Login| Sign Up| Help| Contact|

Patent Searching and Data


Title:
ALGAL AND FUNGAL GENES AND THEIR USES FOR TAURINE BIOSYNTHESIS IN CELLS
Document Type and Number:
WIPO Patent Application WO/2017/176277
Kind Code:
A1
Abstract:
The present invention describes an approach to produce taurine or increase hypotaurine or taurine production in prokaryotes or eukaryotes. More particularly, the invention relates to genetic transformation of organisms with algal, microalgal or fungal genes that encode proteins that catalyze the conversion of sulfur-containing compounds such as sulfate or cysteine to taurine. The invention describes methods for the use of polynucleotides for cysteine dioxygenase-like (CDOL), sulfinoalanine decarboxylase-like (SADL), cysteine sulfate/decarboxylase or a portion of the cysteine synthetase/PLP decarboxylase (partCS/PLP-DC) polypeptide in bacteria, alga, yeast, or plants to produce taurine or increase hypotaurine or taurine. The preferred embodiment of the invention is in plants but other organisms may be used. Taurine production or increased levels of hypoataurine or taurine in plants could be used as nutraceutical, pharmaceutical, or therapeutic compounds or as a supplement in animal feed or for animal feed or as an enhancer for plant growth or yield

Inventors:
TURANO FRANK J (US)
Application Number:
PCT/US2016/026465
Publication Date:
October 12, 2017
Filing Date:
April 07, 2016
Export Citation:
Click for automatic bibliography generation   Help
Assignee:
PLANT SENSORY SYSTEMS LLC (US)
International Classes:
A61K31/145; C12N9/02; C12N15/82; C12P13/12
Domestic Patent References:
WO2016028508A12016-02-25
Foreign References:
US20120222148A12012-08-30
US20130333068A12013-12-12
Other References:
HONJOH ET AL.: "Enhancement of menadione stress tolerance in yeast by accumulation of hypotaurine and taurine: co-expression of cDNA clones, from Cyprinus carpio, for cysteine dioxygenase and cysteine sulfinate decarboxylase in Saccharomyces cerevisiae", AMINO ACIDS, vol. 38, no. Iss. 4, 26 July 2009 (2009-07-26), pages 1 173 - 1183
YEW ET AL.: "The genome of newly classified Ochroconis mirabilis: Insights into fungal adaptation to different living conditions", BMC GENOMICS, vol. 17, 3 February 2016 (2016-02-03), pages 1 - 17, XP021232226
YOUSSEFIAN ET AL.: "Increased Cysteine Biosynthesis Capacity of Transgenic Tobacco Overexpressing an O-Acetylserine(thiol) Lyase Modifies Plant Responses to Oxidative Stress", PLANT PHYSIOLOGY, vol. 126, no. 3, 1 July 2001 (2001-07-01), pages 1001 - 1011, XP002483940
Attorney, Agent or Firm:
IHNEN, Jeffrey L. et al. (Figg Ernst, & Manbeck, P.C.,607 14th Street, N.W.,Suite 80, Washington District of Columbia, US)
Download PDF:
Claims:
CLAIMS

1. A cell comprising either:

(a) two expression units, wherein

(i) a first expression unit that comprises a first promoter operably linked to a first polynucleotide which encodes cysteine dioxygenase-like (CDOL) protein; and

(ii) a second expression unit that comprises a second promoter operably linked to a second polynucleotide which encodes a portion of the cysteine synthetase/PLP decarboxylase (partCS PLP-DC) protein; or

(b) a single expression unit which comprises a promoter operably linked to a polynucleotide which encodes CDOL operably linked to partCS PLP-DC or CS/PLP-DC,

wherein the two expression units or the single expression unit is expressed in the cell and wherein the cell produces taurine.

2. The cell of claim 1, wherein CDOL comprises the nucleotide sequence SEQ ID NO:l.

3. The cell of claim 1, wherein partCS/PLP-DC comprises nucleotides 1 through 3 plus 1414 through 2958 of the sequence SEQ ID NO:9.

4. The cell of claim 1, wherein CS/PLP-DC comprises nucleotide sequence SEQ ID NO: 9,

5. The cell of claim 1, wherein CDOL comprises the amino acid sequence SEQ ID NO:5.

6. The cell of claim 1, wherein partCS/PLP-DC comprises amino acids 1 plus 472 through 985 of the sequence SEQ ID NO:15.

7. The cell of claim 1, wherein CS/PLP-DC comprises amino acid sequence SEQ ID NO:15.

8. The cell of claim 1 which is a prokaryotic cell.

9. A method of producing hypo taurine or taurine, comprising growing the prokaryotic cell of claim 1 under conditions which permit expression of the first and second polynucleotides of the two expression units or expression of the polynucleotide of the single expression unit, thereby producing taurine.

10. A pharmaceutical composition comprising a concentrate, extract or secretion of the prokaryotic cell of claim 1.

11. A nutritional supplement comprising a concentrate, extract or secretion of the transgenic prokaryotic cell of claim 1.

12. A food or food supplement comprising a concentrate, extract or secretion of the transgenic prokaryotic cell of claim 1.

13. An animal feed or feed supplement comprising a concentrate, extract or secretion of the transgenic prokaryotic cell of claim 1.

14. A plant growth or yield enhancer comprising a concentrate, extract or secretion of the transgenic prokaryotic cell of claim 1.

15. The cell of claim 1 which is a eukaryotic cell.

16. The cell of claim 1 which is a plant cell.

17. The plant cell of claim 16, wherein at least one of the first or second promoter is a constitutive promoter.

18. The plant cell of claim 16, wherein at least one of the first or second promoters is a non- constitutive promoter.

19. The plant cell of claim 16, wherein the non-constitutive promoter is selected from the group consisting of a tissue-preferred promoter, a tissue-specific promoter, a cell type- specific promoter, an inducible promoter, seed-specific promoter, a plant GAD promoter, plant sulphate transporter (SULTR) promoter, and a plant glutamate receptor (GLR) promoter.

20. The plant cell of claim 16, which comprises the two expression units, wherein the first polynucleotide further encodes a first peptide sequence and the second polynucleotide further encodes a second peptide sequence, wherein the first and second peptide sequences transport the CDOL and partCS/PLP-DC to a specific location in the cell, wherein the specific location is selected from the group consisting of an apoplast, a vacuole, a plastid, a chloroplast, a proplastid, an etioplast, a chromoplast, a mitochondrion, a peroxisome, a glyoxysome, a nucleus, a lysosome, an endomembrane system, an endoplasmic reticulum, a vesicle, and a Golgi apparatus.

21. The cell of claim 16, which comprises the single expression unit, wherein the polynucleotide further encodes a peptide sequence which will transport the fused CDOL linked to partCS/PLP-DC or CS/PLP-DC to a specific location in the cell, wherein the specific location is selected from the group consisting of an apoplast, a vacuole, a plastid, a chloroplast, a proplastid, an etioplast, a chromoplast, a mitochondrion, a peroxisome, a glyoxysome, a nucleus, a lysosome, an endomembrane system, an endoplasmic reticulum, a vesicle, and a Golgi apparatus.

22. The plant cell of claim 16, wherein the plant cell is selected from the group consisting of acacia, alfalfa, aneth, apple, apricot, artichoke, arugula, asparagus, avocado, banana, barley, beans, beech, beet, Bermuda grass, blackberry, blueberry, Blue grass, broccoli, brussels sprouts, cabbage, camelina, canola, cantaloupe, carinata, carrot, cassava, cauliflower, celery, cherry, chicory, cilantro, citrus, Clementines, coffee, corn, cotton, cucumber, duckweed, Douglas fir, eggplant, endive, escarole, eucalyptus, fennel, fescue, figs, forest trees, garlic, gourd, grape, grapefruit, honey dew, jatropha, jicama, kiwifruit, lettuce, leeks, lemon, lime, Loblolly pine, maize, mango, melon, mushroom, nectarine, nut, oat, okra, onion, orange, an ornamental plant, palm, papaya, parsley, pea, peach, peanut, pear, pepper, persimmon, pine, pineapple, plantain, plum, pomegranate, poplar, potato, pumpkin, quince, radiata pine, radicchio, radish, rapeseed, raspberry, rice, rye, rye grass, scallion, sorghum, Southern pine, soybean, spinach, squash, strawberry, sugarbeet, sugarcane, sunflower, sweet potato, sweetgum, switchgrass, tangerine, tea, tobacco, tomato, turf, turnip, a vine, watermelon, wheat, yams, and zucchini.

23. The method of claim 16, wherein the plant cell is in vitro.

24. The method of claim 16, wherein the plant cell is in vivo.

25. A transgenic plant comprising the plant cell of claim 1.

26. A transgenic plant of claim 25, wherein the plant has increased growth or yield.

27. A method of treating the trangenic plant of claim 25 with an exogenous application of a sulfur-containing compound or sulfate thereby resulting in the production of taurine or increased hypotaurine or taurine in the plant or increased yield.

28. A method of producing a crop of transgenic plants having taurine or increased levels of hypotaurine or taurine comprising multiplying the transgenic seed from plants of claim 25 or breeding plants grown from said transgenic seed to obtain a crop of transgenic plants having taurine or an increased level of hypotaurine or taurine.

29. A pharmaceutical composition comprising a concentrate, extract or secretion of the transgenic plant of claim 25, a plant organ of said transgenic plant, seed of said transgenic plant, wherein the concentrate, extract or secretion contains hypotaurine or taurine.

30. A nutritional supplement comprising a concentrate, extract or secretion of the transgenic transgenic plant of claim 25, a plant organ of said transgenic plant, seed of said transgenic plant, wherein the concentrate, extract or secretion contains hypotaurme or taurine.

A food or food supplement comprising a concentrate, extract or secretion of the of the transgenic plant of claim 25, a plant organ of said transgenic plant, seed of said transgenic plant, wherein the concentrate, extract or secretion contains hypo taurine or taurine.

An animal feed or animal feed supplement comprising a concentrate, extract or secretion of the of the transgenic plant of claim 25, a plant organ of said transgenic plant, seed of said transgenic plant, wherein the concentrate, extract or secretion contains hypotaurine or taurine.

33. A feed of claim 32, wherein the feed is an aquafeed.

Description:
ALGAL AND FUNGAL GENES AND THEIR

USES FOR TAURINE BIOSYNTHESIS IN CELLS

SEQUENCE SUBMISSION

[0010] The present application is being filed along with a Sequence Listing in electronic format. The Sequence Listing is 3834117PCT SequenneListing.txt, created on 5 April 2016 and is 69 kb in size. The information in the electronic format of the Sequence Listing is incorporated herein by reference in their entirety

FIELD OF THE INVENTION

[0011] The present invention is in the field of recombinant production of taurine. The present invention includes the production of taurine in bacteria, microbes, yeast, fungi, plants and animals to increase taurine levels. The invention also relates to methods to increase taurine levels in the cells and to use the said cells or extracts, the plant or plant organs that contain the invention, or the bacteria or yeast that contain the invention to produce plant growth enhancers, food, animal feed, aquafeed, food or drink supplements, animal-feed supplements, dietary supplements, or health supplements.

BACKGROUND OF THE INVENTION

Taurine is an essential compound for animals

[0012] Taurine is essential for human neonatal development (2) and plays an important role in brain development (3, 4). Taurine is involved in the modulation of intracellular calcium homeostasis (5, 6) and may balance glutamate activity, protecting neurons against glutamate excitotoxicity (7, 8). Taurine is also an osmoregulator (9). Taurine is essential for heart function (10), protects the integrity of hepatic tissue (11), and plays a role in photoprotection (12).

Taurine as a dietary supplement

[0013] Taurine is biosynthesized in most animals and can be found in meat and seafood. Those who do not produce sufficient levels of taurine must acquire it through dietary supplement. Dietary taurine is required for the normal development and growth of cats, (13, 14) human infants, (15) and carnivorous fish.(16-24) Taurine also improves the health and/or growth of other fish species(25-28) and shrimp.(29) Taurine is a feed attractant for fish.(21, 30)

Taurine as a pharmaceutical or therapeutic

[0014] Taurine is used as a pharmaceutical and therapeutic. Taurine has been used in the treatment of cardiovascular diseases (31, 32), elevated blood pressure (33), seizure disorders (34), hepatic disorders (35), and alcoholism (36) and may be useful in the treament of diabetes (37), Alzheimer's disease (38), and ocular disorders (39). Taurine has been shown to prevent obesity (40) and control cholesterol (41, 42). Taurine acts as an antioxidant and protects against toxicity of various substances (43-45). Taurine has been shown to prevent oxidative stress induced by exercise (46), and is used in energy drinks to improve performance (47). Taurine can also be used in topical applications to treat dermatological conditions (48).

Taurine as a plant growth stimulator

[0015] Exogenous application of taurine has been reported to increase crop harvest, yield, and biomass (49). Applications of taurine by foliar spray, soil and roots application, and seed immersion increase crop production and seedling growth (49). Exogenous applications of taurine have also been shown to increase photosynthetic capacity of isolated plant cells (protoplasts and chloroplasts) (49).

Metabolic pathways that synthesize taurine

[0016] Several metabolic pathways that synthesize taurine and hypo taurine have been identified in animals and bacteria (Figure 1). These genes and their corresponding gene products have been described in the literature (50-52). A recent study has shown that several algal and microalgal species can synthesize taurine (53). The authors suggest that these organisms may contain the genes for taurine synthesis. However, they never disclose the sequences of the genes or the sequences of the corresponding peptides. Nor do they provide validation through genetic (knock-out or complementation) or biochemical means of the function of the genes or their corresponding peptides.

[0017] This invention describes the use of algal, microalgal, fungal (yeast), diatom and unicellular organism genes and their corresponding peptides for taurine synthesis in cells. The genes include cysteine dioxygenase-like (CDOL), sulfinoalanine decarboxylase-like (SADL), cysteine synthetase/PLP decarboxylase (CS/PLP-DC) or a portion of the cysteine

synthetase/PLP decarboxylase (partCS/PLP-DC). The invention also describes how to use the cells, fractions of the cells, or extracts from the cells for a variety of purposes, including as an additive, feed ingredient, extract or meal. This invention describes the use of polynucleotides and their corresponding polypeptides not derived from vertebrates, invertebrates or bacteria. This invention does not include genes or peptides from animals or bacterial, which are described in the prior art (50-52) and include cysteine dioxygenase (CDO), sulfinoalanine decarboxylase (SAD), glutamate decarboxylase (GAD), hypotaurine dehydrogenase (HTDeHase), cysteamine dioxygenase (ADO), cysteine lyase (cysteine sulfite lyase or cysteine hydrogen-sulfide-lyase). sulfoacetaldehyde acetyltransferase (SA), taurine-pyruvate aminotransferase (TPAT) and taurine dioxygenase (TDO).

SUMMARY OF THE INVENTION

[0018] The invention provides methods and compositions for taurine production in organisms. More particularly, the invention encompasses the use of polynucleotides from algal sources that can be used in bacteria, fungi (yeast) or plants. The invention provides methods for transforming plants and constructing vector constructs and other nucleic acid molecules for use therein. The invention also provides methods for transforming bacteria, yeast, fungi, and unicellular algae and constructing vector constructs and other nucleic acid molecules for use therein. The transgenic plants, bacteria, yeast, fungi, or unicellular algae will have increased levels of taurine for use as animal feed, food, or as a supplement in animal feed or food or to enhance plant growth or yield.

[0010] The invention provides isolated cells comprising exogenous DNA which expresses enzymes of taurine biosynthetic pathways. In one embodiment, an isolated cell comprises two separate expression cassettes. A first expression cassette comprises a first promoter operably linked to a first polynucleotide, and a second expression cassette comprises a second promoter operably linked to a second polynucleotide. In some embodiments, the first polynucleotide encodes cysteine dioxygenase-like (CDOL) and the second polynucleotide encodes sulfinoalanine decarboxylase-like (SADL). In other embodiments the first polynucleotide encodes CDOL and the second polynucleotide encodes cysteine synthetase/PLP decarboxylase (CS/PLP-DC) or a portion of the cysteine synthetase/PLP decarboxylase (partCS/PLP-DC).

[0011] Some isolated cells of the invention comprise exogenous DNA which comprises a single expression cassette. The single expression cassette comprises a promoter operably linked to a polynucleotide which encodes (i) CS/PLP-DC; (ii) SADL; (iii) partCS/PLP-DC; (iv) CDOL operably linked to SADL; (v) CDOL operably linked to CS/PLP-DC; or (vi) CDOL operably linked to partCS/PLP-DC.

[0012] The invention also provides plant storage organs comprising isolated cells of the invention; transgenic seeds with a genome comprising exogenous DNA encoding one or more of (i) CS/PLP-DC; (ii) SADL; (iii) partCS/PLP-DC; (iv) CDOL and SADL; (v) CDOL and CS/PLP-DC; or (vi) CDOL and partCS/PLP-DC, and transgenic plants grown from the transgenic seeds. [0013] The invention provides methods of altering a property of a transgenic plant of the invention by contacting the transgenic plant with an agent which increases sulfur or nitrogen concentration in cells of the transgenic plant.

[0014] The invention also provides nutritional supplements, feed supplements, and pharmaceutical compositions comprising an extract or meal from a transgenic plant of the invention, or comprising a component, which can be one or more of the plant storage organs, transgenic seeds, and transgenic plants of the invention.

[0015] In one embodiment of the invention polynucleotides encoding functional (i) CS/PLP- DC; (ii) SADL; (iii) partCS/PLP-DC; (iv) CDOL and SADL; (v) CDOL and CS/PLP-DC; or (vi) CDOL and partCS/PLP-DC enzymes are used to transform yeast, fungal, bacterial or algal cells. Inventive methods produce cells that have taurine production for nutritional, pharmaceutical, or therapeutic value, feed, food or drink. Cells are genetically modified in accordance with the invention to include polynucleotides that encode a CDOL, SADL, partCS/PLP-DC, or CS/PLP-DC enzyme that functions in the formation of hypotaurine or taurine in the cell.

[0016] Another embodiment of the invention describes the use of polynucleotides that encode polypeptides for functional (i) CS/PLP-DC; (ii) SADL; (iii) partCS/PLP-DC; (iv) CDOL and SADL; (v) CDOL and CS/PLP-DC; or (vi) CDOL and partCS/PLP-DC expressed in eukaryotes or prokaryotes or in eukaryotic or prokaryotic cells, for hypotaurine or taurine production.

[0017] The inventive methods produce plants that have the advantage of increased levels of sulfur-containing compounds, specifically taurine, resulting in algae or plants with increased nutritional value or algae or plant material with improved characteristics for food or feed production, including hypotaurine or taurine production, higher levels of taurine, lower taurine leaching rates, and increased bioavailability of taurine.

BRIEF DESCRIPTION OF THE FIGURE

[0018] Figure 1 shows the novel algal, fungi (yeast), diatom and other unicellular organism genes and their corresponding proteins (CDOL, SADL, partCS/PLP-DC, or CS/PLP-DC) in relation to the known animal and bacterial taurine biosynthetic pathways. In animals, cysteine and oxygen are converted into 3-sulfinoalanine by cysteine dioxygenase (CDO). 3- sulfinoalanine is converted into hypotaurine by sulfinoalanine decarboxylase (SAD) or glutamate decarboxylase (GAD). Hypotaurine is converted into taurine either by the activity of hypotaurine dehydrogenase (HTDeHase) or by a spontaneous conversion. Cysteamine (2- aminoethanethiol) and oxygen are converted into hypotaurine by cysteamine dioxygenase (ADO), and hypotaurine is converted into taurine. Alternatively cysteine and sulfite are converted into cysteate and hydrogen sulfide by cysteine lyase (cysteine sulfite lyase or cysteine hydrogen-sulfide-lyase). Cysteate is converted into taurine by SAD or GAD. In bacteria, the compound 2-sulfoacetaldehyde is synthesized from acetyl phosphate and sulfite by sulfoacetaldehyde acetyltransferase (SA). Alanine and 2-sulfoacetaldehyde are converted into taurine and pyruvate by taurine-pyruvate aminotransferase (TP AT). In addition, sulfoacetaldehyde and ammonia (or ammonium) are converted into taurine and water in the presence of ferrocytochrome C by taurine dehydrogenase. Sulfite, aminoacetaldehyde, carbon dioxide and succinate are converted into taurine, 2-oxoglutarate and oxygen by taurine dioxygenase (TDO).

[0019] Figure 2 shows the superposition of two chromatograms: one from the amino acid extract of a representative transgenic plant containing the CDOL-linker-partCS/PLP-DC (lower line at Tau) and the other is a reference, i.e., the amino acid standards (higher line at Tau). The numbers represent the retention times, and the peaks for hypotaurine (HT), taurine (Tau), and arginine (Arg) are labeled.

DETAILED DESCRIPTION OF THE INVENTION

[0020] The present invention provides methods and materials for the production of taurine (2-aminoethanesulfonic acid) in cells and living organisms. In preferred embodiments, the invention provides methods for the genetic transformation of organisms, preferably plants, with genes that encode proteins that catalyze the conversion of sulfur-compounds such as sulfate or cysteine to taurine. The invention also provides methods of using plants, bacteria, fungi or yeast bacteria, yeast, fungi, or unicellular algae with increased levels of endogenous taurine or taurine derivatives such as hypotaurine as a food- or feed-supplement, dietary supplement, as a component of a health supplement or therapy or for plant growth or yield.

[0021] The present invention describes the methods for the synthesis of DNA constructs for taurine production from polynucleotides and vectors and the methods for making transformed organisms including plants, photosynthetic organisms, microbes, invertebrates, and vertebrates. The present invention is unique in that it describes a method to produce plants that have advantages of enhanced taurine production or hypotaurine and that result in plants with increased nutritional, pharmaceutical, or therapeutic value or with enhanced plant growth characteristics.

[0022] The present invention describes the insertion of the taurine biosynthetic pathway in organisms where the pathway does not exist or has not clearly been identified. The invention describes methods for the use of polynucleotides that encode functional CDOL, SADL, partCS/PLP-DC, or CS/PLP-DC in plants. The preferred embodiment of the invention is in plants but other organisms may be used.

Enzymes of taurine biosynthetic pathways

[0023] Examples of amino acid sequences of enzymes of algal and fungal taurine biosynthetic pathways are provided in the sequence listing: SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, and SEQ ID NO:8 (CDOL); SEQ ID NO: 15 and SEQ ID NO: 16 (CS/PLP-DC); SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19 and SEQ ID NO:20 (SADL);. The invention is not limited to the use of these amino acid sequences. Those of ordinary skill in the art know that organisms of a wide variety of species commonly express and utilize homologous proteins, which include the insertions, substitutions and/or deletions discussed above, and effectively provide similar function. For example, the amino acid sequences for CDOL from Chlamydomonas reinhardtii or Fragilariopsis cylindrus, SADL from Guillardia theta or Cyanidioschyzon merolae, or CS/PLP-DC from Micromonas pusilla or Ostreococcus tauri may differ to a certain degree from the amino acid sequences of CDOL, SADL or CS/PLP-DC in another species and yet have similar functionality with respect to catalytic and regulatory function. Amino acid sequences comprising such variations are included within the scope of the present invention and are considered substantially or sufficiently similar to a reference amino acid sequence. Although it is not intended that the present invention be limited by any theory by which it achieves its advantageous result, it is believed that the identity between amino acid sequences that is necessary to maintain proper functionality is related to maintenance of the tertiary structure of the polypeptide such that specific interactive sequences will be properly located and will have the desired activity, and it is contemplated that a polypeptide including these interactive sequences in proper spatial context will have activity.

[0024] Another manner in which similarity may exist between two amino acid sequences is where there is conserved substitution between a given amino acid of one group, such as a non- polar amino acid, an uncharged polar amino acid, a charged polar acidic amino acid, or a charged polar basic amino acid, with an amino acid from the same amino acid group. For example, it is known that the uncharged polar amino acid serine may commonly be substituted with the uncharged polar amino acid threonine in a polypeptide without substantially altering the functionality of the polypeptide. Whether a given substitution will affect the functionality of the enzyme may be determined without undue experimentation using synthetic techniques and screening assays known to one with ordinary skill in the art.

[0025] One of ordinary skill in the art will recognize that changes in the amino acid sequences, such as individual substitutions, deletions or additions to a nucleic acid, peptide, polypeptide, or protein sequence which alters, adds or deletes a single amino acid or a small percentage of amino acids in the encoded sequence is "sufficiently similar" when the alteration results in the substitution of an amino acid with a chemically similar amino acid. Thus, any number of amino acid residues selected from the group of integers consisting of from 1 to 15 can be so altered. Thus, for example, 1, 2, 3, 4, 5, 7 or 10 alterations can be made. Conservatively modified variants typically provide similar biological activity as the unmodified polypeptide sequence from which they are derived. For example, CDOL, SADL or CS/PLP-DC activity is generally at least 40%, 50%, 60%, 70%, 80% or 90%, preferably 60-90% of the native protein for the native substrate. Tables of conserved substitution provide lists of functionally similar amino acids.

[0026] The following three groups each contain amino acids that are conserved substitutions for one another: (1) Alanine (A), Serine (S), Threonine (T); (2) Aspartic acid (D), Glutamic acid (E); and (3) Asparagine (N), Glutamine (Q);

Suitable polynucleotides for CDOL, SADL, and CS/PLP-DC

[0027] As examples, suitable polynucleotides encoding enzymes of taurine biosynthetic pathways are described below. The invention is not limited to use of these sequences, however. In fact, any nucleotide sequence which encodes an enzyme of a taurine biosynthetic pathway can be used in an expression vector to produce that enzyme recombinantly.

[0028] Suitable polynucleotides for CDOL are provided in SEQ ID NO:l, SEQ ID NO:2, SEQ ID NO:3 and SEQ ID NO:4. Other suitable polynucleotides for use in accordance with the invention may be obtained by the identification of polynucleotides that selectively hybridize to the polynucleotides of SEQ ED NO:l, SEQ ID NO:2, SEQ ID NO:3 or SEQ ID NO:4. by hybridization under low stringency conditions, moderate stringency conditions, or high stringency conditions. Still other suitable polynucleotides for use in accordance with the invention may be obtained by the identification of polynucleotides that have substantial identity of the nucleic acid of SEQ ID NO:l, SEQ ID NO:2, SEQ ID NO:3 or SEQ ID NO:4 when it used as a reference for sequence comparison or polynucleotides that encode polypeptides that have substantial identity to amino acid sequence of SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7 or SEQ ID NO: 8 when it used as a reference for sequence comparison.

[0029] Suitable polynucleotides for SADL are provided in SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13 and SEQ ID NO: 14. Other suitable polynucleotides for use in accordance with the invention may be obtained by the identification of polynucleotides that selectively hybridize to the polynucleotides of SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13 or SEQ ID NO: 14 by hybridization under low stringency conditions, moderate stringency conditions, or high stringency conditions. Still other suitable polynucleotides for use in accordance with the invention may be obtained by the identification of polynucleotides that have substantial identity of the nucleic acid of SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13 or SEQ ID NO: 14 when it used as a reference for sequence comparison or polynucleotides that encode polypeptides that have substantial identity to amino acid sequence of SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19 or SEQ ID NO:20 when it is used as a reference for sequence comparison.

[0030] Suitable polynucleotides for CS/PLP-DC are provided in SEQ ID NO:9 and SEQ ID NO: 10. Other suitable polynucleotides for use in accordance with the invention may be obtained by the identification of polynucleotides that selectively hybridize to the polynucleotides of SEQ ID NO: 9 or SEQ ID NO: 10 by hybridization under low stringency conditions, moderate stringency conditions, or high stringency conditions. Still other suitable polynucleotides for use in accordance with the invention may be obtained by the identification of polynucleotides that have substantial identity of the nucleic acid of SEQ ID NO:9 or SEQ ID NO: 10 when it used as a reference for sequence comparison or polynucleotides that encode polypeptides that have substantial identity to amino acid sequence of SEQ ID NO: 15 or SEQ ID NO: 16 when it used as a reference for sequence comparison.

[0031] Another embodiment of the invention is a polynucleotide {e.g., a DNA construct) that encodes a protein that functions as a CDOL, SADL, or CS/PLP-DC and selectively hybridizes to either SEQ ID NO:l, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:9, SEQ ID NO: 10, SEQ ID NO:ll, SEQ ID NO: 12, SEQ ID NO:13, or SEQ ID NO:14 respectively. Selectively hybridizing sequences typically have at least 40% sequence identity, preferably 60- 90% sequence identity, and most preferably 100% sequence identity with each other.

[0032] Another embodiment of the invention is a polynucleotide that encodes a polypeptide that has substantial identity to the amino acid sequence of SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, or SEQ ID NO:20. Substantial identity of amino acid sequences for these purposes normally means sequence identity of between 50-100%, preferably at least 55%, preferably at least 60%, more preferably at least 70%, 80%, 90%, and most preferably at least 95%.

[0033] The process of encoding a specific amino acid sequence may involve DNA sequences having one or more base changes (i.e., insertions, deletions, substitutions) that do not cause a change in the encoded amino acid, or which involve base changes which may alter one or more amino acids, but do not eliminate the functional properties of the polypeptide encoded by the DNA sequence.

[0034] It is therefore understood that the invention encompasses more than the specific polynucleotides encoding the proteins described herein. For example, modifications to a sequence, such as deletions, insertions, or substitutions in the sequence, which produce "silent" changes that do not substantially affect the functional properties of the resulting polypeptide are expressly contemplated by the present invention. Furthermore, because of the degeneracy of the genetic code, a large number of functionally identical nucleic acids encode any given protein. For instance, the codons GCA, GCC, GCG and GCU all encode the amino acid alanine. Thus, at every position where an alanine is specified by a codon, the codon can be altered to any of the corresponding codons described without altering the encoded polypeptide. Such nucleic acid variations are "silent variations" and represent one species of conservatively modified variation. Every nucleic acid sequence herein that encodes a polypeptide also describes every possible silent variation of the nucleic acid. One of ordinary skill in the art will recognize that each amino acid has more than one codon, except for methionine and tryptophan that ordinarily have the codons AUG and UGG, respectively. It is known by those of ordinary skill in the art, "universal" code is not completely universal. Some mitochondrial and bacterial genomes diverge from the universal code, e.g., some termination codons in the universal code specify amino acids in the mitochondria or bacterial codes. Thus each silent variation of a nucleic acid, which encodes a polypeptide of the present invention, is implicit in each described polypeptide sequence and incorporated in the descriptions of the invention.

[0035] It is understood that alterations in a nucleotide sequence, which reflect the degeneracy of the genetic code, or which result in the production of a chemically equivalent amino acid at a given site, are contemplated. Thus, a codon for the amino acid alanine, a hydrophobic amino acid, may be substituted by a codon encoding another less hydrophobic residue, such as glycine, or a more hydrophobic residue, such as valine, leucine, or isoleucine. Similarly, changes which result in substitution of one negatively charged residue for another, such as aspartic acid for glutamic acid, or one positively charged residue for another, such as lysine for arginine, can also be expected to produce a biologically equivalent product.

[0036] Nucleotide changes which result in alteration of the amino-terminal and carboxy- terminal portions of the encoded polypeptide molecule would also not generally be expected to alter the activity of the polypeptide. In some cases, it may in fact be desirable to make mutations in the sequence in order to study the effect of alteration on the biological activity of the polypeptide. Each of the proposed modifications is well within the routine skill in the art.

[0037] When the nucleic acid is prepared or altered synthetically, one of ordinary skill in the art can take into account the known codon preferences for the intended host where the nucleic acid is to be expressed. For example, although nucleic acid sequences of the present invention may be expressed in both monocotyledonous and dicotyledonous plant species, sequences can be modified to account for the specific codon preferences and GC-content preferences of monocotyledonous plants or dicotyledonous plants, as these preferences have been shown to differ (54).

Cloning techniques

[0038] For purposes of promoting an understanding of the principles of the invention, reference will now be made to particular embodiments of the invention and specific language will be used to describe the same. The materials, methods and examples are illustrative only and not limiting. Unless mentioned otherwise, the techniques employed or contemplated herein are standard methodologies well known to one of ordinary skill in the art. Specific terms, while employed below and defined at the end of this section, are used in a descriptive sense only and not for purposes of limitation. The practice of the present invention will employ, unless otherwise indicated, conventional techniques of botany, microbiology, tissue culture, molecular biology, chemistry, biochemistry and recombinant DNA technology, which are within the skill of the art (55-62).

[0039] A suitable polynucleotide for use in accordance with the invention may be obtained by cloning techniques using cDNA or genomic libraries, DNA, or cDNA from bacteria which are available commercially or which may be constructed using standard methods known to persons of ordinary skill in the art. Suitable nucleotide sequences may be isolated from DNA libraries obtained from a wide variety of species by means of nucleic acid hybridization or amplification methods, such as polymerase chain reaction (PGR) procedures, using as probes or primers nucleotide sequences selected in accordance with the invention. [0040] Furthermore, nucleic acid sequences may be constructed or amplified using chemical synthesis. The product of amplification is termed an amplicon. Moreover, if the particular nucleic acid sequence is of a length that makes chemical synthesis of the entire length impractical, the sequence may be broken up into smaller segments that may be synthesized and ligated together to form the entire desired sequence by methods known in the art. Alternatively, individual components or DNA fragments may be amplified by PGR and adjacent fragments can be amplified together using fusion-PCR (63), overlap-PCR (64) or chemical (de novo) synthesis (65-69) using a vendor (e.g. GE life technologies, GENEART, Gen9, GenScript) by methods known in the art.

[0041] A suitable polynucleotide for use in accordance with the invention may be constructed by recombinant DNA technology, for example, by cutting or splicing nucleic acids using restriction enzymes and mixing with a cleaved (cut with a restriction enzyme) vector with the cleaved insert (DNA of the invention) and ligated using DNA ligase. Alternatively amplification techniques, such as PGR, can be used, where restriction sites are incorporated in the primers that otherwise match the nucleotide sequences (especially at the 3' ends) selected in accordance with the invention. The desired amplified recombinant molecule is cut or spliced using restriction enzymes and mixed with a cleaved vector and ligated using DNA ligase. In another method, after amplification of the desired recombinant molecule, DNA linker sequences are ligated to the 5' and 3' ends of the desired nucleotide insert with ligase, the DNA insert is cleaved with a restriction enzyme that specifically recognizes sequences present in the linker sequences and the desired vector. The cleaved vector is mixed with the cleaved insert, and the two fragments are ligated using DNA ligase. In yet another method, the desired recombinant molecule is amplified with primers that have recombination sites (e.g. Gateway) incorporated in the primers, that otherwise match the nucleotide sequences selected in accordance with the invention. The desired amplified recombinant molecule is mixed with a vector containing the recombination site and recombinase, the two molecules are ligated together by recombination.

[0042] The recombinant expression cassette or DNA construct includes a promoter that directs transcription in a plant cell, operably linked to the polynucleotide encoding a CDOL, SADL, partCS/PLP-DC, or CS/PLP-DC. In various aspects of the invention described herein, a variety of different types of promoters are described and used. As used herein, a polynucleotide is "operably linked" to a promoter or other nucleotide sequence when it is placed into a functional relationship with the promoter or other nucleotide sequence. The functional relationship between a promoter and a desired polynucleotide insert typically involves the polynucleotide and the promoter sequences being contiguous such that transcription of the polynucleotide sequence will be facilitated. Two nucleic acid sequences are further said to be operably linked if the nature of the linkage between the two sequences does not (1) result in the introduction of a frame-shift mutation; (2) interfere with the ability of the promoter region sequence to direct the transcription of the desired nucleotide sequence, or (3) interfere with the ability of the desired nucleotide sequence to be transcribed by the promoter sequence region. Typically, the promoter element is generally upstream (i.e., at the 5' end) of the nucleic acid insert coding sequence.

[0043] While a promoter sequence can be ligated to a coding sequence prior to insertion into a vector, in other embodiments, a vector is selected that includes a promoter operable in the host cell into which the vector is to be inserted. In addition, certain preferred vectors have a region that codes a ribosome binding site positioned between the promoter and the site at which the DNA sequence is inserted so as to be operatively associated with the DNA sequence of the invention to produce the desired polypeptide, i.e., the DNA sequence of the invention in-frame.

Suitable Peptide Linkers

[0044] Peptide linkers are known to those skilled in the art to connect protein domains or peptides. In general, linkers that contain the amino acids glycine and serine are useful linkers. (70, 71) Other suitable linkers that can be used in the invention include, but are not limited to, those described by Kuusinen et. al. (72) Robinson and Sauer, (73) Armstrong & Gouaux, (74) Arai et. al, (75) Wriggers et. al, (76) and Reddy et. al (77).

Suitable promoters

[0045] A wide variety of promoters are known to those of ordinary skill in the art as are other regulatory elements that can be used alone or in combination with promoters. A wide variety of promoters that direct transcription in plants cells can be used in connection with the present invention. For purposes of describing the present invention, promoters are divided into two types, namely, constitutive promoters and non-constitutive promoters. Constitutive promoters are classified as providing for a range of constitutive expression. Thus, some are weak constitutive promoters, and others are strong constitutive promoters. Non-constitutive promoters include tissue-preferred promoters, tissue-specific promoters, cell-type specific promoters, and inducible-promoters.

[0046] Of particular interest in certain embodiments of the present invention are inducible- promoters that respond to various forms of environmental stresses, or other stimuli, including, for example, mechanical shock, heat, cold, salt, flooding, drought, salt, anoxia, pathogens, such as bacteria, fungi, and viruses, and nutritional deprivation, including deprivation during times of flowering and/or fruiting, and other forms of plant stress. For example, the promoter selected in alternate forms of the invention, can be a promoter is induced by one or more, but not limiting to one of the following, abiotic stresses such as wounding, cold, dessication, ultraviolet-B (78), heat shock (79) or other heat stress, drought stress or water stress. The promoter may further be one induced by biotic stresses including pathogen stress, such as stress induced by a virus (80) or fungi (81, 82), stresses induced as part of the plant defense pathway (83) or by other environmental signals, such as light (84), carbon dioxide (85, 86), hormones or other signaling molecules such as auxin, hydrogen peroxide and salicylic acid (87, 88), sugars and gibberellin (89) or abscissic acid and ethylene (90).

[0047] In other embodiments of the invention, tissue-specific promoters are used. Tissue- specific expression patterns as controlled by tissue- or stage-specific promoters that include, but is not limited to, fiber-specific, green tissue-specific, root-specific, stem-specific, and flower- specific. Examples of the utilization of tissue-specific expression includes, but is not limited to, the expression in leaves of the desired peptide for the protection of plants against foliar pathogens, the expression in roots of the desired peptide for the protection of plants against root pathogens, and the expression in roots or seedlings of the desired peptide for the protection of seedlings against soil-borne pathogens. In many cases, however, protection against more than one type of pathogen may be sought, and expression in multiple tissues will be desirable.

[0048] Of particular interest in certain embodiments of the present invention seed-specific promoters are used. Examples of the utilization of seed-specific promoters for expression includes, but is not limited to, napin, (91) sunflower seed-specific promoter, (92) AtFAD2, (93) phaseolin, (94) beta-conglycinin, (95) zein, (96) and rice glutelin. (97)

[0049] Although some promoters from dicotyledons have been shown to be operational in monocotyledons and vice versa, ideally dicotyledonous promoters are selected for expression in dicotyledons, and monocotyledonous promoters are selected for expression in monocotyledons. There are also promoters that control expression of genes in green tissue or for genes involved in photosynthesis from both monocotyledons and dicotyledons such as the maize from the phosphenol carboxylase gene. (98) There are suitable promoters for root specific expression (99, 100). A promoter selected can be an endogenous promoter, i.e. a promoter native to the species and or cell type being transformed. Alternatively, the promoter can be a foreign promoter, which promotes transcription of a length of DNA of viral, microbes, bacterial or eukaryotic origin, invertebrates, vertebrates including those from plants and plant viruses. For example, in certain preferred embodiments, the promoter may be of viral origin, including a cauliflower mosaic virus promoter (CaMV), such as CaMV 35S orl9S, a figwort mosaic virus promoter (FMV 35S), or the coat protein promoter of tobacco mosaic virus (TMV). The promoter may further be, for example, a promoter for the small subunit of ribulose-1, 3-biphosphate carboxylase. Promoters of bacterial origin (microbe promoters) include the octopine synthase promoter, the nopaline synthase promoter and other promoters derived from native Ti plasmids. (101)

[0050] The promoters may further be selected such that they require activation by other elements known to those of ordinary skill in the art, so that production of the protein encoded by the nucleic acid sequence insert may be regulated as desired. In one embodiment of the invention, a DNA construct comprising a non-constitutive promoter operably linked to a polynucleotide encoding the desired polypeptide of the invention is used to make a transformed plant that selectively increases the level of the desired polypeptide of the invention in response to a signal. The term "signal" is used to refer to a condition, stress or stimulus that results in or causes a non-constitutive promoter to direct expression of a coding sequence operably linked to it. To make such a plant in accordance with the invention, a DNA construct is provided that includes a non-constitutive promoter operably linked to a polynucleotide encoding the desired polypeptide of the invention. The construct is incorporated into a plant genome to provide a transformed plant that expresses the polynucleotide in response to a signal.

[0051] In alternate embodiments of the invention, the selected promoter is a tissue-preferred promoter, a tissue-specific promoter, a cell-type- specific promoter, an inducible promoter or other type of non-constitutive promoter. It is readily apparent that such a DNA construct causes a plant transformed thereby to selectively express the gene for the desired polypeptide of the invention. Therefore under specific conditions or in certain tissue- or cell-types the desired polypeptide will be expressed. The result of this expression in the plant depends upon the activity of the promoter and in some cases the conditions of the cell or cells in which it is expressed.

[0052] It is understood that the non-constitutive promoter does not continuously produce the transcript or RNA of the invention. But in this embodiment the selected promoter for inclusion of the invention advantageously induces or increases transcription of gene for the desired polypeptide of the invention in response to a signal, such as an environmental cue or other stress signal including biotic and/or abiotic stresses or other conditions. [0053] In another embodiment of the invention, a DNA construct comprising a plant GAD promoter operably linked to polynucleotides that encode the desired polypeptide of the invention is used to make a transformed plant that selectively increases the transcript or RNA of the desired polypeptide of the invention in the same cells, tissues, and under the environmental conditions that express a plant glutamate decarboxylase. It is understood to those of ordinary skill in the art that the regulatory sequences that comprise a plant promoter driven by RNA polymerase II reside in the region approximately 2900 to 1200 basepairs up-stream (5') of the translation initiation site or start codon (ATG). For example, the full-length promoter for the nodule-enhanced PEP carboxylase from alfalfa is 1277 basepairs prior to the start codon (102), the full-length promoter for cytokinin oxidase from orchid is 2189 basepairs prior to the start codon (103), the full-length promoter for ACC oxidase from peach is 2919 basepairs prior to the start codon (104), full-length promoter for cytokinin oxidase from orchid is 2189 basepairs prior to the start codon, full-length promoter for glutathione peroxidasel from Citrus sinensis is 1600 basepairs prior to the start codon (105), and the full-length promoter for glucuronosyltransferase from cotton is 1647 basepairs prior to the start codon (106). Most full-length promoters are 1700 basepairs prior to the start codon. The accepted convention is to describe this region (promoter) as -1700 to -1, where the numbers designate the number of basepairs prior to the "A" in the start codon. Other plant specific promoters include but are not limited to glutamate deacarboxylase (GAD), Arabidopsis thaliana glutamate receptors (AtGLRs or AtGluRs) or plant sulphate transporter promoter (51) or use amplification, such as PGR, techniques with the incorporation of restriction or recombination sites to clone the plant promoters 5' to the desired polynucleotide.

Plastid Transit Peptides

[0054] A wide variety of plastid transit peptides are known to those of ordinary skill in the art that can be used in connection with the present invention. Suitable transit peptides which can be used to target any CDOL, SADL, partCS/PLP-DC or CS/PLP-DC polypeptide to a plastid include, but are not limited, to those described herein and in U.S. Patent Nos. 8,779,237 (107), 8,674,180 (108), 8,420,888 (109), and 8,138,393 (110) and in Lee et al. (Ill) and von Heijne et al. (112) Cloning a nucleic acid sequence that encodes a transit peptide upstream and in-frame of a nucleic acid sequence that encodes a polypeptide involves standard molecular techniques that are well-known in the art. Suitable vectors

[0055] A wide variety of vectors may be employed to transform a plant, plant cell or other cells with a construct made or selected in accordance with the invention, including high- or low- copy number plasmids, phage vectors and cosmids. Such vectors, as well as other vectors, are well known in the art. Representative T-DNA vector systems (101, 113) and numerous expression cassettes and vectors and in vitro culture methods for plant cell or tissue transformation and regeneration of plants are known and available. (114) The vectors can be chosen such that operably linked promoter and polynucleotides that encode the desired polypeptide of the invention are incorporated into the genome of the plant. Although the preferred embodiment of the invention is expression in plants or plant cells, other embodiments may include expression in prokaryotic or eukaryotic photosynthetic organisms, yeast, fungi, algae, microalgae, microbes, invertebrates or vertebrates.

[0056] It is known by those of ordinary skill in the art that there exist numerous expression systems available for expression of a nucleic acid encoding a protein of the present invention. There are many commercially available recombinant vectors to transform a host plant or plant cell. Standard molecular and cloning techniques (59, 62, 115) are available to make a recombinant expression cassette that expresses the polynucleotide that encodes the desired polypeptide of the invention. No attempt to describe in detail the various methods known for the expression of proteins in prokaryotes or eukaryotes will be made. In brief, the expression of isolated nucleic acids encoding a protein of the present invention will typically be achieved by operably linking, for example, the DNA or cDNA to a promoter, followed by incorporation into an expression vector. The vectors can be suitable for replication and integration in either prokaryotes or eukaryotes. Typical expression vectors contain transcription and translation terminators, initiation sequences, and promoters useful for regulation of the expression of the DNA encoding a protein of the present invention. To obtain high-level expression of a cloned gene, it is desirable to construct expression vectors that contain, at the minimum, a strong promoter, such as ubiquitin, to direct transcription, a ribosome-binding site for translational initiation, and a transcription/translation terminator.

[0057] One of ordinary skill to the art recognizes that modifications could be made to a protein of the present invention without diminishing its biological activity. Some modifications may be made to facilitate the cloning, expression, targeting or to direct the location of the polypeptide in the host, or for the purification or detection of the polypeptide by the addition of a "tag" as a fusion protein. Such modifications are well known to those of skill in the art and include, for example, a methionine added at the amino terminus to provide an initiation site, additional amino acids (tags) placed on either terminus to create a tag, additional nucleic acids to insert a restriction site or a termination.

[0058] In addition to the selection of a suitable promoter, the DNA constructs requires an appropriate transcriptional terminator to be attached downstream of the desired gene of the invention for proper expression in plants. Several such terminators are available and known to persons of ordinary skill in the art. These include, but are not limited to, the toil from CaMV and E9 from rbcS. Another example of a terminator sequence is the polyadenlyation sequence from the bovine growth hormone gene. A wide variety of available terminators known to function in plants can be used in the context of this invention. Vectors may also have other control sequence features that increase their suitability. These include an origin of replication, enhancer sequences, ribosome binding sites, RNA splice sites, polyadenylation sites, selectable markers and RNA stability signal. Origin of replication is a gene sequence that controls replication of the vector in the host cell. Enhancer sequences cooperate with the promoter to increase expression of the polynucleotide insert coding sequence. Enhancers can stimulate promoter activity in host cell. An example of specific polyadenylation sequence in higher eukaryotes is ATTTA. Examples of plant polyadenylation signal sequences are AATAAA or AATAAT. RNA splice sites are sequences that ensure accurate splicing of the transcript. Selectable markers usually confer resistance to an antibiotic, herbicide or chemical or provide color change, which aid the identification of transformed organisms. The vectors also include a RNA stability signal, which are 3 '-regulatory sequence elements that increase the stability of the transcribed RNA. (116, 117)

Terminators

[0059] Terminators are typically located downstream (3') of the gene, after the stop codon (TGA, TAG or TAA). Terminators play an important role in the processing and stability of RNA as well as in translation. Most, but not all terminators, contain a polyadenylation sequence or cleavage site. Examples of specific polyadenylation sequences are AAUAAA or AAUAAU. These sequences are known as the near upstream elements (NUEs). (118) NUEs usually reside approximately 30 bp away from a GU-rich region (119-121) which is known as far upstream elements (FUEs). The FUEs enhance processing at the polyadenylation sequence or cleavage site, which is usually a CA or UA in a U-rich region. (122) Within the terminator, elements exist that increase the stability of the transcribed RNA,(116, 117, 123) and may also control gene expression. (124) [0060] In addition, polynucleotides that encode a CDOL, SADL, partCS/PLP-DC or CS/PLP-DC can be placed in the appropriate plant expression vector used to transform plant cells. The polypeptide can then be isolated from plant callus or the transformed cells can be used to regenerate transgenic plants. Such transgenic plants can be harvested, and the appropriate tissues can be subjected to large-scale protein extraction and purification techniques.

[0061] The vectors may include another polynucleotide insert that encodes a peptide or polypeptide used as a "tag" to aid in purification or detection of the desired protein. The additional polynucleotide is positioned in the vector such that upon cloning and expression of the desired polynucleotide a fusion, or chimeric, protein is obtained. The tag may be incorporated at the amino or carboxy terminus. If the vector does not contain a tag, persons with ordinary skill in the art know that the extra nucleotides necessary to encode a tag can be added with the ligation of linkers, adaptors, or spacers or by PGR using designed primers. After expression of the peptide the tag can be used for purification using affinity chromatography, and if desired, the tag can be cleaved with an appropriate enzyme. The tag can also be maintained, not cleaved, and used to detect the accumulation of the desired polypeptide in the protein extracts from the host using western blot analysis. In another embodiment, a vector includes the polynucleotide for the tag that is fused in-frame to the polynucleotide that encodes a functional CDOL, SADL, partCS/PLP-DC or CS/PLP-DC to form a fusion protein. The tags that may be used include, but are not limited to, Arg-tag, calmodulin-binding peptide, cellulose-binding domain, DsbA, c-myc-tag, glutathione S- transferase, FLAG-tag, HAT-tag, His-tag, maltose-binding protein, NusA, S-tag, SBP-tag, Strep-tag, and thioredoxin (Trx-Tag). These are available from a variety of manufacturers Clontech Laboratories, Takara Bio Company GE Healthcare, Invitrogen, Novagen Promega and QIAGEN.

[0062] The vector may include another polynucleotide that encodes a signal polypeptide or signal sequence ("subcellular location sequence") to direct the desired polypeptide in the host cell, so that the polypeptide accumulates in a specific cellular compartment, subcellular compartment, or membrane. The specific cellular compartments include the apoplast, vacuole, plastids chloroplast, mitochondrion, peroxisomes, secretory pathway, lysosome, endoplasmic reticulum, nucleus or Golgi apparatus. A signal polypeptide or signal sequence is usually at the amino terminus and normally absent from the mature protein due to protease that removes the signal peptide when the polypeptide reaches its final destination. Signal sequences can be a primary sequence located at the N-terminus (125-128), C-terminus (129, 130) or internal (131- 133) or tertiary structure (133). If a signal polypeptide or signal sequence to direct the polypeptide does not exist on the vector, it is expected that those of ordinary skill in the art can incorporate the extra nucleotides necessary to encode a signal polypeptide or signal sequence by the ligation of the appropriate nucleotides or by PCR. Those of ordinary skill in the art can identify the nucleotide sequence of a signal polypeptide or signal sequence using computational tools. There are numerous computational tools available for the identification of targeting sequences or signal sequence. These include, but are not limited to, TargetP (134, 135), iPSORT (136), SignalP (137), PrediSi (138), ELSpred (139) HSLpred (140) and PSLpred (141), MultiLoc (142), SherLoc (143), ChloroP (144), MITOPROT (145), Predotar (146) and 3D-PSSM (147). Additional methods and protocols are discussed in the literature (142).

Transformation of host cells

[0063] Transformation of a plant can be accomplished in a wide variety of ways within the scope of a person of ordinary skill in the art. In one embodiment, a DNA construct is incorporated into a plant by (i) transforming a cell, tissue or organ from a host plant with the DNA construct; (ii) selecting a transformed cell, cell callus, somatic embryo, or seed which contains the DNA construct; (iii) regenerating a whole plant from the selected transformed cell, cell callus, somatic embryo, or seed; and (iv) selecting a regenerated whole plant that expresses the polynucleotide. Many methods of transforming a plant, plant tissue or plant cell for the construction of a transformed cell are suitable. Once transformed, these cells can be used to regenerate transgenic plants. (148)

[0064] Those of ordinary skill in the art can use different plant gene transfer techniques found in references for, but not limited to, the electroporation, (149-153) microinjection, (154, 155) lipofection, (156) liposome or spheroplast fusions, (157-159) Agrobacterium, (160) direct gene transfer, (161) T-DNA mediated transformation of monocots, (162) T- DNA mediated transformation of dicots, (163, 164) microprojectile bombardment or ballistic particle acceleration, (165-168) chemical transfection including CaCl 2 precipitation, polyvinyl alcohol, or poly-L-ornifhine, (169) silicon carbide whisker methods, (170, 171) laser methods, (172, 173) sonication methods, (174-176) polyethylene glycol methods, (177) vacuum infiltration (178) and transbacter. (179) Other methods to edit, incorporate or move genes into plant genomes include, but are not limited to, Zinc-finger nucleases (ZFNs),(180, 181) transcription activator like effector nucleases (TALENs) and clustered regularly interspaced short palindromic repeats/Cas (CRISPR/Cas). (182-185)

[0065] In one embodiment of the invention, a transformed host cell may be cultured to produce a transformed plant. In this regard, a transformed plant can be made, for example, by transforming a cell, tissue or organ from a host plant with an inventive DNA construct; selecting a transformed cell, cell callus, somatic embryo, or seed which contains the DNA construct; regenerating a whole plant from the selected transformed cell, cell callus, somatic embryo, or seed; and selecting a regenerated whole plant that expresses the polynucleotide.

[0066] A wide variety of host cells may be used in the invention, including prokaryotic and eukaryotic host cells. These cells or organisms may include yeast, fungi, algae, microalgae, microbes, invertebrate, vertebrates or photosynthetic organisms. Preferred host cells are eukaryotic, preferably plant cells, such as those derived from monocotyledons, such as duckweed, corn, rice, sugarcane, wheat, bent grass, rye grass, Bermuda grass, Blue grass, and Fescue, or dicotyledons, including canola, cotton, camelina, lettuce, rapeseed, radishes, cabbage, sugarbeet, peppers, broccoli, potatoes and tomatoes, and legumes such as soybeans and bush beans.

[0067] One embodiment of the invention is a method for the production of hypo taurine or taurine by the following steps:

1. operably link a promoter to the 5' end of the polynucleotide for a functional CDOL gene product;

2. insert the polynucleotide construct (from step 1 above) into a vector;

3. transform the vector containing the CDOL construct into a plant or plant cell;

4. operably link a promoter to the 5' end of the polynucleotide for the functional SADL gene product;

5. insert the polynucleotide construct (from step 4 above) into a vector; and

6. transform the vector containing the SADL construct into a plant or plant cell carrying a CDOL construct or one that expresses a functional CDOL gene product. [0068] Another embodiment of the invention is a method for the production of hypotaurine or taurine by the following steps:

1. operably link a promoter to the 5' end of the polynucleotide for the functional CDOL gene product;

2. insert the polynucleotide construct (from step 1 above) into a vector;

3. transform the vector containing the CDOL construct into a plant or plant cell;

4. operably link a promoter to the 5' end of the polynucleotide for the functional SADL gene product;

5. insert the polynucleotide construct (from step 4 above) into a vector;

6. transform the vector containing the SADL construct into a plant or plant cell; and

7. Sexually cross a plant (or fuse cells) carrying a CDOL construct or one that expresses a functional CDOL with a plant (or cells) carrying a SADL construct or one that expresses a functional SADL gene product.

[0069] Another embodiment of the invention is a method for the production of hypotaurine or taurine by the following steps:

1. In the same vector, operably link a promoter to the 5' end of the polynucleotide for the functional CDOL gene product;

2. operably link a promoter to the 5' end of the polynucleotide for the functional SADL gene product;

3. insert the two polynucleotides into the vector in such a manner that both polynucleotides are expressed by one promoter or each polynucleotide is expressed by one promoter; and

4. transform the vector containing the CDOL and SADL constructs into a plant or plant cell.

[0070] Another embodiment of the invention is a method for the production of hypotaurine or taurine by the following steps:

1. operably link a promoter to the 5' end of the polynucleotide for a functional CDOL gene product;

2. insert the polynucleotide construct (from step 1 above) into a vector;

3. transform the vector containing the CDOL construct into a plant or plant cell;

4. operably link a promoter to the 5' end of the polynucleotide for the functional partCS/PLP-DC gene product; 5. insert the polynucleotide construct (from step 4 above) into a vector; and

6. transform the vector containing the partCS/PLP-DC construct into a plant or plant cell carrying a CDOL construct or one that expresses a functional CDOL gene product.

[0071] Another embodiment of the invention is a method for the production of hypotaurine or taurine by the following steps:

1. operably link a promoter to the 5' end of the polynucleotide for the functional CDOL gene product;

2. insert the polynucleotide construct (from step 1 above) into a vector;

3. transform the vector containing the CDOL construct into a plant or plant cell;

4. operably link a promoter to the 5' end of the polynucleotide for the functional partCS/PLP-DC gene product;

5. insert the polynucleotide construct (from step 4 above) into a vector;

6. transform the vector containing the partCS/PLP-DC construct into a plant or plant cell; and

7. Sexually cross a plant (or fuse cells) carrying a CDOL construct or one that expresses a functional CDOL with a plant (or cells) carrying a partCS/PLP-DC construct or one that expresses a functional partCS/PLP-DC gene product.

[0072] Another embodiment of the invention is a method for the production of hypotaurine or taurine by the following steps:

1. In the same vector, operably link a promoter to the 5' end of the polynucleotide for the functional CDOL gene product;

2. operably link a promoter to the 5' end of the polynucleotide for the functional partCS/PLP-DC gene product;

3. insert the two polynucleotides into the vector in such a manner that both polynucleotides are expressed by one promoter or each polynucleotide is expressed by one promoter; and

4. transform the vector containing the CDOL and partCS/PLP-DC constructs into a plant or plant cell.

[0073] Another embodiment of the invention is a method for the production of hypotaurine or taurine by the following steps: 1, operably link a promoter to the 5' end of the polynucleotide for a functional CDOL gene product;

2. insert the polynucleotide construct (from step 1 above) into a vector; transform the vector containing the CDOL construct into a plant or plant cell.

[0074] Another embodiment of the invention is a method for the production of hypotaurine or taurine by the following steps:

1. operably link a promoter to the 5' end of the polynucleotide for a functional SADL gene product;

2. insert the polynucleotide construct (from step 1 above) into a vector; transform the vector containing the SADL construct into a plant or plant cell.

[0075] Another embodiment of the invention is a method for the production of hypotaurine or taurine by the following steps:

1. operably link a promoter to the 5' end of the polynucleotide for a functional CS/PLP-DC gene product;

2. insert the polynucleotide construct (from step 1 above) into a vector; transform the vector containing the CS/PLP-DC construct into a plant or plant cell.

Suitable plants

[0076] The methods described above may be applied to transform a wide variety of plants, including decorative or recreational plants or crops, but are particularly useful for treating commercial and ornamental crops. Examples of plants that may be transformed in the present invention include, but are not limited to, Acacia, alfalfa, algae, aneth, apple, apricot, artichoke, arugula, asparagus, avocado, banana, barley, beans, beech, beet, Bermuda grass, bent grass, blackberry, blueberry, Blue grass, broccoli, Brussels sprouts, cabbage, camelina, canola, cantaloupe, carinata, carrot, cassava, cauliflower, celery, cherry, chicory, cilantro, citrus, Clementines, coffee, corn, cotton, cucumber, duckweed, Douglas fir, eggplant, endive, escarole, eucalyptus, fennel, fescue, figs, forest trees, garlic, gourd, grape, grapefruit, honey dew, jicama, kiwifruit, lettuce, leeks, lemon, lime, Loblolly pine, maize, mango, melon, mushroom, nectarine, nut, oat, okra, onion, orange, an ornamental plant, papaya, parsley, pea, peach, peanut, pear, pepper, persimmon, pine, pineapple, plantain, plum, pomegranate, poplar, potato, pumpkin, quince, radiata pine, radicchio, radish, rapeseed, raspberry, rice, rye, rye grass, seaweed, scallion, sorghum, Southern pine, soybean, spinach, squash, strawberry, sugarbeet, sugarcane, sunflower, sweet potato, sweetgum, switchgrass, tangerine, tea, tobacco, tomato, turf, turnip, a vine, watermelon, wheat, yams, and zucchini. Other suitable hosts include bacteria, fungi, algae and other photosynthetic organisms, and animals including vertebrate and invertebrates.

[0077] Once transformed, the plant may be treated with other "active agents" either prior to or during the exposure of the plant to stress to further decrease the effects of plant stress. "Active agent," as used herein, refers to an agent that has a beneficial effect on the plant or increases production of amino acid production by the plant. For example, the agent may have a beneficial effect on the plant with respect to nutrition, and the resistance against, or reduction of, the effects of plant stress. Some of these agents may be precursors of end products for reaction catalyzed by CDOL, SADL, CS/PLP-DC, or partCS/PLP-DC. These compounds could promote growth, development, biomass and yield, and change in metabolism. In addition to the twenty amino acids that are involved in protein synthesis specifically sulfur containing amino acids methionine, and cysteine, other amino acids such as glutamate, glutamine, serine, alanine and glycine, sulfur containing compounds such as fertilizer, sulfite, sulfide, sulfate, taurine, hypo taurine, cysteate, 2-sulfacetaldehyde, homotaurine, homocysteine, cystathionine, N-acetyl thiazolidine 4 carboxylic acid (ATCA), glutathione, or bile, or other non-protein amino acids, such as GABA, citrulline and ornithine, or other nitrogen containing compounds such as polyamines may also be used to activate CDOL, SADL, CS/PLP-DC, or partCS/PLP-DC. Depending on the type of gene construct or recombinant expression cassette, other metabolites and nutrients may be used to activate CDOL, SADL, CS/PLP-DC, or partCS/PLP-DC. These include, but are not limited to, sugars, carbohydrates, lipids, oligopeptides, mono- (glucose, arabinose, fructose, xylose, and ribose) di- (sucrose and trehalose) and polysaccharides, carboxylic acids (succinate, malate and fumarate) and nutrients such as phosphate, molybdate, or iron.

[0078] Accordingly, the active agent may include a wide variety of fertilizers, pesticides and herbicides known to those of ordinary skill in the art. (186) Other greening agents fall within the definition of "active agent" as well, including minerals such as calcium, magnesium and iron. The pesticides protect the plant from pests or disease and may be either chemical or biological and include fungicides, bactericides, insecticides and anti-viral agents as known to those of ordinary skill in the art.

[0079] In some embodiments properties of a transgenic plant are altered using an agent which increases sulfur concentration in cells of the transgenic plant, such as fertilizer, sulfur, sulfite, sulfide, sulfate, taurine, hypotaurine, homotaurine, cysteate, 2-sulfacetaldehyde, N-acetyl thiazolidine 4 carboxylic acid (ATCA), glutathione, and bile. In other embodiments, the agent increases nitrogen concentration. Amino acids either naturally occurring in proteins (e.g., cysteine, methionine, glutamate, glutamine, serine, alanine, or glycine) or which do no naturally occur in proteins (e.g., GABA, citrulline, or ornithine) and/or polyamines can be used for this purpose.

Expression in Prokaryotes

[0080] The use of prokaryotes including bacteria, archaebacteria and eubacteria, may be used for this invention, including proteobacteria such as members of Alphaproteobacteria, Betaproteobacteria, Gammaproteobacteria, Deltaproteobacteria, and Epsilonproteobacteria, Other bacteria strains include, but are not limited to, Bacillus, (187) Salmonella, Lactococcus, Streptococcus, Brevibacterium and coryneform bacteria. Some bacteria that can be used for the invention include, but are not limited to, Bacillus subtilis, Brevibacterium ammoniagene, Corynebacterium crenatum, Corynebacterim pekinese, Corynebacterium glutamicumas, Erwinia citreus, Erwinia herbicola, Escherichia coli, Fusarium venenatum Gluconobacter oxydans, Propionibacterium freudenreicheii, and Propionibacterium denitrificans. (188) Commonly used prokaryotic control sequences include promoters for transcription initiation, optionally with an operator, along with ribosome binding site sequences. Commonly used prokaryotic promoters include the beta lactamase (189), lactose (189), and tryptophan (190) promoters. The vectors usually contain selectable markers to identify transfected or transformed cells. Some commonly used selectable markers include the genes for resistance to ampicillin, tetracycline, or chloramphenicol. The vectors are typically a plasmid or phage. Bacterial cells are transfected or transformed with the plasmid vector DNA. Phage DNA can be infected with phage vector particles or transfected with naked phage DNA. The plasmid and phage DNA for the vectors are commercially available from numerous vendors known to those of ordinary skill in the art. Those of ordinary skill in the art know the molecular techniques and DNA vectors that are used in bacterial systems. (191-193)

Algae and microalgae

[0081] The use of algae and microscopic algae (microphytes or microalgae) can be used for this invention. These include, but are not limited to, diatoms, green algae (Chlorophyta), Euglenophyta, Dinoflagellata, Chrysophyta, Phaeophyta, red algae (Rhodophyta), and Cyanobacteria. Protocols for transfromation as well as commonly used vectors with control sequences include promoters for transcription initiation, optionally with an operator, together with ribosome binding site sequences are known to those of ordinary skill in the art. Vectors that are used in algal or microalgal systems are known to those of ordinary skill in the art. (194-201)

Expression in non-plant eiikaryotes

[0082] The present invention can be expressed in a variety of eukaryotic expression systems such as yeast, insect cell lines, and mammalian cells which are known to those of ordinary skill in the art. For each host system there are suitable vectors that are commercially available (e.g., Agilent Technologies, DNA2.0, GE Healthcare Life Sciences, New England Biolabs, ThermoFisher). The vectors usually have expression control sequences, such as promoters, an origin of replication, enhancer sequences, termination sequences, ribosome binding sites, RNA splice sites, polyadenylation sites, transcriptional terminator sequences, and selectable markers. Synthesis of heterologous proteins and fermentation of products in yeast is well known to those of ordinary skill in the art. (202, 203) Yeast and fungi that can be used include, but are not limited to, Ashbya gossypii, Blakeslea trispora, Candida flareri, Eremothecium ashbyii, Mortierella isabellina, Pichia pastoris, Saccharomyces cerevisiae and Saccyom mycaesess. Molecular protocols for transformation, and the vectors required for expression in these systems, are known to those of ordinary skill in the art. (204-208)

[0083] Invertebrate and vertebrate cells may also be used to express proteins of the present invention. Insect cell lines that include, but are not limited to, black-fly larvae, mosquito larvae, silkworm, armyworm, moth, and Drosophila cell lines can be used to express proteins of the present invention using baculovirus-derived vectors. (209) Mammalian cell systems including, but not limited to, monolayers of cells or cell suspensions may also be used to express proteins of the present invention. (210) A number of suitable host cell lines capable of expressing intact proteins have been developed in the art, including the HEK293, BHK21, and CHO cell lines. For each host system there are suitable vectors that are commercially available (BD Boosciences, New England Biolabs, ThermoFisher).

[0084] A protein of the present invention, once expressed in any of the non-plant eukaryotic systems can be isolated from the organism by lysing the cells and applying standard protein isolation techniques to the lysates or the pellets. The monitoring of the purification process can be accomplished by using western blot techniques or radioimmunoassay or other standard immunoassay techniques. Pharmaceutical compositions

[0085] The invention provides pharmaceutical compositions that comprise extracts of one or more transgenic plants described above. Plant extracts containing hypo taurine or taurine can be used to synthesize or manufacture taurine derivatives (211, 212), taurine-conjugates (213) or taurine-polymers (214) that may have a wide range of commercial and medicinal applications. (215) Some taurine derivatives can function as organogelators (216) or dyes (217) and can be used in nanosensor synthesis. (218) Some taurine derivatives have anticonvulsant (211) or anticancer (219) properties. Other taurine derivatives are used in the treatment of alcoholism. (220, 221) Taurine-conjugated carboxyethylester-polyrotaxanes increase anticoagulant activity. (222) Taurine-containing polymers may increase wound healing. (223, 224) Taurine linked polymers such as poly gamma-glutamic acid-sulfonates are biodegradable and may have applications in the development of drug delivery systems, environmental materials, tissue engineering, and medical materials. (225) Extracts from taurine-containing plants may be used in pharmaceutical or medicinal compositions to deliver taurine, hypotaurine, taurine-conjugates, or taurine- polymers for use in the treatment of congestive heart failure, high blood pressure, hepatitis, high cholesterol, fibrosis, epilepsy, autism, attention deficit-hyperactivity disorder, retinal degeneration, diabetes, and alcoholism. It is also used to improve mental performance and as an antioxidant.

[0086] Pharmaceutically acceptable vehicles of taurine, taurine derivatives, taurine- conjugates, or taurine-polymers are tablets, capsules, gel, ointment, film, patch, powder or dissolved in liquid form.

Nutritional Supplements and Feeds

[0087] Transgenic plants containing hypotaurine or taurine may be consumed or used to make extracts for nutritional supplements. Transgenic plant parts that contain hypotaurine or taurine may be used for human consumption. The plant parts may include but are not limited to leaves, stalks, stems, tubers, stolons, roots, petioles, cotyledons, seeds, fruits, grain, strover, nuts, flowers, petioles, pollen, buds, or pods. Extracts from transgenic plants containing hypotaurine or taurine may be used as nutritional supplements, as an antioxidant or to improve physical or mental performance. The extracts may be used in the form of a liquid, powder, capsule or tablet. Other transgenic cells, bacterial, algal, microalgal, fungal, yeast or insects (or insects or larvae) containing taurine may be used in the form of a liquid, powder, capsule or tablet.

[0088] Transgenic plants containing hypotaurine or taurine may be used as fish or animal feed or used to make extracts for the supplementation of animal feed. Transgenic plant parts that contain hypotaurine or taurine may be used as animal or fish feed. The plant parts include but are not limited to leaves, stalks, stems, tubers, stolons, roots, petioles, cotyledons, seeds, fruits, grain, strover, nuts, flowers, petioles, buds, pods, or husks. Extracts from transgenic plants containing taurine may be used as feed supplements in the form of a liquid, powder, capsule or tablet. Other transgenic cells, bacterial, algal, microalgal, fungal, yeast or insects (or insects or larvae) containing hypotaurine or taurine may be used as animal or fish feed.

Enhancer of plant growth or yield

[0089] Transgenic plant parts that contain hypotaurine or taurine may be used as an enhancer for plant growth or yield. The plant parts include, but are not limited to, leaves, stalks, stems, tubers, stolons, roots, petioles, cotyledons, seeds, fruits, grain, strover, nuts, flowers, petioles, buds, pods, or husks. Extracts from transgenic plants containing hypotaurine or taurine may be used as plant enhancers in the form of a liquid, powder, capsule or tablet. Other transgenic cells, bacterial, algal, microalgal, fungal, yeast or insects (or insects or larvae) containing hypotaurine or taurine may be used as plant enhancers.

[0090] Taurine could be purified from the cells or from extracts of the cells or from media from which the cells were grown. The extracted taurine could be used as a food or feed additive, nutrient, pharmaceutical or an enhancer of plant growth or yield. Prokaryotic or eukaryotic cells with the invention can be grown in culture or by fermentation to produce hyptotaurine or taurine. Methods to produce chemical compounds by batch fermentation, fed-batch

fermentation, continuous fermentation or in tanks or ponds are well known to one with ordinary skill in the art. (226-237)

[0091] Methods such as centrifugation, filtration, crystallization, ion exchange,

electrodialysis, solvent extraction, decolorization or evaporation to purify or separate chemical compounds from cells or from liquids or media that grew cells are well known to one with ordinary skill in the art. These methods can be used by one with ordinary skill in the art to purify or separate taurine from cells with the invention, or from liquids or media from which cell suspensions or cell cultures containing the invention were grown. (227, 230, 238-242)

DEFINITIONS

[0092] The term "polynucleotide" refers to a natural or synthetic linear and sequential array of nucleotides and/or nucleosides, including deoxyribonucleic acid, ribonucleic acid, and derivatives thereof. It includes chromosomal DNA, self-replicating plasmids, infectious polymers of DNA or RNA and DNA or RNA that performs a primarily structural role. Unless otherwise indicated, nucleic acids or polynucleotide are written left to right in 5' to 3' orientation, Nucleotides are referred to by their commonly accepted single-letter codes. Numeric ranges are inclusive of the numbers defining the range.

[0093] The terms "amplified" and "amplification" refer to the construction of multiple copies of a nucleic acid sequence or multiple copies complementary to the nucleic acid sequence using at least one of the nucleic acid sequences as a template. Amplification can be achieved by chemical synthesis using any of the following methods, such as solid-phase phosphoramidate technology or the polymerase chain reaction (PCR). Other amplification systems include the ligase chain reaction system, nucleic acid sequence based amplification, Q-Beta Replicase systems, transcription-based amplification system, and strand displacement amplification. The product of amplification is termed an amplicon.

[0094] As used herein "promoter" includes reference to a region of DNA upstream from the start of transcription and involved in recognition and binding of RNA polymerase, either I, II or III, and other proteins to initiate transcription. Promoters include necessary nucleic acid sequences near the start site of transcription, such as, in the case of a polymerase II type promoter, a TATA element. A promoter also optionally includes distal enhancer or repressor elements, which can be located as far as several thousand base pairs from the start site of transcription.

[0095] The term "plant promoter" refers to a promoter capable of initiating transcription in plant cells.

[0096] The term "microbe promoter" refers to a promoter capable of initiating transcription in microbes.

[0097] The term "foreign promoter" refers to a promoter, other than the native, or natural, promoter, which promotes transcription of a length of DNA of viral, bacterial or eukaryotic origin, including those from microbes, plants, plant viruses, invertebrates or vertebrates.

[0098] The term "microbe" refers to any microorganism (including both eukaryotic and prokaryotic microorganisms), such as fungi, yeast, bacteria, actinomycetes, algae and protozoa, as well as other unicellular structures.

[0099] The term "plant" includes whole plants, and plant organs, and progeny of same. Plant organs comprise, e.g., shoot vegetative organs/structures (e.g. leaves, stems and tubers), roots, flowers and floral organs/structures (e.g. bracts, sepals, petals, stamens, carpels, anthers and ovules), seed (including embryo, endosperm, and seed coat) and fruit (the mature ovary), plant tissue (e.g. vascular tissue, ground tissue, and the like) and cells (e.g. guard cells, egg cells, trichomes and the like). The class of plants that can be used in the method of the invention is generally as broad as the class of higher and lower plants amenable to transformation techniques, including angiosperms (monocotyledonous and dicotyledonous plants), gymnosperms, ferns, and multicellular algae. It includes plants of a variety of ploidy levels, including aneuploid, polyploid, diploid, haploid and hemizygous.

[0100] The term "plant storage organ" includes roots, seeds, tubers, fruits, and specialized stems.

[0101] The term "constitutive" refers to a promoter that is active under most environmental and developmental conditions, such as, for example, but not limited to, the CaMV 35S promoter and the nopaline synthase terminator.

[0102] The term "tissue-preferred promoter" refers to a promoter that is under developmental control or a promoter that preferentially initiates transcription in certain tissues.

[0103] The term "tissue-specific promoter" refers to a promoter that initiates transcription only in certain tissues.

[0104] The term "cell-type specific promoter" refers to a promoter that primarily initiates transcription only in certain cell types in one or more organs.

[0105] The term "seed-specific promoter" refers to a promoter that primarily initiates transcription only in the seeds.

[0106] The term "inducible promoter" refers to a promoter that is under environmental control.

[0107] The terms "encoding" and "coding"" refer to the process by which a polynucleotide, through the mechanisms of transcription and translation, provides the information to a cell from which a series of amino acids can be assembled into a specific amino acid sequence to produce a functional polypeptide, such as, for example, an active enzyme or ligand binding protein.

[0108] The terms "polypeptide," "peptide," "protein" and "gene product" are used interchangeably herein to refer to a polymer of amino acid residues. The terms apply to amino acid polymers in which one or more amino acid residue is an artificial chemical analogue of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers. Amino acids may be referred to by their commonly known three-letter or one-letter symbols. Amino acid sequences are written left to right in amino to carboxy orientation, respectively. Numeric ranges are inclusive of the numbers defining the range. [0109] The terms "residue," "amino acid residue," and "amino acid" are used interchangeably herein to refer to an amino acid that is incorporated into a protein, polypeptide, or peptide. The amino acid may be a naturally occurring amino acid and may encompass known analogs of natural amino acids that can function in a similar manner as the naturally occurring amino acids.

[0110] The terms "cysteine dioxygenase" and "CDO" refer to the protein (EC:1.13.11.20) that catalyzes the following reaction:

cysteine + oxygen = 3-sulfinoalanine

[0111] NOTE: 3-sulfinoalanine is another name for cysteine sulfinic acid, cysteine sulfinate, 3-sulphino-L-alanine, 3-sulfino-alanine, 3-sulfino-L-alanine, L-cysteine sulfinic acid, L-cysteine sulfinic acid, cysteine hydrogen sulfite ester or alanine 3-sulfinic acid.

[0112] The terms "sulfinoalanine decarboxylase" and "SAD" refer to the protein (4.1.1.29) that catalyzes the following reaction:

3-sulfinoalanine = hypotaurine + C0 2

[0113] NOTE: SAD is another name for cysteine-sulfinate decarboxylase, L-cysteine sulfinic acid decarboxylase, cysteine-sulfinate decarboxylase, CADCase/CSADCase, CSAD, cysteic decarboxylase, cysteine sulfinic acid decarboxylase, cysteine sulfinate decarboxylase, sulfoalanine decarboxylase, sulphinoalanine decarboxylase, and 3-sulfino-L-alanine carboxylase.

[0114] NOTE: the SAD reaction is also catalyzed by GAD (4.1.1.15) (glutamic acid decarboxylase or glutamate decarboxylase).

[0115] Other names for hypotaurine are 2-aminoethane sulfinate, 2-aminoethylsulfinic acid, and 2-aminoethanesulfinic acid.

[0116] Other names for taurine are 2-aminoethane sulfonic acid, aminoethanesulfonate, L- taurine, taurine ethyl ester, and taurine ketoisocaproic acid 2-aminoethane sulfinate.

[0117] The terms "cysteamine dioxygenase" and "ADO" refer to the protein (EC 1.13.11.19) that catalyzes the following reaction:

2-aminoethanethiol + 0 2 = hypotaurine

[0118] ADO is another name for 2-aminoethanethiol : oxygen oxidoreductase, persulfurase, cysteamine oxygenase, and cysteamine : oxygen oxidoreductase.

[0119] Other names for 2-aminoethanethiol are cysteamine or 2-aminoethane- 1 -thiol, b- mercaptoethylamine, 2-mercaptoethylamine, decarboxycysteine, and thioethanolamine. [0120] The terms "taurine-pyruvate aminotransferase" and "TPAT" refer to the protein (EC 2.6.1.77) that catalyzes the following reaction:

L-alanine + 2-sulfoacetaldehyde = taurine + pyruvate

[0121] TPAT is another name for taurine transaminase or Tpa.

[0122] The terms "sulfoacetaldehyde acetyltransferase" and "SA" refer to the protein (EC:2.3.3.15) that catalyzes the following reaction:

acetyl phosphate + sulfite = sulfoacetaldehyde + orthophosphate

[0123] SA is another name for acetyl-phosphate : sulfite S-acetyltransferase or Xsc.

[0124] The terms "taurine dehydrogenase" and "TDeHase" refer to the protein (EC: 1.4.2.-) that catalyzes the following reaction:

ammonia + 2-sulfoacetaldehyde = taurine + water

[0125] TDeHase is another name for taurine : oxidoreductase, taurine:ferricytochrome-c oxidoreductase, tauX or tauY.

[0126] The terms "taurine dioxygenase" and "TDO" refer to the protein (EC:1.14.11.17) that catalyzes the following reaction:

sulfite + aminoacetaldehyde + succinate + C0 2 = taurine + 2-oxoglutarate + 0 2

[0127] TDO is another name for 2-aminoethanesulfonate dioxygenase, alpha-ketoglutarate- dependent taurine dioxygenase, taurine, 2-oxoglutarate:0 2 oxidoreductase or tauD.

[0128] 2-oxoglutarate is another name for alpha-ketoglutarate .

[0129] The term "functional" with reference to CDOL, SADL, CS/PLP-DC, ADO, TPAT, SA, ssTDeHase, IsTDeHase or CS PLP-DC refers to peptides, proteins or enzymes that catalyze the CDOL, SADL, GAD, ADO, TPAT, SA, TDeHase or CS/PLP-DC reactions, respectively.

[0130] The terms "cysteine synthetase/PLP decarboxylase" and "CS/PLP-DC" refer to the protein that catalyzes the following reactions:

cysteine + oxygen = hypotaurine

cysteine + oxygen = taurine

O-acetyl-L-serine + hydrogen sulfide = hypotaurine

O-acetyl-L-serine + hydrogen sulfide = taurine

[0131] The terms "portion of the cysteine synthetase/PLP decarboxylase" and "partCS/PLP- DC" refers to the protein that catalyzes a decarboxylase reaction which cleaves carbon-carbon bonds and includes, but is not limited to, the following substrate and end-products:

Aspartate = beta-alanine + C0 2

Glutamate = 4-aminobutanoate + C0 2 Cysteic acid = 2-aminoethane sulfonate + C0 2

[0132] Note: another name for 4-aminobutanoate is gamma-aminobutyric acid (GABA).

[0133] The term "recombinant" includes reference to a cell or vector that has been modified by the introduction of a heterologous nucleic acid. Recombinant cells express genes that are not normally found in that cell or express native genes that are otherwise abnormally expressed, underexpressed, or not expressed at all as a result of deliberate human intervention, or expression of the native gene may have reduced or eliminated as a result of deliberate human intervention.

[0134] The term "recombinant expression cassette" refers to a nucleic acid construct, generated recombinantly or synthetically, with a series of specified nucleic acid elements, which permit transcription of a particular nucleic acid in a target cell. The recombinant expression cassette can be incorporated into a plasmid, chromosome, mitochondrial DNA, plastid DNA, virus, or nucleic acid fragment. Typically, the recombinant expression cassette portion of an expression vector includes, among other sequences, a nucleic acid to be transcribed, and a promoter.

[0135] The term "transgenic plant" includes reference to a plant, which comprises within its genome a heterologous polynucleotide. Generally, the heterologous polynucleotide is integrated within the genome such that the polynucleotide is passed on to successive generations. The heterologous polynucleotide may be integrated into the genome alone or as part of a recombinant expression cassette. "Transgenic" is also used to include any cell, cell line, callus, tissue, plant part or plant, the genotype of which has been altered by the presence of heterologous nucleic acid including those transgenic plants altered or created by sexual crosses or asexual propagation from the initial transgenic plant. The term "transgenic" does not encompass the alteration of the genome by conventional plant breeding methods or by naturally occurring events such as random cross-fertilization, non-recombinant viral infection, non- recombinant bacterial transformation, non-recombinant transposition, or spontaneous mutation.

[0136] The term "vector" includes reference to a nucleic acid used in transfection or transformation of a host cell and into which can be inserted a polynucleotide.

[0137] The term "selectively hybridizes" includes reference to hybridization, under stringent hybridization conditions, of a nucleic acid sequence to a specified nucleic acid target sequence to a detectably greater degree (e.g., at least 2-fold over background) than its hybridization to non-target nucleic acid sequences and to the substantial exclusion of non-target nucleic acids. Selectively hybridizing sequences typically have about at least 40% sequence identity, preferably 60-90% sequence identity, and most preferably 100% sequence identity (i.e., complementary) with each other.

[0138] The terms "stringent conditions" and "stringent hybridization conditions" include reference to conditions under which a probe will hybridize to its target sequence, to a detectably greater degree than other sequences (e.g., at least 2-fold over background). Stringent conditions are sequence-dependent and will be different in different circumstances. By controlling the stringency of the hybridization and/or washing conditions, target sequences can be identified which can be up to 100% complementary to the probe (homologous probing). Alternatively, stringency conditions can be adjusted to allow some mismatching in sequences so that lower degrees of similarity are detected (heterologous probing). Optimally, the probe is approximately 500 nucleotides in length, but can vary greatly in length from less than 500 nucleotides to equal to the entire length of the target sequence.

[0139] Typically, stringent conditions will be those in which the salt concentration is less than about 1.5 M Na ion, typically about 0.01 to 1.0 M Na ion concentration (or other salts) at pH 7.0 to 8.3 and the temperature is at least about 30°C for short probes (e.g., 10 to 50 nucleotides) and at least about 60°C for long probes (e.g., greater than 50 nucleotides). Stringent conditions may also be achieved with the addition of destabilizing agents such as formamide or Denhardt solution. Low stringency conditions include hybridization with a buffer solution of 30 to 35% formamide, 1 M NaCl, 1% SDS (sodium dodecyl sulfate) at 37°C, and a wash in IX to 2X SSC (20X SSC = 3.0 M NaCl/0.3 M trisodium citrate) at 50 to 55°C. Moderate stringency conditions include hybridization in 40 to 45% formamide, 1 M NaCl, 1% SDS at 37°C, and a wash in 0.5X to IX SSC at 55 to 60°C. High stringency conditions include hybridization in 50% formamide, 1 M NaCl, 1% SDS at 37°C, and a wash in 0.1X SSC at 60 to 65 °C. Specificity is typically the function of post-hybridization washes, the critical factors being the ionic strength and temperature of the final wash solution. For DNA-DNA hybrids, the T m can be approximated (243), where the T m = 81.5°C + 16.6 (log M) + 0.41 (%GC) - 0.61 (% form) - 500/L; where M is the molarity of monovalent cations, %GC is the percentage of guanosine and cytosine nucleotides in the DNA, % form is the percentage of formamide in the hybridization solution, and L is the length of the hybrid in base pairs. T m is the temperature (under defined ionic strength and pH) at which 50% of a complementary target sequence hybridizes to a perfectly matched probe. T m is reduced by about 1°C for each 1% of mismatching; thus, T m , hybridization and/or wash conditions can be adjusted to hybridize to sequences of the desired identity. For example, if sequences with >90% identity are sought, the T ra can be decreased 10°C. Generally, stringent conditions are selected to be about 5°C lower than the thermal melting point (T m ) for the specific sequence and its complement at a defined ionic strength and pH. However, severely stringent conditions can utilize a hybridization and/or wash at 1, 2, 3 or 4°C lower than the thermal melting point (T m ); moderately stringent conditions can utilize a hybridization and/or wash at 6, 7, 8, 9 or 10°C lower than the thermal melting point (T m ); low stringency conditions can utilize a hybridization and/or wash at 11, 12, 13, 14, 15 or 20°C lower than the thermal melting point (T m ). Using the equation, hybridization and wash compositions, and desired T m , those of ordinary skill in the art will understand that variations in the stringency of hybridization and/or wash solutions are inherently described. An extensive guide to the hybridization of nucleic acids is found in the scientific literature (115, 244). Unless otherwise stated, in the present application high stringency is defined as hybridization in 4X SSC, 5X Denhardt solution (5 g Ficoll, 5 g polyvinypyrrolidone, 5 g bovine serum albumin in 500ml of water), 0.1 mg/ml boiled salmon sperm DNA, and 25 mM Na phosphate at 65 °C, and a wash in 0.1X SSC, 0.1% SDS at 65°C.

[0140] The following terms are used to describe the sequence relationships between two or more nucleic acids or polynucleotides or polypeptides: "reference sequence," "comparison window," "sequence identity," "percentage of sequence identity," and "substantial identity."

[0141] The term "reference sequence" is a defined sequence used as a basis for sequence comparison. A reference sequence may be a subset or the entirety of a specified sequence; for example, as a segment of a full-length cDNA or gene sequence, or the complete cDNA or gene sequence.

[0142] The term "comparison window" includes reference to a contiguous and specified segment of a polynucleotide sequence, where the polynucleotide sequence may be compared to a reference sequence and the portion of the polynucleotide sequence in the comparison window may comprise additions or deletions (i.e., gaps) when it is compared to the reference sequence for optimal alignment. The comparison window is usually at least 20 contiguous nucleotides in length, and optionally can be 30, 40, 50, 100 or longer. Those of ordinary skill in the art understand that the inclusion of gaps in a polynucleotide sequence alignment introduces a gap penalty, and it is subtracted from the number of matches.

[0143] Methods of alignment of nucleotide and amino acid sequences for comparison are well known to those of ordinary skill in the art. The local homology algorithm, BESTFTT, (245) can perform an optimal alignment of sequences for comparison using a homology alignment algorithm called GAP (246), search for similarity using Tfasta and Fasta (247), by computerized implementations of these algorithms widely available on-line or from various vendors (Intelligenetics, Genetics Computer Group). CLUSTAL allows for the alignment of multiple sequences (248-250) and program PileUp can be used for optimal global alignment of multiple sequences (251). The BLAST family of programs can be used for nucleotide or protein database similarity searches. BLASTN searches a nucleotide database using a nucleotide query. BLASTP searches a protein database using a protein query. BLASTX searches a protein database using a translated nucleotide query that is derived from a six-frame translation of the nucleotide query sequence (both strands). TBLASTN searches a translated nucleotide database using a protein query that is derived by reverse-translation. TBLASTX search a translated nucleotide database using a translated nucleotide query.

[0144] GAP (246) maximizes the number of matches and minimizes the number of gaps in an alignment of two complete sequences. GAP considers all possible alignments and gap positions and creates the alignment with the largest number of matched bases and the fewest gaps. It also calculates a gap penalty and a gap extension penalty in units of matched bases. Default gap creation penalty values and gap extension penalty values in Version 10 of the Wisconsin Genetics Software Package are 8 and 2, respectively. The gap creation and gap extension penalties can be expressed as an integer selected from the group of integers consisting of from 0 to 100. GAP displays four figures of merit for alignments: Quality, Ratio, Identity, and Similarity. The Quality is the metric maximized in order to align the sequences. Ratio is the quality divided by the number of bases in the shorter segment. Percent Identity is the percent of the symbols that actually match. Percent Similarity is the percent of the symbols that are similar. Symbols that are across from gaps are ignored. A similarity is scored when the scoring matrix value for a pair of symbols is greater than or equal to 0.50, the similarity threshold. The scoring matrix used in Version 10 of the Wisconsin Genetics Software Package is BLOSUM62. (252)

[0145] Unless otherwise stated, sequence identity or similarity values refer to the value obtained using the BLAST 2.0 suite of programs using default parameters. (253) As those of ordinary skill in the art understand that BLAST searches assume that proteins can be modeled as random sequences and that proteins comprise regions of nonrandom sequences, short repeats, or enriched for one or more amino acid residues, called low-complexity regions. These low- complexity regions may be aligned between unrelated proteins even though other regions of the protein are entirely dissimilar. Those of ordinary skill in the art can use low-complexity filter programs to reduce number of low-complexity regions that are aligned in a search. These filter programs include, but are not limited to, the SEG (254, 255) and XNU. (256)

[0146] The terms "sequence identity" and "identity" are used in the context of two nucleic acid or polypeptide sequences and include reference to the residues in the two sequences, which are the same when aligned for maximum correspondence over a specified comparison window. When the percentage of sequence identity is used in reference to proteins it is recognized that residue positions which are not identical often differ by conservative amino acid substitutions, where amino acid residues are substituted for other amino acid residues with similar chemical properties (e.g., charge or hydrophobicity) and therefore do not change the functional properties of the molecule. Where sequences differ in conserved substitutions, the percent sequence identity may be adjusted upwards to correct for the conserved nature of the substitution. Sequences, which differ by such conservative substitutions, are said to have "sequence similarity" or "similarity." Scoring for a conservative substitution allows for a partial rather than a full mismatch, (257) thereby increasing the percentage sequence similarity.

[0147] The term "percentage of sequence identity" means the value determined by comparing two optimally aligned sequences over a comparison window, wherein the portion of the polynucleotide sequence in the comparison window may comprise gaps (additions or deletions) when compared to the reference sequence for optimal alignment. The percentage is calculated by determining the number of positions at which the identical nucleic acid base or amino acid residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison and multiplying the result by 100 to yield the percentage of sequence identity.

[0148] The term "substantial identity" of polynucleotide sequences means that a polynucleotide comprises a sequence that has between 50-100% sequence identity, preferably at least 50% sequence identity, preferably at least 60% sequence identity, preferably at least 70%, more preferably at least 80%, more preferably at least 90%, and most preferably at least 95%, compared to a reference sequence using one of the alignment programs described using standard parameters. One of ordinary skill in the art will recognize that these values can be appropriately adjusted to determine corresponding identity of proteins encoded by two nucleotide sequences by taking into account codon degeneracy, amino acid similarity, reading frame positioning and the like. Substantial identity of amino acid sequences for these purposes normally means sequence identity of between 50-100%. Another indication that nucleotide sequences are substantially identical is if two molecules hybridize to each low stringency conditions, moderate stringency conditions or high stringency conditions. Yet another indication that two nucleic acid sequences are substantially identical is if the two polypeptides immunologically cross-react with the same antibody in a western blot, immunoblot or ELISA assay.

[0149] The terms "substantial identity" in the context of a peptide indicates that a peptide comprises a sequence with between 55-100% sequence identity to a reference sequence preferably at least 55% sequence identity, preferably 60% preferably 70%, more preferably 80%, most preferably at least 90% or 95% sequence identity to the reference sequence over a specified comparison window. Preferably, optimal alignment is conducted using the homology alignment algorithm (246). Thus, a peptide is substantially identical to a second peptide, for example, where the two peptides differ only by a conserved substitution. Another indication that amino acid sequences are substantially identical is if two polypeptides immunologically cross- react with the same antibody in a western blot, immunoblot or EOSA assay. In addition, a peptide can be substantially identical to a second peptide when they differ by a non-conservative change if the epitope that the antibody recognizes is substantially identical.

[0150] All patents, patent applications, and references cited in this disclosure are expressly incorporated herein by reference. The above disclosure generally describes the present invention. A more complete understanding can be obtained by reference to the following specific examples, which are provided for purposes of illustration only and are not intended to limit the scope of the invention

REFERENCES

[0151] 1. Abbott PC, Hurt C, & Tyner WE (201 1) What's driving food prices in 2011? Farm Foundation Issue Report (July).

[0152] 2. Sturman JA (1988) Taurine in development. Journal of Nutrition 118:1169-1176.

[0153] 3. Sturman JA & Hayes KC (1980) The biology of taurine in nutrition and development. Advances in Nutritional Research 3:231-299.

[0154] 4. Chen XC, Pan ZL, Liu DS, & Han X (1998) Effect of taurine on human fetal neuron cells: Proliferation and differentiation. Advances in Experimental Medicine and Biology 442:397-403.

[0155] 5. El Idrissi A & Trenkner E (1999) Growth factors and taurine protect against excitotoxicity by stabilizing calcium homeostasis and energy metabolism. Journal of Neuroscience 19:9459-9468.

[0156] 6. El Idrissi A & Trenkner E (2003) Taurine regulates mitochondrial calcium homeostasis. Advances in Experimental Medicine and Biology 526:527-536.

[0157] 7. Trenkner E (1990) Possible role of glutamate with taurine in neuron-glia interaction during cerebellar development. Progress in Clinical and Biological Research 351:133-140. [0158] 8. Wu H et al- (2005) Mode of action of taurine as a neuroprotector. Brain Research 1038:123-131.

[0159] 9. Schaffer S, Takahashi K, & Azuma J (2000) Role of osmoregulation in the actions of taurine. Amino Acids 19:527-546.

[0160] 10. Chapman RA, Suleiman MS, & Earm YE (1993) Taurine and the heart. Cardiovascular Research 27:358-363.

[0161] 11. Tabassuma H, Rehmana H, Banerjeeb BD, Raisuddina S, & Parvez S (2006) Attenuation of tamoxifen-induced hepatotoxicity by taurine in mice. Clinica Chimica Acta 370:129-136.

[0162] 12. Rocket N, et al (2007) The osmolyte taurine protects against ultraviolet B radiation-induced immunosuppression. Journal of Immunology 179:3604-3612.

[0163] 13. Knopf K, Sturman J A, Armstrong M, & Hayes AC (1978) Taurine: An essential nutrient for the cat. Journal of Nutrition 108:773-778.

[0164] 14. Morris JG, Rogers QR, & Pacioretty LM (1990) Taurine: an essential nutrient for cats. Journal of Small Animal Practice 31(10):502-509.

[0165] 15. Chesney R, et al. (1998) The Role of Taurine in Infant Nutrition. Taurine 3, Advances in Experimental Medicine and Biology, eds Schaffer S, Lombardini J, & Huxtable R (Springer US), Vol 442, pp 463-476.

[0166] 16. Gibson GT, et al. (2007) Supplementation of taurine and methionine to all-plant protein diets for rainbow trout (Oncorhynchus mykiss). Aquaculture 269:514-524.

[0167] 17. Buentello A, Jirsa D, Barrows FT, & Drawbridge M (2015) Minimizing fishmeal use in juvenile California yellowtail, Seriola lalandi, diets using non-GM soybeans selectively bred for aquafeeds. Aquaculture 435(0):403-411.

[0168] 18. Rossi W, Moxely D, Buentello A, Pohlenz C, & Gatlin DM (2013) Replacement of fishmeal with novel plant feedstuffs in the diet of red drum Sciaenops ocellatus: an assessment of nutritional value. Aquaculture Nutrition 19:72-81.

[0169] 19. Watson AM, Buentello A, & Place AR (2014) Partial replacement of fishmeal, poultry by-product meal and soy protein concentrate with two non-genetically modified soybean cultivars in diets for juvenile cobia, Rachycentron canadum. Aquaculture 434(0): 129-136.

[0170] 20. Takagia S, et al. (2008) Taurine is an essential nutrient for yellowtail Seriola quinqueradiata fed non-fish meal diets based on soy protein concentrate. Aquaculture 280:198- 205.

[0171] 21. Lunger AN, McLean E, Gaylord TG, Kuhn D, & Craig SR (2007) Taurine supplementation to alternative dietary proteins used in fish meal replacement enhances growth of juvenile cobia {Rachycentron canadum). Aquaculture 271:401-410.

[0172] 22. Watson AM, Barrows FT, & Place AR (2013) Taurine supplementation of plant derived protein and n-3 fatty acids are critical for optimal growth and development of cobia, Rachycentron canadum. Lipids 48(9):899-913.

[0173] 23. Watson AM, Barrows FT, & Place AR (2013) Taurine supplemented plant protein based diets with alternative lipid sources for juvenile gilthead sea bream, Sparus aurata. Journal of Fisheries and Aquaculture 4:59-66. [0174] 24. Park GS, Takeuchi T, Yokoyama M, & Seikai T (2002) Optimal dietary taurine level for growth of juvenile Japanese flounder Paralichthys olivaceus. Fisheries Science 68:824-829.

[0175] 25. Gaylord TG, Teague AM, & Barrows FT (2006) Taurine supplementation of all- plant protein diets for rainbow trout (Oncorhynchus mykiss). Journal of the World Aquaculture Society 37:509-517.

[0176] 26. Salze GP & Davis DA (2015) Taurine: a critical nutrient for future fish feeds. Aquaculture 437:215-229.

[0177] 27. Yang H, Tian L, Huang J, Liang G, & Liu Y (2013) Dietary taurine can improve the hypoxia-tolerance but not the growth performance in juvenile grass carp Ctenopharyngodon idellus. Fish physiology and biochemistry 39(5): 1071-1078.

[0178] 28. Kuz'mina VV, Gavrovskaya LK, Rusanova PV, Kulivatskaya EA, & Ryzhova OV (2011) Effect of taurine on the glycemia level and the activity of hydrolases in the intestinal mucosa in carp (Cyprinus carpio L.). Inland Water Biol 4(2):242-248.

[0179] 29. Yue Y-R, et al. (2012) The effect of dietary taurine supplementation on growth performance, feed utilization and taurine contents in tissues of juvenile white shrimp (Litopenaeus vannamei, Boone, 1931) fed with low-fishmeal diets. Aquaculture Research DOI: 10.111 l/j.l365-2109.2012.03135.x.

[0180] 30. Brotons Martinez J, Chatzifotis S, Divanach P, & Takeuchi T (2004) Effect of dietary taurine supplementation on growth performance and feed selection of sea bass Dicentrarchus labrax fry fed with demand-feeders. Fisheries Science 70(l):74-79.

[0181] 31. Milei J, et al. (1992) Reduction of reperfusion injury with preoperative rapid intravenous infusion of taurine during myocardial revascularization. American Heart Journal 123:339-345.

[0182] 32. Militante JD & Lombardini JB (2002) Treatment of hypertension with oral taurine. Endocrinology 147:3276-3284.

[0183] 33. Fujita T, Ando K, Noda H, Ito Y, & Sato Y (1987) Effects of increased adrenomedullary activity and taurine in young patients with borderline hypertension. Circulation 75:525-532.

[0184] 34. McCown TJ, Givens BS, & Breese GR (1987) Amino acid influences on seizures elicited within the inferior colliculus. Pharmacology and Experimental Therapeutics 243:603- 608.

[0185] 35. Matsuyama Y, Morita T, Higuchi M, & Tsujii T (1983) The effect of taurine administration on patients with acute hepatitis. Progress in Clinical and Biological Research 125:461-468.

[0186] 36. Ikeda H (1977) Effects of taurine on alcohol withdrawal. Lancet 2:509.

[0187] 37. Franconi F, Di Leo MAS, Bennardini F, & Ghirlanda G (2004) Is taurine beneficial in reducing risk factors for diabetes mellitus? Neurochemical Research 29:143-150.

[0188] 38. Paula-Lima AC, De Felice FG, Brito-Moreira J, & Ferreira ST (2005) Activation of GABAA receptors by taurine and muscimol blocks the neurotoxicity of [beta] -amyloid in rat hippocampal and cortical neurons. Neuropharmacology 49:1140-1148. [0189] 39. Nakamori K, et al. (1993) Quantitative evaluation of the effectiveness of taurine in protecting the ocular surface against oxidant. Chemical & Pharmaceutical Bulletin 41:335- 338.

[0190] 40. Zhang M, et al. (2004) Beneficial effects of taurine on serum lipids in overweight or obese non-diabetic subjects. Ammo Acids 26:267-271.

[0191] 41. Yokogoshi H, et al. (1999) Dietary taurine enhances cholesterol degradation and reduces serum and liver cholesterol concentrations in rats fed a high-cholesterol diet. Journal of Nutrition 129:1705-1712.

[0192] 42. Yamamoto K, et al. (2000) Dietary taurine decreases hepatic secretion of cholesterol ester in rats fed a high-cholesterol diet. Pharmacology 60:27-33.

[0193] 43. Green TR, Fellman JH, Eicher AL, & Pratt KL (1991) Antioxidant role and subcellular location of hypotaurine and taurine in human neutrophils. Biochimica et Biophysica Acta 1073:91-97.

[0194] 44. Gurer H, Ozgiines H, Saygin E, & Ercal N (2001) Antioxidant effect of taurine against lead-induced oxidative stress. Archives of Environmental Contamination and Toxicology 41:397-402.

[0195] 45. Das J, Ghosh J, Manna P, & Sil PC (2008) Taurine provides antioxidant defense against NaF-induced cytotoxicity in murine hepatocytes. Pathophysiology 15:181-190.

[0196] 46. Zhang M, et al. (2004) Role of taurine supplementation to prevent exercise- induced oxidative stress in healthy young men. Amino Acids 26:203-207.

[0197] 47. Williams M (2005) Dietary supplements and sports performance: Amino acids. Journal of the International Society of Sports Nutrition 2:63-67.

[0198] 48. da Silva DLP, et al. (2008) Penetration profile of taurine in the human skin and its distribution in skin layers. Pharmaceutical Research 25:1846-1850.

[0199] 49. Suzuki A, Kajita T, & Furushima M (1989) 4877447.

[0200] 50. Honjoh KI, et al. (2010) Enhancement of menadione stress tolerance in yeast by accumulation of hypotaurine and taurine: co-expression of cDNA clones, from Cyprinus carpio, for cysteine dioxygenase and cysteine sulfinate decarboxylase in Saccharomyces cerevisiae. Amino Acids 38:1173-1183.

[0201] 51. Turano FJ, Turano KA, Carlson PS, & Kinnersley AM (2012) US Patent No. 9,267,148.

[0202] 52. Turano FJ, Price MB, & Turano KA (2014).US Patent No. 9,267,148.

[0203] 53. Tevatia R, et al. (2015) The taurine biosynthetic pathway of microalgae. Algal Research 9:21-26.

[0204] 54. Murray EE, Lotzer J, & Eberle M (1989) Codon usage in plant genes. Nucleic Acids Research 17:477-498.

[0205] 55. Langenheim JH & Thimann KV (1982) Botany: Plant Biology and its Relation to Human Affairs (John Wiley & Sons Inc., New York).

[0206] 56. Vasil IK (1984) Cell Culture and Somatic Cell Genetics of Plants: Laboraory Procedures and Their Applications (Academic Press, Orlando). [0207] 57. Stanier R, Ingrahm J, Wheelis M, & Painter P (1986) The Microbial World (Prentice-Hall, New Jersey) 5 Ed.

[0208] 58. Dhringra OD & Sinclair JB (1985) Basic plant pathology methods (CRC Press, Boca Raton, FL).

[0209] 59. Maniatis T, Fritsch EF, & Sambrook J (1985) Molecular Cloning: A Laboratory Manual: DNA Cloning (Cold Spring Harbor, New York).

[0210] 60. Gait (1984) Oligonucleotide Synthesis-A Practical Approach (IRL Press, Washington, D.C.).

[0211] 61. Hames DD & Higgins SJ (1984) Nucleic Acid Hybridization: A Practical Approach ( IRL Press, Washington D.C.).

[0212] 62. Watson JD, Gilman M, Witowski J, & Zoller M (1992) Recombinant DNA (Scientific American Books, New York).

[0213] 63. Szewczyk E, et al. (2006) Fusion PGR and gene targeting in Aspergillus nidulans. Nature Protocols 1:3111-3121.

[0214] 64. Ho SN, Hunt HD, Horton RM, Pullen JK, & Pease LR (1989) Site-directed mutagenesis by overlap extension using the polymerase chain reaction. Gene 77:51-59.

[0215] 65. Fuhrmann M, Oertel W, & Hegemann P (1999) A synthetic gene coding for the green fluorescent protein (GFP) is a versatile reporter in Chlamydomonas reinhardtii. Plant Journal 19:353-361.

[0216] 66. Mandecki W & Boiling TJ (1988) Fokl method of gene synthesis. Gene 68:101- 107.

[0217] 67. Stemmer WP, Crameri,A., Ha,K.D., Brennan,T.M. and Heyneker,H.L. (1995) Single-step assembly of a gene and entire plasmid from large numbers of oligodeoxyribonucleotides. Gene 164:49-53.

[0218] 68. Gao X, Yo P, Keith A, Ragan TJ, & Harris TK (2003) Thermodynamically balanced inside-out (TBIO) PCR-based gene synthesis: a novel method of primer design for high-fidelity assembly of longer gene sequences. Nucleic Acids Research 31:el43.

[0219] 69. Young L & Dong Q (2004) Two-step total gene synthesis method. Nucleic Acids Research 32:e59.

[0220] 70. Trinh R, Gurbaxani B, Morrison SL, & Seyfzadeh M (2004) Optimization of codon pair use within the (GGGGS)3 linker sequence results in enhanced protein expression. Molecular immunology 40(10):717-722.

[0221] 71. Chang TW & Yu L (1999) Genetic engineering. (Google Patents).

[0222] 72. Kuusinen A, Arvola M, & Keinanen K (1995) Molecular dissection of the agonist binding site of an AMPA receptor. EmboJ 14(24):6327-6332.

[0223] 73. Robinson CR & Sauer RT (1998) Optimizing the stability of single-chain proteins by linker length and composition mutagenesis. Proc Natl Acad Sci USA 95(11):5929- 5934.

[0224] 74. Armstrong N & Gouaux E (2000) Mechanisms for activation and antagonism of an AMPA-sensitive glutamate receptor: crystal structures of the GluR2 ligand binding core. Neuron 28(1):165-181. [0225] 75. Arai R, Ueda H, Kitayama A, Kamiya N, & Nagamune T (2001) Design of the linkers which effectively separate domains of a bifunctional fusion protein. Protein Eng 14(8):529-532.

[0226] 76. Wriggers W, Chakravarty S, & Jennings PA (2005) Control of protein functional dynamics by peptide linkers. Biopolymers 80(6):736-746.

[0227] 77. Reddy Chichili VP, Kumar V, & Sivaraman J (2013) Linkers in the structural biology of protein-protein interactions. Protein Sci 22(2): 153-167.

[0228] 78. van Der Krol AR, et al. (1999) Developmental and wound-, cold-, desiccation-, ultraviolet-B-stress-induced modulations in the expression of the petunia zinc finger transcription factor gene ZPT2-2. Plant Physiology 121(4):1153-1162.

[0229] 79. Shinmyo A, et al. (1998) Metabolic engineering of cultured tobacco cells. Biotechnology and Bioengineering 58(2-3):329-332.

[0230] 80. Sohal AK, Pallas JA, & Jenkins GI (1999) The promoter of a Brassica napus lipid transfer protein gene is active in a range of tissues and stimulated by light and viral infection in transgenic Arabidopsis. Plant Molecular Biology 41(l):75-87.

[0231] 81. Cormack RS, et al. (2002) Leucine zipper-containing WRKY proteins widen the spectrum of immediate early elicitor-induced WRKY transcription factors in parsley. Biochimica et Biophysica Acta 1576(l-2):92-100.

[0232] 82. Eulgem T, Rushton PJ, Schmelzer E, Hahlbrock K, & Somssich IE (1999) Early nuclear events in plant defence signalling: rapid gene activation by WRKY transcription factors. EMBO (European Molecular Biology Organization) Journal 18(17):4689-4699.

[0233] 83. Lebel E, et al. (1998) Functional analysis of regulatory sequences controlling PR- 1 gene expression in Arabidopsis. Plant Journal 16(2):223-233.

[0234] 84. Ngai N, Tsai FY, & Coruzzi G (1997) Light-induced transcriptional repression of the pea AS1 gene: identification of cis-elements and transfactors. Plant Journal 12(5):1021- 1034.

[0235] 85. Kucho K, Ohyama K, & Fukuzawa H (1999) CO(2)-responsive transcriptional regulation of CAHl encoding carbonic anhydrase is mediated by enhancer and silencer regions in Chlamydomonas reinhardtii. Plant Physiology 121(4): 1329-1338.

[0236] 86. Kucho K, Yoshioka S, Taniguchi F, Ohyama K, & Fukuzawa H (2003) Cis- acting elements and DNA-binding proteins involved in C02-responsive transcriptional activation of Cahl encoding a periplasmic carbonic anhydrase in Chlamydomonas reinhardtii. Plant Physiology 133(2):783-793.

[0237] 87. Chen W, Chao G, & Singh KB (1996) The promoter of a H202-inducible, Arabidopsis glutathione S-transferase gene contains closely linked OBF- and OBPl-binding sites. Plant Journal 10(6):955-966.

[0238] 88. Chen W & Singh KB (1999) The auxin, hydrogen peroxide and salicylic acid induced expression of the Arabidopsis GST6 promoter is mediated in part by an ocs element. Plant Journal 19(6):667-677.

[0239] 89. Lu CA, Lim EK, & Yu SM (1998) Sugar response sequence in the promoter of a rice alpha-amylase gene serves as a transcriptional enhancer. J Biol Chem 273(17):10120-10131.

[0240] 90. Leubner-Metzger G, Petruzzelli L, Waldvogel R, Vogeli-Lange R, & Meins F, Jr. (1998) Ethylene-responsive element binding protein (EREBP) expression and the transcriptional regulation of class I beta- 1 ,3 -glucanase during tobacco seed germination. Plant Molecular Biology 38(5):785-795.

[0241] 91. Kridl JQ et al. (1991) Isolation and characterization of an expressed napin gene from Brassica rapa. Seed Science Research 1(04):209-219.

[0242] 92. Zavallo D, Lopez Bilbao M, Hopp HE, & Heinz R (2010) Isolation and functional characterization of two novel seed-specific promoters from sunflower (Helianthus annuus L.). Plant cell reports 29(3):239-248.

[0243] 93. Kim MJ, et al. (2006) Seed-specific expression of sesame microsomal oleic acid desaturase is controlled by combinatorial properties between negative cis-regulatory elements in the SeFAD2 promoter and enhancers in the 5 -UTR intron. Molecular genetics and genomics : MGG 276(4):351-368.

[0244] 94. Bustos MM, et al. (1989) Regulation of beta-glucuronidase expression in transgenic tobacco plants by an A T-rich, cis-acting sequence found upstream of a French bean beta-phaseolin gene. Plant Cell l(9):839-853.

[0245] 95. Fuji war a T & Beachy RN (1994) Tissue-specific and temporal regulation of a beta-conglycinin gene: roles of the RY repeat and other cis-acting elements. Plant Mol Biol 24(2):261-272.

[0246] 96. Wienand U, Langridge P, & Feix G (1981) Isolation and characterization of a genomic sequence of maize coding for a zein gene. Molec. Gen. Genet. 182(3):440-444.

[0247] 97. Takaiwa F, Oono K, Wing D, & Kato A (1991) Sequence of three members and expression of a new major subfamily of glutelin genes from rice. Plant Molecular Biology 17(4):875-885.

[0248] 98. Hudspeth RL, Grula JW, Dai Z, Edwards GE, & Ku MS (1992) Expression of maize phosphoenolpyruvate carboxylase in transgenic tobacco : Effects on biochemistry and physiology. Plant Physiology 98(2):458-464.

[0249] 99. de Framond AJ (1991) A metallothionein-like gene from maize (Zea mays). Cloning and characterization. FEBS Letters 290(1-2): 103-106.

[0250] lOO.Hudspeth RL, Hobbs SL, Anderson DM, Rajasekaran K, & Grula JW (1996) Characterization and expression of metallothionein-like genes in cotton. Plant Molecular Biology 31(3):701-705.

[0251] lOl.Herrera-Estrella L, Depicker A, van Montagu M, & Schell J (1983) Expression of chimaeric genes transferred into plant cells using a Ti-plasmid-derived vector. Nature 303:209- 213.

[0252] 102.Pathirana MS, et al. (1997) Analyses of phosphoenolpyruvate carboxylase gene structure and expression in alfalfa nodules. Plant Journal 12(2):293-304.

[0253] 103. Yang SH, Yu H, & Goh CJ (2002) Isolation and characterization of the orchid cytokinin oxidase DSCKXl promoter. Journal of Experimental Botany 53:1899-1907.

[0254] 104. Moon H & Callahan AM (2004) Developmental regulation of peach ACC oxidase promoter-GUS fusions in transgenic tomato fruits. Journal of Experimental Botany 55:1519-1528.

[0255] 105.Avsian-Kretchmer O, Gueta-Dahan Y, Lev-Yadun S, Gollop R, & Ben-Hayyim G (2004) The salt-stress signal transduction pathway that activates the gpxl promoter is mediated by intracellular H202, different from the pathway induced by extracellular H202. Plant Physiology 135(3): 1685-1696.

[0256] 106. Wu A-M, Lv S-Y, & Liu J-Y (2007) Functional analysis of a cotton glucuronosyltransferase promoter in transgenic tobaccos. Cell Research 17:174—183.

[0257] 107.Hatzfeld Y (2014) US 8779237.

[0258] 108.Franklin S, Somanchi A, Espina K, Rudenko G, & Chua P (2014) US 8674180.

[0259] 109. Feng PCC, Malven M, & Flasinski S (2013) US 8420888.

[0260] HO.Manjunath S, et al. (2012) US 8138393.

[0261] lll.Lee DW, et al. (2008) Arabidopsis Nuclear-Encoded Plastid Transit Peptides Contain Multiple Sequence Subgroups with Distinctive Chloroplast-Targeting Sequence Motifs. The Plant Cell 20(6):1603-1622.

[0262] 112. von Heijne G, et al. (1991) CHLPEP: a database of chloroplast transit peptides. Plant Mol Biol Rep 9:104-126.

[0263] 113.An G, Watson BD, Stachel S, Gordon MP, & Nester EW (1985) New cloning vehicles for transformation of higher plants. EMBO (European Molecular Biology Organization) Journal 4:277-284.

[0264] 114.Gruber MY & Cosby WL (1993) Vectors for plant transformation. Methods in Plant Molecular Biology and Biotechnology, eds Glick BR & Thompson JE (CRC Press, Baco Raton, Fla.), pp 89-119.

[0265] 115.Ausubel FM, et al. (1995) Current Protocols in Molecular Biology (Greene Publishing and Wiley-Interscience, New York).

[0266] 116.Newman TC, Ohme-Takagi M, Taylor CB, & Green PJ (1993) DST sequences, highly conserved among plant SAUR genes, target reporter transcripts for rapid decay in tobacco. Plant Cell 5(6):701-714.

[0267] 117. Ohme-Takagi M, Taylor CB, Newman TC, & Green PJ (1993) The effect of sequences with high AU content on mRNA stability in tobacco. Proceedings of the National Academy of Sciences of the United States of America 90(24):11811-11815.

[0268] H8.Nagaya S, Kawamura K, Shinmyo A, & Kato K (2010) The HSP Terminator of Arabidopsis thaliana Increases Gene Expression in Plant Cells. Plant and Cell Physiology 51(2):328-332.

[0269] 119.Mogen BD, MacDonald MH, Graybosch R, & Hunt AG (1990) Upstream sequences other than AAUAAA are required for efficient messenger RNA 3 '-end formation in plants. Plant Cell 2(12):1261-1272.

[0270] 120.Mogen BD, MacDonald MH, Leggewie G, & Hunt AG (1992) Several distinct types of sequence elements are required for efficient mRNA 3' end formation in a pea rbcS gene. Mol Cell Biol 12(12):5406-5414.

[0271] 121.Rothnie HM, Reid J, & Hohn T (1994) The contribution of AAUAAA and the upstream element UUUGUA to the efficiency of mRNA 3 '-end formation in plants. Embo j

13(9):2200-2210.

[0272] 122.Bassett CL (2007) Regulation of Gene Expression in Plants: The Role of Transcript Structure and Processing (Springer Press, New York). [0273] 123. Gutierrez RA, Macintosh GC, & Green PJ (1999) Current perspectives on mRNA stability in plants: multiple levels and mechanisms of control. Trends in Plant Science 4:429- 438.

[0274] 124.1ngelbrecht IL, Herman LM, Dekeyser RA, Van Montagu MC, & Depicker AG (1989) Different 3' end regions strongly influence the level of gene expression in plant cells. Plant Cell 1:671-680.

[0275] 125. von Heijne G (1986) Mitochondrial targeting sequences may form amphiphilic helices. EMBO (European Molecular Biology Organization) Journal 5:1335-1342.

[0276] 126.Swinkels BW, Gould SJ, Bodnar AG, Rachubinski RA, & Subramani S (1991) A novel, cleavable peroxisomal targeting signal at the amino-terminus of the rat 3-ketoacyl-CoA thiolase. EMBO (European Molecular Biology Organization) Journal 10(11):3255-3262.

[0277] 127.Rusch SL & Kendall DA (1995) Protein transport via amino-terminal targeting sequences: Common themes in diverse systems. Molecular Membrane Biology 12(4):295-307.

[0278] 128. Soli J & Tien R (1998) Protein translocation into and across the chloroplastic envelope membranes. Plant Molecular Biology 38:191-207.

[0279] 129. Gould SJ, Keller GA, & Subramani S (1988) Identification of peroxisomal targeting signals located at the carboxy terminus of four peroxisomal proteins. Journal of Cell Biology 107(3):897-905.

[0280] 130.Gould SJ, Keller GA, Hosken N, Wilkinson J, & Subramani S (1989) A conserved tripeptide sorts proteins to peroxisomes. Journal of Cell Biology 108(5): 1657-1664.

[0281] Bl.McCammon MT, McNew J A, Willy PJ, & Goodman JM (1994) An internal region of the peroxisomal membrane protein PMP47 is essential for sorting to peroxisomes. Journal of Cell Biology 124(6):915-925.

[0282] 132.Cokol M, Nair R, & Rost B (2000) Finding nuclear localization signals. EMBO Reports 1(5):411-415.

[0283] 133.Helenius A & Aebi M (2001) Intracellular functions of N-linked glycans. Science 291(5512):2364-2369.

[0284] 134.Emanuelsson O, Brunak S, von Heijne G, & Nielsen H (2007) Locating proteins in the cell using TargetP, SignalP and related tools. Nature Protocols 2(4):953-971.

[0285] 135.Emanuelsson O, Nielsen H, Brunak S, & von Heijne G (2000) Predicting subcellular localization of proteins based on their N-terminal amino acid sequence. Journal of Molecular Biology 300(4): 1005-1016.

[0286] 136.Bannai H, Tamada Y, Maruyama O, Nakai K, & Miyano S (2002) Extensive feature detection of N-terminal protein sorting signals. Bioinformatics 18(2):298-305.

[0287] 137.Bendtsen JD, Nielsen H, von Heijne G, & Brunak S (2004) Improved prediction of signal peptides: SignalP 3.0. Journal of Molecular Biology 340(4):783-795.

[0288] 138.Hiller K, Grote A, Scheer M, Munch R, & Jahn D (2004) PrediSi: prediction of signal peptides and their cleavage positions. Nucleic Acids Research 32(Web Server issue):W375-379.

[0289] 139.Bhasin M & Raghava GP (2004) ESLpred: SVM-based method for subcellular localization of eukaryotic proteins using dipeptide composition and PSI-BLAST. Nucleic Acids Research 32(Web Server issue): W414-419. [0290] 140.Garg A, Bhasin M, & Raghava GP (2005) Support vector machine-based method for subcellular localization of human proteins using amino acid compositions, their order, and similarity search. Journal of Biological Chemistry 280(15): 14427-14432.

[0291] 141. Bhasin M, Garg A, & Raghava GP (2005) PSLpred: prediction of subcellular localization of bacterial proteins. Bioinformatics 21(10):2522-2524.

[0292] 142.Hoglund A, Donnes P, Blum T, Adolph HW, & Kohlbacher O (2006) MultiLoc: prediction of protein subcellular localization using N-terminal targeting sequences, sequence motifs and amino acid composition. Bioinformatics 22(10):1158-1165.

[0293] 143.Shatkay H, et al. (2007) SherLoc: high-accuracy prediction of protein subcellular localization by integrating text and protein sequence data. Bioinformatics 23(11):1410-1417.

[0294] 144.Emanuelsson O, Nielsen H, & von Heijne G (1999) ChloroP, a neural network- based method for predicting chloroplast transit peptides and their cleavage sites. Protein Science 8(5):978-984.

[0295] 145.Claros MG & Vincens P (1996) Computational method to predict mitochondrially imported proteins and their targeting sequences. European Journal of Biochemistry 241(3):779-786.

[0296] 146. Small I, Peeters N, Legeai F, & Lurin C (2004) Predotar: A tool for rapidly screening proteomes for N-terminal targeting sequences. Proteomics 4(6): 1581-1590.

[0297] 147.Kelley LA, acCallum RM, & Sternberg MJ (2000) Enhanced genome annotation using structural profiles in the program 3D-PSSM. Journal of Molecular Biology 299(2):499-520.

[0298] 148.Shahin EA (1985) Totipotency of tomato protoplasts. Theoretical and Applied Genetics 69:235-240.

[0299] 149.Fromm M, Taylor LP, & V. W (1985) Expression of genes transferred into monocot and dicot plant cells by electroporation. Proceedings of the National Academy of Sciences of the United States of America 82:5824-5828.

[0300] 150.Fromm ME, Taylor LP, & Walbot V (1986) Stable transformation of maize after gene transfer by electroporation. Nature 319(6056):791-793.

[0301] 151.Riggs CD & Bates GW (1986) Stable transformation of tobacco by electroporation: evidence for plasmid concatenation. Proceedings of the National Academy of Sciences of the United States of America 83(15):5602-5606.

[0302] 152.D'Halluin K, Bonne E, Bossut M, De Beuckeleer M, & Leemans J (1992) Transgenic maize plants by tissue electroporation. Plant Cell 4:1495-1505.

[0303] 153.Laursen CM, Krzyzek RA, Rick CE, Anderson PC, & Spencer TM (1994) Production of fertile transgenic maize by electroporation of suspension culture cells Plant Molecular Biology 24:51-61

[0304] 154.Crossway A, et al. (1986) Integration of foreign DNA following microinjection of tobacco mesophyll protoplasts. Molecular and General Genetics 202:179-185.

[0305] 155.Griesbach RJ (1983) Protoplast microinjection. Plant Molecular Biology Reporter 1:32-37.

[0306] 156.Sporlein B & Koop H-U (1991) Lipofectin: direct gene transfer to higher plants using cationic liposomes. Theoretical and Applied Genetics 83:1-5. [0307] 157,Ohgawara T, Uchimiya H, & Harada H (1983) Uptake of liposome-encapsulated plasmid DNA by plant protoplasts and molecular fate of foreign DNA Protoplasma 116:145- 148.

[0308] 158.Deshayes A, Herrera-Estrella L, & Caboche M (1985) Liposome-mediated transformation of tobacco mesophyll protoplasts by an Escherichia coli plasmid. EMBO (European Molecular Biology Organization) Journal 4(ll):2731-2737.

[0309] 159.Christou P, Murphy JE, & Swain WF (1987) Stable transformation of soybean by electroporation and root formation from transformed callus. Proceedings of the National Academy of Sciences of the United States of America 84(12):3962-3966.

[0310] 160.Horsch RB, et al. (1985) A Simple and General Method for Transferring Genes into Plants. Science 227:1229-1231.

[0311] 161.Paszkowski J, et al. (1984) Direct gene transfer to plants. Embo J 3(12):2717- 2722.

[0312] 162.Hooykaas-Van Slogteren GM, Hooykaas PJ, & Schilperoort RA (1984) Expression of Ti plasmid genes in monocotyledonous plants infected with Agrobactenum tumefaciens. Nature 311:763-764.

[0313] 163. Rogers SG, Horsch, R.B., and Fraley, R.T. 1986. Gene transfer in plants: Production of transformed plants using Ti-plasmid vectors. (1986) Gene transfer in plants: Production of transformed plants using Ti-plasmid vectors. Methods in Enzymology 118:627- 640.

[0314] 164.Bevan MW & Chilton M-D (1982) T-DNA of the Agrobacterium Ti and Ri plasmids. Annual Review of Genetics 16:357-384.

[0315] 165. Klein TM, et al. (1988) Transfer of foreign genes into intact maize cells with high-velocity microprojectiles. Proceedings of the National Academy of Sciences of the United States of America 85(12):4305-4309.

[0316] 166.Klein TM, Gradziel T, Fromm ME, & Sanford JC (1988) Factors influencing gene delivery into Zea mays cells by high-velocity microprojectiles. Biotechnology 6:559-563.

[0317] 167.McCabe DE, Swain WF, Martinell BJ, & Christou P (1988) Stable transformation of soybean (Glycine max) by particle acceleration. Biotechnology 6:923-926.

[0318] 168. Sanford JC, Smith FD, & Rushell JA (1993) Optimizing the biolistic process for different biological application. The Methods in Enzymology, ed Wu R (Academic Press, Orlando), Vol 217, pp 483-509.

[0319] 169.Freeman JP, et al. (1984) A Comparison of Methods for Plasmid Delivery into Plant Protoplasts. Plant and Cell Physiology 25:1353-1365.

[0320] 170. Frame BR, et al. (1994) Production of fertile transgenic maize plants by silicon carbide whisker-mediated transformation. Plant Journal 6:941-948.

[0321] 171. Thompson J A, Drayton P, Frame B, Wang K, & Dunwell JM (1995) Maize transformation utilizing silicon carbide whiskers: a review. Euphytica 85:75-80.

[0322] 172. Guo Y, Liang H, & Berns MW (1995) Laser-mediated gene transfer in rice. Physiologia Plantarum 93:19-24. [0323] 173.Badr YA, Kereim MA, Yehia MA, Fouad 00, & Bahieldin A (2005) Production of fertile transgenic wheat plants by laser micropuncture. Photochemical & Photobiological Sciences 4:803-807.

[0324] 174.Bao S, Thrall BD, & Miller DL (1997) Transfection of a reporter plasmid into cultured cells by sonoporation in vitro. Ultrasound in Medicine and Biology 23:953-959.

[0325] 175. Finer KR & Finer JJ (2000) Use of Agrobacterium expressing green fluorescent protein to evaluate colonization of sonication-assisted Agrobacterium-mediated transformation- treated soybean cotyledons. Letters in Applied Microbiology 30(5):406-410.

[0326] 176.Amoah BK, Wu H, Sparks C, & Jones HD (2001) Factors influencing Agrobacterium-mediated transient expression of uidA in wheat inflorescence tissue. Journal of Experimental Botany 52(358): 1135-1142.

[0327] 177.Krens FA, Molendijk L, Wullems GJ, & Schilperoort RA (1982) In Vitro transformation of plant protoplasts with Ti-plasmid DNA. Nature 296:72-74.

[0328] 178.Bechtold N & Pelletier G (1998) In planta Agrobacterium-mediated transformation of adult Arabidopsis thaliana plants by vacuum infiltration. Methods in Molecular Biology 82:259-266.

[0329] 179.Broothaerts W, et al (2005) Gene transfer to plants by diverse species of bacteria. Nature 433:629-633.

[0330] 180.Urnov FD, Rebar EJ, Holmes MC, Zhang HS, & Gregory PD (2010) Genome editing with engineered zinc finger nucleases. Nature reviews. Genetics 11(9):636-646.

[0331] 181.Weinthal D, Tovkach A, Zeevi V, & Tzfira T (2010) Genome editing in plant cells by zinc finger nucleases. Trends Plant Sci 15(6):308-321.

[0332] 182.Gaj T, Gersbach CA, & Barbas CF (2013) ZFN, TALEN and CRISPR/Cas-based methods for genome engineering. Trends in biotechnology 31(7):397-405.

[0333] 183.Sprink T, Metje J, & Hartung F (2015) Plant genome editing by novel tools: TALEN and other sequence specific nucleases. Current Opinion in Biotechnology 32:47-53.

[0334] 184.Bortesi L & Fischer R (2015) The CRISPR/Cas9 system for plant genome editing and beyond. Biotechnology Advances 33(l):41-52.

[0335] 185.Kumar V & Jain M (2015) The CRISPR-Cas system for plant genome editing: advances and opportunities. J Exp Bot 66(l):47-57.

[0336] 186.Kirk RE & Othmer DF (1993) Concise Encyclopedia of Chemical Technology (John Wiley & Sons,) 4th Ed pp 433-514

[0337] 187.Mosbach K, Birnbaum S, Hardy K, Davies J, & Bulow L (1983) Formation of proinsulin by immobilized Bacillus subtilis. Nature 302:543-545.

[0338] 188.Demain AL (2007) Reviews: The business of biotechnology. Industrial Biotechnology 3:269-283.

[0339] 189.Chan HW & Wells RD (1974) Structural uniqueness of lactose operator. Nature 252:205-209.

[0340] 190.Goeddel DV, et al. (1980) Synthesis of human fibroblast interferon by E. coli Nucleic Acids Research 8:4057-4074. [0341] 191. Marx CJ & Lidstrom ME (2001) Development of improved versatile broad-host- range vectors for use in methylotrophs and other Gram-negative bacteria. Microbiology 147:2065-2075.

[0342] 192.Atomi H, Imanaka T, & Fukui T (2012) Overview of the genetic tools in the Archaea. Frontiers in microbiology 3:337.

[0343] 193.Farkas JA, Picking JW, & Santangelo TJ (2013) Genetic techniques for the archaea. Anna Rev Genet 47:539-561.

[0344] 194.Rehnstam-Holm A-S & Godhe A (2003) Genetic engineering of algal species. Biotechnology, ed Doelle HW (UNESCO, Eolss Publishers, Oxford, UK).

[0345] 195. Rosa L, Galvan-Cejudo A, & Fernandez E eds (2007) Transgenic Microalgae as Green Cell Factories (Springer Science+Business Media, LLC, New York, NY), Vol 616.

[0346] 196.Leon R & Fernandez E (2007) Nuclear transformation of eukaryotic microalgae: historical overview, achievements and problems. AdvExp Med Biol 616:1-11.

[034η 197.Mikami K, Hirata R, Takahashi M, Uji T, & Saga N (2011) Transient Transformation of Red Algal Cells: Breakthrough Toward Genetic Transformation of Marine Crop Porphyra Species. Genetic Transformation, ed Alvarez M (InTech).

[0348] 198.Umen JG & Olson BJ (2012) Genomics of Volvocine Algae. Advances in botanical research 64:185-243.

[0349] 199.Liu L, et al. (2013) Development of a new method for genetic transformation of the green alga Chlorella ellipsoidea. Molecular biotechnology 54(2):211-219.

[0350] 200.Gimpel JA, Specht EA, Georgianna DR, & Mayfield SP (2013) Advances in microalgae engineering and synthetic biology applications for biofuel production. Current opinion in chemical biology 17(3):489-495.

[0351] 201.Rasala BA, Chao S-S, Pier M, Barrera DJ, & Mayfield SP (2014) Enhanced genetic tools for engineering multigene traits into green algae. PLoS ONE.

[0352] 202.Sherman F (1991) Getting started with yeast. Methods in Enzymology, Guide to Yeast Genetics and Molecular Biology, eds Guthrie C & Fink GR ( Acad. Press, New York), Vol 194, pp 3-21.

[0353] 203. Sherman F, Fink GR, & Hick JB (1982) Methods in Yeast Genetics (Cold Spring Harbor Laboratory, New York).

[0354] 204.Olmedo-Monfil V, CortEs-Penagos C, & Herrera-Estrella A (2004) Three Decades of Fungal Transformation.), Vol 267, pp 297-313.

[0355] 205.Weld RJ, Plummer KM, Carpenter MA, & Ridgway HJ (2006) Approaches to functional genomics in filamentous fungi. Cell Res 16(l):31-44.

[0356] 206.Kawai S, Hashimoto W, & Murata K (2010) Transformation of Saccharomyces cerevisiae and other fungi: Methods and possible underlying mechanism. Bioengineered Bugs l(6):395-403.

[0357] 207.van den Berg MA & Maruthachalam K eds (2015) Genetic Transformation Systems in Fungi, Volume 1 (Springer, New York, NY).

[0358] 208.Rivera AL, Magana-Ortiz D, Gomez-Lim M, Fernandez F, & Loske AM (2014) Physical methods for genetic transformation of fungi and yeast. Physics of life reviews ll(2):184-203. [0359] 209.van Oers MM, Pijlman GP, & Vlak JM (2015) Thirty years of baculovirus-insect cell protein expression: from dark horse to mainstream technology. Journal of General Virology 96(l):6-23.

[0360] 210.Almo SC & Love JD (2014) Better and faster: improvements and optimization for mammalian recombinant protein production. Current Opinion in Structural Biology 26:39- 43.

[0361] 211.Andersen L, Sundman L-O, Inge-Britt Linden I-B, Kontro P, & Simo SO (1984) Synthesis and anticonvulsant properties of some 2-Aminoethanesulfonic acid (Taurine) derivatives. Journal of Pharmaceutical Sciences 73:106-108.

[0362] 212.Herdeis C & Weis CE (1999) 5889183.

[0363] 213.Tserng K-Y, Hachey DL, & Klein PD (1977) An improved procedure for the synthesis of glycine and taurine conjugates of bile acids. Journal of Lipid Research 18:404-407.

[0364] 214.Fong DW & Hoots JE (1992) 5128419.

[0365] 215.Seeberger S, Griffin RJ, Hardcastle IR, & Golding BT (2007) A new strategy for the synthesis of taurine derivatives using the 'safety-catch' principle for the protection of sulfonic acids. Organic and Biomolecular Chemistry 5:132-138.

[0366] 216.Suzuki M, Nakajima Y, Sato T, Shirai H, & Hanabusa K (2006) Fabrication of Ti02 using L-lysine-based organogelators as organic templates: control of the nanostructures. Chemical Communications (4):377-379.

[0367] 217.Mikhalenko SA, Soloveva LI, & Lukyanets EA (2004) Phthalocyanines and related compounds: XXXVIII. Synthesis of symmetric taurine- and choline-substituted phthalocyanines. Russian Journal of General Chemistry 74:1775-1800.

[0368] 218.Capone R, Blake S, Restrepo MR, Yang J, & Mayer M (2007) Designing Nanosensors Based on Charged Derivatives of Gramicidin A. Journal of the American Chemical Society 129:9737-9745.

[0369] 219.Gupta RC, Win T, & Bittner S (2005) Taurine analogues; A new class of therapeutics: Retrospect and prospects Current Medicinal Chemistry 12:2021-2039.

[0370] 220.Johnson BA (2008) Update on neuropharmacological treatments for alcoholism: Scientific basis and clinical findings. Biochemical Pharmacology 75:34-56.

[0371] 221.Tambour S & Quertemont E (2007) Preclinical and clinical pharmacology of alcohol dependence. Fundamental and Clinical Pharmacology 21:9-28.

[0372] 222.Joung YK, Sengoku Y, Ooya T, Park KD, & Yui N (2005) Anticoagulant supramolecular-structured polymers: Synthesis and anticoagulant activity of taurine-conjugated carboxyethylester-polyrotaxanes. Science and Technology of Advanced Materials 6:484-490.

[0373] 223.0zmeric, N, et al. (2000) Chitosan film enriched with an antioxidant agent, taurine, in fenestration defects. Journal of Biomedical Materials Research Part A 51:500 - 503.

[0374] 224.Degim Z, et al. (2002) An investigation on skin wound healing in mice with a taurinechitosan gel formulation. Amino Acids 22:187-198.

[0375] 225.Matsusaki M, Serizawa T, Kishida A, Endo T, & Akashi M (2002) Novel functional biodegradable polymer: Synthesis and anticoagulant activity of poly(y-Glutamic Acid)sulfonate (γ-PGA-sulfonate). Bioconjugate Chemistry 13:23-28. [0376] 226.Roubos JA, van Straten G, & van Boxtel AJB (1999) An evolutionary strategy for fed-batch bioreactor optimization; concepts and performance. Journal of biotechnology 67(2-3):173-187.

[0377] 227. Oka T (1999) Amino acids, production processes. Encyclopedia of Bioprocess Technology: Fermentation, Biocatalysis, and Bioseparation, eds Flickinger MC & Drew SW (Wiley, London).

[0378] 228.Borowitzka MA (1999) Commercial production of microalgae: ponds, tanks, tubes and fermenters. Journal of biotechnology 70(1-3):313-321.

[0379] 229. Hermann T (2003) Industrial production of amino acids by coryneform bacteria. JBiotechnol 104(1-3): 155-172.

[0380] 230.1keda M (2003) Amino acid production processes. Advances in biochemical engineering/biotechnology 79: 1-35.

[0381] 231. Richmond A & Hu Q eds (2013) Handbook of Microalgal Culture: Biotechnology and Applied Phycology (Wiley-Blackwell, Hoboken, New Jersey), 2nd Ed.

[0382] 232.1keda M (2005) Towards bacterial strains overproducing 1-tryptophan and other aromatics by metabolic engineering. Appl Microbiol Biotechnol 69(6):615-626.

[0383] 233.Cardozo KH, et al. (2007) Metabolites from algae with economical impact. Comparative biochemistry and physiology. Toxicology & pharmacology : CBP 146(l-2):60-78.

[0384] 234.Demain AL (2007) The business of biotechnology. Industrial Biotechnology 3:269-283.

[0385] 235.Milledge JJ (2011) Commercial application of microalgae other than as biofuels: a brief review. Reviews in Environmental Science and Biotechnology 10:31-41.

[0386] 236.Xu Q, Li S, Huang H, & Wen J (2012) Key technologies for the industrial production of fumaric acid by fermentation. Biotechnology advances 30(6): 1685-1696.

[0387] 237.Dufosse L, Fouillaud M, Caro Y, Mapari SAS, & Sutthiwong N (2014) Filamentous fungi are large-scale producers of pigments and colorants for the food industry. Current Opinion in Biotechnology 26:56-61.

[0388] 238.Hofler A, et al. (1998) US 5840358

[0389] 239.Lee I, Lee K, Namgoong K, & Lee Y-S (2002) The use of ion exclusion chromatography as approved to the normal ion exchange chromatography to achieve a more efficient lysine recovery from fermentation broth. Enzyme and Microbial Technology 30(6):798- 803.

[0390] 240.Binder M & Uffmann K-E (2002) US 6,465,025.

[0391] 241.Hermann T (2003) Industrial production of amino acids by coryneform bacteria. Journal of biotechnology 104(1-3): 155-172.

[0392] 242.Leuchtenberger W, Huthmacher K, & Drauz K (2005) Biotechnological production of amino acids and derivatives: current status and prospects. Appl Microbiol Biotechnol 69(1): 1-8.

[0393] 243.Meinkoth J & G. W (1984) Hybridization of nucleic acids immobilized on solid supports. Analytical Biochemistry 138:267-284. [0394] 244.Tijssen P (1993) Overview of principles of hybridization and the strategy of nucleic acid probe assays. Laboratory Techniques in Biochemistry and Molecular Biology - Hybridization with Nucleic Acid Probes: Part I, (Elsevier, New York).

[0395] 245. Smith TF & Waterman MS (1981) Comparison of biosequences. Advances in Applied Mathematics 2:482-489.

[0396] 246.Needleman SB & Wunsch CD (1970) A general method applicable to the search for similarities in the amino acid sequence of two proteins. Journal of Molecular Biology 48:443-453.

[0397] 247.Pearson WR & Lipman DJ (1988) Improved tools for biological sequence comparison. Proceedings of the National Academy of Sciences of the United States of America

85:2444-2448.

[0398] 248.Higgins DG & Sharp PM (1989) Fast and sensitive multiple sequence alignments on a microcomputer. Computer Applications in the Biosciences 5(2):151-153.

[0399] 249.Higgins DG, Bleasby AJ, & Fuchs R (1992) CLUSTAL V: improved software for multiple sequence alignment. Computer Applications in the Biosciences 8(2): 189-191.

[0400] 250.Higgins DG & Sharp PM (1988) CLUSTAL: a package for performing multiple sequence alignment on a microcomputer. Gene 73(l):237-244.

[0401] 251. Feng DF & Doolittle RF (1987) Progressive sequence alignment as a prerequisite to correct phylogenetic trees. Journal of Molecular Evolution 25(4):351-360.

[0402] 252.Henikoff S & Henikoff J (1989) Amino acid substitution matrices from protein blocks Proceedings of the National Academy of Sciences of the United States of America 89:10915-10919.

[0403] 253.Altschul SF, et al. (1997) Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Research 25:3389-3402.

[0404] 254.Wootton JC & Federhen S (1993) Statistics of local complexity in amino acid sequences and sequence databases. Computational Chemistry 17:149-163.

[0405] 255.Wootton JC & Federhen S (1996) Analysis of compositionally biased regions in sequence databases. Methods Enzymol 266:554-571.

[0406] 256.Claverie J-M & States DJ (1993) Information enhancement methods for large scale sequence analysis. Computational Chemistry 17:191-201.

[0407] 257. Myers EW & Miller W (1988) Optimal alignments in linear-space. Computer Applications in the Biological Sciences 4:11-17.

[0408] All patents, patent applications, and references cited in this disclosure are expressly incorporated herein by reference. The above disclosure generally describes the present invention. A more complete understanding can be obtained by reference to the following specific examples, which are provided for purposes of illustration only and are not intended to limit the scope of the invention. EXAMPLE 1

Development of a transgenic plant that constitutively expresses CDOL with the native transit peptide and constitutively expresses SADL with the native transit peptide in tandem with independent promoters

[0409] Step 1: Use chemical synthesis to make a DNA construct that contains a constitutive promoter, 35S, fused with the nucleotide sequence for CDOL gene (SEQ ID NO:l, SEQ ID NO:2, SEQ ID NO:3, or SEQ ID NO:4) and a NOS terminator. Clone the DNA construct into a binary vector, such as pCambial300, pCambia2300, or pCambia3200.

[0410] The CDOL gene is as follows:

[0411] a. Derived from SEQ ID NO:l, optimized for expression in Arabidopsis or soybean (dicots) or corn (a monocot), and encoding a CDOL peptide from Chlamydomonas reinhardtii (SEQ ID NO:5); or

[0412] b. Derived from SEQ ID NO: 2, optimized for expression in Arabidopsis or soybean (dicots) or corn (a monocot), and encoding a CDOL peptide from Guillardia theta (SEQ ID NO:6); or

[0413] c. Derived from SEQ ID NO:3, optimized for expression in Arabidopsis or soybean (dicots) or corn (a monocot), and encoding a CDOL peptide from Candida tenuis (SEQ ID NO:7); or

[0414] d. Derived from SEQ ID NO:4, optimized for expression in Arabidopsis or soybean (dicots) or corn (a monocot), and encoding a CDOL peptide from Fragilariopsis cylindrus (SEQ ID NO:8).

[0415] Step 2: Use chemical synthesis to make a DNA construct that contains a constitutive promoter, 35S, fused with the nucleotide sequence for SADL (SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, or SEQ ID NO: 14) and a NOS terminator. Clone the SADL DNA construct into a binary vector that contains the CDOL DNA construct (Stepl).

[0416] The SADL gene is as follows:

[0417] a. Derived from SEQ ID NO: 11 optimized for expression in Arabidopsis or soybean (dicots) or corn (a monocot), and encoding a SADL peptide from Guillardia theta (SEQ ID NO: 17); or

[0418] b. Derived from SEQ ID NO: 12, optimized for expression in Arabidopsis or soybean (dicots) or corn (a monocot), and encoding a SADL peptide from Candida tenuis (SEQ ID NO: 18); or [0419] c. Derived from SEQ ID NO: 13 optimized for expression in Arabidopsis or soybean (dicots) or corn (a monocot), and encoding a SADL peptide from Cyanidioschyzon merolae (SEQ ID NO: 19); or

[0420] d. Derived from SEQ ID NO: 14, optimized for expression in Arabidopsis or soybean (dicots) or corn (a monocot), and encoding a SADL peptide from Thalassiosira oceanica (SEQ ID NO:20).

[0421] Step 3: Transform the DNA construct into Agrobacterium tumefaciens, select for antibiotic resistance, and confirm the presence of the DNA construct.

[0422] Step 4: Transform plant (Arabidopsis, soybean, corn, wheat, sugar beet, rice, camelina or canola), select for antibiotic resistance, and confirm the presence of the DNA constructs in the transgenic plant.

EXAMPLE 2

Development of a transgenic plant that constitutively expresses CDOL with a plant plastid transit peptide and constitutively expresses SADL protein with a plant plastid transit peptide in tandem with independent promoters

[0423] Step 1: Use chemical synthesis to make a DNA construct that contains a constitutive promoter, 35S, fused with the nucleotide sequence for a plastid transit peptide (SEQ ID NO:21), truncated CDOL gene (SEQ ID NO:l, SEQ ID NO:2, SEQ ID NO:3, or SEQ ID NO:4) and a NOS terminator. Clone the DNA construct into a binary vector, such as pCambial300, pCambia2300, or pCambia3200.

[0424] The nucleotide sequence for the plastid transit peptide (SEQ ID NO:21) encodes the peptide SEQ ID NO:22.

[0425] The CDOL gene is as follows:

[0426] a. Derived from SEQ ID NO.l by removing nucleotides 1 through 159 (corresponding to the native transit peptide), optimized for expression in Arabidopsis or soybean (dicots) or corn (a monocot), and encoding a CDOL peptide from Chlamydomonas reinhardtii (SEQ ID NO:5 minus amino acids 1 through 53); or

[0427] b. Derived from SEQ ID NO:2 by removing nucleotides 1 through 279 (corresponding to the native transit peptide), optimized for expression in Arabidopsis or soybean (dicots) or corn (a monocot), and encoding a CDOL peptide from Guillardia theta (SEQ ID NO:6 minus amino acids 1 through 93); or

[0428] c. Derived from SEQ ID NO:3 by removing nucleotides 1 through 126 (corresponding to the native transit peptide), optimized for expression m Arabidopsis or soybean (dicots) or corn (a monocot), and encoding a CDOL peptide from Candida tenuis (SEQ ID NO:7 minus amino acids 1 through 42); or

[0429] d. Derived from SEQ ID NO:4 by removing nucleotides 1 through 180 (corresponding to the native transit peptide), optimized for expression in Arabidopsis or soybean (dicots) or corn (a monocot), and encoding a CDOL peptide from Fragilariopsis cylindrus (SEQ ID NO:8 minus amino acids 1 through 60).

[0430] Step 2: Use chemical synthesis to make a DNA construct that contains a constitutive promoter, 35S, fused with the nucleotide sequence for a plastid transit peptide (SEQ ID NO: 21), truncated SADL (SEQ ID NO:ll, SEQ ID NO: 12, SEQ ID NO:13, or SEQ ID NO: 14) and a NOS terminator. Clone the SADL DNA construct into a binary vector that contains the CDOL DNA construct (Stepl).

[0431] The nucleotide sequence for the plastid transit peptide (SEQ ID NO: 21) encodes the peptide SEQ ID NO: 22. Clone the SADL DNA construct into a binary vector that contains the

CDOL DNA construct (Stepl).

[0432] The SADL gene is as follows:

[0433] a. Derived from SEQ ID NO: 11, by removing nucleotides 1 through 54, (corresponding to the native transit peptide) optimized for expression in Arabidopsis or soybean (dicots) or corn (a monocot), and encoding a truncated SADL peptide from Guillardia theta (SEQ ID NO: 17 minus amino acids 1 through 18); or

[0434] b. Derived from SEQ ID NO: 12, by removing nucleotides 1 through 33 (corresponding to the native transit peptide), optimized for expression in Arabidopsis or soybean (dicots) or corn (a monocot), and encoding a truncated SADL peptide from Candida tenuis (SEQ ID NO:18 minus amino acids 1 through 11); or

[0435] c. Derived from SEQ ID NO: 13, by removing nucleotides 1 through 12, (corresponding to the native transit peptide) optimized for expression in Arabidopsis or soybean (dicots) or corn (a monocot), and encoding a truncated SADL peptide from Cyanidioschyzon merolae (SEQ ID NO: 19 minus amino acids 1 through 4); or

[0436] d. Derived from SEQ ID NO: 14, by removing nucleotides 1 through 18 (corresponding to the native transit peptide), optimized for expression in Arabidopsis or soybean (dicots) or corn (a monocot), and encoding a truncated SADL peptide from Thalassiosira oceanica (SEQ ID NO: 20 minus amino acids 1 through 6).

[0437] Step 3: Transform the DNA construct into Agrobacterium tumefaciens, select for antibiotic resistance, and confirm the presence of the DNA construct. [0438] Step 4: Transform plant (Arabidopsis, soybean, corn, wheat, sugar beet, rice, camelina or canola), select for antibiotic resistance, and confirm the presence of the DNA constructs in the transgenic plant.

EXAMPLE 3

Development of a transgenic plant that constitutively expresses CDOL with the native plastid transit peptide and constitutively expresses CS/PLP-DC with the native transit peptide in tandem with independent promotes

[0439] Step 1: Use chemical synthesis to make a DNA construct that contains a constitutive promoter, 35S, fused with the nucleotide sequence for CDOL gene (SEQ ID NO:l, SEQ ID NO:2, SEQ ID NO:3, or SEQ ID NO:4) and a NOS terminator. Clone the DNA construct into a binary vector, such as pCambial300, pCambia2300, or pCambia3200.

[0440] The CDOL gene is as follows:

[0441] a. Derived from SEQ ID NO:l, optimized for expression in Arabidopsis or soybean (dicots) or corn (a monocot), and encoding a CDOL peptide from Chlamydomonas reinhardtii (SEQ ID NO:5); or

[0442] b. Derived from SEQ ID NO:2, optimized for expression in Arabidopsis or soybean (dicots) or corn (a monocot), and encoding a CDOL peptide from Guillardia theta (SEQ ID NO:6); or

[0443] c. Derived from SEQ ID NO:3, optimized for expression in Arabidopsis or soybean (dicots) or corn (a monocot), and encoding a CDOL peptide from Candida tenuis (SEQ ID NO:7); or

[0444] d. Derived from SEQ ID NO:4, optimized for expression in Arabidopsis or soybean (dicots) or corn (a monocot), and encoding a CDOL peptide from Fragilariopsis cylindrus (SEQ ID NO:8).

[0445] Step 2: Use chemical synthesis to make a DNA construct that contains a constitutive promoter, 35S, fused with the nucleotide sequence for CS/PLP-DC (SEQ ID NO:9 or SEQ ID NO: 10) and a NOS terminator. Clone the CS/PLP-DC DNA construct into a binary vector that contains the CDOL DNA construct (Stepl).

[0446] The CS/PLP-DC gene is as follows:

[0447] a. Derived from SEQ ID NO: 9 optimized for expression in Arabidopsis or soybean (dicots) or corn (a monocot), and encoding CS/PLP-DC peptide from Micromonas pusilla (SEQ ID NO: 15; or [0448] b. Derived from SEQ ID NO: 10, optimized for expression in Arabidopsis or soybean (dicots) or corn (a monocot), and encoding CS/PLP-DC peptide from Ostreococcus tauri (SEQ ID NO: 16).

[0449] Step 3: Transform the DNA construct into Agrobacterium t mefaciens, select for antibiotic resistance, and confirm the presence of the DNA construct.

[0450] Step 4: Transform plant (Arabidopsis, soybean, corn, wheat, sugar beet, rice, camelina or canola), select for antibiotic resistance, and confirm the presence of the DNA constructs in the transgenic plant.

EXAMPLE 4

Development of a transgenic plant that constitutively expresses CDOL with a plant plastid transit peptide and constitutively expresses CS/PLP-DC with a plant plastid transit peptide in tandem with independent promoters

[0451] Step 1: Use chemical synthesis to make a DNA construct that contains a constitutive promoter, 35S, fused with the nucleotide sequence for a plastid transit peptide (SEQ ID NO:21), truncated CDOL gene (SEQ ID NO:l, SEQ ID NO:2, SEQ ID NO:3, or SEQ ID NO:4) and a NOS terminator. Clone the DNA construct into a binary vector, such as pCambial300, pCambia2300 or pCambia3200.

[0452] The nucleotide sequence for the plastid transit peptide (SEQ ID NO:21) encodes the peptide SEQ ID NO:22.

[0453] The CDOL gene is as follows:

[0454] a. Derived from SEQ ID NO:l by removing nucleotides 1 through 159 (corresponding to the native transit peptide), optimized for expression in Arabidopsis or soybean (dicots) or corn (a monocot), and encoding a CDOL peptide from Chlamydomonas reinhardtii (SEQ ID NO:5 minus amino acids 1 through 53); or

[0455] b. Derived from SEQ ID NO:2 by removing nucleotides 1 through 279 (corresponding to the native transit peptide), optimized for expression in Arabidopsis or soybean (dicots) or corn (a monocot), and encoding a CDOL peptide from Guillardia theta (SEQ ID NO:6 minus amino acids 1 through 93); or

[0456] c. Derived from SEQ ID NO:3 by removing nucleotides 1 through 126 (corresponding to the native transit peptide), optimized for expression in Arabidopsis or soybean (dicots) or corn (a monocot), and encoding a CDOL peptide from Candida tenuis (SEQ ID NO:7 minus amino acids 1 through 42); or [0457] d. Derived from SEQ ID NO:4 by removing nucleotides 1 through 180 (corresponding to the native transit peptide), optimized for expression in Arabidopsis or soybean (dicots) or corn (a monocot), and encoding a CDOL peptide from Fragilariopsis cylindrus (SEQ ID NO:8 minus amino acids 1 through 60).

[0458] Step 2: Use chemical synthesis to make a DNA construct that contains a constitutive promoter, 35S, fused with the nucleotide sequence for a plastid transit peptide (SEQ ID NO: 21), CS/PLP-DC (SEQ ID NO:9 or SEQ ID NO: 10) and a NOS terminator. The nucleotide sequence for the plastid transit peptide (SEQ ID NO: 21) encodes the peptide SEQ ID NO: 22. Clone the CS/PLP-DC DNA construct into a binary vector that contains the CDOL DNA construct (Stepl).

[0459] The CS/PLP-DC gene is as follows:

[0460] a. Derived from SEQ ID NO:9, by removing nucleotides 1 through 234, (corresponding to the native transit peptide) optimized for expression in Arabidopsis or soybean (dicots) or corn (a monocot), and encoding a CS/PLP-DC peptide from Micromonas pusilla (SEQ ID NO: 15 minus amino acids 1 through 78); or

[0461] b. Derived from SEQ ID NO: 10, by removing nucleotides 1 through 69 (corresponding to the native transit peptide), optimized for expression in Arabidopsis or soybean (dicots) or corn (a monocot), and encoding a CS/PLP-DC peptide from Ostreococcus tauri (SEQ ID NO: 16 minus amino acids 1 through 23).

[0462] Step 3: Transform the DNA construct into Agrobacterium tumefaciens, select for antibiotic resistance, and confirm the presence of the DNA construct.

[0463] Step 4: Transform plant (Arabidopsis, soybean, corn, wheat, sugar beet, rice, camelina or canola), select for antibiotic resistance, and confirm the presence of the DNA constructs in the transgenic plant.

EXAMPLE 5

Development of a transgenic plant that constitutively expresses CDOL with the native plastid transit peptide and constitutively expresses partCS/PLP-DC with a plant plastid transit peptide in tandem with independent promotes

[0464] Step 1: Use chemical synthesis to make a DNA construct that contains a constitutive promoter, 35S, fused with the nucleotide sequence for CDOL gene (SEQ ID NO:l, SEQ ID NO:2, SEQ ID NO:3, or SEQ ID NO:4) and a NOS terminator. Clone the DNA construct into a binary vector, such as pCambial300, pCambia2300, or pCambia3200. [0465] The CDOL gene is as follows:

[0466] a. Derived from SEQ ID NO:l, optimized for expression in Arabidopsis or soybean (dicots) or corn (a monocot), and encoding a CDOL peptide from Chlamydomonas reinhardtii (SEQ ID NO:5); or

[0467] b. Derived from SEQ ID NO:2, optimized for expression in Arabidopsis or soybean (dicots) or corn (a monocot), and encoding a CDOL peptide from Guillardia theta (SEQ ID NO:6); or

[0468] c. Derived from SEQ ID NO:3, optimized for expression in Arabidopsis or soybean (dicots) or corn (a monocot), and encoding a CDOL peptide from Candida tenuis (SEQ ID NO:7); or

[0469] d. Derived from SEQ ID NO:4, optimized for expression in Arabidopsis or soybean (dicots) or corn (a monocot), and encoding a CDOL peptide from Fragilariopsis cylindrus (SEQ ID NO:8).

[0470] Step 2: Use chemical synthesis to make a DNA construct that contains a constitutive promoter, 35S, fused with the nucleotide sequence for a plastid transit peptide (SEQ ID NO: 21), CS/PLP-DC (SEQ ID NO:9 or SEQ ID NO: 10) and a NOS terminator. The nucleotide sequence for the plastid transit peptide (SEQ ID NO: 21) encodes the peptide SEQ ID NO: 22. Clone the partCPLP-DC DNA construct into a binary vector that contains the CDOL DNA construct (Stepl).

[0471] The partCS/PLP-DC gene is as follows:

[0472] a. Derived from SEQ ID NO:9, by removing nucleotides 1 through 1413, (corresponding to the native transit and cysteine synthetase peptides) optimized for expression in Arabidopsis or soybean (dicots) or corn (a monocot), and encoding a partCS/PLP-DC peptide from Micromonas pusilla (SEQ ID NO: 15 minus amino acids 1 through 471); or

[0473] b. Derived from SEQ ID NO: 10, by removing nucleotides 1 through 1068 (corresponding to the native transit peptide and cysteine synthetase peptides), optimized for expression in Arabidopsis or soybean (dicots) or corn (a monocot), and encoding a partCS/PLP-DC peptide from Ostreococcus tauri (SEQ ID NO: 16 minus amino acids 1 through 356).

[0474] Step 3: Transform the DNA construct into Agrobacterium tumefaciens, select for antibiotic resistance, and confirm the presence of the DNA construct. Step 4: Transform plant (Arabidopsis, soybean, corn, wheat, sugar beet, rice, camelina or canola), select for antibiotic resistance, and confirm the presence of the DNA constructs in the transgenic plant.

EXAMPLE 6

Development of a transgenic plant that constitutively expresses CDOL with a plant plastid transit peptide and constitutively expresses partCS/PLP-DC protein with a plant plastid transit peptide in tandem with independent promoters

[0475] Step 1: Use chemical synthesis to make a DNA construct that contains a constitutive promoter, 35S, fused with the nucleotide sequence for a plastid transit peptide (SEQ ID NO:21), truncated CDOL gene (SEQ ID NO:l, or SEQ ID NO:2, SEQ ID NO:3 or SEQ ID NO:4) and a NOS terminator. Clone the DNA construct into a binary vector, such as pCambial300, pCambia2300 or pCambia3200.

[0476] The nucleotide sequence for the plastid transit peptide (SEQ ID NO:21) encodes the peptide SEQ ID NO:22.

[0477] The CDOL gene is as follows:

[0478] a. Derived from SEQ ID NO:l by removing nucleotides 1 through 159 (corresponding to the native transit peptide), optimized for expression in Arabidopsis or soybean (dicots) or corn (a monocot), and encoding a CDOL peptide from Chlamydomonas reinhardtii (SEQ ID NO:5 minus amino acids 1 through 53); or

[0479] b. Derived from SEQ ID NO:2 by removing nucleotides 1 through 279 (corresponding to the native transit peptide), optimized for expression in Arabidopsis or soybean (dicots) or corn (a monocot), and encoding a CDOL peptide from Guillardia theta (SEQ ID NO:6 minus amino acids 1 through 93); or

[0480] c. Derived from SEQ ID NO:3 by removing nucleotides 1 through 126 (corresponding to the native transit peptide), optimized for expression in Arabidopsis or soybean (dicots) or corn (a monocot), and encoding a CDOL peptide from Candida tenuis (SEQ ID NO:7 minus amino acids 1 through 42); or

[0481] d. Derived from SEQ ID NO:4 by removing nucleotides 1 through 180 (corresponding to the native transit peptide), optimized for expression in Arabidopsis or soybean (dicots) or corn (a monocot), and encoding a CDOL peptide from Fragilariopsis cylindrus (SEQ ID NO:8 minus amino acids 1 through 60). [0482] Step 2: Use chemical synthesis to make a DNA construct that contains a constitutive promoter, 35S, fused with the nucleotide sequence for a plastid transit peptide (SEQ ID NO: 21), partCS/PLP-DC (SEQ ID NO:9 or SEQ ID NO: 10) and a NOS terminator. The nucleotide sequence for the plastid transit peptide (SEQ ID NO: 21) encodes the peptide SEQ ID NO: 22. Clone the partCPLP-DC DNA construct into a binary vector that contains the CDOL DNA construct (Stepl).

[0483] The partCS/PLP-DC gene is as follows:

[0484] a. Derived from SEQ ID NO:9, by removing nucleotides 1 through 1413, (corresponding to the native transit and cysteine synthetase peptides ) optimized for expression in Arabidopsis or soybean (dicots) or corn (a monocot), and encoding a partCS/PLP-DC peptide from Micromonas pusilla (SEQ ID NO: 15 minus amino acids 1 through 471); or

[0485] b. Derived from SEQ ID NO: 10 by removing nucleotides 1 through 1068 (corresponding to the native transit and cysteine synthetase peptides), optimized for expression in Arabidopsis or soybean (dicots) or corn (a monocot), and encoding a partCS/PLP-DC peptide from Ostreococcus tauri (SEQ ED NO: 16 minus amino acids 1 through 356).

[0486] Step 3: Transform the DNA construct into Agrobacterium tumefaciens, select for antibiotic resistance, and confirm the presence of the DNA construct.

[0487] Step 4: Transform plant (Arabidopsis, soybean, corn, wheat, sugar beet, rice, camelina or canola), select for antibiotic resistance, and confirm the presence of the DNA constructs in the transgenic plant.

EXAMPLE 8

Development of a transgenic plant that constitutively expresses CS/PLP-DC protein with the native transit peptide

[0488] Step 1: Use chemical synthesis to make a DNA construct that contains a constitutive promoter, 35S, fused with the nucleotide sequence for CS/PLP-DC (SEQ ID NO:9 or SEQ ID NO: 10) and a NOS terminator. Clone the DNA construct into a binary vector, such as pCambial300, pCambia2300, or pCambia3200.

[0489] The CS/PLP-DC gene is as follows: [0490] a. Derived from SEQ ID NO:9 optimized for expression in Arabidopsis or soybean (dicots) or corn (a monocot), and encoding CS/PLP-DC peptide from Micromonas pusilla (SEQ ID NO: 15; or

[0491] b. Derived from SEQ ID NO: 10, optimized for expression in Arabidopsis or soybean (dicots) or corn (a monocot), and encoding CS/PLP-DC peptide from Ostreococcus tauri (SEQ ID NO: 16).

[0492] Step 2: Transform the DNA construct into Agrobacterium tumefaciens, select for antibiotic resistance, and confirm the presence of the DNA construct.

[0493] Step 4: Transform plant (Arabidopsis, soybean, corn, wheat, sugar beet, rice, camelina or canola), select for antibiotic resistance, and confirm the presence of the DNA constructs in the transgenic plant.

EXAMPLE 8

Development of a transgenic plant that constitutively CS/PLP-DC protein with a plant plastid transit peptide

[0494] Step 1: Use chemical synthesis to make a DNA construct that contains a constitutive promoter, 35S, fused with the nucleotide sequence for a plastid transit peptide (SEQ ID NO: 21),

CS/PLP-DC (SEQ ID NO:9 or SEQ ID NO: 10) and a NOS terminator. Clone the DNA construct into a binary vector, such as pCambial300, pCambia2300, or pCambia3200.

[0495] The nucleotide sequence for the plastid transit peptide (SEQ ID NO:21) encodes the peptide SEQ ID NO:22.

[0496] The CS/PLP-DC gene is as follows:

[0497] a. Derived from SEQ ID NO:9, by removing nucleotides 1 through 234, (corresponding to the native transit peptide) optimized for expression in Arabidopsis or soybean (dicots) or corn (a monocot), and encoding a CS/PLP-DC peptide from Micromonas pusilla (SEQ ID NO: 15 minus amino acids 1 through 78); or

[0498] b. Derived from SEQ ID NO: 10, by removing nucleotides 1 through 69 (corresponding to the native transit peptide), optimized for expression in Arabidopsis or soybean (dicots) or corn (a monocot), and encoding a CS/PLP-DC peptide from Ostreococcus tauri (SEQ ID NO: 16 minus amino acids 1 through 23).

[0499] Step 2: Transform the DNA construct into Agrobacterium tumefaciens, select for antibiotic resistance, and confirm the presence of the DNA construct. [0500] Step 3: Transform plant (Arabidopsis, soybean, corn, wheat, sugar beet, rice, camelina or canola), select for antibiotic resistance, and confirm the presence of the DNA constructs in the transgenic plant.

EXAMPLE 9

Development of a transgenic plant that constitutively expresses CDOL with the native transit peptide fused (without a linker) to a SADL using chemical synthesis

[0501] Step 1: Use chemical synthesis to make a DNA construct that contains a constitutive promoter, 35S, fused with nucleotide sequence for a CDOL gene (SEQ ID NO:l, SEQ ID NO:2, SEQ ID NO:3, or SEQ ID NO:4), and SADL gene (SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, or SEQ ID NO: 14) all in-frame and a NOS terminator. Clone the DNA construct into a binary vector, such as pCambial300, pCambia2300 or pCambia3200.

[0502] The CDOL gene is as follows:

[0503] a. Derived from SEQ ID NO:l, without the stop codon, optimized for expression in Arabidopsis or soybean (dicots) or com (a monocot), and encoding a CDOL peptide from Chlamydomonas reinhardtii (SEQ ID NO:5); or

[0504] b. Derived from SEQ ID NO: 2, without the stop codon, optimized for expression in Arabidopsis or soybean (dicots) or corn (a monocot), and encoding a CDOL peptide from Guillardia theta (SEQ ID NO:6); or

[0505] c. Derived from SEQ ID NO:3, without the stop codon, optimized for expression in Arabidopsis or soybean (dicots) or corn (a monocot), and encoding a CDOL peptide from Candida tenuis (SEQ ID NO:7); or

[0506] d. Derived from SEQ ID NO:4, without the stop codon, optimized for expression in Arabidopsis or soybean (dicots) or corn (a monocot), and encoding a CDOL peptide from Fragilariopsis cylindrus (SEQ ID NO:8).

[0507] The SADL gene is as follows:

[0508] a. Derived from SEQ ID NO: 11 by removing nucleotides 1 through 54, (corresponding to the native transit peptide) optimized for expression in Arabidopsis or soybean (dicots) or corn (a monocot), and encoding a truncated SADL peptide from Guillardia theta (SEQ ID NO: 17 minus amino acids 1 through 18); or

[0509] b. Derived from SEQ ID NO: 12 by removing nucleotides 1 through 33 (corresponding to a putative transit peptide), optimized for expression in Arabidopsis or soybean (dicots) or corn (a monocot), and encoding a truncated SADL peptide from Candida tenuis (SEQ ID NO: 18 minus amino acids 1 through 11); or

[0510] c. Derived from SEQ ID NO:13 by removing nucleotides 1 through 12, (corresponding to the native transit peptide) optimized for expression in Arabidopsis or soybean (dicots) or corn (a monocot), and encoding a truncated SADL peptide from Cyanidioschyzon merolae (SEQ ID NO: 19 minus amino acids 1 through 4); or

[0511] d. Derived from SEQ ID NO: 14 by removing nucleotides 1 through 18 (corresponding to the native transit peptide), optimized for expression in Arabidopsis or soybean (dicots) or corn (a monocot), and encoding a truncated SADL peptide from Thalassiosira oceanica (SEQ ID NO:20 minus amino acids 1 through 6).

[0512] Step 2: Transform the DNA construct into Agrobacterium tumefaciens, select for antibiotic resistance, and confirm the presence of the DNA construct.

[0513] Step 3: Transform plant (Arabidopsis, soybean, corn, wheat, sugar beet, rice, camelina or canola), select for antibiotic resistance, and confirm the presence of the DNA constructs in the transgenic plant.

EXAMPLE 10

Development of a transgenic plant that constitutively expresses CDOL without transit peptide fused with a linker to partCS/PLP-DC using chemical synthesis

[0514] Step 1: Use chemical synthesis to make a DNA construct that contains a constitutive promoter, 35S, fused with nucleotide sequence of a CDOL gene (SEQ ID NO:l, SEQ ID NO: 2, SEQ ID NO:3, or SEQ ID NO:4) without the transit peptide, linker (SEQ ID NO:23), SADL gene (SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, or SEQ ID NO: 14) without the transit peptide all in-frame and a NOS terminator. Clone the DNA construct into a binary vector, such as pCambial300, pCambia2300 or pCambia3200.

[0515] The CDOL gene is as follows:

[0516] a. Derived from SEQ ID NO:l by removing nucleotides 4 through 159 (corresponding to the native transit peptide) and without the stop codon, optimized for expression in Arabidopsis or soybean (dicots) or corn (a monocot), and encoding a CDOL peptide from Chlamydomonas reinhardtii (SEQ ID NO:5 minus amino acids 2 through 53); or

[0517] b. Derived from SEQ ID NO:2 by removing nucleotides 4 through 279 (corresponding to the native transit peptide) and without the stop codon, optimized for expression in Arabidopsis or soybean (dicots) or corn (a monocot), and encoding a CDOL peptide from G illardia theta (SEQ ID NO:6 minus amino acids 2 through 93); or

[0518] c. Derived from SEQ ID NO:3 by removing nucleotides 4 through 126 (corresponding to the native transit peptide) and without the stop codon, optimized for expression in Arabidopsis or soybean (dicots) or corn (a monocot), and encoding a CDOL peptide from Candida tenuis (SEQ ID NO:7 minus amino acids 2 through 42); or

[0519] d. Derived from SEQ ID NO:4 by removing nucleotides 4 through 180 (corresponding to the native transit peptide) and without the stop codon, optimized for expression in Arabidopsis or soybean (dicots) or corn (a monocot), and encoding a CDOL peptide from Fragilariopsis cylindrus (SEQ ID NO: 8 minus amino acids 2 through 60).

[0520] The partCS/PLP-DC gene is as follows:

[0521] a. Derived from SEQ ID NO:9 by removing nucleotides 1 through 1413 (corresponding to the native transit and cysteine synthetase peptides), optimized for expression in Arabidopsis or soybean (dicots) or corn (a monocot), and encoding a partCS/PLP-DC peptide from Micromonas pusilla (SEQ ID NO: 15 minus amino acids 1 through 471); or

[0522] b. Derived from SEQ ID NO: 10 truncated by removing nucleotides 1 through 1068 (corresponding to the native transit and cysteine synthetase peptides), optimized for expression in Arabidopsis or soybean (dicots) or corn (a monocot), and encoding a partCS/PLP-DC peptide from Ostreococcus tauri (SEQ ED NO: 16 minus amino acids 1 through 356).

[0523] Step 2: Transform the DNA construct into Agrobacterium tumefaciens, select for antibiotic resistance, and confirm the presence of the DNA construct.

[0524] Step 3: Transform plant (Arabidopsis, soybean, corn, wheat, sugar beet, rice, camelina or canola), select for antibiotic resistance, and confirm the presence of the DNA constructs in the transgenic plant.

EXAMPLE 11

Development of a transgenic plant that constitutively expresses CDOL with a plant transit peptide fused with a linker to SADL using chemical synthesis

[0525] Step 1: Use chemical synthesis to make a DNA construct that contains a constitutive promoter, 35S, fused with nucleotide sequence of a plastid transit peptide (SEQ ID NO:21), CDOL gene (SEQ ID NO:l, SEQ ID NO:2, SEQ ID NO:3, or SEQ ID NO:4), linker (SEQ ID NO:23), SADL gene (SEQ ID NO:ll, SEQ ID NO:12, SEQ ID NO:13, or SEQ ID NO: 14) all in-frame and a NOS terminator. Clone the DNA construct into a binary vector, such as pCambial300, pCambia2300 or pCambia3200.

[0526] The nucleotide sequence for the plastid transit peptide (SEQ ID NO:21) encodes the peptide SEQ ID NO:22.

[0527] The CDOL gene is as follows:

[0528] a. Derived from SEQ ID NO:l by removing nucleotides 1 through 159 (corresponding to the native transit peptide) and removing the stop codon, optimized for expression in Arabidopsis or soybean (dicots) or corn (a monocot), and encoding a CDOL peptide from Chlamydomonas reinhardtii (SEQ ID NO:5 minus amino acids 1 through 53); or

[0529] b. Derived from SEQ ID NO:2 by removing nucleotides 1 through 279 (corresponding to the native transit peptide) and removing the stop codon, optimized for expression in Arabidopsis or soybean (dicots) or corn (a monocot), and encoding a CDOL peptide from Guillardia theta (SEQ ID NO:6 minus amino acids 1 through 93); or

[0530] c. Derived from SEQ ID NO:3 by removing nucleotides 1 through 126 (corresponding to the native transit peptide) and removing the stop codon, optimized for expression in Arabidopsis or soybean (dicots) or corn (a monocot), and encoding a CDOL peptide from Candida tenuis (SEQ ID NO:7 minus amino acids 1 through 42); or

[0531] d. Derived from SEQ ID NO:4 by removing nucleotides 1 through 180 (corresponding to the native transit peptide) and removing the stop codon, optimized for expression in Arabidopsis or soybean (dicots) or corn (a monocot), and encoding a CDOL peptide from Fragilariopsis cylindrus (SEQ ID NO: 8 minus amino acids 1 through 60).

[0532] The SADL gene is as follows:

[0533] a. Derived from SEQ ID NO: 11 by removing nucleotides 1 through 54, (corresponding to the native transit peptide) optimized for expression in Arabidopsis or soybean (dicots) or corn (a monocot), and encoding a truncated SADL peptide from Guillardia theta (SEQ ID NO: 17 minus amino acids 1 through 18); or

[0534] b. Derived from SEQ ID NO: 12 by removing nucleotides 1 through 33 (corresponding to the native transit peptide), optimized for expression in Arabidopsis or soybean (dicots) or corn (a monocot), and encoding a truncated SADL peptide from Candida tenuis (SEQ ID NO: 18 minus amino acids 1 through 11); or

[0535] c. Derived from SEQ ID NO: 13 by removing nucleotides 1 through 12, (corresponding to the native transit peptide) optimized for expression in Arabidopsis or soybean (dicots) or corn (a monocot), and encoding a truncated SADL peptide from Cyanidioschyzon merolae (SEQ ID NO: 19 minus amino acids 1 through 4); or

[0536] d. Derived from SEQ ID NO: 14 by removing nucleotides 1 through 18 (corresponding to the native transit peptide), optimized for expression in Arabidopsis or soybean (dicots) or corn (a monocot), and encoding a truncated SADL peptide from Thalassiosira oceanica (SEQ ID NO:20 minus amino acids 1 through 6).

[0537] Step 2: Transform the DNA construct into Agrobacterium tumefaciens, select for antibiotic resistance, and confirm the presence of the DNA construct.

[0538] Step 3: Transform plant (Arabidopsis, soybean, corn, wheat, sugar beet, rice, camelina or canola), select for antibiotic resistance, and confirm the presence of the DNA constructs in the transgenic plant.

EXAMPLE 12

Development of a transgenic plant that constitutively expresses CDOL without transit peptide fused with a linker to SADL using chemical synthesis

[0539] Step 1: Use chemical synthesis to make a DNA construct that contains a constitutive promoter, 35S, fused with nucleotide sequence of a CDOL gene (SEQ ID NO:l, SEQ ID NO:2, SEQ ID NO:3, or SEQ ID NO:4) without the transit peptide, linker (SEQ ID NO:23), SADL gene (SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO:13, or SEQ ID NO: 14) without the transit peptide, all in-frame and a NOS terminator. Clone the DNA construct into a binary vector, such as pCambial300, pCambia2300 or pCambia3200.

[0540] The CDOL gene is as follows:

[0541] a. Derived from SEQ ID NO:l by removing nucleotides 4 through 159 (corresponding to the native transit peptide) and without the stop codon, optimized for expression in Arabidopsis or soybean (dicots) or corn (a monocot), and encoding a CDOL peptide from Chlamydomonas reinhardtii (SEQ ID NO:5 minus amino acids 2 through 53); or

[0542] b. Derived from SEQ ID NO:2 by removing nucleotides 4 through 279 (corresponding to the native transit peptide) and without the stop codon, optimized for expression in Arabidopsis or soybean (dicots) or corn (a monocot), and encoding a CDOL peptide from Guillardia theta (SEQ ID NO:6 minus amino acids 2 through 93); or

[0543] c. Derived from SEQ ID NO:3 by removing nucleotides 4 through 126 (corresponding to the native transit peptide) and without the stop codon, optimized for expression in Arabidopsis or soybean (dicots) or corn (a monocot), and encoding a CDOL peptide from Candida tenuis (SEQ ID NO:7 minus amino acids 2 through 42); or

[0544] d. Derived from SEQ ID NO:4 by removing nucleotides 4 through 180 (corresponding to the native transit peptide) and without the stop codon, optimized for expression in Arabidopsis or soybean (dicots) or corn (a monocot), and encoding a CDOL peptide from Fragilariopsis cylindrus (SEQ ID NO: 8 minus amino acids 2 through 60).

[0545] The SADL gene is as follows:

[0546] a. Derived from SEQ ID NO: 11 by removing nucleotides 1 through 54, (corresponding to the native transit peptide) optimized for expression in Arabidopsis or soybean (dicots) or corn (a monocot), and encoding a truncated SADL peptide from Guillardia theta (SEQ ID NO: 17 minus amino acids 1 through 18); or

[0547] b. Derived from SEQ ID NO:12, by removing nucleotides 1 through 33 (corresponding to the native transit peptide), optimized for expression in Arabidopsis or soybean (dicots) or corn (a monocot), and encoding a truncated SADL peptide from Candida tenuis (SEQ ID NO: 18 minus amino acids 1 through 11); or

[0548] c. Derived from SEQ ID NO: 13, by removing nucleotides 1 through 12, (corresponding to the native transit peptide) optimized for expression in Arabidopsis or soybean (dicots) or corn (a monocot), and encoding a truncated SADL peptide from Cyanidioschyzon merolae (SEQ ID NO: 19 minus amino acids 1 through 4); or

[0549] d. Derived from SEQ ID NO: 14, without the native transit peptide by removing nucleotides 1 through 18 (corresponding to the native transit peptide), optimized for expression in Arabidopsis or soybean (dicots) or com (a monocot), and encoding a truncated SADL peptide from Thalassiosira oceanica (SEQ ID NO:20 minus amino acids 1 through 6).

[0550] Step 2: Transform the DNA construct into Agrobacterium tumefaciens, select for antibiotic resistance, and confirm the presence of the DNA construct. [0551] Step 3: Transform plant (Arabidopsis, soybean, corn, wheat, sugar beet, rice, camelina or canola), select for antibiotic resistance, and confirm the presence of the DNA constructs in the transgenic plant.

EXAMPLE 13

Development of a transgenic plant that constitutively expresses CDOL with a plant transit peptide fused with a linker to CS/PLP-DC using chemical synthesis

[0552] Step 1: Use chemical synthesis to make a DNA construct that contains a constitutive promoter, 35S, fused with nucleotide sequence a plastid transit peptide (SEQ ID NO:21), CDOL gene (SEQ ID NO:l, SEQ ID NO:2, SEQ ID NO:3, or SEQ ID NO:4), linker (SEQ ID NO:23), CS/PLP-DC gene (SEQ ID NO:9, or SEQ ID NO: 10) without the native transit peptide all in- frame and a NOS terminator. Clone the DNA construct into a binary vector, such as pCambial300, pCambia2300 or pCambia3200.

[0553] The nucleotide sequence for the plastid transit peptide (SEQ ID NO:21) encodes the peptide SEQ ID NO:22.

[0554] The CDOL gene is as follows:

[0555] a. Derived from SEQ ID NO:l by removing nucleotides 1 through 159 (corresponding to the native transit peptide) and removing the stop codon, optimized for expression in Arabidopsis or soybean (dicots) or corn (a monocot), and encoding a CDOL peptide from Chlamydomonas reinhardtii (SEQ ID NO:5 minus amino acids 1 through 53); or

[0556] b. Derived from SEQ ID NO:2 by removing nucleotides 1 through 279 (corresponding to the native transit peptide) and removing the stop codon, optimized for expression in Arabidopsis or soybean (dicots) or corn (a monocot), and encoding a CDOL peptide from Guillardia theta (SEQ ID NO: 6 minus amino acids 1 through 93); or

[0557] c. Derived from SEQ ID NO:3 by removing nucleotides 1 through 126 (corresponding to the native transit peptide) and removing the stop codon, optimized for expression in Arabidopsis or soybean (dicots) or corn (a monocot), and encoding a CDOL peptide from Candida tenuis (SEQ ID NO:7 minus amino acids 1 through 42); or [0558] d. Derived from SEQ ID NO:4 by removing nucleotides 1 through 180 (corresponding to the native transit peptide) and removing the stop codon, optimized for expression in Arabidopsis or soybean (dicots) or corn (a monocot), and encoding a CDOL peptide from Fragilariopsis cylindrus (SEQ ID NO: 8 minus amino acids 1 through 60).

[0559] The CS/PLP-DC gene is as follows:

[0560] a. Derived from SEQ ID NO:9 by removing nucleotides 1 through 234, (corresponding to the native transit peptide) optimized for expression in Arabidopsis or soybean (dicots) or corn (a monocot), and encoding a CS/PLP-DC peptide from Micromonas pusilla (SEQ ID NO: 15 minus amino acids 1 through 78); or

[0561] b. Derived from SEQ ID NO: 10 by removing nucleotides 1 through 69 (corresponding to the native transit peptide), optimized for expression in Arabidopsis or soybean (dicots) or corn (a monocot), and encoding a CS/PLP-DC peptide from Ostreococcus tauri (SEQ ID NO: 16 minus amino acids 1 through 23).

[0562] Step 2: Transform the DNA construct into Agrobacterium tumefaciens, select for antibiotic resistance, and confirm the presence of the DNA construct.

[0563] Step 3: Transform plant (Arabidopsis, soybean, corn, wheat, sugar beet, rice, camelina or canola), select for antibiotic resistance, and confirm the presence of the DNA constructs in the transgenic plant.

EXAMPLE 14

Development of a transgenic plant that constitutively expresses CDOL with a native transit peptide fused with a linker to partCS/PLP-DC using chemical synthesis

[0564] Step 1: Use chemical synthesis to make a DNA construct that contains a constitutive promoter, 35S, fused with nucleotide sequence of the CDOL gene (SEQ ID NO:l, SEQ ID NO:2, SEQ ID NO:3, or SEQ ID NO:4), linker (SEQ ID NO:23), partCS/PLP-DC gene (SEQ ID NO:9, or SEQ ID NO: 10) all in-frame and a NOS terminator. Clone the DNA construct into a binary vector, such as pCambial300, pCambia2300 or pCambia3200.

[0565] The nucleotide sequence for the plastid transit peptide (SEQ ID NO:21) encodes the peptide SEQ ID NO:22.

[0566] The CDOL gene is as follows:

[0567] a. Derived from SEQ ID NO:l without the stop codon, optimized for expression in Arabidopsis or soybean (dicots) or corn (a monocot), and encoding a CDOL peptide from Chlamydomonas reinhardtii (SEQ ID NO:5); or [0568] b. Derived from SEQ ID NO:2 without the stop codon, optimized for expression in Arabidopsis or soybean (dicots) or corn (a monocot), and encoding a CDOL peptide from Guillardia theta (SEQ ID NO:6); or

[0569] c. Derived from SEQ ID NO:3 without the stop codon, optimized for expression in Arabidopsis or soybean (dicots) or corn (a monocot), and encoding a CDOL peptide from Candida tenuis (SEQ ID NO:7); or

[0570] d. Derived from SEQ ID NO:4 without the stop codon, optimized for expression in Arabidopsis or soybean (dicots) or corn (a monocot), and encoding a CDOL peptide from Fragilariopsis cylindrus (SEQ ID NO:8).

[0571] The partCS/PLP-DC gene is as follows:

[0572] a. Derived from SEQ ID NO:9 by removing nucleotides 1 through 1413 (corresponding to the native transit and cysteine synthetase peptides), optimized for expression in Arabidopsis or soybean (dicots) or corn (a monocot), and encoding a partCS/PLP-DC peptide from Micromonas pusilla (SEQ ID NO: 15 minus amino acids 1 through 471); or

[0573] b. Derived from SEQ ID NO: 10 truncated by removing nucleotides 1 through 1068 (corresponding to the native transit and cysteine synthetase peptides), optimized for expression in Arabidopsis or soybean (dicots) or corn (a monocot), and encoding a partCS/PLP-DC peptide from Ostreococcus tauri (SEQ ID NO: 16 minus amino acids 1 through 356).

[0574] Step 2: Transform the DNA construct into Agrobacterium tumefaciens, select for antibiotic resistance, and confirm the presence of the DNA construct.

[0575] Step 3: Transform plant (Arabidopsis, soybean, corn, wheat, sugar beet, rice, camelina or canola), select for antibiotic resistance, and confirm the presence of the DNA constructs in the transgenic plant.

EXAMPLE 15

Development of a transgenic plant that constitutively expresses CDOL with a plant transit peptide fused with a linker to pa rtCS/PLP-DC using chemical synthesis

[0576] Step 1: Use chemical synthesis to make a DNA construct that contains a constitutive promoter, 35S, fused with nucleotide sequence a plastid transit peptide (SEQ ID NO:21), CDOL gene (SEQ ID NO:l, SEQ ID NO:2, SEQ ID NO:3, or SEQ ID NO:4), linker (SEQ ID NO:23), partCS/PLP-DC gene (SEQ ID NO:9, or SEQ ID NO: 10) all in-frame and a NOS terminator. Clone the DNA construct into a binary vector, such as pCambial300, pCambia2300 or pCambia3200.

[0577] The nucleotide sequence for the plastid transit peptide (SEQ ID NO:21) encodes the peptide SEQ ID NO:22.

[0578] The CDOL gene is as follows:

[0579] a. Derived from SEQ ID NO:l by removing nucleotides 1 through 159 (corresponding to a putative transit peptide) and removing the stop codon, optimized for expression in Arabidopsis or soybean (dicots) or corn (a monocot), and encoding a CDOL peptide from Chlamydomonas reinhardtii (SEQ ID NO:5 minus amino acids 1 through 53); or

[0580] b. Derived from SEQ ID NO:2 by removing nucleotides 1 through 279 (corresponding to the native transit) and removing the stop codon, optimized for expression in Arabidopsis or soybean (dicots) or corn (a monocot), and encoding a CDOL peptide from Guillardia theta (SEQ ID NO:6 minus amino acids 1 through 93); or

[0581] c. Derived from SEQ ID NO:3 by removing nucleotides 1 through 126 (corresponding to the native transit) and removing the stop codon, optimized for expression in Arabidopsis or soybean (dicots) or corn (a monocot), and encoding a CDOL peptide from Candida tenuis (SEQ ID NO:7 minus amino acids 1 through 42); or

[0582] d. Derived from SEQ ID NO:4 by removing nucleotides 1 through 180 (corresponding to the native transit) and removing the stop codon, optimized for expression in Arabidopsis or soybean (dicots) or corn (a monocot), and encoding a CDOL peptide from Fragilariopsis cylindrus (SEQ ID NO: 8 minus amino acids 1 through 60).

[0583] The partCS/PLP-DC gene is as follows:

[0584] a. Derived from SEQ ID NO:9 by removing nucleotides 1 through 1413, (corresponding to the native transit and cysteine synthetase peptides ) optimized for expression in Arabidopsis or soybean (dicots) or corn (a monocot), and encoding a partCS/PLP-DC peptide from Micromonas pusilla (SEQ ID NO: 15 minus amino acids 1 through 471); or

[0585] b. Derived from SEQ ID NO: 10 by removing nucleotides 1 through 1068 (corresponding to the native transit and cysteine synthetase peptides), optimized for expression in Arabidopsis or soybean (dicots) or corn (a monocot), and encoding a partCS/PLP-DC peptide from Ostreococcus tauri (SEQ ID NO: 16 minus amino acids 1 through 356).

[0586] Step 2: Transform the DNA construct into Agrobacterium tumefaciens, select for antibiotic resistance, and confirm the presence of the DNA construct.

[0587] Step 3: Transform plant (Arabidopsis, soybean, corn, wheat, sugar beet, rice, camelina or canola), select for antibiotic resistance, and confirm the presence of the DNA constructs in the transgenic plant.

EXAMPLE 16

Development of a transgenic plant that constitutively expresses CDOL without a transit peptide and constitutively expresses SADL protein without a transit peptide in tandem with independent promoters

[0588] Step 1: Use chemical synthesis to make a DNA construct that contains a constitutive promoter, 35S, fused with a CDOL gene (SEQ ID NO:l, SEQ ID NO:2, SEQ ID NO:3, or SEQ ID NO:4) with the native transit peptide removed and a NOS terminator. Clone the DNA construct into a binary vector, such as pCambial300, pCambia2300, or pCambia3200.

[0589] The CDOL gene is as follows:

[0590] a. Derived from SEQ ID NO:l by removing nucleotides 4 through 159 (corresponding to the native transit peptide), optimized for expression in Arabidopsis or soybean (dicots) or corn (a monocot), and encoding a CDOL peptide from Chlamydomonas reinhardtii (SEQ ID NO:5 minus amino acids 2 through 53); or

[0591] b. Derived from SEQ ID NO:2 by removing nucleotides 4 through 279 (corresponding to the native transit peptide), optimized for expression in Arabidopsis or soybean (dicots) or corn (a monocot), and encoding a CDOL peptide from Guillardia theta (SEQ ID NO:6 minus amino acids 2 through 93); or

[0592] c. Derived from SEQ ID NO:3 by removing nucleotides 4 through 126 (corresponding to the native transit peptide), optimized for expression in Arabidopsis or soybean (dicots) or corn (a monocot), and encoding a CDOL peptide from Candida tenuis (SEQ ID NO:7 minus amino acids 2 through 42); or

[0593] d. Derived from SEQ ID NO:4 by removing nucleotides 4 through 180 (corresponding to the native transit peptide), optimized for expression in Arabidopsis or soybean (dicots) or corn (a monocot), and encoding a CDOL peptide from Fragilariopsis c lindrus (SEQ ID NO:8 minus amino acids 2 through 60).

[0594] Step 2: Use chemical synthesis to make a DNA construct that contains a constitutive promoter, 35S, fused with a SADL (SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, or SEQ ID NO: 14) with the native transit peptide removed and a NOS terminator. Clone the SADL DNA construct into a binary vector that contains the CDOL DNA construct (Stepl).

[0595] The SADL gene is as follows:

[0596] a. Derived from SEQ ID NO: 11 by removing nucleotides 4 through 54, (corresponding to a putative transit peptide) optimized for expression in Arabidopsis or soybean (dicots) or corn (a monocot), and encoding a truncated SADL peptide from Guillardia theta (SEQ ID NO: 17 minus amino acids 2 through 18); or

[0597] b. Derived from SEQ ID NO: 12 by removing nucleotides 4 through 33 (corresponding to the native transit peptide), optimized for expression in Arabidopsis or soybean (dicots) or corn (a monocot), and encoding a truncated SADL peptide from Candida tenuis (SEQ ID NO:18 minus amino acids 2 through 11); or

[0598] c. Derived from SEQ ID NO: 13 by removing nucleotides 4 through 12, (corresponding to the native transit peptide) optimized for expression in Arabidopsis or soybean (dicots) or corn (a monocot), and encoding a truncated SADL peptide from Cyanidioschyzon merolae (SEQ ID NO: 19 minus amino acids 2 through 4); or

[0599] d. Derived from SEQ ID NO: 14 by removing nucleotides 4 through 18 (corresponding to the native transit peptide), optimized for expression in Arabidopsis or soybean (dicots) or corn (a monocot), and encoding a truncated SADL peptide from Thalassiosira oceanica (SEQ ID NO: 20 minus amino acids 2 through 6).

[0600] Step 3: Transform the DNA construct into Agrobacterium tumefaciens, select for antibiotic resistance, and confirm the presence of the DNA construct.

[0601] Step 4: Transform plant (Arabidopsis, soybean, corn, wheat, sugar beet, rice, camelina or canola), select for antibiotic resistance, and confirm the presence of the DNA constructs in the transgenic plant. EXAMPLE 17

Development of a transgenic plant that constitutively expresses CDOL without a transit peptide and constitutively expresses CS/PLP-DC protein without transit peptide in tandem with independent promoters

[0602] Step 1: Use chemical synthesis to make a DNA construct that contains a constitutive promoter, 35S, fused with the CDOL gene (SEQ ID NO:l, SEQ ID NO:2, SEQ ID NO:3, or SEQ ID NO:4) with the native transit peptide removed and a NOS terminator. Clone the DNA construct into a binary vector, such as pCambial300, pCambia2300 or pCambia3200.

[0603] The CDOL gene is as follows:

[0604] a. Derived from SEQ ID NO:l by removing nucleotides 4 through 159 (corresponding to the native transit peptide), optimized for expression in Arabidopsis or soybean (dicots) or corn (a monocot), and encoding a CDOL peptide from Chlamydomonas reinhardtii (SEQ ID NO:5 minus amino acids 2 through 53); or

[0605] b. Derived from SEQ ID NO:2 by removing nucleotides 4 through 279 (corresponding to the native transit peptide), optimized for expression in Arabidopsis or soybean (dicots) or corn (a monocot), and encoding a CDOL peptide from Guillardia theta (SEQ ID NO: 6 minus amino acids 2 through 93); or

[0606] c. Derived from SEQ ID NO:3 by removing nucleotides 4 through 126 (corresponding to the native transit peptide), optimized for expression in Arabidopsis or soybean (dicots) or corn (a monocot), and encoding a CDOL peptide from Candida tenuis (SEQ ID NO: 7 minus amino acids 2 through 42); or

[0607] d. Derived from SEQ ID NO:4 by removing nucleotides 4 through 180 (corresponding to the native transit peptide), optimized for expression in Arabidopsis or soybean (dicots) or corn (a monocot), and encoding a CDOL peptide from Fragilariopsis cylindrus (SEQ ID NO:8 minus amino acids 2 through 60).

[0608] Step 2: Use chemical synthesis to make a DNA construct that contains a constitutive promoter, 35S, fused with the CS/PLP-DC (SEQ ID NO:9 or SEQ ID NO: 10) with the native transit peptide removed and a NOS terminator. The nucleotide sequence for the plastid transit peptide (SEQ ID NO: 21) encodes the peptide SEQ ID NO: 22. Clone the CS/PLP-DC DNA construct into a binary vector that contains the CDOL DNA construct (Stepl).

[0609] The CS/PLP-DC gene is as follows:

[0610] a. Derived from SEQ ID NO:9 by removing nucleotides 4 through 234, (corresponding to the native transit peptide) optimized for expression in Arabidopsis or soybean (dicots) or corn (a monocot), and encoding a CS/PLP-DC peptide from Micromonas pusilla (SEQ ID NO: 15 minus amino acids 2 through 78); or

[0611] b. Derived from SEQ ID NO: 10 by removing nucleotides 4 through 69 (corresponding to the native transit peptide), optimized for expression in Arabidopsis or soybean (dicots) or corn (a monocot), and encoding a CS/PLP-DC peptide from Ostreococcus tauri (SEQ ID NO:16 minus amino acids 2 through 23).

[0612] Step 3: Transform the DNA construct into Agrobacterium tumefaciens, select for antibiotic resistance, and confirm the presence of the DNA construct.

[0613] Step 4: Transform plant (Arabidopsis, soybean, corn, wheat, sugar beet, rice, camelina or canola), select for antibiotic resistance, and confirm the presence of the DNA constructs in the transgenic plant.

EXAMPLE 18

Development of a transgenic plant that constitutively expresses CDOL without a transit peptide and constitutively expresses partCS/PLP-DC protein without a transit peptide in tandem with independent promoters

[0614] Step 1: Use chemical synthesis to make a DNA construct that contains a constitutive promoter, 35S, fused with CDOL gene (SEQ ID NO:l, or SEQ ID NO:2, SEQ ID NO:3 or SEQ ID NO:4) with the native transit peptide removed and a NOS terminator. Clone the DNA construct into a binary vector, such as pCambial300, pCambia2300 or pCambia3200.

[0615] The CDOL gene is as follows:

[0616] a. Derived from SEQ ID NO:l by removing nucleotides 4 through 159 (corresponding to the native transit peptide), optimized for expression in Arabidopsis or soybean (dicots) or corn (a monocot), and encoding a CDOL peptide from Chlamydomonas reinhardtii (SEQ ID NO:5 minus amino acids 2 through 53); or

[0617] b. Derived from SEQ ID NO:2 by removing nucleotides 4 through 279 (corresponding to the native transit peptide), optimized for expression in Arabidopsis or soybean (dicots) or corn (a monocot), and encoding a CDOL peptide from Guillardia theta (SEQ ID NO:6 minus amino acids 2 through 93): or

[0618] c. Derived from SEQ ID NO:3 by removing nucleotides 4 through 126 (corresponding to the native transit peptide), optimized for expression in Arabidopsis or soybean (dicots) or corn (a monocot), and encoding a CDOL peptide from Candida tenuis (SEQ ID NO:7 minus amino acids 2 through 42); or [0619] d. Derived from SEQ ID NO:4 by removing nucleotides 4 through 180 (corresponding to the native transit peptide), optimized for expression in Arabidopsis or soybean (dicots) or corn (a monocot), and encoding a CDOL peptide from Fragilariopsis cylindrus (SEQ ID NO: 8 minus amino acids 2 through 60).

[0620] Step 2: Use chemical synthesis to make a DNA construct that contains a constitutive promoter, 35S, fused with the partCS/PLP-DC (SEQ ID NO:9 or SEQ ID NO: 10) and a NOS terminator. Clone the partCPLP-DC DNA construct into a binary vector that contains the CDOL DNA construct (Stepl).

[0621] The partCS/PLP-DC gene is as follows:

[0622] a. Derived from SEQ ID NO:9 by removing nucleotides 4 through 1413, (corresponding to the native transit and cysteine synthetase peptides) optimized for expression in Arabidopsis or soybean (dicots) or corn (a monocot), and encoding a partCS/PLP-DC peptide from Micromonas pusilla (SEQ ID NO: 15 minus amino acids 2 through 471); or

[0623] b. Derived from SEQ ID NO: 10 by removing nucleotides 4 through 1068 (corresponding to the native transit and cysteine synthetase peptides), optimized for expression in Arabidopsis or soybean (dicots) or corn (a monocot), and encoding a partCS/PLP-DC peptide from Ostreococcus tauri (SEQ ID NO: 16 minus amino acids 2 through 356).

[0624] Step 3: Transform the DNA construct into Agrobacterium tumefaciens, select for antibiotic resistance, and confirm the presence of the DNA construct.

[0625] Step 4: Transform plant (Arabidopsis, soybean, corn, wheat, sugar beet, rice, camelina or canola), select for antibiotic resistance, and confirm the presence of the DNA constructs in the transgenic plant.

EXAMPLE 19

Development of a transgenic plant that constitutively expresses partCS/PLP-DC without a transit peptide

[0626] Step 1: Use chemical synthesis to make a DNA construct that contains a constitutive promoter, 35S, fused with nucleotide sequence for partCS/PLP-DC (SEQ ID NO:9 or SEQ ID NO: 10) and a NOS terminator. Clone the DNA construct into a binary vector, such as pCambial300, pCambia2300, or pCambia3200.

[062η The partCS/PLP-DC gene is as follows: [0628] a. Derived from SEQ ID NO:9 by removing nucleotides 4 through 1413, (corresponding to a putative transit and cysteine synthetase peptides ) optimized for expression in Arabidopsis or soybean (dicots) or corn (a monocot), and encoding a partCS/PLP-DC peptide from Micromonas pusilla (SEQ ID NO: 15 minus amino acids 2 through 471); or

[0629] b. Derived from SEQ ID NO:10 by removing nucleotides 4 through 1068 (corresponding to a putative transit and cysteine synthetase peptides), optimized for expression in Arabidopsis or soybean (dicots) or corn (a monocot), and encoding a partCS/PLP-DC peptide from Ostreococcus tauri (SEQ ID NO: 16 minus amino acids 2 through 356).

[0630] Step 2: Transform the DNA construct into Agrobacterium tumefaciens, select for antibiotic resistance, and confirm the presence of the DNA construct.

[0631] Step 3: Transform plant (Arabidopsis, soybean, corn, wheat, sugar beet, rice, camelina or canola), select for antibiotic resistance, and confirm the presence of the DNA constructs in the transgenic plant.

EXAMPLE 20

Development of a transgenic plant that constitutively expresses partCS/PLP-DC with a plant plastid transit peptide

[0632] Step 1: Use chemical synthesis to make a DNA construct that contains a constitutive promoter, 35S, fused with the nucleotide sequence for a plastid transit peptide (SEQ ID NO: 21), partCS/PLP-DC (SEQ ID NO:9 or SEQ ID NO: 10) and a NOS terminator. Clone the DNA construct into a binary vector, such as pCambial300, pCambia2300, or pCambia3200.

[0633] The nucleotide sequence for the plastid transit peptide (SEQ ID NO: 21) encodes the peptide SEQ ID NO: 22.

[0634] The partCS/PLP-DC gene is as follows:

[0635] a. Derived from SEQ ID NO:9 by removing nucleotides 1 through 1413 (corresponding to a putative transit and cysteine synthetase peptides) optimized for expression in Arabidopsis or soybean (dicots) or com (a monocot), and encoding a partCS/PLP-DC peptide from Micromonas pusilla (SEQ ID NO: 15 minus amino acids 1 through 471); or

[0636] b. Derived from SEQ ID NO: 10 by removing nucleotides 1 through 1068 (corresponding to the native transit and cysteine synthetase peptides), optimized for expression in Arabidopsis or soybean (dicots) or corn (a monocot), and encoding a partCS/PLP-DC peptide from Ostreococcus tauri (SEQ ID NO: 16 minus amino acids 1 through 356).

[0637] Step 2: Transform the DNA construct into Agrobacterium tumefaciens, select for antibiotic resistance, and confirm the presence of the DNA construct.

[0638] Step 3: Transform plant (Arabidopsis, soybean, corn, wheat, sugar beet, rice, camelina or canola), select for antibiotic resistance, and confirm the presence of the DNA constructs in the transgenic plant.

EXAMPLE 21

Development of a transgenic plant that constitutively expresses CS/PLP-DC without a transit peptide

[0639] Step 1: Use chemical synthesis to make a DNA construct that contains a constitutive promoter, 35S, fused with nucleotide sequence for CS/PLP-DC (SEQ ID NO:9 or SEQ ID NO: 10) with the native transit peptide removed and a NOS terminator. Clone the DNA construct into a binary vector, such as pCambial300, pCambia2300, or pCambia3200.

[0640] The CS/PLP-DC gene is as follows:

[0641] a. Derived from SEQ ID NO:9 by removing nucleotides 4 through 234, (corresponding to the native transit peptide) optimized for expression in Arabidopsis or soybean (dicots) or corn (a monocot), and encoding a CS/PLP-DC peptide from Micromonas pusilla (SEQ ID NO: 15 minus amino acids 2 through 78); or

[0642] b. Derived from SEQ ID NO: 10 without the native transit peptide by removing nucleotides 4 through 69 (corresponding to the native transit peptide), optimized for expression in Arabidopsis or soybean (dicots) or corn (a monocot), and encoding a CS PLP-DC peptide from Ostreococcus tauri (SEQ ED NO:16 minus amino acids 2 through 23).

[0643] Step 2: Transform the DNA construct into Agrobacterium tumefaciens, select for antibiotic resistance, and confirm the presence of the DNA construct.

[0644] Step 3: Transform plant (Arabidopsis, soybean, corn, wheat, sugar beet, rice, camelina or canola), select for antibiotic resistance, and confirm the presence of the DNA constructs in the transgenic plant. EXAMPLE 22

Development of a bacterial strain that expresses CDOL without a native transit peptide fused (without a linker) to a SADL without a native transit peptide using chemical synthesis

[0645] Step 1: Use chemical synthesis to make a DNA construct that contains a CDOL gene (SEQ ID NO:l, SEQ ID NO:2, SEQ ID NO:3, or SEQ ID NO:4) without the native transit peptide, and SADL gene (SEQ ID NO:ll, SEQ ID NO:12, SEQ ID NO:13, or SEQ ID NO:14) without the native transit peptide all in-frame. Clone the DNA construct into a bacterial expression vector, such as pKK233-2, pGEX (GE Healthcare), pET (Novagen) and pMAL™ (New England biolabs) in a manner that the construct is operably functional.

[0646] The CDOL gene is as follows:

[0647] a. Derived from SEQ ID NO:l by removing nucleotides 4 through 159 (corresponding to the native transit peptide) and without the stop codon, optimized for expression in Arabidopsis or soybean (dicots) or corn (a monocot), and encoding a CDOL peptide from Chlamydomonas reinhardtii (SEQ ID NO:5 minus amino acids 2 through 53); or

[0648] b. Derived from SEQ ID NO:2 by removing nucleotides 4 through 279 (corresponding to the native transit peptide) and without the stop codon, optimized for expression in Arabidopsis or soybean (dicots) or corn (a monocot), and encoding a CDOL peptide from Guillardia theta (SEQ ID NO:6 minus amino acids 2 through 93); or

[0649] c. Derived from SEQ ID NO:3 by removing nucleotides 4 through 126 (corresponding to the native transit peptide) and without the stop codon, optimized for expression in Arabidopsis or soybean (dicots) or corn (a monocot), and encoding a CDOL peptide from Candida tenuis (SEQ ID NO:7 minus amino acids 2 through 42); or [0650] d. Derived from SEQ ID NO:4 by removing nucleotides 4 through 180 (corresponding to the native transit peptide) and without the stop codon, optimized for expression in Arabidopsis or soybean (dicots) or corn (a monocot), and encoding a CDOL peptide from Fragilariopsis cylindrus (SEQ ID NO: 8 minus amino acids 2 through 60).

[0651] The SADL gene is as follows:

[0652] a. Derived from SEQ ID NO: 11 by removing nucleotides 1 through 54, (corresponding to the native transit peptide) optimized for expression in Arabidopsis or soybean (dicots) or corn (a monocot), and encoding a truncated SADL peptide from

Guillardia theta (SEQ ID NO:17 minus amino acids 1 through 18); or

[0653] b. Derived from SEQ ID NO: 12, by removing nucleotides 1 through 33

(corresponding to the native transit peptide), optimized for expression in Arabidopsis or soybean (dicots) or corn (a monocot), and encoding a truncated SADL peptide from

Candida tenuis (SEQ ID NO:18 minus amino acids 1 through 11); or

[0654] c. Derived from SEQ ID NO: 13, by removing nucleotides 1 through 12,

(corresponding to the native transit peptide) optimized for expression in Arabidopsis or soybean (dicots) or corn (a monocot), and encoding a truncated SADL peptide from

Cyanidioschyzon merolae (SEQ ID NO: 19 minus amino acids 1 through 4); or

[0655] d. Derived from SEQ ID NO: 14, without the native transit peptide by removing nucleotides 1 through 18 (corresponding to the native transit peptide), optimized for expression in Arabidopsis or soybean (dicots) or corn (a monocot), and encoding a truncated SADL peptide from Thalassiosira oceanica (SEQ ID NO:20 minus amino acids 1 through 6).

[0656] Step 2: Transform the DNA construct into bacteria Escherichia coli, B.sublits, select for antibiotic resistance, and confirm the presence of the DNA construct.

EXAMPLE 23

Development of a bacterial strain that express CDOL without transit peptide fused with a linker to SADL without a native transit peptide using chemical synthesis

[0657] Step 1: Use chemical synthesis to make a DNA construct that contains a CDOL gene (SEQ ID NO:l, SEQ ID NO:2, SEQ ID NO:3, or SEQ ID NO:4) without the transit peptide, linker (SEQ ID NO:23), and SADL gene (SEQ ID NO:ll, SEQ ID NO: 12, SEQ ID NO:13, or SEQ ID NO: 14) without the transit peptide all in-frame. Clone the DNA construct into a bacterial expression vector, such as pKK233-2, pGEX (GE Healthcare), pET (Novagen) and pMAL™ (New England biolabs) in a manner that the construct is operably functional.

[0658] The CDOL gene is as follows:

[0659] a. Derived from SEQ ID NO:l by removing nucleotides 4 through 159 (corresponding to the native transit peptide) and without the stop codon, optimized for expression in Arabidopsis or soybean (dicots) or com (a monocot), and encoding a CDOL peptide from Chlamydomonas reinhardtii (SEQ ID NO:5 minus amino acids 2 through 53); or

[0660] b. Derived from SEQ ID NO:2 by removing nucleotides 4 through 279 (corresponding to the native transit peptide) and without the stop codon, optimized for expression in Arabidopsis or soybean (dicots) or corn (a monocot), and encoding a CDOL peptide from Guillardia theta (SEQ ID NO:6 minus amino acids 2 through 93); or

[0661] c. Derived from SEQ ID NO:3 by removing nucleotides 4 through 126 (corresponding to the native transit peptide) and without the stop codon, optimized for expression in Arabidopsis or soybean (dicots) or corn (a monocot), and encoding a CDOL peptide from Candida tenuis (SEQ ID NO:7 minus amino acids 2 through 42); or

[0662] d. Derived from SEQ ID NO:4 by removing nucleotides 4 through 180 (corresponding to the native transit peptide) and without the stop codon, optimized for expression in Arabidopsis or soybean (dicots) or corn (a monocot), and encoding a CDOL peptide from Fragilariopsis cylindrus (SEQ ID NO: 8 minus amino acids 2 through 60).

The SADL gene is as follows:

[0664] a. Derived from SEQ ID NO:ll by removing nucleotides 1 through 54,

(corresponding to the native transit peptide) optimized for expression in Arabidopsis or soybean (dicots) or corn (a monocot), and encoding a truncated SADL peptide from

Guillardia theta (SEQ ID NO: 17 minus amino acids 1 through 18); or

[0665] b. Derived from SEQ ID NO: 12, by removing nucleotides 1 through 33

(corresponding to the native transit peptide), optimized for expression in Arabidopsis or soybean (dicots) or corn (a monocot), and encoding a truncated SADL peptide from

Candida tenuis (SEQ ID NO: 18 minus amino acids 1 through 11); or

[0666] c. Derived from SEQ ID NO: 13, by removing nucleotides 1 through 12,

(corresponding to the native transit peptide) optimized for expression in Arabidopsis or soybean (dicots) or corn (a monocot), and encoding a truncated SADL peptide from

Cyanidioschyzon merolae (SEQ ID NO: 19 minus amino acids 1 through 4); or

[0667] d. Derived from SEQ ID NO: 14, without the native transit peptide by removing nucleotides 1 through 18 (corresponding to the native transit peptide), optimized for expression in Arabidopsis or soybean (dicots) or corn (a monocot), and encoding a truncated SADL peptide from Thalassiosira oceanica (SEQ ID NO: 20 minus amino acids 1 through 6).

[0668] Step 2: Transform the DNA construct into bacteria Escherichia coli, B.sublits, select for antibiotic resistance, and confirm the presence of the DNA construct.

EXAMPLE 24

Development of a bacterial strain that express CDOL without transit peptide fused with a linker to partCS/PLP-DC without a native transit peptide using chemical synthesis

[0669] Step 1: Use chemical synthesis to make a DNA construct with a CDOL gene (SEQ ID NO:l, SEQ ID NO:2, SEQ ID NO:3, or SEQ ID NO:4) without the transit peptide, linker (SEQ ID NO:23), and partCS/PLP-DC (SEQ ID NO:9 or SEQ ID NO: 10) all in-frame. Clone the DNA construct into a bacterial expression vector, such as pKK233-2, pGEX (GE Healthcare), pET (Novagen) and pMAL™ (New England biolabs) in a manner that the construct is operably functional.

[0670] The CDOL gene is as follows:

[0671] a. Derived from SEQ ID NO:l by removing nucleotides 4 through 159 (corresponding to the native transit peptide) and without the stop codon, optimized for expression in Arabidopsis or soybean (dicots) or corn (a monocot), and encoding a CDOL peptide from Chlamydomonas reinhardtii (SEQ ID NO:5 minus amino acids 2 through 53); or

[0672] b. Derived from SEQ ID NO:2 by removing nucleotides 4 through 279 (corresponding to the native transit peptide) and without the stop codon, optimized for expression in Arabidopsis or soybean (dicots) or corn (a monocot), and encoding a CDOL peptide from Guillardia theta (SEQ ID NO:6 minus amino acids 2 through 93); or

[0673] c. Derived from SEQ ID NO:3 by removing nucleotides 4 through 126 (corresponding to the native transit peptide) and without the stop codon, optimized for expression in Arabidopsis or soybean (dicots) or corn (a monocot), and encoding a CDOL peptide from Candida tenuis (SEQ ID NO:7 minus amino acids 2 through 42); or

[0674] d. Derived from SEQ ID NO:4 by removing nucleotides 4 through 180 (corresponding to the native transit peptide) and without the stop codon, optimized for expression in Arabidopsis or soybean (dicots) or corn (a monocot), and encoding a CDOL peptide from Fragilariopsis cylindrus (SEQ ID NO: 8 minus amino acids 2 through 60).

[0675] The partCS/PLP-DC gene is as follows:

[0676] a. Derived from SEQ ID NO:9, by removing nucleotides 1 through 1413, (corresponding to the native transit and cysteine synthetase peptides) optimized for expression in Arabidopsis or soybean (dicots) or corn (a monocot), and encoding a partCS/PLP-DC peptide from Micromonas pusilla (SEQ ID NO: 15 minus amino acids 1 through 471); or

[0677] b. Derived from SEQ ID NO: 10, by removing nucleotides 1 through 1068 (corresponding to the native transit peptide and cysteine synthetase peptides), optimized for expression in Arabidopsis or soybean (dicots) or corn (a monocot), and encoding a partCS/PLP-DC peptide from Ostreococcus tauri (SEQ ID NO: 16 minus amino acids 1 through 356).

[0678] Step 2: Transform the DNA construct into bacteria Escherichia coli, B.sublits., select for antibiotic resistance, and confirm the presence of the DNA construct.

Development of a bacterial strain that express CDOL without transit peptide fused with a linker to CS/PLP-DC without a native transit peptide using chemical synthesis

[0679] Step 1: Use chemical synthesis to make a DNA construct with a CDOL gene (SEQ ID NO:l, SEQ ID NO:2, SEQ ID NO:3, or SEQ ID NO:4) without the transit peptide, linker (SEQ ID NO:23), and CS/PLP-DC (SEQ ID NO:9 or SEQ ID NO: 10) all in-frame. Clone the DNA construct into a bacterial expression vector, such as pKK233-2, pGEX (GE Healthcare), pET (Novagen) and pMAL™ (New England biolabs) in a manner that the construct is operably functional.

[0680] The CDOL gene is as follows:

[0681] a. Derived from SEQ ID NO:l by removing nucleotides 4 through 159 (corresponding to the native transit peptide) and without the stop codon, optimized for expression in Arabidopsis or soybean (dicots) or corn (a monocot), and encoding a CDOL peptide from Chlamydomonas reinhardtii (SEQ ID NO:5 minus amino acids 2 through 53); or

[0682] b. Derived from SEQ ID NO:2 by removing nucleotides 4 through 279 (corresponding to the native transit peptide) and without the stop codon, optimized for expression in Arabidopsis or soybean (dicots) or corn (a monocot), and encoding a CDOL peptide from Guillardia theta (SEQ ID NO:6 minus amino acids 2 through 93); or

[0683] c. Derived from SEQ ID NO:3 by removing nucleotides 4 through 126 (corresponding to the native transit peptide) and without the stop codon, optimized for expression in Arabidopsis or soybean (dicots) or corn (a monocot), and encoding a CDOL peptide from Candida tenuis (SEQ ID NO:7 minus amino acids 2 through 42); or

[0684] d. Derived from SEQ ID NO:4 by removing nucleotides 4 through 180 (corresponding to the native transit peptide) and without the stop codon, optimized for expression in Arabidopsis or soybean (dicots) or corn (a monocot), and encoding a CDOL peptide from Fragilariopsis cylindrus (SEQ ID NO: 8 minus amino acids 2 through 60).

[0685] The CS/PLP-DC gene is as follows:

[0686] a. Derived from SEQ ID NO:9 by removing nucleotides 1 through 234, (corresponding to the native transit peptide) optimized for expression in Arabidopsis or soybean (dicots) or corn (a monocot), and encoding a CS PLP-DC peptide from Micromonas pusilla (SEQ ID NO: 15 minus amino acids 1 through 78); or

[0687] b. Derived from SEQ ID NO: 10 without the native transit peptide by removing nucleotides 1 through 69 (corresponding to the native transit peptide), optimized for expression in Arabidopsis or soybean (dicots) or corn (a monocot), and encoding a CS/PLP-DC peptide from Ostreococcus tauri (SEQ ID NO: 16 minus amino acids 1 through 23).

[0688] Step 2: Transform the DNA construct into bacteria Escherichia coli, B.sublits , select for antibiotic resistance, and confirm the presence of the DNA construct.

EXAMPLE 26

Development of a bacterial strain that expresses CS/PLP-DC without a native transit peptide using chemical synthesis

[0689] Step 1: Use chemical synthesis to make a DNA construct with a CS/PLP-DC (SEQ ID NO:9 or SEQ ID NO: 10). Clone the DNA construct into a bacterial expression vector, such as pKK233-2, pGEX (GE Healthcare), pET (Novagen) and pMAL™ (New England biolabs) in a manner that the construct is operably functional.

[0690] The CS/PLP-DC gene is as follows: [0691] a. Derived from SEQ ID N0:9 by removing nucleotides 4 through 234, (corresponding to the native transit peptide) optimized for expression in Arabidopsis or soybean (dicots) or corn (a monocot), and encoding a CS/PLP-DC peptide from Micromonas pusilla (SEQ ID NO: 15 minus amino acids 2 through 78); or

[0692] b. Derived from SEQ ID NO: 10 without the native transit peptide by removing nucleotides 4 through 69 (corresponding to the native transit peptide), optimized for expression in Arabidopsis or soybean (dicots) or corn (a monocot), and encoding a CS/PLP-DC peptide from Ostreococcus tauri (SEQ ID NO: 16 minus amino acids 2 through 23).

[0693] Step 2: Transform the DNA construct into bacteria Escherichia coli, B.sublits., select for antibiotic resistance, and confirm the presence of the DNA construct.

EXAMPLE 27

Development of a transgenic plant that expresses in the seed CDOL without transit peptide fused with a linker to partCS/PLP-DC using chemical synthesis

[0694] Step 1: Use chemical synthesis to make a DNA construct that contains a seed-specific promoter, beta-conglycinin, fused with nucleotide sequence of a CDOL gene (SEQ ID NO:l, SEQ ID NO:2, SEQ ID NO:3, or SEQ ID NO:4) without the transit peptide, linker (SEQ ID NO:23), SADL gene (SEQ ID NO:ll, SEQ ID NO: 12, SEQ ID NO: 13, or SEQ ID NO: 14) without the transit peptide all in-frame and a beta-conglycinin terminator. Clone the DNA construct into a binary vector, such as pCambial300, pCambia2300 or pCambia3200.

[0695] The CDOL gene is as follows:

[0696] a. Derived from SEQ ID NO:l by removing nucleotides 4 through 159 (corresponding to the native transit peptide) and without the stop codon, optimized for expression in Arabidopsis or soybean (dicots) or corn (a monocot), and encoding a CDOL peptide from Chlamydomonas reinhardtii (SEQ ID NO:5 minus amino acids 2 through 53); or

[0697] b. Derived from SEQ ID NO:2 by removing nucleotides 4 through 279 (corresponding to the native transit peptide) and without the stop codon, optimized for expression in Arabidopsis or soybean (dicots) or corn (a monocot), and encoding a CDOL peptide from Guillardia theta (SEQ ID NO:6 minus amino acids 2 through 93); or [0698] c. Derived from SEQ ID NO:3 by removing nucleotides 4 through 126 (corresponding to the native transit peptide) and without the stop codon, optimized for expression in Arabidopsis or soybean (dicots) or corn (a monocot), and encoding a CDOL peptide from Candida tenuis (SEQ ID NO:7 minus amino acids 2 through 42); or

[0699] d. Derived from SEQ ID NO:4 by removing nucleotides 4 through 180 (corresponding to the native transit peptide) and without the stop codon, optimized for expression in Arabidopsis or soybean (dicots) or corn (a monocot), and encoding a CDOL peptide from Fragilariopsis cylindrus (SEQ ID NO:8 minus amino acids 2 through 60).

[0700] The partCS/PLP-DC gene is as follows:

[0701] a. Derived from SEQ ID NO:9 by removing nucleotides 1 through 1413 (corresponding to the native transit and cysteine synthetase peptides), optimized for expression in Arabidopsis or soybean (dicots) or corn (a monocot), and encoding a partCS/PLP-DC peptide from Micromonas pusilla (SEQ ID NO: 15 minus amino acids 1 through 471); or

[0702] b. Derived from SEQ ID NO: 10 truncated by removing nucleotides 1 through 1068 (corresponding to the native transit and cysteine synthetase peptides), optimized for expression in Arabidopsis or soybean (dicots) or corn (a monocot), and encoding a partCS/PLP-DC peptide from Ostreococcus tauri (SEQ ID NO: 16 minus amino acids 1 through 356).

[0703] Step 2: Transform the DNA construct into Agrobacterium tumefaciens, select for antibiotic resistance, and confirm the presence of the DNA construct.

[0704] Step 3: Transform plant (Arabidopsis, soybean, corn, wheat, sugar beet, rice, camelina or canola), select for antibiotic resistance, and confirm the presence of the DNA constructs in the transgenic plant.

EXAMPLE 28

Develop a plant with taurine.

[0705] This example demonstrates the use of an CDOL fused to SADL with a linker (CDOL- linker-partCS/PLP-DC) (such as from Example #27) to produce hypotaurine and taurine in a seed. Transformed Arabidopsis plants were confirmed by selection and PCR analysis. Transgenic plants were grown in soil (Metro Mix 360) for 60 days. The growth conditions were maintained at 20-21°C, under cool white fluorescent lights (120 umol of photons per m 2 per s) with a 16-h light/8-h dark cycle. Plants were grown to maturity. Dried seeds were harvested. Free amino acids were extracted from the dry brown seeds to determine the level of taurine using high-performance liquid chromatography (HPLC). The results are shown in Figure 2 which shows hypotaurine and taurine levels in the transgenic line were 6.95% and 5.2%, respectively, of the total extracted free amino acids.

EXAMPLE 29

Develop aquafeed using plant tissue with taurine.

[0706] Grow plants from seed of transgenic plants that constitutively express CDOL fused to SADL with a linker (CDOL-linker-SADL) (such as from Example # 10). Collect the seeds, grind to a powder and use as an additive in feed.

EXAMPLE 30

Develop bacteria with taurine.

[0707] Grow bacteria with CS/PLP-DC (such as from Example #26) and induce gene expression with the appropriate inducer as suggested the vector. Collect the cells and confirm that the cells express the CS/PLP-DC peptide (-96.6 kDa) using western blot analysis and that have increased taurine using HPLC analysis.

EXAMPLE 31

Develop aquafeed using bacterial cells with taurine.

[0708] Grow bacteria with CS/PLP-DC (such as from Example #26) and induce gene expression with the appropriate inducer associated with the vector. Collect the cells and process for use as an additive to feed.

[0709] The use of the terms "a" and "an" and "the" and similar referents in the context of describing the invention (especially in the context of the following claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. The terms "comprising," "having," "including," and "containing" are to be construed as open-ended terms (i.e., meaning "including, but not limited to,") unless otherwise noted. Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., "such as") provided herein, is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention unless otherwise claimed. No language in the specification should be construed as indicating any non- claimed element as essential to the practice of the invention.

[0710] Embodiments of this invention are described herein, including the best mode known to the inventors for carrying out the invention. Variations of those embodiments may become apparent to those of ordinary skill in the art upon reading the foregoing description. The inventors expect skilled artisans to employ such variations as appropriate, and the inventors intend for the invention to be practiced otherwise than as specifically described herein. Accordingly, this invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above-described elements in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or otherwise clearly contradicted by context. Embodiments of this invention are described herein, including the best mode known to the inventors for carrying out the invention. Variations of those embodiments may become apparent to those of ordinary skill in the art upon reading the foregoing description. The inventors expect skilled artisans to employ such variations as appropriate, and the inventors intend for the invention to be practiced otherwise than as specifically described herein. Accordingly, this invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above- described elements in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or otherwise clearly contradicted by context.