdE CAA80874 415917 Veillonella parvula

mmdD CAA80875 415918 Veillonella parvula

mmdB CAA80876 415919 Veillonella parvula

6.2.1. a CoA synthetase

Activation of malonate to malonyl-CoA is catalyzed by a CoA synthetase in EC class 6.2.1. a. CoA synthetase enzymes that catalyze this reaction have not been described in the literature to date. Several CoA synthetase enzymes described above can also be applied to catalyze step K of Figure 10. These enzymes include acetyl-CoA synthetase (Table 16, 25) and ADP forming CoA synthetases (Table 17).

6.4.1.a Carboxylase

Pyruvate carboxylase (EC 6.4.1.1) converts pyruvate to oxaloacetate at the cost of one ATP (step H). Exemplary pyruvate carboxylase enzymes are encoded by PYCI (Walker et al, Biochem. Biophys. Res. Commun. 176: 1210-1217 (1991) and PYC2 (Walker et al, supra) in Saccharomyces cerevisiae, and pyc in Mycobacterium smegmatis (Mukhopadhyay and Purwantini, Biochim. Biophys. Acta 1475: 191-206 (2000)).

Protein ( ,cn Bank ID GI Number Organism

PYCI NP 011453 6321376 Saccharomyces cerevisiae

PYC2 NP 009777 6319695 Saccharomyces cerevisiae

Pyc YP 890857.1 118470447 Mycobacterium smegmatis EXAMPLE IV

Pathways for Producing Cytosolic Acetyl-CoA from Mitochondrial Acetyl-CoA

A mechanism for transporting acetyl-CoA from the mitochondrion to the cytosol can facilitate deployment of a cytosolic fatty alcohol, fatty aldehyde or fatty acid production pathway that originates from acetyl-CoA. Exemplary mechanisms for exporting acetyl-CoA include those depicted in Figures 4 and 5, which can involve forming citrate from acetyl-CoA and oxaloacetate in the mitochondrion, exporting the citrate from the mitochondrion to the cytosol, and converting the citrate to oxaloacetate and either acetate or acetyl-CoA. In certain embodiments, provided herein are methods for engineering a eukaryotic organism to increase its availability of cytosolic acetyl-CoA by introducing enzymes capable of carrying out the transformations depicted in any one of Figures 4 and 5. Exemplary enzymes capable of carrying out the required transformations are also disclosed herein.

The production of cytosolic acetyl-CoA from mitochondrial acetyl-CoA can be accomplished by a number of pathways, for example, in three to five enzymatic steps. In one exemplary pathway, mitochondrial acetyl-CoA and oxaloacetate are combined into citrate by a citrate synthase and the citrate is exported out of the mitochondrion by a citrate or citrate/oxaloacetate transporter. Enzymatic conversion of the citrate in the cytosol results in cytosolic acetyl-CoA and oxaloacetate. The cytosolic oxaloacetate can then optionally be transported back into the mitochondrion by an oxaloacetate transporter and/or a

citrate/oxaloacetate transporter. In another exemplary pathway, the cytosolic oxaloacetate is first enzymatically converted into malate in the cytosol and then optionally transferred into the mitochondrion by a malate transporter and/or a malate/citrate transporter. Mitochondrial malate can then be converted into oxaloacetate with a mitochondrial malate dehydrogenase.

In yet another exemplary pathway, mitochondrial acetyl-CoA can be converted to cytosolic acetyl-CoA via a citramalate intermediate. For example, mitochondrial acetyl-CoA and pyruvate are converted to citramalate by citramalate synthase. Citramalate can then be transported into the cytosol by a citramalate or dicarboxylic acid transporter. Cytosolic acetyl-CoA and pyruvate are then regenerated from citramalate, directly or indirectly, and the pyruvate can re-enter the mitochondria. Along these lines, several exemplary acetyl-CoA pathways for the production of cytosolic acetyl-CoA from mitochondrial acetyl-CoA are shown in Figures 4 and 5. In one embodiment, mitochondrial oxaloacetate is combined with mitochondrial acetyl-CoA to form citrate by a citrate synthase. The citrate is transported outside of the mitochondrion by a citrate transporter, a citrate/oxaloacetate transporter or a citrate/malate transporter. Cytosolic citrate is converted into cytosolic acetyl-CoA and oxaloacetate by an ATP citrate lyase. In another pathway, cytosolic citrate is converted into acetate and oxaloacetate by a citrate lyase. Acetate can then be converted into cytosolic acetyl-CoA by an acetyl-CoA synthetase or transferase. Alternatively, acetate can be converted by an acetate kinase to acetyl phosphate, and the acetyl phosphate can be converted to cytosolic acetyl-CoA by a

phosphotransacetylase . Exemplary enzyme candidates for acetyl-CoA pathway enzymes are described below.

The conversion of oxaloacetate and mitochondrial acetyl-CoA is catalyzed by a citrate synthase (Figures 4 and 5, step A). In certain embodiments, the citrate synthase is expressed in a mitochondrion of a non-naturally occurring eukaryotic organism provided herein.

Transport of citrate from the mitochondrion to the cytosol can be carried out by several transport proteins. Such proteins either export citrate directly (i.e., citrate transporter, Figures 4 and 5, step B) to the cytosol or export citrate to the cytosol while simultaneously transporting a molecule such as malate (i.e., citrate/malate transporter, Figure 4, step C) or oxaloacetate (i.e., citrate/oxaloacetate transporter Figure 5, step C) from the cytosol into the mitochondrion as shown in Figures 4 and 5. Exemplary transport enzymes that carry out these transformations are provided in the table below.

Protein ( ,cn Bank ID GI number Organism

CTP1 NP 009850.1 6319768 Saccharomyces cerevisiae S288c

YALI0F26323p XP 505902.1 50556988 Yarrowia lipolytica ATEG 09970 EAU29419.1 114187719 Aspergillus terreus NIH2624

KLLA0E18723g XP 454797.1 50309571 Kluyveromyces lactis NRRL Y-1140

CTRG 02320 XP 002548023.1 255726194 Candida tropicalis MYA-3404

ANI 1 1474094 XP 001395080.1 145245625 Aspergillus niger CBS 513.88

YHM2 NP 013968.1 6323897 Saccharomyces cerevisiae S288c

DTC CAC84549.1 19913113 Arabidopsis thaliana

DTC1 CAC84545.1 19913105 Nicotiana tabacum

DTC2 CAC84546.1 19913107 Nicotiana tabacum

DTC3 CAC84547.1 19913109 Nicotiana tabacum

DTC4 CAC84548.1 19913111 Nicotiana tabacum

DTC AAR06239.1 37964368 Citrus junos

ATP citrate lyase (ACL, EC 2.3.3.8, Figures 4 and 5, step D), also called ATP citrate synthase, catalyzes the ATP-dependent cleavage of citrate to oxaloacetate and acetyl-CoA.

In certain embodiments, ATP citrate lyase is expressed in the cytosol of a eukaryotic

organism. ACL is an enzyme of the RTCA cycle that has been studied in green sulfur

bacteria Chlorobium limicola and Chlorobium tepidum. The alpha(4)beta(4) heteromeric enzyme from Chlorobium limicola was cloned and characterized in E. coli (Kanao et al., Eur. J. Biochem. 269:3409-3416 (2002). The C. limicola enzyme, encoded by aclAB, is

irreversible and activity of the enzyme is regulated by the ratio of ADP/ATP. The

Chlorobium tepidum a recombinant ACL from Chlorobium tepidum was also expressed in E. coli and the holoenzyme was reconstituted in vitro, in a study elucidating the role of the alpha and beta subunits in the catalytic mechanism (Kim and Tabita, J. Bacteriol. 188:6544-6552 (2006). ACL enzymes have also been identified in Balnearium lithotrophicum,

Sulfurihydrogenibium subterraneum and other members of the bacterial phylum Aquificae (Hugler et al., Environ. Microbiol. 9:81-92 (2007)). This activity has been reported in some fungi as well. Exemplary organisms include Sordaria macrospora (Nowrousian et al., Curr. Genet. 37:189-93 (2000)), Aspergillus nidulans and Yarrowia lipolytica (Hynes and Murray, Eukaryotic Cell, July: 1039-1048, (2010) , and Aspergillus niger (Meijer et al. J. Ind.

Microbiol. Biotechnol. 36: 1275-1280 (2009). Other candidates can be found based on

sequence homology. Information related to these enzymes is tabulated below.

Protein ( ,cn Bank ID GI Number Organism

aclA BAB21376.1 12407237 Chlorobium limicola

aclB BAB21375.1 12407235 Chlorobium limicola

aclA AAM72321.1 21647054 Chlorobium tepidum

aclB AAM72322.1 21647055 Chlorobium tepidum

aclB ABI50084.1 114055039 Sulfurihydrogenibium

subterraneum Protein ( ,cn Bank ID GI Number Organism

aclA AAX76834.1 62199504 Sulfurimonas denitrificans aclB AAX76835.1 62199506 Sulfurimonas denitrificans acll XP 504787.1 50554757 Yarrowia lipolytica

acl2 XP 503231.1 50551515 Yarrowia lipolytica

SPBC1703.07 NP 596202.1 19112994 Schizosaccharomyces pombe

SPAC22A12.16 NP 593246.1 19114158 Schizosaccharomyces pombe acll CAB76165.1 7160185 Sordaria macrospora

acl2 CAB76164.1 7160184 Sordaria macrospora

aclA CBF86850.1 259487849 Aspergillus nidulans

aclB CBF86848 259487848 Aspergillus nidulans

In some organisms the conversion of citrate to oxaloacetate and acetyl-CoA proceeds through a citryl-CoA intermediate and is catalyzed by two separate enzymes, citryl-CoA synthetase (EC 6.2.1.18) and citryl-CoA lyase (EC 4.1.3.34) (Aoshima, M., Appl. Microbiol. Biotechnol. 75:249-255 (2007). Citryl-CoA synthetase catalyzes the activation of citrate to citryl-CoA. The Hydrogenobacter thermophilus enzyme is composed of large and small subunits encoded by ccsA and ccsB, respectively (Aoshima et al., Mol. Micrbiol. 52:751-761 (2004)). The citryl-CoA synthetase of Aquifex aeolicus is composed of alpha and beta subunits encoded by sucCl and sucDl (Hugler et al., Environ. Microbiol. 9:81-92 (2007)). Citryl-CoA lyase splits citryl-CoA into oxaloacetate and acetyl-CoA. This enzyme is a homotrimer encoded by ccl in Hydrogenobacter thermophilus (Aoshima et al., Mol.

Microbiol. 52:763-770 (2004)) and aq_150 in Aquifex aeolicus (Hugler et al., supra (2007)). The genes for this mechanism of converting citrate to oxaloacetate and citryl-CoA have also been reported recently in Chlorobium tepidum (Eisen et al., PNAS 99(14): 9509-14 (2002)).

Citrate lyase (EC 4.1.3.6, Figures 4 and 5, step E) catalyzes a series of reactions resulting in the cleavage of citrate to acetate and oxaloacetate. In certain embodiments, citrate lyase is expressed in the cytosol of a eukaryotic organism. The enzyme is active under anaerobic conditions and is composed of three subunits: an acyl-carrier protein (ACP, gamma), an ACP transferase (alpha), and an acyl lyase (beta). Enzyme activation uses covalent binding and acetylation of an unusual prosthetic group, 2'-(5"-phosphoribosyl)-3-'- dephospho-CoA, which is similar in structure to acetyl-CoA. Acylation is catalyzed by CitC, a citrate lyase synthetase. Two additional proteins, CitG and CitX, are used to convert the apo enzyme into the active holo enzyme (Schneider et al., Biochemistry 39:9438-9450 (2000)). Wild type E. coli does not have citrate lyase activity; however, mutants deficient in molybdenum cofactor synthesis have an active citrate lyase (Clark, FEMS Microbiol. Lett. 55:245-249 (1990)). The E. coli enzyme is encoded by citEFD and the citrate lyase synthetase is encoded by citC (Nilekani and SivaRaman, Biochemistry 22:4657-4663 (1983)). The Leuconostoc mesenteroides citrate lyase has been cloned, characterized and expressed in E. coli (Bekal et al., J. Bacteriol. 180:647-654 (1998)). Citrate lyase enzymes have also been identified in enterobacteria that utilize citrate as a carbon and energy source, including Salmonella typhimurium and Klebsiella pneumoniae (Bott, Arch. Microbiol. 167: 78-88

(1997); Bott and Dimroth, Mol. Microbiol. 14:347-356 (1994)). The aforementioned proteins are tabulated below.

Protein ( ,cn Bank ID GI Number Organism

citF AAC73716.1 1786832 Escherichia coli

cite AAC73717.2 87081764 Escherichia coli

citD AAC73718.1 1786834 Escherichia coli

citC AAC73719.2 87081765 Escherichia coli

citG AAC73714.1 1786830 Escherichia coli

citX AAC73715.1 1786831 Escherichia coli

citF CAA71633.1 2842397 Leuconostoc mesenteroides citE CAA71632.1 2842396 Leuconostoc mesenteroides citD CAA71635.1 2842395 Leuconostoc mesenteroides citC CAA71636.1 3413797 Leuconostoc mesenteroides citG CAA71634.1 2842398 Leuconostoc mesenteroides citX CAA71634.1 2842398 Leuconostoc mesenteroides citF NP 459613.1 16763998 Salmonella typhimurium citE AAL19573.1 16419133 Salmonella typhimurium citD NP 459064.1 16763449 Salmonella typhimurium citC NP 459616.1 16764001 Salmonella typhimurium citG NP 459611.1 16763996 Salmonella typhimurium citX NP 459612.1 16763997 Salmonella typhimurium citF CAA56217.1 565619 Klebsiella pneumoniae citE CAA56216.1 565618 Klebsiella pneumoniae citD CAA56215.1 565617 Klebsiella pneumoniae citC BAH66541.1 238774045 Klebsiella pneumoniae Protein ( ,cn Bank ID GI Number Organism

citG CAA56218.1 565620 Klebsiella pneumoniae citX AAL60463.1 18140907 Klebsiella pneumoniae

The acylation of acetate to acetyl-CoA is catalyzed by enzymes with acetyl-CoA synthetase activity (Figures 4 and 5, step F). In certain embodiments, acetyl-CoA synthetase is expressed in the cytosol of a eukaryotic organism. Two enzymes that catalyze this reaction are AMP-forming acetyl-CoA synthetase (EC 6.2.1.1) and ADP-forming acetyl-CoA synthetase (EC 6.2.1.13). AMP-forming acetyl-CoA synthetase (ACS) is the predominant enzyme for activation of acetate to acetyl-CoA. Exemplary ACS enzymes are found in E. coli (Brown et al., J. Gen. Microbiol. 102:327-336 (1977)), Ralstonia eutropha (Priefert and Steinbuchel, J. Bacteriol. 174:6590-6599 (1992)), Methanothermobacter thermautotrophicus (Ingram-Smith and Smith, Archaea 2:95-107 (2007)), Salmonella enterica (Gulick et al., Biochemistry 42:2866-2873 (2003)) and Saccharomyces cerevisiae (Jogl and Tong,

Biochemistry 43: 1425-1431 (2004)).

ADP-forming acetyl-CoA synthetase (ACD, EC 6.2.1.13) is another candidate enzyme that couples the conversion of acyl-CoA esters to their corresponding acids with the concurrent synthesis of ATP. Several enzymes with broad substrate specificities have been described in the literature. ACD I from Archaeoglobus fulgidus, encoded by AF1211, was shown to operate on a variety of linear and branched-chain substrates including acetyl-CoA, propionyl-CoA, butyryl-CoA, acetate, propionate, butyrate, isobutyryate, isovalerate, succinate, fumarate, phenylacetate, indoleacetate (Musfeldt et al., J. Bacteriol. 184:636-644 (2002)). The enzyme from Haloarcula marismortui (annotated as a succinyl-CoA synthetase) accepts propionate, butyrate, and branched-chain acids (isovalerate and isobutyrate) as substrates, and was shown to operate in the forward and reverse directions (Brasen et al., Arch. Microbiol. 182:277-287 (2004)). The ACD encoded by PAE3250 from hyperthermophilic crenarchaeon Pyrobaculum aerophilum showed the broadest substrate range of all characterized ACDs, reacting with acetyl-CoA, isobutyryl-CoA (preferred substrate) and phenylacetyl-CoA (Brasen et al., supra (2004)). The enzymes from A.

fulgidus, H. marismortui and P. aerophilum have all been cloned, functionally expressed, and characterized in E. coli (Musfeldt et al., supra; Brasen et al., supra (2004)). Additional candidates include the succinyl-CoA synthetase encoded by sucCD in E. coli (Buck et al., Biochemistry 24:6245-6252 (1985)) and the acyl-CoA ligase from Pseudomonas putida (Fernandez- Valverde et al., Appl. Environ. Microbiol. 59: 1149-1154 (1993)). Information related to these proteins and genes is shown below.

An alternative method for adding the CoA moiety to acetate is to apply a pair of enzymes such as a phosphate-transferring acyltransferase and an acetate kinase (Figures 4 and 5, Step F). This activity enables the net formation of acetyl-CoA with the simultaneous consumption of ATP. In certain embodiments, phosphotransacetylase is expressed in the cytosol of a eukaryotic organism. An exemplary phosphate -transferring acyltransferase is phosphotransacetylase, encoded by pta. The pta gene from E. coli encodes an enzyme that can convert acetyl-CoA into acetyl-phosphate, and vice versa (Suzuki, T.

Biochim.Biophys.Acta 191 :559-569 (1969)). This enzyme can also utilize propionyl-CoA instead of acetyl-CoA forming propionate in the process (Hesslinger et al. Mol.Microbiol 27:477-492 (1998)). Homologs exist in several other organisms including Salmonella enterica and Chlamydomonas reinhardtii.

Protein ( ,cn Bank ID GI number Organism

Pta NP 416800.1 16130232 Escherichia coli

Pta NP_461280.1 16765665 Salmonella enterica subsp. enterica serovar Typhimurium str. LT2

PAT2 XP 001694504.1 159472743 Chlamydomonas reinhardtii

PAT1 XP 001691787.1 159467202 Chlamydomonas reinhardtii An exemplary acetate kinase is the E. coli acetate kinase, encoded by ackA (Skarstedt and Silverstein J.5 0/. Chem. 251 :6775-6783 (1976)). Homologs exist in several other organisms including Salmonella enterica and Chlamydomonas reinhardtii. Information related to these proteins and genes is shown below:

In some embodiments, cytosolic oxaloacetate is transported back into a

mitochondrion by an oxaloacetate transporter. Oxaloacetate transported back into a mitochondrion can then be used in the acetyl-CoA pathways described herein. Transport of oxaloacetate from the cytosol to the mitochondrion can be carried out by several transport proteins. Such proteins either import oxaloacetate directly (i.e., oxaloacetate transporter) to the mitochondrion or import oxaloacetate to the cytosol while simultaneously transporting a molecule such as citrate (i.e., citrate/oxaloacetate transporter) from the mitochondrion into the cytosol as shown in Figure 5. Exemplary transport enzymes that carry out these transformations are provided in the table below.

Protein ( ,cn Bank ID GI number Organism

OAC1 NP 012802.1 6322729 Saccharomyces cerevisiae S288c

Kluyveromyces lactis NRRL Y-

KLLA0B12826g XP 452102.1 50304305 1140

YALI0E04048g XP 503525.1 50552101 Yarrowia lipolytica

CTRG 02239 XP 002547942.1 255726032 Candida tropicalis MYA-3404

DICl NP 013452.1 6323381 Saccharomyces cerevisiae S288c

YALI0B03344g XP 500457.1 50545838 Yarrowia lipolytica

CTRG 02122 XP 002547815.1 255725772 Candida tropicalis MYA-3404

PAS chr4 0877 XP 002494326.1 254574434 Pichia pastoris GS115

DTC CAC84549.1 19913113 Arabidopsis thaliana

DTC1 CAC84545.1 19913105 Nicotiana tabacum

DTC2 CAC84546.1 19913107 Nicotiana tabacum

DTC3 CAC84547.1 19913109 Nicotiana tabacum

DTC4 CAC84548.1 19913111 Nicotiana tabacum

DTC AAR06239.1 37964368 Citrus junos In some embodiments, cytosolic oxaloacetate is first converted to malate by a cytosolic malate dehydrogenase (Figures 4, step H). Cytosolic malate is transported into a mitochondrion by a malate transporter or a citrate/malate transporter (Figure 4, step I).

Mitochondrial malate is then converted to oxaloacetate by a mitochondrial malate

dehydrogenase (Figure 4, step J). Mitochondrial oxaloacetate can then be used in the acetyl- CoA pathways described herein. Exemplary examples of each of these enzymes are provided below.

Oxaloacetate is converted into malate by malate dehydrogenase (EC 1.1.1.37, Figures 4, step H). When malate is the dicarboxylate transported from the cytosol to mitochondrion, expression of both a cytosolic and mitochondrial version of malate dehydrogenase, e.g., as shown in Figure 3, can be used. S. cerevisiae possesses three copies of malate

dehydrogenase, MDHl (McAlister-Henn and Thompson, J. Bacteriol. 169:5157-5166 (1987), MDH2 (Minard and McAlister-Henn, Mol. Cell. Biol. 11 :370-380 (1991); Gibson and McAlister-Henn, J. Biol. Chem. 278:25628-25636 (2003)), and MDH3 (Steffan and

McAlister-Henn, J. Biol. Chem. 267:24708-24715 (1992)), which localize to the

mitochondrion, cytosol, and peroxisome, respectively. Close homologs to the cytosolic malate dehydrogenase, MDH2, from S. cerevisiae are found in several organisms including Kluyveromyces lactis and Candida tropicalis. E. coli is also known to have an active malate dehydrogenase encoded by mdh.

Transport of malate from the cytosol to the mitochondrion can be carried out by several transport proteins. Such proteins either import malate directly (i.e., malate

transporter) to the mitochondrion or import malate to the cytosol while simultaneously transporting a molecule such as citrate (i.e., citrate/malate transporter) from the

mitochondrion into the cytosol as shown in Figure 4. Exemplary transport enzymes that carry out these transformations are provided in the table below. Protein ( ,cn Bank ID GI number Organism

OAC1 NP 012802.1 6322729 Saccharomyces cerevisiae S288c

KLLA0B12826g XP 452102.1 50304305 Kluyveromyces lactis NRRL Y-1140

YALI0E04048g XP 503525.1 50552101 Yarrowia lipolytica

CTRG 02239 XP 002547942.1 255726032 Candida tropicalis MYA-3404

DICl NP 013452.1 6323381 Saccharomyces cerevisiae S288c

YALI0B03344g XP 500457.1 50545838 Yarrowia lipolytica

CTRG 02122 XP 002547815.1 255725772 Candida tropicalis MYA-3404

PAS chr4 0877 XP 002494326.1 254574434 Pichia pastoris GS115

DTC CAC84549.1 19913113 Arabidopsis thaliana

DTC1 CAC84545.1 19913105 Nicotiana tabacum

DTC2 CAC84546.1 19913107 Nicotiana tabacum

DTC3 CAC84547.1 19913109 Nicotiana tabacum

DTC4 CAC84548.1 19913111 Nicotiana tabacum

DTC AAR06239.1 37964368 Citrus junos

Malate can be converted into oxaloacetate by malate dehydrogenase (EC 1.1.1.37, Figure 4, step J). When malate is the dicarboxylate transported from the cytosol to

mitochondrion, in certain embodiments, both a cytosolic and mitochondrial version of malate dehydrogenase is expressed, as shown in Figures 3 and 4. S. cerevisiae possesses three copies of malate dehydrogenase, MDH1 (McAlister-Henn and Thompson, J. Bacteriol.

169:5157-5166 (1987), MDH2 (Minard and McAlister-Henn, Mol. Cell. Biol. 11 :370-380 (1991); Gibson and McAlister-Henn, J. Biol. Chem. 278:25628-25636 (2003)), and MDH3 (Steffan and McAlister-Henn, J. Biol. Chem. 267:24708-24715 (1992)), which localize to the mitochondrion, cytosol, and peroxisome, respectively. Close homologs to the mitochondrial malate dehydrogenase, MDH1, from S. cerevisiae are found in several organisms including Kluyveromyces lactis, Yarrowia lipolytica, Candida tropicalis. E. coli is also known to have an active malate dehydrogenase encoded by mdh.

Protein ( ,cn Bank ID GI Number Organism

MDH1 NP 012838 6322765 Saccharomyces cerevisiae

MDH2 NP 014515 116006499 Saccharomyces cerevisiae

MDH3 NP 010205 6320125 Saccharomyces cerevisiae

Mdh NP 417703.1 16131126 Escherichia coli

KLLA0F25960g XP 456236.1 50312405 Kluyveromyces lactis NRRL Y-1140

YALI0D16753g XP 502909.1 50550873 Yarrowia lipolytica

CTRG 00226 XP 002545445.1 255721021 Candida tropicalis MYA-3404 EXAMPLE V

Utilization of Pathway Enzymes with a Preference for NADH

The production of acetyl-CoA from glucose can generate at most four reducing equivalents in the form of NADH. A straightforward and energy efficient mode of maximizing the yield of reducing equivalents is to employ the Embden-Meyerhof-Parnas glycolysis pathway (EMP pathway). In many carbohydrate utilizing organisms, one NADH molecule is generated per oxidation of each glyceraldehyde-3 -phosphate molecule by means of glyceraldehyde-3 -phosphate dehydrogenase. Given that two molecules of glyceraldehyde- 3 -phosphate are generated per molecule of glucose metabolized via the EMP pathway, two NADH molecules can be obtained from the conversion of glucose to pyruvate.

Two additional molecules of NADH can be generated from conversion of pyruvate to acetyl-CoA given that two molecules of pyruvate are generated per molecule of glucose metabolized via the EMP pathway. This could be done by employing any of the following enzymes or enzyme sets to convert pyruvate to acetyl-CoA: I. NAD-dependant pyruvate dehydrogenase;

II. Pyruvate formate lyase and NAD-dependant formate dehydrogenase;

III . Pyruvate : ferredoxin oxidoreductase and NADH : ferredoxin oxidoreductase;

IV. Pyruvate decarboxylase and an NAD-dependant acylating acetylaldehyde dehydrogenase; V. Pyruvate decarboxylase, NAD-dependant acylating acetaldehyde dehydrogenase, acetate kinase, and phosphotransacetylase; and

VI. Pyruvate decarboxylase, NAD-dependant acylating acetaldehyde dehydrogenase, and acetyl-CoA synthetase.

Overall, four molecules of NADH can be attained per glucose molecule metabolized. In one aspect, the fatty alcohol pathway requires three reduction steps from acetyl-CoA. Therefore, it can be possible that each of these three reduction steps will utilize NADPH or NADH as the reducing agents, in turn converting these molecules to NADP or NAD, respectively. Therefore, in some aspects, it can be desireable that all reduction steps are NADH-dependant in order to maximize the yield of fatty alcohols, fatty aldehydes or fatty acis. High yields of fatty alcohols, fatty aldehydes and fatty acids can thus be accomplished by:

Identifying and implementing endogenous or exogenous fatty alcohol, fatty aldehyde or fatty acid pathway enzymes with a stronger preference for NADH than other reducing equivalents such as NADPH,

I. Attenuating one or more endogenous fatty alcohol, fatty aldehyde or fatty acid pathway enzymes that contribute NADPH-dependant reduction activity,

II. Altering the cofactor specificity of endogenous or exogenous fatty alcohol, fatty aldehyde or fatty acid pathway enzymes so that they have a stronger preference for NADH than their natural versions, or

III. Altering the cofactor specificity of endogenous or exogenous fatty alcohol, fatty aldehyde or fatty acid pathway enzymes so that they have a weaker preference for NADPH than their natural versions.

The individual enzyme or protein activities from the endogenous or exogenous DNA sequences can be assayed using methods well known in the art. For example, the genes can be expressed in E. coli and the activity of their encoded proteins can be measured using cell extracts. Alternatively, the enzymes can be purified using standard procedures well known in the art and assayed for activity. Spectrophotometric based assays are particularly effective.

Several examples and methods of altering the cofactor specificity of enzymes are known in the art. For example, Khoury et al. (Protein Sci. 2009 October; 18(10): 2125-2138) created several xylose reductase enzymes with an increased affinity for NADH and decreased affinity for NADPH. Ehsani et al (Biotechnology and Bioengineering, Volume 104, Issue 2, pages 381-389, 1 October 2009) drastically decreased activity of 2,3-butanediol

dehydrogenase on NADH while increasing activity on NADPH. Machielsen et al

(Engineering in Life Sciences, Volume 9, Issue 1, pages 38-44, February 2009) dramatically increased activity of alcohol dehydrogenase on NADH. Khoury et al (Protein Sci. 2009 October; 18(10): 2125-2138) list in Table I several previous examples of successfully changing the cofactor preference of over 25 other enzymes. Additional descriptions can be found in Lutz et al, Protein Engineering Handbook, Volume 1 and Volume 2, 2009, Wiley- VCH Verlag GmbH & Co. KGaA, in particular, Chapter 31 : Altering Enzyme Substrate and Cofactor Specificity via Protein Engineering.

EXAMPLE VI

Determining Cofactor Preference of Pathway Enzymes This example describes an experimental method for determining the cofactor preference of an enzyme.

Cofactor preference of enzymes for each of the pathway steps can be determined by cloning the individual genes on a plasmid behind a constitutive or inducible promoter and transforming into a host organism such as Escherichia coli. For example, genes encoding enzymes that catalyze pathway steps from: 1) acetoacetyl-CoA to 3-hydroxybutyryl-CoA, 2) 3-hydroxybutyryl-CoA to 3-hydroxybutyraldehyde, 3) 3-hydroxybutyraldehyde to 1 ,3- butanediol (wherein Ri is Ci; R 3 is OH) can be assembled onto the pZ-based expression vectors as described below.

Replacement of the Stuffer Fragment in the pZ-based Expression Vectors. Vector backbones were obtained from Dr. Rolf Lutz of Expressys (http://www.expressys.de/). The vectors and strains are based on the pZ Expression System developed by Lutz and Bujard (Nucleic Acids Res 25, 1203-1210 (1997)). The pZE131uc, pZA331uc, pZS* 131uc and pZE221uc contain the luciferase gene as a stuffer fragment. To replace the luciferase stuffer fragment with a lacZ-alpha fragment flanked by appropriate restriction enzyme sites, the luciferase stuffer fragment is removed from each vector by digestion with EcoRI and Xbal. The lacZ-alpha fragment is PCR amplified from pUC19 with the following primers: lacZalpha-RI

5 ' GACGAATTCGCTAGCAAGAGGAGAAGTCGACATGTCCAATTCACTGGCC GTCGTTTTAC3 ' (SEQ ID NO: ) lacZalpha 3'BB

5 '-GACCCTAGGAAGCTTTCTAGAGTCGACCTATGCGGCATCAGAGCAGA-3 ' (SEQ ID NO: ) This generates a fragment with a 5' end of EcoRI site, Nhel site, a Ribosomal Binding Site, a Sail site and the start codon. On the 3' end of the fragment are the stop codon, Xbal, Hindlll, and Avrll sites. The PCR product is digested with EcoRI and Avrll and ligated into the base vectors digested with EcoRI and Xbal (Xbal and Avrll have compatible ends and generate a non-site). Because Nhel and Xbal restriction enzyme sites generate compatible ends that can be ligated together (but generate a site after ligation that is not digested by either enzyme), the genes cloned into the vectors can be "Biobricked" together

(http://openwetware.Org/wiki/Synthetic_Biology:BioBricks) . Briefly, this method enables joining an unlimited number of genes into the vector using the same 2 restriction sites (as long as the sites do not appear internal to the genes), because the sites between the genes are destroyed after each addition. These vectors can be subsequently modified using the

Phusion® Site-Directed Mutagenesis Kit (NEB, Ipswich, MA, USA) to insert the spacer sequence AATTAA between the EcoRI and Nhel sites. This eliminates a putative stem loop structure in the RNA that bound the RBS and start codon. All vectors have the pZ designation followed by letters and numbers indicating the origin of replication, antibiotic resistance marker and promoter/regulatory unit. The origin of replication is the second letter and is denoted by E for ColEl, A for pl5A and S for pSClOl (as well as a lower copy number version of pSClOl designated S*) - based origins. The first number represents the antibiotic resistance marker (1 for Ampicillin, 2 for Kanamycin, 3 for Chloramphenicol). The final number defines the promoter that regulated the gene of interest (1 for PLtetO-1, 2 for PLlacO-1 and 3 for PAllacO-1). For the work discussed here we employed three base vectors, pZS* 13S, pZA33S and pZE13S, modified for the biobricks insertions as discussed above.

Plasmids containing genes encoding pathway enzymes can then transformed into host strains containing lacIQ, which allow inducible expression by addition of isopropyl β-D-l- thiogalactopyranoside (IPTG). Activities of the heterologous enzymes are tested in in vitro assays, using strain E. coli MG1655 lacIQ as the host for the plasmid constructs containing the pathway genes. Cells can be grown aerobically in LB media (Difco) containing the appropriate antibiotics for each construct, and induced by addition of IPTG at 1 mM when the optical density (OD600) reached approximately 0.5. Cells can be harvested after 6 hours, and enzyme assays conducted as discussed below. In Vitro Enzyme Assays. To obtain crude extracts for activity assays, cells can be harvested by centrifugation at 4,500 rpm (Beckman-Coulter, Allegera X-15R) for 10 min. The pellets are resuspended in 0.3 mL BugBuster (Novagen) reagent with benzonase and lysozyme, and lysis proceeds for about 15 minutes at room temperature with gentle shaking. Cell-free lysate is obtained by centrifugation at 14,000 rpm (Eppendorf centrifuge 5402) for 30 min at 4°C. Cell protein in the sample is determined using the method of Bradford et al, Anal. Biochem. 72:248-254 (1976), and specific enzyme assays conducted as described below. Activities are reported in Units/mg protein, where a unit of activity is defined as the amount of enzyme required to convert 1 micromol of substrate in 1 minute at room temperature.

Pathway steps can be assayed in the reductive direction using a procedure adapted from several literature sources (Durre et al., FEMS Microbiol. Rev. 17:251-262 (1995); Palosaari and Rogers, Bacteriol. 170:2971-2976 (1988) and Welch et al., Arch. Biochem. Biophys. 273:309-318 (1989). The oxidation of NADH or NADPH can be followed by reading absorbance at 340 nM every four seconds for a total of 240 seconds at room temperature. The reductive assays can be performed in 100 mM MOPS (adjusted to pH 7.5 with KOH), 0.4 mM NADH or 0.4 mM NADPH, and from 1 to 50 μιηοΐ of cell extract. For carboxylic acid reductase-like enzymes, ATP can also be added at saturating concentrations. The reaction can be started by adding the following reagents: 100 μιηοΐ of 100 mM acetoacetyl-CoA, 3-hydroxybutyryl-CoA, 3-hydroxybutyrate, or 3-hydroxybutyraldehyde. The spectrophotometer is quickly blanked and then the kinetic read is started. The resulting slope of the reduction in absorbance at 340 nM per minute, along with the molar extinction coefficient of NAD(P)H at 340 nM (6000) and the protein concentration of the extract, can be used to determine the specific activity.

EXAMPLE VII

Methods for Increasing NADPH Availability

In some aspects of the invention, it can be advantageous to employ pathway enzymes that have activity using NADPH as the reducing agent. For example, NADPH-dependant pathway enzymes can be highly specific for MI-FAE cycle, MD-FAE cycle and/or termination pathway intermediates or can possess favorable kinetic properties using NADPH as a substrate. If one or more pathway steps is NADPH dependant, several alternative approaches to increase NADPH availability can be employed. These include:

1) Increasing flux relative to wild-type through the oxidative branch of the

pentose phosphate pathway comprising glucose-6-phosphate dehydrogenase, 6-phosphogluconolactonase, and 6-phosphogluconate dehydrogenase

(decarboxylating). This will generate 2 NADPH molecules per glucoses- phosphate metabolized. However, the decarboxylation step will reduce the maximum theoretical yield of 1,3-butanediol.

2) Increasing flux relative to wild-type through the Entner Doudoroff pathway comprising glucose-6-phosphate dehydrogenase, 6-phosphogluconolactonase, phosphogluconate dehydratase, and 2-keto-3-deoxygluconate 6-phosphate aldolase.

3) Introducing a soluble transhydrogenase to convert NADH to NADPH.

4) Introducing a membrane-bound transhydrogenase to convert NADH to

NADPH.

5) Employing an NADP-dependant glyceraldehyde-3 -phosphate dehydrogenase.

6) Employing any of the following enzymes or enzyme sets to convert pyruvate to acetyl-CoA

a) NADP-dependant pyruvate dehydrogenase;

b) Pyruvate formate lyase and NADP-dependant formate dehydrogenase; c) Pyruvate : ferredoxin oxidoreductase and NADPH : ferredoxin oxidoreductase;

d) Pyruvate decarboxylase and an NADP-dependant acylating acetylaldehyde dehydrogenase;

e) Pyruvate decarboxylase, NADP-dependant acetaldehyde dehydrogenase, acetate kinase, and phosphotransacetylase; and

f) Pyruvate decarboxylase, NADP-dependant acetaldehyde

dehydrogenase,and acetyl-CoA synthetase; and optionally attenuating NAD-dependant versions of these enzymes.

7) Altering the cofactor specificity of a native glyceraldehyde-3 -phosphate

dehydrogenase, pyruvate dehydrogenase, formate dehydrogenase, or acylating acetylaldehyde dehydrogenase to have a stronger preference for NADPH than their natural versions. 8) Altering the cofactor specificity of a native glyceraldehyde-3 -phosphate

dehydrogenase, pyruvate dehydrogenase, formate dehydrogenase, or acylating acetylaldehyde dehydrogenase to have a weaker preference for NADH than their natural versions.

The individual enzyme or protein activities from the endogenous or exogenous DNA sequences can be assayed using methods well known in the art. For example, the genes can be expressed in E. coli and the activity of their encoded proteins can be measured using cell extracts as described in the previous example. Alternatively, the enzymes can be purified using standard procedures well known in the art and assayed for activity. Spectrophotometric based assays are particularly effective.

Several examples and methods of altering the cofactor specificity of enzymes are known in the art. For example, Khoury et al (Protein Sci. 2009 October; 18(10): 2125-2138) created several xylose reductase enzymes with an increased affinity for NADH and decreased affinity for NADPH. Ehsani et al (Biotechnology and Bioengineering, Volume 104, Issue 2, pages 381-389, 1 October 2009) drastically decreased activity of 2,3-butanediol

dehydrogenase on NADH while increasing activity on NADPH. Machielsen et al

(Engineering in Life Sciences, Volume 9, Issue 1, pages 38-44, February 2009) dramatically increased activity of alcohol dehydrogenase on NADH. Khoury et al (Protein Sci. 2009 October; 18(10): 2125-2138) list in Table I several previous examples of successfully changing the cofactor preference of over 25 other enzymes. Additional descriptions can be found in Lutz et al, Protein Engineering Handbook, Volume 1 and Volume 2, 2009, Wiley- VCH Verlag GmbH & Co. KGaA, in particular, Chapter 31 : Altering Enzyme Substrate and Cofactor Specificity via Protein Engineering. Enzyme candidates for these steps are provided below.

Glucose-6-phosphate dehydrogenase

Protein ( ,cn Bank ID GI Number Organism

ZWF1 NP 014158.1 6324088 Saccharomyces cerevisiae S288c

ZWF1 XP 504275.1 50553728 Yarrowia lipolytica

Zwf XP 002548953.1 255728055 Candida tropicalis MYA-3404

Zwf XP 001400342.1 145233939 Aspergillus niger CBS 513.88 Protein ( ,cn Bank ID GI Number Organism

KLLA0D19855g XP 453944.1 50307901 Kluyveromyces lactis NRRL Y-1140

6-Phosphogluconolactonase

Protein ( ,cn Bank ID GI Number Organism

SOL3 NP 012033.2 82795254 Saccharomyces cerevisiae S288c

SOL4 NP 011764.1 6321687 Saccharomyces cerevisiae S288c

YALI0E11671g XP 503830.1 50552840 Yarrowia lipolytica

YALI0C19085g XP 501998.1 50549055 Yarrowia lipolytica

ANI 1 656014 XP 001388941.1 145229265 Aspergillus niger CBS 513.88

CTRG 00665 XP 002545884.1 255721899 Candida tropicalis MYA-3404

CTRG 02095 XP 002547788.1 255725718 Candida tropicalis MYA-3404

KLLA0A05390g XP 451238.1 50302605 Kluyveromyces lactis NRRL Y-1140

KLLA0C08415g XP 452574.1 50305231 Kluyveromyces lactis NRRL Y-1140

6-Phosphogluconate dehydrogenase (decarboxylating)

Protein ( ,oii Bank ID GI Number Organism

GND1 NP 012053.1 6321977 Saccharomyces cerevisiae S288c

GND2 NP 011772.1 6321695 Saccharomyces cerevisiae S288c

ANI 1 282094 XP 001394208.2 317032184 Aspergillus niger CBS 513.88

ANI 1 2126094 XP 001394596.2 317032939 Aspergillus niger CBS 513.88

YALI0B15598g XP 500938.1 50546937 Yarrowia lipolytica

CTRG 03660 XP 002549363.1 255728875 Candida tropicalis MYA-3404

KLLA0A09339g XP 451408.1 50302941 Kluyveromyces lactis NRRL Y-1140

Phosphogluconate dehydratase

Protein ( ,oii ank ID GI Number Organism

Edd AAC74921.1 1788157 Escherichia coli K-12 MG1655

Edd AAG29866.1 Zymomonas mobilis subsp. mobilis

11095426 ZM4

Edd YP 350103.1 77460596 Pseudomonas fluorescens PfO-1

ANI 1 2126094 XP 001394596.2 317032939 Aspergillus niger CBS 513.88

YALI0B15598g XP 500938.1 50546937 Yarrowia lipolytica

CTRG 03660 XP 002549363.1 255728875 Candida tropicalis MYA-3404

KLLA0A09339g XP 451408.1 50302941 Kluyveromyces lactis NRRL Y-1140 2-Keto-3-deoxygluconate 6-phosphate aldolase

Soluble transhydrogenase

5 Membrane-bound transhydrogenase

NADP-dependant glyceraldehyde-3-phosphate dehydrogenase

NAD-dependant glyceraldehyde-3-phosphate dehydrogenase

Protein ( ,oii Bank ID GI Number Organism

TDH1 NP 012483.1 6322409 Saccharomyces cerevisiae s288c

TDH2 NP 012542.1 6322468 Saccharomyces cerevisiae s288c

TDH3 NP 011708.1 632163 Saccharomyces cerevisiae s288c Protein ( ,cn Bank ID GI Number Organism

KLLA0A11858g XP 451516.1 50303157 Kluyveromyces lactis NRRL Y-1140

KLLA0F20988g XP 456022.1 50311981 Kluyveromyces lactis NRRL Y-1140

ANI 1 256144 XP 001397496.1 145251966 Aspergillus niger CBS 513.88

YALI0C06369g XP 501515.1 50548091 Yarrowia lipolytica

CTRG 05666 XP 002551368.1 255732890 Candida tropicalis MYA-3404

Mutated LpdA from E. coli K-12 MG1655 described in Biochemistry, 1993, 32 (1 1), pp 2737-2740:

MSTEIKTQVVVLGAGPAGYSAAFRCADLGLETVIVERYNTLGGVCLNVGCIP

5 SKALLHVAKVIEEAKALAEHGIVFGEPKTDIDKIRTWKEKVINQLTGGLAGMAKGRK VKVVNGLGKFTGANTLEVEGENGKTVINFDNAIIAAGSRPIQLPFIPHEDPRIWDSTD ALELKEVPERLLVMGGGIIGLEMGTVYHALGSQIDVVVRKHQVIRAADKDIVKVFTK RISKKFNLMLETKVTAVEAKEDGIYVTMEGKKAPAEPQRYDAVLVAIGRVPNGK L DAGKAGVEVDDRGFIRVDKQLRTNVPHIFAIGDIVGQPMLAHKGVHEGHVAAEVIA

0 GKKHYFDPKVIPSIAYTEPEVAWVGLTEKEAKEKGIS YETATFPWAASGRAIASDCA DGMTKLIFDKESHRVIGGAIVGTNGGELLGEIGLAIEMGCDAEDIALTIHAHPTLHES VGLAAEVFEGSITDLPNPKAKK (SEQ ID NO: )

Mutated LpdA from E. coli K-12 MG1655 described in Biochemistry, 1993, 32 (1 1), pp 2737-2740: 5 MSTEIKTQ VVVLGAGPAGYS AAFRCADLGLETVIVERYNTLGGVCLNVGCIP

SKALLHVAKVIEEAKALAEHGIVFGEPKTDIDKIRTWKEKVINQLTGGLAGMAKGRK VKVVNGLGKFTGANTLEVEGENGKTVINFDNAIIAAGSRPIQLPFIPHEDPRIWDSTD ALELKEVPERLLVMGGGIIALEMATVYHALGSQIDVVVRKHQVIRAADKDIVKVFTK RISKKFNLMLETKVTAVEAKEDGIYVTMEGKKAPAEPQRYDAVLVAIGRVPNGKNL

0 DAGKAGVEVDDRGFIRVDKQLRTNVPHIFAIGDIVGQPMLAHKGVHEGHVAAEVIA GKKHYFDPKVIPSIAYTEPEVAWVGLTEKEAKEKGISYETATFPWAASGRAIASDCA DGMTKLIFDKESHRVIGGAIVGTNGGELLGEIGLAIEMGCDAEDIALTIHAHPTLHES VGLAAEVFEGSITDLPNPKAKKK (SEQ ID NO: ) 5 NADP-dependant formate dehydrogenase

Mutant Candida bodinii enzyme described in Journal of Molecular Catalysis B:

Enzymatic, Volume 61, Issues 3-4, December 2009, Pages 157-161 : MKIVLVLYDAGKHAADEEKLYGCTENKLGIANWLKDQGHELITTSDKEGETS ELDKHIPDADIIITTPFHPAYITKERLDKAK LKLVVVAGVGSDHIDLDYINQTGKKIS VLEVTGSNVVSVAEHVVMTMLVLVR FVPAHEQIINHDWEVAAIAKDAYDIEGKTI ATIGAGRIGYRVLERLLPFNPKELLYYQRQALPKEAEEKVGARRVENIEELVAQADIV TVNAPLHAGTKGLINKELLSKFK GAWLVNTARGAICVAEDVAAALESGQLRGYGG DVWFPQPAPKDHPWRDMRNKYGAGNAMTPHYSGTTLDAQTRYAEGTK ILESFFT GKFDYRPQDIILLNGEYVTKAYGKHDK (SEQ ID NO: )

Mutant Candida bodinii enzyme described in Journal of Molecular Catalysis B:

Enzymatic, Volume 61, Issues 3-4, December 2009, Pages 157-161 :

MKIVLVLYDAGKHAADEEKLYGCTENKLGIANWLKDQGHELITTSDKEGETS ELDKHIPDADIIITTPFHPAYITKERLDKAKNLKLVVVAGVGSDHIDLDYINQTGKKIS VLEVTGSNVVSVAEHVVMTMLVLVRNFVPAHEQIINHDWEVAAIAKDAYDIEGKTI ATIGAGRIGYRVLERLLPFNPKELLYYSPQALPKEAEEKVGARRVENIEELVAQADIV TVNAPLHAGTKGLINKELLSKFK GAWLVNTARGAICVAEDVAAALESGQLRGYGG DVWFPQPAPKDHPWRDMRNKYGAGNAMTPHYSGTTLDAQTRYAEGTKNILESFFT GKFDYRPQDIILLNGEYVTKAYGKHDKK (SEQ ID NO : )

Mutant Saccharomyces cerevisiae enzyme described in Biochem J. 2002 November l :367(Pt. 3):841-847:

MSKGKVLLVLYEGGKHAEEQEKLLGCIENELGIRNFIEEQGYELVTTIDKDPE PTSTVDRELKDAEIVITTPFFPAYISRNRIAEAPNLKLCVTAGVGSDHVDLEAANERKI TVTEVTGSNVVSVAEHVMATILVLIRNYNGGHQQAINGEWDIAGVAKNEYDLEDKII STVGAGRIGYRVLERLVAFNPKKLLYYARQELPAEAINRLNEASKLFNGRGDIVQRV EKLEDMVAQSDVVTINCPLHKDSRGLFNKKLISHMKDGAYLVNTARGAICVAEDVA EAVKSGKLAGYGGDVWDKQPAPKDHPWRTMDNKDHVGNAMTVHISGTSLDAQKR YAQGVK ILNSYFSKKFDYRPQDIIVQNGSYATRAYGQK (SEQ ID NO: ).

NADPHrferredoxin oxidoreductase

5 NADP-dependant acylating acetylaldehyde dehydrogenase

Exemplary genes encoding pyruvate dehydrogenase, pyruvate :ferredoxin oxidoreductase, pyruvate formate lyase, pyruvate decarboxylase, acetate kinase,

phosphotransacetylase and acetyl-CoA synthetase are described above in Example II.

Example VIII

Engineering Saccharomyces cerevisiae for Chemical Production

Eukaryotic hosts have several advantages over prokaryotic systems. They are able to support post-translational modifications and host membrane-anchored and organelle-specific enzymes. Genes in eukaryotes typically have introns, which can impact the timing of gene expression and protein structure.

An exemplary eukaryotic organism well suited for industrial chemical production is Saccharomyces cerevisiae. This organism is well characterized, genetically tractable and industrially robust. Genes can be readily inserted, deleted, replaced, overexpressed or underexpressed using methods known in the art. Some methods are plasmid-based whereas others modify the chromosome (Guthrie and Fink. Guide to Yeast Genetics and Molecular and Cell Biology, Part B, Volume 350, Academic Press (2002); Guthrie and Fink, Guide to Yeast Genetics and Molecular and Cell Biology, Part C, Volume 351, Academic Press

(2002) ).

Plasmid-mediated gene expression is enabled by yeast episomal plasmids (YEps). YEps allow for high levels of expression; however they are not very stable and they require cultivation in selective media. They also have a high maintenance cost to the host metabolism. High copy number plasmids using auxotrophic (e.g., URA3, TRP1, HIS3,

LEU2) or antibiotic selectable markers (e.g., Zeo R or Kan R ) can be used, often with strong, constitutive promoters such as PGK1 or ACT1 and a transcription terminator-polyadenylation region such as those from CYCl or AOX. Many examples are available for one well-versed in the art. These include pVV214 (a 2 micron plasmid with URA3 selectable marker) and pVV200 (2 micron plasmid with TRP1 selectable marker) (Van et al., Yeast 20:739-746

(2003) ). Alternatively, low copy plasmids such as centromeric or CEN plamids can be used. Again, many examples are available for one well-versed in the art. These include pRS313 and pRS315 (Sikorski and Hieter, Genetics 122: 19-27 (1989) both of which require that a promoter (e.g., PGK1 or ACT1) and a terminator (e.g., CYCl, AOX) are added. For industrial applications, chromosomal overexpression of genes is preferable to plasmid-mediated overexpression. Mikkelsen and coworkers have identified 11 integration sites on highly expressed regions of the S. cerevisiae genome on chromosomes X, XI and XII (Mikkelsen et al, Met Eng 14: 104-11 (2012)). The sites are separated by essential genes, minimizing the possibility of recombination between sites. Tools for inserting genes into eukaryotic organisms such as S. cerevisiae are known in the art. Particularly useful tools include yeast integrative plasmids (Yips), yeast artificial chromosomes (YACS) and gene targeting/homologous recombination. Note that these tools can also be used to insert, delete, replace, underexpress or otherwise alter the genome of the host. Yeast integrative plasmids (Yips) utilize the native yeast homologous recombination system to efficiently integrate DNA into the chromosome. These plasmids do not contain an origin of replication and can therefore only be maintained after chromosomal integration. An exemplary construct includes a promoter, the gene of interest, a terminator, and a selectable marker with a promoter, flanked by FRT sites, loxP sites, or direct repeats enabling the removal and recycling of the resistance marker. The method entails the synthesis and amplification of the gene of interest with suitable primers, followed by the digestion of the gene at a unique restriction site, such as that created by the EcoRI and Xhol enzymes (Vellanki et al., Biotechnol Lett. 29:313-318 (2007)). The gene of interest is inserted at the EcoRI and Xhol sites into a suitable expression vector, downstream of the promoter. The gene insertion is verified by PCR and DNA sequence analysis. The recombinant plasmid is then linearized and integrated at a desired site into the chromosomal DNA of S. cerevisiae using an appropriate transformation method. The cells are plated on the YPD medium with an appropriate selection marker and incubated for 2-3 days. The transformants are analyzed for the requisite gene insert by colony PCR. To remove the antibiotic marker from a construct flanked by loxP sites, a plasmid containing the Cre recombinase is introduced. Cre recombinase promotes the excision of sequences flanked by loxP sites. (Gueldener et al, Nucleic Acids Res 30:e23 (2002)). The resulting strain is cured of the Cre plasmid by successive culturing on media without any antibiotic present. Alternately, the Cre

recombinase plasmid has a URA selection marker and the plasmid is efficiently removed by growing cells on 5-FOA which acts as a counter-selection for URA. This method can also be employed for a scarless integration instead of using loxP. One skilled in the art can integrate using URA as a marker, select for integration by growing on URA-minus plates, and then select for URA mutants by growing on 5-FOA plates. 5-FOA is converted to the toxic 5- fluoruracil by the URA gene product. Alternatively, the FLP-FRT system can be used to integrate genes into the chromosome. This system involves the recombination of sequences between short Flipase Recognition Target (FRT) sites by the Flipase recombination enzyme (FLP) derived from the 2μ plasmid of the yeast Saccharomyces cerevisiae (Sadowski, P. D., Prog.Nucleic.Acid.Res.Mol.Biol. 51 :53-91 (1995); Zhu and Sadowski J.Biol.Chem.

270:23044-23054 (1995)). Similarly, gene deletion methodologies will be carried out as described in refs. Baudin et al. Nucleic.Acids Res. 21 :3329-3330 (1993); Brachmann et al, Yeast 14: 115-132 (1998); Giaever et al, Nature 418:387-391 (2002); Longtine et al, Yeast 14:953-961 (1998) Winzeler et al, Science 285:901-906 (1999).

Another approach for manipulating the yeast chromosome is gene targeting. This approach takes advantage of the fact that double stranded DNA breaks in yeast are repaired by homologous recombination. Linear DNA fragments flanked by targeting sequences can thus be efficiently integrated into the yeast genome using the native homologous

recombination machinery. In addition to the application of inserting genes, gene targeting approaches are useful for genomic DNA manipulations such as deleting genes, introducing mutations in a gene, its promoter or other regulatory elements, or adding a tag to a gene.

Yeast artificial chromosomes (YACs) are artificial chromosomes useful for pathway construction and assembly. YACs enable the expression of large sequences of DNA (100- 3000 kB) containing multiple genes. The use of YACs was recently applied to engineer flavenoid biosynthesis in yeast (Naesby et al, Microb Cell Fact 8:49-56 (2009)). In this approach, YACs were used to rapidly test randomly assembled pathway genes to find the best combination.

The expression level of a gene can be modulated by altering the sequence of a gene and/or its regulatory regions. Such gene regulatory regions include, for example, promoters, enhancers, introns, and terminators. Functional disruption of negative regulatory elements such as repressors and/or silencers also can be employed to enhance gene expression. RNA based tools can also be employed to regulate gene expression. Such tools include RNA aptamers, riboswitches, antisense RNA, ribozymes and riboswitches.

For altering a gene's expression by its promoter, libraries of constitutive and inducible promoters of varying strengths are available. Strong constitutive promoters include pTEFl, pADHl and promoters derived from glycolytic pathway genes. The pGAL promoters are well-studied inducible promoters activated by galactose and repressed by glucose. Another commonly used inducible promoter is the copper inducible promoter pCUPl (Farhi et al, Met Eng 13:474-81 (2011)). Further variation of promoter strengths can be introduced by mutagenesis or shuffling methods. For example, error prone PCR can be applied to generate synthetic promoter libraries as shown by Alper and colleagues (Alper et al, PNAS

102: 12678-83 (2005)). Promoter strength can be characterized by reporter proteins such as beta-galactosidase, fluorescent proteins and luciferase.

The placement of an inserted gene in the genome can alter its expression level. For example, overexpression of an integrated gene can be achieved by integrating the gene into repeating DNA elements such as ribosomal DNA or long terminal repeats. For exogenous expression in yeast or other eukaryotic cells, genes can be expressed in the cytosol without the addition of leader sequence, or can be targeted to mitochondrion or other organelles, or targeted for secretion, by the addition of a suitable targeting sequence such as a mitochondrial targeting or secretion signal suitable for the host cells. Thus, it is understood that appropriate modifications to a nucleic acid sequence to remove or include a targeting sequence can be incorporated into an exogenous nucleic acid sequence to impart desirable properties. Genetic modifications can also be made to enhance polypeptide synthesis. For example, translation efficiency is enhanced by substituting ribosome binding sites with an optimal or consensus sequence and/or altering the sequence of a gene to add or remove secondary structures. The rate of translation can also be increased by substituting one coding sequence with another to better match the codon preference of the host.

EXAMPLE IX

Termination Pathways for Making Fatty Alcohols, Aldehydes and Acids

This example describes enzymes for converting intermediates of the MI-FAE cycle or MD-FAE cycle to products of interest such as fatty alcohols, fatty aldehydes, and fatty acids. Pathways are shown in Figures 1 and 7. Enzymes for catalyzing steps A-G are disclosed in Example I. This example describes enzymes suitable for catalyzing steps H-N.

Enzymes include: A. Thiolase, B. 3-Ketoacyl-CoA reductase, C. β-Hydroxyl-ACP dehydratase, D. Enoyl-CoA reductase, E. Acyl-CoA reductase (aldehyde forming), F.

Alcohol dehydrogenase, G. Acyl-CoA reductase (alcohol forming), H. acyl-CoA hydrolase, transferase or synthetase, J. Acyl-ACP reductase, K. Acyl-CoA:ACP acyltransferase, L. Thioesterase, N. Aldehyde dehydrogenase (acid forming) or carboxylic acid reductase.

Pathways for converting an MI-FAE cycle intermediate to an fatty alcohol, fatty aldehyde or fatty acid product are shown in the table below. These pathways are also referred to herein as "termination pathways".

Product Termination pathway enzymes from Figure 1

Acid H

K/L

E/N

K/J/N

Aldehyde E

K/J H/N

K L/N

Alcohol E/F

K/J/F

H/N/F

K/L/N/F

G

Product specificity can be fine-tuned using one or more enzymes shown in Figures 1 and 6. Chain length is controlled by one or more enzymes of the elongation pathway in conjunction with one more enzymes of the termination pathway as described above. The structure of the product is controlled by one or more enzymes of the termination pathway. Examples of selected termination pathway enzymes reacting with various pathway intermediates are shown in the table below. Additional examples are described herein.

Step H. Acyl-CoA hydrolase, transferase or synthase

Acyl-CoA hydrolase, transferase and synthase enzymes convert acyl-CoA moieties to their corresponding acids. Such an enzyme can be utilized to convert, for example, a fatty acyl-CoA to a fatty acid, a 3-hydroxyacyl-CoA to a 3 -hydroxy acid, a 3-oxoacyl-CoA to a 3- oxoacid, or an enoyl-CoA to an enoic acid. CoA hydrolase or thioesterase enzymes in the 3.1.2 family hydro lyze acyl-CoA molecules to their corresponding acids. Several CoA hydrolases with different substrate ranges are suitable for hydro lyzing acyl-CoA, 3-hydroxyacyl-CoA, 3-oxoacyl-CoA and enoyl-CoA substrates to their corresponding acids. For example, the enzyme encoded by acotl2 from Rattus norvegicus brain (Robinson et al, Biochem. Biophys. Res. Commun.

71 : 959-965 (1976)) can react with butyryl-CoA, hexanoyl-CoA and malonyl-CoA. The human dicarboxylic acid thioesterase, encoded by acotS, exhibits activity on glutaryl-CoA, adipyl-CoA, suberyl-CoA, sebacyl-CoA, and dodecanedioyl-CoA (Westin et al,

J.Biol. Chem. 280:38125-38132 (2005)). The closest E. coli homolog to this enzyme, tesB, can also hydrolyze a range of CoA thiolesters (Naggert et al., J Biol Chem 266: 11044-11050 (1991)). A similar enzyme has also been characterized in the rat liver (Deana R., Biochem Int 26:767-773 (1992)). Additional enzymes with hydrolase activity in E. coli include ybgC, paal, and ybdB (Kuznetsova, et al, FEMS Microbiol Rev, 2005, 29(2):263-279; Song et al, J Biol Chem, 2006, 281(16): 11028-38). Though its sequence has not been reported, the enzyme from the mitochondrion of the pea leaf has a broad substrate specificity, with demonstrated activity on acetyl-CoA, propionyl-CoA, butyryl-CoA, palmitoyl-CoA, oleoyl-CoA, succinyl- CoA, and crotonyl-CoA (Zeiher et al, Plant. Physiol. 94:20-27 (1990)) The acetyl-CoA hydrolase, ACH1, from S. cerevisiae represents another candidate hydrolase (Buu et al, J.Biol.Chem. 278: 17203-17209 (2003)) . Additional enzymes with aryl-CoA hydrolase activity include the palmitoyl-CoA hydrolase of Mycobacterium tuberculosis (Wang et al, Chem.Biol. 14:543-551 (2007)) and the acyl-CoA hydrolase of E. coli encoded by entH (Guo et al, Biochemistry 48: 1712-1722 (2009)). Additional CoA hydrolase enzymes are described above.

Gene name GenBank Accession # GI# Organism

acotl2 NP 570103.1 18543355 Rattus norvegicus

tesB NP 414986 16128437 Escherichia coli

acot8 CAA15502 3191970 Homo sapiens

acot8 NP 570112 51036669 Rattus norvegicus

tesA NP 415027 16128478 Escherichia coli

ybgC NP 415264 16128711 Escherichia coli

paal NP 415914 16129357 Escherichia coli

ybdB NP 415129 16128580 Escherichia coli

ACH1 NP 009538 6319456 Saccharomvces cerevisiae

Rv0098 NP 214612.1 15607240 Mycobacterium tuberculosis entH AAC73698.1 1786813 Escherichia coli Co A hydrolase enzymes active on 3-hydroxyacyl-CoA, 3-oxoacyl-CoA and enoyl- CoA intermediates are also well known in the art. For example, an enzyme for converting enoyl-CoA substrates to their corresponding acids is the glutaconate CoA-transferase from Acidaminococcus fermentans. This enzyme was transformed by site-directed mutagenesis into an acyl-CoA hydrolase with activity on glutaryl-CoA, acetyl-CoA and 3-butenoyl-CoA (Mack et al, FEBS.Lett. 405:209-212 (1997)). Another suitable enzyme is the fadM thioesterase III of E. coli. This enzyme is involved in oleate beta-oxidation and the preferred substrate is 3,5-tetradecadienoyl-CoA (Nie et al, Biochem 47:7744-51 (2008)).

3-Hydroxyisobutyryl-CoA hydrolase is active on 3-hydroxyacyl-CoA substrates

(Shimomura et al., J Biol Chem. 269: 14248-14253 (1994)). Genes encoding this enzyme include hibch of Rattus norvegicus (Shimomura et al, Methods Enzymol. 324:229-240 (2000)) and Homo sapiens (Shimomura et al, supra). Similar gene candidates can also be identified by sequence homology, including hibch of Saccharomyces cerevisiae and

BC 2292 of Bacillus cereus. An exemplary 3-oxoacyl-CoA hydrolase is MKS2 of Solanum lycopersicum (Yu et al, Plant Physiol 154:67-77 (2010)). The native substrate of this enzyme is 3-oxo-myristoyl-CoA, which produces a C14 chain length product.

CoA transferases catalyze the reversible transfer of a CoA moiety from one molecule to another. Several transformations require a CoA transferase to activate carboxylic acids to their corresponding acyl-CoA derivatives. CoA transferase enzymes have been described in the open literature and represent suitable candidates for these steps. These are described below.

The gene products of catl, cat2, and cat3 of Clostridium kluyveri have been shown to exhibit succinyl-CoA, 4-hydroxybutyryl-CoA, and butyryl-CoA transferase activity, respectively (Seedorf et al, Proc.Natl.Acad.Sci U.S.A 105:2128-2133 (2008); Sohling et al, J Bacteriol. 178:871-880 (1996)). Similar CoA transferase activities are also present in

Trichomonas vaginalis, Trypanosoma brucei, Clostridium aminobutyricum and

Porphyromonas gingivalis (Riviere et al, J.Biol. Chem. 279:45337-45346 (2004); van Grmsven et a J.Biol.Chem. 283: 1411-1418 (2008)).

A fatty acyl-CoA transferase that utilizes acetyl-CoA as the CoA donor is acetoacetyl- CoA transferase, encoded by the E. coli atoA (alpha subunit) and atoD (beta subunit) genes (Korolev et al, Acta Cry stallogr. D.Biol. Cry stallogr. 58:2116-2121 (2002); Vanderwinkel et al, 33:902-908 (1968)). This enzyme has a broad substrate range on substrates of chain length C3-C6 (Sramek et al, Arch Biochem Biophys 171 : 14-26 (1975)) and has been shown to transfer the CoA moiety to acetate from a variety of branched and linear 3-oxo and acyl- CoA substrates, including isobutyrate (Matthies et al, Appl Environ.Microbiol 58: 1435-1439 (1992)), valerate (Vanderwinkel et al, Biochem.Biophys.Res.Commun. 33:902-908 (1968)) and butanoate (Vanderwinkel et al, Biochem.Biophys.Res.Commun. 33:902-908 (1968)). This enzyme is induced at the transcriptional level by acetoacetate, so modification of regulatory control may be necessary for engineering this enzyme into a pathway (Pauli et al., Eur. J Biochem. 29:553-562 (1972)). Similar enzymes exist in Corynebacterium glutamicum ATCC 13032 (Duncan et al, 68:5186-5190 (2002)), Clostridium acetobutylicum (Cary et al, Appl Environ Microbiol 56: 1576-1583 (1990); Wiesenborn et al, Appl Environ Microbiol 55:323-329 (1989)), and Clostridium saccharoperbutylacetonicum (Kosaka et al.,

Biosci.Biotechnol Biochem. 71 :58-68 (2007)). Gene GI # Accession No. Organism

atoA 2492994 P76459.1 Escherichia coli

atoD 2492990 P76458.1 Escherichia coli

actA 62391407 YP 226809.1 Corynebacterium glutamicum

cg0592 62389399 YP 224801.1 Corynebacterium glutamicum

ctfA 15004866 NP 149326.1 Clostridium acetobutylicum

ctfB 15004867 NP 149327.1 Clostridium acetobutylicum

ctfA 31075384 AAP42564.1 Clostridium saccharoperbutylacetonicum ctfB 31075385 AAP42565.1 Clostridium saccharoperbutylacetonicum

Beta-ketoadipyl-CoA transferase, also known as succinyl-CoA:3:oxoacid-CoA transferase, is active on 3-oxoacyl-CoA substrates. This enzyme is encoded by pcal and pcaJ in Pseudomonas putida (Kaschabek et al, J Bacteriol. 184:207-215 (2002)). Similar enzymes are found in Acinetobacter sp. ADP1 (Kowalchuk et al, Gene 146:23-30 (1994)), Streptomyces coelicolor and Pseudomonas knackmussii (formerly sp. B13) (Gobel et al, J Bacteriol. 184:216-223 (2002); Kaschabek et al, J Bacteriol. 184:207-215 (2002)).

Additional exemplary succinyl-CoA:3:oxoacid-CoA transferases have been characterized in in Helicobacter pylori (Corthesy-Theulaz et al, J Biol.Chem. 272:25659-25667 (1997)), Bacillus subtilis (Stols et al., Protein Expr.Purif. 53:396-403 (2007)) and Homo sapiens

(Fukao, T., et al, Genomics 68:144-151 (2000); Tanaka, H., et al, Mol Hum Reprod 8:16-23 (2002)). Genbank information related to these genes is summarized below.

The conversion of acyl-CoA substrates to their acid products can be catalyzed by a CoA acid-thiol ligase or CoA synthetase in the 6.2.1 family of enzymes. CoA synthases that convert ATP to ADP (ADP-forming) are reversible and react in the direction of acid formation, whereas AMP forming enzymes only catalyze the activation of an acid to an acyl- CoA. For fatty acid formation, deletion or attenuation of AMP forming enzymes will reduce backflux. ADP-forming acetyl-CoA synthetase (ACD, EC 6.2.1.13) is an enzyme that couples the conversion of acyl-CoA esters to their corresponding acids with the concomitant synthesis of ATP. ACD I from Archaeoglobus fulgidus, encoded by AF1211, was shown to operate on a variety of linear and branched-chain substrates including isobutyrate, isopentanoate, and fumarate (Musfeldt et al, J Bacteriol. 184:636-644 (2002)). A second reversible ACD in Archaeoglobus fulgidus, encoded by AF1983, was also shown to have a broad substrate range (Musfeldt and Schonheit, J Bacteriol. 184:636-644 (2002)). The enzyme from Haloarcula marismortui (annotated as a succinyl-CoA synthetase) accepts propionate, butyrate, and branched-chain acids (isovalerate and isobutyrate) as substrates, and was shown to operate in the forward and reverse directions (Brasen et al., Arch Microbiol 182:277-287 (2004)). The ACD encoded by PAE3250 from hyperthermophilic crenarchaeon Pyrobaculum aerophilum showed the broadest substrate range of all characterized ACDs, reacting with acetyl-CoA, isobutyryl-CoA (preferred substrate) and phenylacetyl-CoA (Brasen et al, supra). Directed evolution or engineering can be used to modify this enzyme to operate at the physiological temperature of the host organism. The enzymes from A. fulgidus, H. marismortui and P. aerophilum have all been cloned, functionally expressed, and characterized in E. coli (Brasen and Schonheit, supra; Musfeldt and Schonheit, J Bacteriol.

184:636-644 (2002)). An additional candidate is succinyl-CoA synthetase, encoded by sucCD of E. coli and LSC1 and LSC2 genes of Saccharomyces cerevisiae. These enzymes catalyze the formation of succinyl-CoA from succinate with the concomitant consumption of one ATP in a reaction which is reversible in vivo (Buck et al., Biochemistry 24:6245-6252 (1985)). The acyl CoA ligase from Pseudomonas putida has been demonstrated to work on several aliphatic substrates including acetic, propionic, butyric, valeric, hexanoic, heptanoic, and octanoic acids and on aromatic compounds such as phenylacetic and phenoxyacetic acids (Fernandez- Valverde et al, Appl.Environ.Microbiol. 59: 1149-1154 (1993)). A related enzyme, malonyl CoA synthetase (6.3.4.9) from Rhizobium leguminosarum could convert several diacids, namely, ethyl-, propyl-, allyl-, isopropyl-, dimethyl-, cyclopropyl-, cyclopropylmethylene-, cyclobutyl-, and benzyl-malonate into their corresponding

monothioesters (Pohl et al, J.Am.Chem.Soc. 123:5822-5823 (2001)). Protein ( ,cn Bank ID GI Number Organism

AF121 1 NP 070039.1 1 1498810 Archaeoglobus fulgidus

AF1983 NP 070807.1 1 1499565 Archaeoglobus fulgidus scs YP 135572.1 55377722 Haloarcula marismortui

PAE3250 NP 560604.1 18313937 Pyrobaculum aerophilum str.

IM2

sucC NP 415256.1 16128703 Escherichia coli

sucD AAC73823.1 1786949 Escherichia coli

LSC 1 NP 014785 6324716 Saccharomyces cerevisiae

LSC2 NP 01 1760 6321683 Saccharomyces cerevisiae paaF AAC24333.2 2271 1873 Pseudomonas putida matB AAC83455.1 3982573 Rhizobium leguminosarum

Step J. Acyl-ACP reductase

The reduction of an acyl-ACP to its corresponding aldehyde is catalyzed by an acyl- ACP reductase (AAR). Such a transformation is depicted in step J of Figures 1 and 7.

Suitable enzyme candidates include the orfl594 gene product of Synechococcus elongatus PCC7942 and homologs thereof (Schirmer et al, Science, 329: 559-62 (2010)). The S elongates PCC7942 acyl-ACP reductase is coexpressed with an aldehyde decarbonylase in an operon that appears to be conserved in a majority of cyanobacterial organisms. This enzyme, expressed in E. coli together with the aldehyde decarbonylase, conferred the ability to produce alkanes. The P. marinus AAR was also cloned into E. coli and, together with a decarbonylase, demonstrated to produce alkanes (US Application 201 1/0207203).

Step K. Acyl-CoA:ACP acyltransferase

The transfer of an acyl-CoA to an acyl-ACP is catalyzed by acyltransferase enzymes in EC class 2.3.1. Enzymes with this activity are described above. Step L. Thioesterase

Acyl-ACP thioesterase enzymes convert an acyl-ACP to its corresponding acid. Such a transformation is required in step L of Figure 1. Exemplary enzymes include the FatA and FatB isoforms of Arabidopsis thaliana (Salas et al, Arch Biochem Biophys 403:25-34 (2002)). The activities of these two proteins vary with carbon chain length, with FatA preferring oleyl-ACP and FatB preferring palmitoyl-ACP. A number of thioesterases with different chain length specificities are listed in WO 2008/113041 and are included in the table below. For example, it has been shown previously that expression of medium chain plant thioesterases like FatB from Umbellularia californica in E. coli results in accumulation of high levels of medium chain fatty acids, primarily laurate (C12:0). Similarly, expression of Cuphea palustris FatBl thioesterase in E. coli led to accumulation of C8-10:0 products (Dehesh et al, Plant Physiol 110:203-10 (1996)). Similarly, Carthamus tinctorius thioesterase expressed in E. coli leads to >50 fold elevation in C 18: 1 chain termination and release as free fatty acid (Knutzon et al, Plant Physiol 100: 1751-58 (1992)). Methods for altering the substrate specificity of thioesterases are also known in the art (for example, EP1605048).

Step N. Aldehyde dehydrogenase (acid forming) or carboxylic acid reductase

The conversion of an aldehyde to an acid is catalyzed by an acid-forming aldehyde dehydrogenase. Several Saccharomyces cerevisiae enzymes catalyze the oxidation of aldehydes to acids including ALD1 (ALD6), ALD2 and ALD3 (Navarro-Avino et al, Yeast 15:829-42 (1999); Quash et al, Biochem Pharmacol 64: 1279-92 (2002)). The mitochondrial proteins ALD4 and ALD5 catalyze similar transformations (Wang et al, J Bacteriol 180:822- 30 (1998); Boubekeur et al, Eur J Biochem 268:5057-65 (2001)). HFD1 encodes a hexadecanal dehydrogenase. Exemplary acid- forming aldehyde dehydrogenase enzymes are listed in the table below. Protein ( ,cn Bank ID GI number Organism

ALD2 NP 013893.1 6323822 Saccharomyces cerevisiae s288c

ALD3 NP 013892.1 6323821 Saccharomyces cerevisiae s288c

ALD4 NP 015019.1 6324950 Saccharomyces cerevisiae s288c

ALD5 NP 010996.2 330443526 Saccharomyces cerevisiae s288c

ALD6 NP 015264.1 6325196 Saccharomyces cerevisiae s288c

HFD1 NP 013828.1 6323757 Saccharomyces cerevisiae s288c

Ca019.8361 XP 710976.1 68490403 Candida albicans

CaO 19.742 XP 710989.1 68490378 Candida albicans

YALI0C03025 CAG81682.1 49647250 Yarrowia lipolytica

ANI 1 1334164 XP 001398871.1 145255133 Aspergillus niger

ANI 1 2234074 XP 001392964.2 317031176 Aspergillus niger

ANI 1 226174 XP 001402476.1 145256256 Aspergillus niger

ALDH P41751.1 1169291 Aspergillus niger

KLLA0D09999 CAH00602.1 49642640 Kluyveromyces lactis

The conversion of an acid to an aldehyde is thermodynamically unfavorable and typically requires energy-rich cofactors and multiple enzymatic steps. For example, in butanol biosynthesis conversion of butyrate to butyraldehyde is catalyzed by activation of butyrate to its corresponding acyl-CoA by a CoA transferase or ligase, followed by reduction to butyraldehyde by a CoA-dependent aldehyde dehydrogenase. Alternately, an acid can be activated to an acyl-phosphate and subsequently reduced by a phosphate reductase. Direct conversion of the acid to aldehyde by a single enzyme is catalyzed by a bifunctional carboxylic acid reductase enzyme in the 1.2.1 family. Exemplary enzymes that catalyze these transformations include carboxylic acid reductase, alpha-aminoadipate reductase and retinoic acid reductase.

Carboxylic acid reductase (CAR), found in Nocardia iowensis, catalyzes the magnesium, ATP and NADPH-dependent reduction of carboxylic acids to their

corresponding aldehydes (Venkitasubramanian et al., J Biol.Chem. 282:478-485 (2007)). The natural substrate of this enzyme is benzoic acid and the enzyme exhibits broad acceptance of aromatic and aliphatic substrates including fatty acids of length C12-C18

(Venkitasubramanian et al., Biocatalysis in Pharmaceutical and Biotechnology Industries. CRC press (2006); WO 2010/135624). CAR requires post-translational activation by a phosphopantetheine transferase (PPTase) that converts the inactive apo-enzyme to the active holo-enzyme (Hansen et al., Appl.Environ.Microbiol 75:2765-2774 (2009)). The Nocardia CAR enzyme was cloned and functionally expressed in E. coli (Venkitasubramanian et al., J Biol.Chem. 282:478-485 (2007)). Co-expression of the npt gene, encoding a specific PPTase, improved activity of the enzyme. A related enzyme from Mycobacterium sp. strain JLS catalyzes the reduction of fatty acids of length C12-C16. Variants of this enzyme with enhanced activity on fatty acids are described in WO 2010/135624. Alpha-aminoadipate reductase (AAR, EC 1.2.1.31), participates in lysine biosynthesis pathways in some fungal species. This enzyme naturally reduces alpha-aminoadipate to alpha-aminoadipate semialdehyde. The carboxyl group is first activated through the ATP-dependent formation of an adenylate that is then reduced by NAD(P)H to yield the aldehyde and AMP. Like CAR, this enzyme utilizes magnesium and requires activation by a PPTase. Enzyme candidates for AAR and its corresponding PPTase are found in Saccharomyces cerevisiae (Morris et al, Gene 98: 141-145 (1991)), Candida albicans (Guo et al, Mol.Genet.Genomics 269:271-279 (2003)), and Schizosaccharomyces pombe (Ford et al, Curr.Genet. 28: 131-137 (1995)). The AAR from S. pombe exhibited significant activity when expressed in E. coli (Guo et al, Yeast 21 : 1279-1288 (2004)). The AAR from Penicillium chrysogenum accepts S-carboxymethyl-L- cysteine as an alternate substrate, but did not react with adipate, L-glutamate or

diaminopimelate (Hijarrubia et al., J Biol.Chem. 278:8250-8256 (2003)). The gene encoding the P. chrysogenum PPTase has not been identified to date and no high-confidence hits were identified by sequence comparison homology searching.

Additional car and npt genes can be identified based on sequence homology. GenBank

Gene name GI o. Organism

Accession No.

fadD9 121638475 YP 978699.1 Mycobacterium bovis BCG

BCG 2812c 121638674 YP 978898.1 Mycobacterium bovis BCG

Nocardia farcinica IFM nfa20150 54023983 YP_118225.1

10152

Nocardia farcinica IFM nfa40540 54026024 YP 120266.1

10152

Streptomyces griseus

SGR 6790 YP OO 1828302.1 182440583 subsp. griseus NBRC

13350

Streptomyces griseus

SGR 665 YP_001822177.1 182434458 subsp. griseus NBRC

13350

Mycobacterium smegmatis

MSMEG 2956 YP_887275.1 118473501

MC2 155

Mycobacterium smegmatis

MSMEG 5739 YP_889972.1 118469671

MC2 155

Mycobacterium smegmatis

MSMEG_2648 YP_886985.1 118471293

MC2 155

Mycobacterium avium

MAP 1040c NP_959974.1 41407138 subsp. paratuberculosis K- 10

Mycobacterium avium

MAP2899c NP_961833.1 41408997 subsp. paratuberculosis K- 10

Mycobacterium marinum

MMAR_2117 YP OO 1850422.1 183982131

M

Mycobacterium marinum

MMAR 2936 YP 001851230.1 183982939

M

Mycobacterium marinum

MMAR 1916 YP OO 1850220.1 183981929

M

Tsukamurella

Tpau_1373 YP 003646340.1 296139097 paurometabola DSM

20162

Tsukamurella

Tpau_1726 YP 003646683.1 296139440 paurometabola DSM

20162

CPCC7001 1320 ZP 05045132.1 254431429 Cyanobium PCC7001

Dictyostelium discoideum

DDBDRAFT O 187729 XP 636931.1 66806417

AX4

An additional enzyme candidate found in Streptomyces griseus is encoded by the griC and griD genes. This enzyme is believed to convert 3-amino-4-hydroxybenzoic acid to 3- amino-4-hydroxybenzaldehyde as deletion of either griC or griD led to accumulation of extracellular 3-acetylamino-4-hydroxybenzoic acid, a shunt product of 3-amino-4- hydroxybenzoic acid metabolism (Suzuki, et al, J. Antibiot. 60(6):380-387 (2007)). Co- expression of griC and griD with SGR 665, an enzyme similar in sequence to the Nocardia iowensis npt, can be beneficial.

EXAMPLE X

PRODUCTION OF 1,3-BUTANEDIOL FROM GLUCOSE IN SACCHAROMYCES

CEREVISIAE

This example illustrates the construction and biosynthetic production of 1,3-BDO from glucose in Saccharomyces cerevisiae.

The pathway for 1,3-BDO production is comprised of two MI-FAE cycle enzymes (thiolase and 3-oxoacyl-CoA reductase), in conjunction with termination pathway enzymes (acyl-CoA reductase (aldehyde forming) and alcohol dehydrogenase). The 1,3-BDO pathway engineered into S. cerevisiae is composed of four enzymatic steps which transform acetyl- CoA to 1,3-BDO. The first step entails the condensation of two molecules of acetyl-CoA into acetoacetyl-CoA by an acetoacetyl-CoA thiolase enzyme (THL). In the second step, acetoacetyl-CoA is reduced to 3-hydroxybutyryl-CoA by acetoacetyl-CoA reductase, also called 3-hydroxybutyryl-CoA dehydrogenase (HBD). 3-hydroxybutyryl-CoA reductase (ALD) catalyzes formation of the aldehyde from the acyl-CoA. Further reduction of 3- hydroxybutyraldehyde to 1,3-BDO is catalyzed by 1,3-BDO dehydrogenase (ADH).

To enable 13-BDO production in the cytosol, two acetyl-CoA forming pathways were engineered into S. cerevisiae. The first pathway entails conversion of pyruvate to acetyl-CoA by pyruvate decarboxylase (Figure 2E), acetaldehyde dehydrogenase (Figure 2F) and acetyl- CoA synthetase (Figure 2B). The second pathway is pyruvate formate lyase (Figure 2H).

For each enzymatic step of the 1,3-BDO pathway, a list of applicable genes was assembled for corroboration. The genes cloned and assessed in this study are presented below in Table 1 , along with the appropriate references and URL citations to the polypeptide sequence.

Table 1

Genes were cloned via PCR from the genomic DNA of the native or wild-type organism. Primers used to amplify the pathway genes are (from 5' to 3'; underlined sequences are gene specific):

Thl 1502:

FP:TCTAATCTAAGTTTTCTAGAACTAGTAAAGATGAGAGATGTAGTAATA GTAAGTGCTGTA (SEQ ID NO:)

RP:GATATCGAATTCCTGCAGCCCGGGGGATCCTTAGTCTCTTTCAACTACG AGAGCTGTT (SEQ ID NO:) Thl 1491 :

FP :TCTAATCTAAGTTTTCTAGAACTAGTAAAGATGAAAAATTGTGTCATCG TCAGTG (SEP ID NO: 11)

RP:GATATCGAATTCCTGCAGCCCGGGGGATCCTTAATTCAACCGTTCAAT CACCATCGCAAT (SEQ ID NO :)

Thl 560:

FP:AATCTAAGTTTTCTAGAACTAGTAAAGATGAAAGAAGTTGTAATAGCT AGTGCAGTAA (SEQ ID NO:)

RP:TATCGAATTCCTGCAGCCCGGGGGATCCTTAATGGTGATGGTGATGAT GGCACTTTTCTA (SEP ID NO:)

Thl 1512:

FP:TCTAATCTAAGTTTTCTAGAACTAGTAAAGATGAGCACCCCGTCCATCG TCA (SEQ ID NO:)

PR:GATATCGAATTCCTGCAGCCCGGGGGATCCCTAAAGGCTCTCGATGCA CATCGCC (SEP ID NO :)

Thl 1501 :

FP:TAAGCTAGCAAGAGGAGAAGTCGACATGACTGACGTTGTCATCGTATC CGC (SEQ ID NP:)

RP: GCCTCTAGGAAGCTTTCTAGATTATTATTTGCGCTCGACTGCCAGC (SEQ ID NP:)

Hbd 1495:

FP:AAGCATACAATCAACTATCTCATATACAATGAAAAAGATTTTTGTACTT GGAGCA (SEQ ID NP:)

RP :AAAAATCATAAATCATAAGAAATTCGCTTATTTAGAGTAATCATAGAA TCCTTTTCCTGA (SEQ ID NP :)

Aid 707:

FP:AATCTAAGTTTTCTAGAACTAGTAAAGATGAACACAGAAAACATTGAA CAAGCCAT (SEQ ID NP:)

RP:TATCGAATTCCTGCAGCCCGGGGGATCCCTAAGCCTCCCAAGTCCGTA ATGAGAACCCTT (SEQ ID NP:)

Adh 28:

FP:CCAAGCATACAATCAACTATCTCATATACAATGGAGAATTTTAGATTTA ATGCATATACA (SEQ ID NP:) RP :AATAAAAATCATAAATCATAAGAAATTCGCTTAAAGGGACATTTCTAA AATTTTATATAC (SEQ ID NO:)

1845 A is a sequence variant of the wild type (1845) enzyme. The variation is a point mutation in the residue Leu-641 (L641P), described in Starai and coworkers (Starai et al, J Biol Chem 280: 26200-5 (2005)). The function of the mutation, e.g., is to prevent post- translational regulation by acetylation and maintain the Acs enzyme in its active state.

Shuttle plasmids shown in Table 2 were constructed for expression of heterologous genes in S. cerevisiae. Plasmids d9, dlO, and dl 1 are empty plasmid controls with the selection marker of Ura, His, and Leu, respectively. Plasmids dl2 or dl3 contains a single ALD or ADH gene with the URA3 selection marker. Plasmids dl4, dl6, and dl7 contains hbd and thil genes with the HIS3 selection marker.

Table 2

Yeast host BY4741 [MATa his3A0 leu2A0 metl5A0 ura3A0] was chosen as the host strain for this work as a wild-type laboratory strain with the appropriate auxotrophic markers to host the pathway plasmids. BY4741 was transformed with plasmids containing 1,3-BDO pathway genes alone or along with plasmids that contain PDH bypass genes or pflAB genes. Vector backbones used in this example include p427TEF yeast expression vectors, the pY3H bridging vectors (Sunrise Science) and pESC yeast epitope tagging vectors (Agilent Technologies). The pY3H vector containing a TEFl promoter, CYC terminator and URA3 selection marker from S. cerevisiae was used to build dual-promoter plasmids with different selection markers. ADH1 promoter and terminator sequences from S. cerevisiae were inserted upstream of the TEFl promoter so the two transcriptional units are in a back-to-back orientation. The SV40 nuclear localization signal sequence was removed during the cloning process. The resulting plasmid was named pY3Hd9. To construct plasmids with a different selection marker, the URA3 gene in pY3Hd9 was replaced with the HIS3 or LEU2 gene from S. cerevisiae to produce pY3Hdl0 and pY3Hdl 1, respectively. Two of the four 1,3-BDO pathway genes— Hbd and Thl (see Table 103 for gene numbers)— were cloned into the dual- promoter plasmid with the HIS3 marker such that the expression of the Hbd genes is controlled by the ADH1 promoter while the expression of the Thl gene is controlled by the TEFl promoter (pY3Hdl4 ~ 17). Aid and Adh genes were cloned into the dual-promoter plasmid with the LEU2 selection marker such that the ADH1 promoter drives the adh genes and the TEFl promoter drives the aid genes (pY3Hd8). The PflAB genes or the PDH bypass genes (ALD6 and acs) were cloned into the dual-promoter plasmid with the URA3 marker where pflA or ALD6 is controlled under the ADH1 promoter and pflB or acs is controlled under the TEFl promoter. Yeast transformation was done using Frozen-EZ Yeast

Transformation (Zymo Research).

Tables 3 and 4 show the combinations of plasmids and experimental conditions tested.

Table 3

Table 4

Plasmid l; Plasmid 2i pl asmid 3: gene 1 gene 2 gene 3 gene 4 gene 5 gene 6 lAeroationi Note d9 dll ; aerobic ; EVC d8 dl7 1495 560 28 707 ; aerobic ; BDO d8 dl7 d5 1495 560 28 707 1849 1845A ; aerobic ; BDO + PDH d8 dl4 1495 1502 28 707 ; aerobic ; BDO d8 dl4 d5 1495 1502 28 707 1849 1845A ; aerobic ; BDO + PDH : In Table 3, colonies were inoculated in 5 ml of 2% glucose medium with

corresponding amino acid dropouts and cultured at 30 degree for approximately 48 hrs. Cells were briefly spun down and re-suspended in 2 ml fresh 2% glucose medium with tween-80 and ergosterol added. Resuspended cultures were added to 10 ml fresh glucose medium in 20 ml bottles to obtain a starting OD of 0.2. For anaerobic cultures, the bottles containing

cultures were vacuumed and filled with nitrogen. For micro-aerobic growth, a 23 G needle

was inserted. All the cultures were incubated at 30 degree with shaking for 24 hours. In Table 4, the experiment was carried out in a 96-well plate and cells grown aerobically in 1.2 ml of medium with varying glucose and acetate concentrations (5% glucose, 10% glucose, 5% glucose + 50 mM acetate, and 10% glucose+50 mM acetate).

Concentrations of glucose, 1,3-BDO, alcohols, and other organic acid byproducts in the culture supernatant were determined by HPLC using an HPX-87H column (BioRad).

MI-FAE cycle and termination pathway genes were tested with or without pflAB or PDH bypass. As shown in FIGS. 9-11, these constructs produced 0.3 - 3.35 mM 1,3-BDO in yeast S. cerevisiae BY4741, and ethanol was produced in the tested samples tested. The PDH bypass (here, overexpression of ALD6 and acs or acsm genes) improved production of 1,3- BDO.

EXAMPLE XI

ENZYMATIC ACTIVITY OF 1,3-BUTANEDIOL PATHWAY ENZYMES

This example describes the detection of 1,3-BDO pathway enzyme activity using in vitro assays.

Activity of the heterologous enzymes was tested in in vitro assays, using an internal yeast strain as the host for the plasmid constructs containing the pathway genes. Cells were grown aerobically in yeast media containing the appropriate amino acid for each construct. To obtain crude extracts for activity assays, cells were harvested by centrifugation. The pellets were resuspended in 0.1 mL 100 mM Tris pH 7.0 buffer containing protease inhibitor cocktail. Lysates were prepared using the method of bead beating for 3 min. Following bead beating, the solution was centrifuged at 14,000 rpm (Eppendorf centrifuge 5402) for 15 min at 4°C. Cell protein in the sample was determined using the method of Bradford et al., Anal. Biochem. 72:248-254 (1976), and specific enzyme assays conducted as described below.

Thiolase

Thiolase enzymes catalyze the condensation of two acetyl-CoA to form acetoacetyl- CoA. In the reaction, coenzyme A (CoA) is released and the free CoA can be detected using 5,5'-dithiobis-2-nitrobenzoic acid (DTNB) which absorbs at 410 nm upon reaction with CoA. Five thiolases were tested (see example X, Table 1). Estimated specific activity in E.coli crude lysates is shown in Figure 12. Among the Thl that showed expressed protein, 1512 and 1502 demonstrated the highest specific activity for acetyl-CoA condensation activity n E.coli crude lysates.

Both 1491 and 560 were cloned in dual promoter yeast vectors with 1495, which is the 3-hydroxybutyryl-CoA dehydrogenase (see Figure 13). These thiolases were evaluated for acetyl-CoA condensation activity, and the data is shown in Figure 13. The results indicate that both 560 and 1491 demonstrate an initial burst of activity that is too fast to measure. However, after the initial enzyme rate, the condensation rate of 560 is greater than 1491. Thus, there is protein expression and active enzyme with the yeast dual promoter vectors as indicated by active thiolase activity observed in crude lysates. 3-Hydroxybutyryl-CoA Dehydrogenase (Hbd)

Acetoacetyl-CoA is metabolized to 3-hydroxybutyryl-CoA by 3-hydroxybutyryl- CoA dehydrogenase. The reaction requires oxidation of NADH, which can be monitored by fluorescence at an excitation wavelength at 340 nm and an emission at 460 nm. . The oxidized form, NAD+, does not fluoresce. This detection strategy was used for all of the dehydrogenase steps. 1495, the Hbd from Clostridium beijerinckii, was assayed in the dual promoter yeast vectors that contained either 1491 (vector id = pY3Hdl7) or 560 (vector id = pY3Hdl6). See Table 1 for GenBank identifiers of each enzyme. The time course data is shown in Figure 14.

The Hbd rate of 1495 containing 560 was much faster than 1491. The results provided in Figure 15 show that the Hbd prefers NADH over NADPH. The Hbd enzyme appears to display the fastest catalytic activity among the four pathway enzymes in crude lysates. The Hbd enzyme, i.e. a 3-ketoacyl-CoA reductase, is an example of a MI-FAE cycle or MD-FAE cycle enzyme that preferentially reacts with an NADH cofactor.

Aldehyde Devhdrogenase (Aid) An aldehyde reductase converts 3-hydroxybutyryl-CoA to 3- hydroxybutyraldehyde. This reaction requires NAD(P)H oxidation, which can be used to monitor enzyme activity. The Aid from Lactobacillus brevis (Gene ID 707) was cloned in a dual vector that contained the alcohol dehydrogenase from Clostridium

saccharoperbutylacetonicum (Gene ID 28). These two enzymes were cloned in another dual promoter yeast vector containing a Leu marker. The Aid activity data for crude lysates is shown in Figure 16 with a 707 lysate from E. coli used as a standard. The results indicate the 707 showed enzyme activity in yeast lysates that is comparable to the lysate from bacteria. In addition, the 707 gene product prefers NADH to NADPH as the cofactor. The 707 gene product, i.e. an acy-CoA reductase

(aldehyde forming), is an example of a termination pathway enzyme that preferentially reacts with an NADH cofactor.

Alcohol Dehydrogenase (Adh)

1,3-BDO is formed by an alcohol dehydrogenase (Adh), which reduces 3- hydroxybutyraldehyde in the presence of NAD(P)H. The oxidation of NAD(P)H can be used to monitor the reaction as described above.

The evaluation of ADH (Gene 28) in the dual promoter vector with ALD (Gene 707) is shown in Figure 17 with butyraldehyde, a surrogate substrate for 3 -hydroxybutyraldehyde. The data indicate that Gene 28 have Adh activity similar to the no insert control (EV) with butyraldehyde and NADPH. This is likely caused by endogenous ADH enzymes present in yeast that may function in the same capability as 28.

In summary, candidates for the Thl, Hbd, Aid, and Adh to produce 1,3-BDO showed enzyme activity in yeast crude lysates for the dual promoter vectors constructed.

Throughout this application various publications have been referenced. The disclosures of these publications in their entireties, including GenBank and GI number publications, are hereby incorporated by reference in this application in order to more fully describe the state of the art to which this invention pertains. Although the invention has been described with reference to the examples provided above, it should be understood that various modifications can be made without departing from the spirit of the invention.