METHODS FOR HIGH TAURINE PRODUCTION IN UNICELLULAR ORGANISMS

Abstract

The present invention describes an approach to produce or increase hypotaurine or taurine production in unicellular organisms. More particularly, the invention relates to genetic modification of unicellular organisms that include bacteria, algal, microalgal, diatoms, yeast, or fungi. The invention relates to methods to increase taurine levels in the cells by binding taurine or decreasing taurine degradation. The invention can be used in organisms that contain native or heterologous (transgenic) taurine biosynthetic pathways or cells that have taurine by enrichment. The invention also relates to methods to increase taurine levels in the cells and to use the said cells or extracts or purifications from the cells that contain the invention to produce plant growth enhancers, food, animal feed, aquafeed, food or drink supplements, animal-feed supplements, dietary supplements, health supplements or taurine.

Description

SEQUENCE SUBMISSION

[0001] The present application is being filed along with a Sequence Listing in electronic format. The Sequence Listing is entitled 3834118PCTSequenceListing.txt, created on 21 Apr. 2016 and is 106 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

[0002] The present invention is in the field of recombinant production of taurine. The present invention includes the production of taurine in unicellular organisms. Unicellular organisms include prokaryotic and single-cell eukaryotic organisms, bacteria, microbes, archaea, protozoa, yeast, unicellular algae and unicellular fungi. The invention also relates to methods to increase taurine levels in the cells by binding taurine or decreasing taurine degradation. The invention includes use in organisms that contain native or heterologous taurine biosynthetic pathways or cells that have taurine by enrichment. The invention also relates to methods to increase taurine levels in the cells and to use the said cells or extracts or purifications from the cells 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.

[0003] The publications and other materials used herein to illuminate the background of the invention or provide additional details respecting the practice, are incorporated by reference, and for convenience are respectively grouped in the Bibliography.

BACKGROUND OF THE INVENTION

Taurine is an Essential Compound for Animals

[0004] Taurine is essential for human neonatal development (1) and plays an important role in brain development (2, 3). Taurine is involved in the modulation of intracellular calcium homeostasis (4, 5) and may balance glutamate activity, protecting neurons against glutamate excitotoxicity (6, 7). Taurine is also an osmoregulator (8). Taurine is essential for heart function (9), protects the integrity of hepatic tissue (10), and plays a role in photoprotection (11).

Taurine as a Dietary Supplement

[0005] 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, (12, 13) human infants, (14) and carnivorous fish. (15-23) Taurine also improves the health and/or growth of other fish species (24-27) and shrimp. (28) Taurine is a feed attractant for fish. (20, 29)

Taurine as a Pharmaceutical or Therapeutic

[0006] Taurine is used as a pharmaceutical and therapeutic. Taurine has been used in the treatment of cardiovascular diseases, (30, 31) elevated blood pressure, (32) seizure disorders, (33) hepatic disorders, (34) and alcoholism (35) and may be useful in the treatment of diabetes, (36) Alzheimer's disease, (37) and ocular disorders. (38) Taurine has been shown to prevent obesity (39) and control cholesterol. (40, 41) Taurine acts as an antioxidant and protects against toxicity of various substances. (42-44) Taurine has been shown to prevent oxidative stress induced by exercise (45) and is used in energy drinks to improve performance. (46) Taurine can also be used in topical applications to treat dermatological conditions. (47)

Taurine as a Plant Growth Stimulator

[0007] Exogenous application of taurine has been reported to increase crop harvest, yield, and biomass. (48) Applications of taurine by foliar spray, soil and roots application, and seed immersion increase crop production and seedling growth. (48) Exogenous applications of taurine have also been shown to increase photosynthetic capacity of isolated plant cells (protoplasts and chloroplasts). (48)

Metabolic Pathways that Synthesize Taurine

[0008] Several metabolic pathways that synthesize taurine and hypotaurine have been identified in animals. The genes and their corresponding gene products and methods for the use of genes and the corresponding peptides to make taurine in cells have been described in the literature. (49-51) Briefly, 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-like 1 (GADL1). (52, 53) 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. (54)

[0009] A recent study has shown that several algal and microalgal species can synthesize taurine. (55) In addition, a recent invention identifies algal, microalgal, fungal, yeast, and diatoms genes and their corresponding peptides and describes their use to synthesize taurine in cells. (56) The genes and corresponding peptides 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 present invention could be used with these organisms and prior art to increase taurine levels in the cell.

Taurine Enrichment

[0010] Other studies have shown that multicellular organisms such as rotifers that contain no or low levels of taurine can be enriched with taurine by diffusion (dissolved method), (57-59) or with liposomes. (60) Taurine enrichment methods could also be used with unicellular organisms and in combination with the present invention to increase taurine levels in the cell.

Periplasmic-Binding or Taurine-Binding Proteins

[0011] In bacteria, periplasmic binding proteins or substrate-binding proteins, bind specific molecules as part of a multicomponent (peptide) system that is involved in the binding and transportation of specific molecules from the periplasmic space, outside, of the bacterium to the inside of the cell. (61-63) In the ABC transporter system, the substrate-binding protein delivers the bound molecule to transporter proteins on the bacterial membrane where the bound molecule is released into the cell in an energy-dependent manner. In the absence of membrane-bound proteins or energy-dependent releasing peptides (ATP-binding proteins) the substrate molecules remain bound to the substrate-binding protein. In the tripartite ATP-independent periplasmic (TRAP) transporter systems, the substrate-binding protein delivers the bound molecule to membrane bound protein complex (with two peptides) and releases the bound molecule into the cell in an A IP-independent process. In the absence of membrane-bound proteins the substrate molecule remains bound to the substrate-binding protein. Methods to increase pools of sulfonic acids, such as taurine, by expressing only the substrate-binding protein from an ABC transporter or TRAP system, TauA or TauK, respectively, in the cells has been described for use in plant tissues. (51, 64) The present invention describes methods to express substrate-specific binding proteins in the cell of a unicellular organism to increase taurine in the cell.

Sulfonic Acid or Taurine Degradation

[0012] In the absence of sulfur, bacteria utilize the sulfonic acid uptake and degradation pathway or the taurine uptake and degradation pathway to mobilize carbon, nitrogen or sulfur. (65-68). Genes and their corresponding peptides involved in the uptake and degradation of taurine are usually on the same operon and are induced in the absence of nitrogen (69, 70) or sulfur (65) or in the presence of taurine. (68, 71). The genes for the degradation enzymes and their corresponding gene products are the TauX and TauY genes (70) that encode taurine dehydrogenase (TDH), the TauD gene (65) that encodes taurine dioxygenase (TDO), the Tpa gene (72) that encodes taurine-pyruvate aminotransferase (TPAT) or the SssuDE (SsuD or SsuE) genes (66) that encode the two-component alkanesulfonate monooxygenase (2CASM).

Transcriptional Regulators

[0013] Translational regulators, Cbl or TauR, control the expression and induction of the taurine degradation pathways in bacteria. (65, 72) Cbl is a LysR-type transcriptional regulator of the sulfonic acid uptake and degradation pathway or the taurine uptake and degradation pathway in several bacteria. (73, 74) The Cbl gene is found in Proteobacteria including members of the Alphaproteobacteria, Betaproteobacteria, and Gammaproteobacteria. In bacteria that lack Cbl or Cbl-like transcriptional regulators there is a MocR subfamily of activators, which include TauR, that control the taurine uptake and degradation system. The TauR is found in Rhizobiales and Rhodobacterales of the Alphaproteobacteria, in Burkholderiaceae and Comamonadaceae of the Betaproteobacteria, in Enterobacteriales, Oceanospirillales and Psychromonadales from the Gammaproteobacteria, and in Rhizobiales and Rhodobacter of the Alphaproteobacteria. This invention describes how to decrease the expression of these genes or decrease the activities of their corresponding proteins in the cell of a unicellular organism to increase taurine in the cell.

SUMMARY OF THE INVENTION

[0014] The invention provides methods and compositions for taurine production in unicellular organisms. More particularly, the invention encompasses the use of polynucleotides for substrate-binding proteins, such as the TauA or TauK genes, to increase taurine in cells or the use of polynucleotides for peptides that degrade taurine. This invention describes methods to use cells with increased taurine pools of the sulfonic acids, such as taurine, by binding taurine in the cell with specific bacterial substrate-binding proteins or by blocking or inhibiting taurine degradation. This invention also describes approaches to block taurine degradation by methods of silencing, mutating or knocking out genes for enzymes in taurine degradation pathway(s) including the TauX or TauY genes that encode TDH, the TauD gene that encodes TDO, the SsuD or SsuE genes that encode 2CASM, or the Tpa gene that encodes TPAT, or by methods of silencing, mutating or knocking out the Cbl gene that encodes LysR-type transcriptional regulator or the TauR gene that encodes a MocR transcriptional regulator. This invention describes the use of polynucleotides for taurine-binding proteins or taurine degradation proteins and their corresponding peptides in unicellular organisms that are capable of producing taurine due to the presence of endogenous (native) or heterologous (gene transfer) taurine biosynthetic pathways or in cells enriched with taurine.

[0015] The invention also describes methods for the use of polynucleotides for substrate-binding proteins, such as the TauA or TauK genes, to produce peptides that bind taurine to increase taurine in cells in a unicellular organism that contains taurine by insertion of heterologous polynucleotides or genes (via insertion or transformation) from animal, algal, microalgal, fungal, yeast, diatom and unicellular organisms and their corresponding peptides for taurine synthesis in cells. The genes include CDO, CDOL, SAD, SADL, GADL1, CS/PLP-DC or partCS/PLP-DC.

[0016] This invention also describes approaches to block taurine degradation by methods of silencing, mutating or knocking out genes for enzymes in taurine degradation pathway(s) including the TauX or TauY genes that encode TDH, the TauD gene that encodes TDO, the SsuD or SsuE genes that encode 2CASM, or the Tpa gene that encodes TPAT, or by methods of silencing, mutating or knocking out genes for the Cbl gene that encodes LysR-type transcriptional regulator or the TauR gene that encodes a MocR transcriptional regulator. This invention also describes the use of polynucleotides for proteins that degrade taurine in a unicellular organism that contains taurine by insertion of heterologous polynucleotides or genes (via insertion or transformation) from animal, algal, microalgal, fungal, yeast, diatom, and unicellular organisms genes and their corresponding peptides for taurine synthesis in cells. The genes include CDO, CDOL, SAD, SADL, GADL1, CS/PLP-DC or partCS/PLP-DC.

[0017] This invention also describes the use of polynucleotides for substrate-binding proteins to increase taurine in cells and the use of methods and polynucleotides to silence, mutate or knock out genes for enzymes in taurine degradation pathway(s) in the same unicellular organism and contains taurine by insertion of heterologous polynucleotides or genes (via insertion or transformation) from animal, algal, microalgal, fungal, yeast, diatom and unicellular organisms genes and their corresponding peptides for taurine synthesis in cells. The genes include CDO, CDOL, SAD, SADL, GADL1, CS/PLP-DC or partCS/PLP-DC.

[0018] The invention provides methods for transforming unicellular organisms and constructing vector constructs and other nucleic acid molecules for use therein. The invention also provides methods for transforming unicellular organisms such as bacteria, yeast, fungi, and unicellular algae and constructing vector constructs and other nucleic acid molecules for use therein. The invention also provides methods for mutating the unicellular organisms such as bacteria, yeast, fungi, and unicellular algae and constructing vector constructs and other nucleic acid molecules for use therein. The transgenic or mutant 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.

[0019] In addition this invention describes methods to bind taurine in cells to increase taurine in unicellular organisms that produce taurine. (55) This invention describes methods to block taurine degradation by silencing, mutating or knocking out genes for enzymes in the taurine degradation pathway(s). The invention can be used to increase taurine in cells of unicellular organisms that produce taurine through a native or endogenous taurine (55) or heterologous pathway (50, 56) or in cells enriched with taurine. (57-60)

[0020] The invention provides isolated cells comprising DNA which does not express a functional taurine degradation enzyme, some isolated cells of the invention comprise (1) exogenous DNA which disrupts the expression of the gene or renders the corresponding peptide for the degradation enzyme non-functional (ii) a basepair mutation that disrupts the expression of the gene or renders the corresponding peptide for the degradation enzyme non-functional, or (iii) a deletion of the entire polynucleotide or a portion of the polynucleotide which disrupts the expression of the gene or renders the corresponding peptide for the degradation enzyme non-functional. The non-functional DNA could be due to changes in the promoter, a portion of the coding region, coding region, or terminator to a polynucleotide which encodes taurine degradation enzyme, that includes TauX, TauY, TauD, Tpa, SsuD, or SsuE or translational activators of those genes including Cbl or TauR genes in a manner where the genes products are not functional. The invention also provides isolated cells comprising non-functional genes or gene products of taurine degradation enzymes from the suppression or decreased accumulation of the corresponding RNA due to antisense RNA or RNA interference.

[0021] The invention provides isolated cells comprising exogenous DNA which expresses enzymes of taurine biosynthetic pathways and DNA which does not express a functional taurine degradation enzyme. In one embodiment, an isolated cell comprises three separate expression cassettes. A first expression cassette comprises a first promoter operably linked to a first polynucleotide, a second expression cassette comprises a second promoter operably linked to a second polynucleotide and a third cassette contains DNA which does not express a functional taurine degradation enzyme. In some embodiments, the first polynucleotide encodes CDO or CDOL and the second polynucleotide encodes SAD, SADL or GADL1. In other embodiments the first polynucleotide encodes CDO or CDOL and the second polynucleotide encodes CS/PLP-DC or partCS/PLP-DC. The third polynucleotide comprises the promoter, a portion of the coding region, coding region, or terminator to genes for a taurine degradation enzyme that does not express a functional TauX, TauY, TauD, Tpa, SsuD, or SsuE or translational activators including Cbl or TauR genes in a manner where the genes are not expressed or the gene products are not functional.

[0022] The invention provides isolated cells comprising exogenous DNA which expresses enzymes of taurine biosynthetic pathways and taurine binding protein. In one embodiment, an isolated cell comprises three separate expression cassettes. A first expression cassette comprises a first promoter operably linked to a first polynucleotide, a second expression cassette comprises a second promoter operably linked to a second polynucleotide and a third expression cassette comprises a third promoter operably linked to a third polynucleotide. In some embodiments, the first polynucleotide encodes CDO or CDOL and the second polynucleotide encodes SAD, SADL or GADL1. In other embodiments the first polynucleotide encodes CDO or CDOL and the second polynucleotide encodes CS/PLP-DC or partCS/PLP-DC. The third polynucleotide encodes a taurine binding protein (TauA or TauK).

[0023] Some isolated cells of the invention comprise exogenous DNA which comprises a single expression cassette and DNA which does not express a functional taurine degradation enzyme. In one embodiment, an isolated cell comprises one single expression cassette. The 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; (vi) CDOL operably linked to partCS/PLP-DC, (vii) CDO operably linked to SADL; (viii) CDO operably linked to CS/PLP-DC; (ix) CDO operably linked to partCS/PLP-DC; or (x) CDOL operably linked to SAD in a cell that comprises the promoter, coding region, or terminator to taurine degradation enzyme that does not express a functional TauX, TauY, TauD, Tpa, SsuD, or SsuE or translational activators including Cbl or TauR genes in a manner where the genes are not expressed or the gene products are not functional,

[0024] The invention provides isolated cells comprising exogenous DNA which expresses enzymes of taurine biosynthetic pathways and a taurine binding protein. 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 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 or GADL1; (v) CDOL operably linked to CS/PLP-DC; (vi) CDOL operably linked to partCS/PLP-DC, (vii) CDO operably linked to SADL or GADL1; (viii) CDO operably linked to CS/PLP-DC; (ix) CDO operably linked to partCS/PLP-DC; or (x) CDOL operably linked to SAD. The second polynucleotide comprises a promoter operably linked to a polynucleotide which encodes a taurine binding protein (TauA or TauK).

[0025] Some isolated cells of the invention comprise exogenous DNA which comprises a double expression which expresses enzymes of taurine biosynthetic pathways and taurine binding protein in a cell and DNA which does not express a functional taurine degradation enzyme. 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 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 or GADL1; (v) CDOL operably linked to CS/PLP-DC; (vi) CDOL operably linked to partCS/PLP-DC, (vii) CDO operably linked to SADL or GADL1; (viii) CDO operably linked to CS/PLP-DC; (ix) CDO operably linked to partCS/PLP-DC; or (x) CDOL operably linked to SAD. The second polynucleotide comprises a promoter operably linked to a polynucleotide which encodes a taurine binding protein (TauA or TauK) in a cell that comprises polynucleotide to the promoter, coding region, or terminator to taurine degradation enzyme that does not express a functional TauX, TauY, TauD, Tpa, SsuD, or SsuE or translational activators including Cbl or TauR genes in a manner where the genes are not expressed or the gene products are not functional.

[0026] The invention also describes how to use the cells, fractions of the cells, or extracts from the cells for the present invention 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 that either bind or degrade taurine.

[0027] The invention provides methods of increasing taurine in the cell of the invention by growing or treating the cell with an agent that increases sulfur or nitrogen concentration in the cell of the invention.

[0028] The invention also provides nutritional supplements, feed supplements, and pharmaceutical compositions comprising an extract or meal from the cell of the invention,

BRIEF DESCRIPTION OF THE FIGURE

[0029] FIG. 1 shows a unicellular organism (outer black rectangle) with genes and their corresponding taurine biosynthetic proteins (CDO, CDOL, SAD, SADL, GADL1, partCS/PLP-DC, or CS/PLP-DC) in relation to the known animal, yeast, fungal, or algal taurine biosynthetic pathways. Othrologs or paralogs of these genes may occur in some unicellular organisms such as algae. (55) In animals, cysteine and oxygen are converted into 3-sulfinoalanine by CDO. 3-sulfinoalanine is converted into hypotaurine by SAD or GADL1. The indicated genes could be heterologous gene(s) from animals, yeast, fungi, algae or microalgae transferred into the unicellular organism. Alternatively the indicated genes could be their orthologs or homologs that are native or endogenous to the unicellular organism. If there is no native taurine synthetic gene than the animals, yeast, fungi, algae or microalgae can be transferred into the unicellular organism. The technology to increase taurine in the unicellular organism is described in the dashed rectangle, these include (i) genes and the corresponding taurine-substrate binding protein (gray circle bound to Tau), (ii) silenced, mutated, or knocked-out genes (large gray X) for TauD (TDO), TauX or TauY (TDH), Tpa (TPAT), or SsuE or SsuF (2CASM) and their corresponding taurine degradation proteins, or ii) silenced, mutated or knocked-out (large gray X in open oval) genes for cbl or TauR, translational activators. In the absence of functional cbl or TauR gene products, translational activators for the expression of genes and their corresponding products for the taurine degradation pathway(s) (dashed lines) will not be induced or expressed.

DETAILED DESCRIPTION OF THE INVENTION

[0030] 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 unicellular organisms, with genes that encode proteins that bind taurine or with silenced or knocked out genes for taurine degradation. The invention also provides methods of using algae, microalgae, bacteria, fungi, yeast, or unicellular cellular organisms 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.

[0031] The present invention describes the methods for the synthesis of DNA constructs from polynucleotides and vectors and the methods for making transformed organisms including unicellular organisms, microbes, fungi, yeast, algae and microalgae that produce taurine due to the presence of peptides that bind taurine. The present invention is unique in that it describes a method to produce taurine that have advantages of enhanced taurine production and that result in cells with increased nutritional, pharmaceutical, or therapeutic value. The invention can be used in cells enriched with taurine, that contain a native taurine biosynthetic pathway(s), or that contain taurine from the insertion of a heterologous pathway by transformation or gene transfer.

[0032] The present invention describes the methods for the synthesis of DNA constructs to inhibit taurine degradation from polynucleotides and vectors and the methods for making transformed organisms including unicellular organisms, microbes, fungi yeast, algae and microalgae. The present invention is unique in that it describes a method to produce taurine that has advantages of enhanced taurine production or hypotaurine and that result in cells with increased nutritional, pharmaceutical, or therapeutic value

[0033] 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 unicellular organisms, microbes, fungi yeast, algae and microalgae that produce taurine due to the presence of peptides that bind and do not degrade taurine. The present invention is unique in that it describes a method to produce taurine that has advantages of enhanced taurine production or hypotaurine and that result in cells with increased nutritional, pharmaceutical, or therapeutic value.

[0034] The present invention describes the insertion of the polynucleotides that encode functional taurine binding proteins (TauA or TauK) or polynucleotides silenced or knocked-out genes for proteins involved in taurine degradation (TauD, SsuD, SsuE, TauX, TauY, or Tpa) or transcriptional regulators (cbl or TauR) for taurine degradation in unicellular organisms, or their use in taurine biosynthetic pathway in unicellular 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 CDO, CDOL, SAD, SADL, GADL1, partCS/PLP-DC, or CS/PLP-DC in unicellular organisms. The preferred embodiment of the invention is in bacteria but other organisms may be used.

Enzymes of Taurine Biosynthetic Pathways

[0035] Examples of amino acid sequences of enzymes of taurine biosynthetic pathways are provided in the sequence listing: SEQ ID NO:2 (CDO), SEQ ID NO:4 (CDOL), SEQ ID NO:6 (SAD), SEQ ID NO:8 (SADL), SEQ ID NO:10 (GADL1), and SEQ ID NO:12 (CS/PLP-DC). 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 CDO, SAD, or GADL from Danio rerio, CDOL from Chlamydomonas reinhardtii, SADL from Guillardia theta, or CS/PLP-DC from Micromonas pusilla may differ to a certain degree from the amino acid sequences of CDO, CDOL, SAD, SADL, GADL1, 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.

Substrate Binding Proteins

[0036] Examples of amino acid sequences of substrate binding proteins or periplasmic binding proteins that bind taurine are provided in the sequence listing: SEQ ID NO:17 (TauA) and SEQ ID NO:19 (TauK), 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 TauA from Escherichia coli or TauK from Roseobacter denitrificans may differ to a certain degree from the amino acid sequences of TauA or TauK 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.

Enzymes of Taurine Degradation Pathways

[0037] Examples of amino acid sequences of substrate binding proteins or periplasmic binding proteins that bind taurine are provided in the sequence listing: SEQ ID NO:21 (TDO), SEQ ID NO:23 or SEQ ID NO:27 (SsuD), SEQ ID NO:25 or SEQ ID NO:29 (SsuE), SEQ ID NO:31 (TauX), SEQ ID NO:33 (TauY) and SEQ ID NO:35 (Tpa). 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 TDO, SsuD or SsuE from Escherichia coli, SsuD or SsuE from Corynebacterium glutamicum, TauX, TauY, or Tpa from Roseobacter denitrificans may differ to a certain degree from the amino acid sequences of TDO, SsuD, SsuE, TauX, TauY, or Tpa 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.

Translational Regulators

[0038] Examples of amino acid sequences of translational regulators are provided in the sequence listing: SEQ ID NO:37 or SEQ ID NO:39 (cbl), or SEQ ID NO:41 or SEQ ID NO:43 (TauR). 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 cbl from Escherichia coli, or cbl from Corynebacterium glutamicum or TauR from Corynebacterium glutamicum or Rhodobacteraceae species may differ to a certain degree from the amino acid sequences of cbl or TauR 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.

[0039] 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.

[0040] 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, CDO, CDOL, SAD, SADL, GADL1, CS/PLP-DC, TauD (TDO), TauX or TauY (TDH), Tpa (TPAT), SsuD or SsuE (2CASM), cbl, or TauR activity is generally at least 40%, 50%, 60%, 70%, 80% or 90%.COPYRGT., preferably 60-90% of the native protein for the native substrate.

[0041] The following three groups each contain amino acids that are conserved substitutions for one another: (1) Alanine (A), Serine (S), Threonine (1); (2) Aspartic acid (D), Glutamic acid (E); and (3) Asparagine (N), Glutamine (Q).

Suitable Polynucleotides for CDO, CDOL, SAD, SADL, GADL1, CS/PLP-DC, TauA, TauK, SsuD, SsuE, TauX, TauY, Tpa, cbl and TauR

[0042] As examples, suitable polynucleotides encoding enzymes of taurine biosynthetic and degradation pathways, taurine specific substrate binding proteins, and translational regulators of taurine degradation 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 recombinant protein with CDO, CDOL, SAD. SADL, GADL1, or CS/PLP-DC activity in a unicellular organism with a taurine-binding protein or lacks degradation taurine pathway(s) or lacks regulators of the degradation taurine pathway.

[0043] A suitable polynucleotide for CDO is provided in SEQ ID NO:1. 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:1 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:1 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:2 when it used as a reference for sequence comparison.

[0044] A suitable polynucleotide for CDOL is provided in SEQ ID NO:3. 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:3 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:3 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:4 when it used as a reference for sequence comparison.

[0045] A suitable polynucleotide for SAD is provided in SEQ ID NO:5. 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:5 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:5 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:6 when it is used as a reference for sequence comparison.

[0046] A suitable polynucleotide for SADL is provided in SEQ ID NO:7. 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:7 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:7 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:8 when it is used as a reference for sequence comparison.

[0047] A suitable polynucleotide for GADL1 is provided in SEQ ID NO:9. 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 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 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:10 when it used as a reference for sequence comparison.

[0048] A suitable polynucleotide for CS/PLP-DC is provided in SEQ ID NO:11. 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 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 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:12 when it used as a reference for sequence comparison.

[0049] Suitable polynucleotides for a taurine-binding protein are provided in SEQ ID NO:16 and SEQ ID NO:18. 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:16 or SEQ ID NO:18 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:16 or SEQ ID NO:18 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 or SEQ ID NO:19 when it used as a reference for sequence comparison.

[0050] A suitable polynucleotide for TDO is provided in SEQ ID NO:20. 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:20 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:20 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:21 when it used as a reference for sequence comparison.

[0051] Suitable polynucleotides for a SsuD are provided in SEQ ID NO:22 and SEQ ID NO:26. 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:22 or SEQ ID NO:26 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:22 or SEQ ID NO:26 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:23 or SEQ ID NO:27 when it used as a reference for sequence comparison.

[0052] Suitable polynucleotides for a SsuE are provided in SEQ ID NO:24 and SEQ ID NO:28. 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:24 or SEQ ID NO:28 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:24 or SEQ ID NO:28 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:25 or SEQ ID NO:29 when it used as a reference for sequence comparison.

[0053] A suitable polynucleotide for TauX is provided in SEQ ID NO:30. 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:30 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:30 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:31 when it used as a reference for sequence comparison.

[0054] A suitable polynucleotide for TauY is provided in SEQ ID NO:32. 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:32 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:32 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:33 when it used as a reference for sequence comparison.

[0055] A suitable polynucleotide for Tpa is provided in SEQ ID NO:34. 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:34 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:34 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:35 when it used as a reference for sequence comparison.

[0056] Suitable polynucleotides for a cbl are provided in SEQ ID NO:36 and SEQ ID NO:38. 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:36 or SEQ ID NO:38 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:36 or SEQ ID NO:38 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:37 or SEQ ID NO:39 when it used as a reference for sequence comparison.

[0057] A suitable polynucleotide for TauR is provided in SEQ ID NO:40 and SEQ ID NO:42. 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:40 or SEQ ID NO:42 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:40 or SEQ ID NO:42 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:41 or SEQ ID NO:43 when it used as a reference for sequence comparison.

[0058] Another embodiment of the invention is a polynucleotide (e.g., a DNA construct) that encodes a protein that functions as a CDO, CDOL, SAD, SADL, GADL1, CS/PLP-DC, TauA, TauK, TauD, SsuD, SsuE, TauX, TauY, Tpa, cbl and TauR selectively hybridizes to either SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:40 or SEQ ID NO:42, 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.

[0059] 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:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:41 or SEQ ID NO:43. 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%.

[0060] 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.

[0061] 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.

[0062] 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.

[0063] 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.

[0064] 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 different species, sequences can be modified to account for the specific codon preferences and GC-content preferences of the organism, as these preferences have been shown to differ. (75-80)

Cloning Techniques

[0065] 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, mycology, physiology, tissue culture, molecular biology, chemistry, biochemistry, biotechnology, and recombinant DNA technology, which are within the skill of the art. (81-88)

[0066] 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, algae, microalgae, diatoms, yeast or fungi 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 (PCR) procedures, using as probes or primers nucleotide sequences selected in accordance with the invention.

[0067] 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 PCR and adjacent fragments can be amplified together using fusion-PCR, (89) overlap-PCR (90) or chemical (de novo) synthesis (91-95) using a vendor (e.g. DNA2.0, GE life technologies, GENEART, Gen9, GenScript) by methods known in the art.

[0068] 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 PCR, 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 fused together by recombination.

[0069] The recombinant expression cassette or DNA construct includes a promoter that directs transcription in an unicellular organism, operably linked to the polynucleotide encoding a CDO, CDOL, SAD, SADL, GADL1, CS/PLP-DC, partCS/PLP-DC, TauA, or TauK. 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.

[0070] 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

[0071] 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. (96, 97) Other suitable linkers that can be used in the invention include, but are not limited to, those described by Kuusinen et. al. (98) Robinson and Sauer, (99) Armstrong & Gouaux, (100) Arai et. al., (101) Wriggers et. al., (102) and Reddy et. al. (103)

Suitable Promoters

[0072] 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 unicellular organisms can be used in connection with the present invention. (104-106) The features (binding sites and regulatory elements) necessary for the identification and use of functional bacterial promoters are known to those of ordinary skill in the art (107-109) For purposes of describing the present invention, promoters are divided into two types, namely, constitutive promoters and non-constitutive promoters. (105, 110) Constitutive promoters are classified as providing for a range of constitutive expression. Some are weak constitutive promoters, and others are strong constitutive promoters. (111) Other promoters are considered non-constitutive promoters. (112-116) A selected promoter 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. The promoter may be of viral origin, including a cauliflower mosaic virus promoter (CaMV 35S), (111) and SV40 promoters from viruses have been used to express target genes. (117) 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 unicellular organism 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 unicellular organism 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 unicellular organism to provide a transformed organism that expresses the polynucleotide in response to a signal. 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 the gene for the desired polypeptide of the invention in response to a signal, such as a chemical or environmental cue or other stress signal including biotic and/or abiotic stresses or other conditions.

Plastid Transit Peptides

[0073] 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 CDO, CDOL, SAD, SADL, GADL1, CS/PLP-DC, partCS/PLP-DC, TauA, or TauK polypeptide to a plastid include, but are not limited, to those described herein and in U.S. Pat. Nos. 8,779,237, (118) 8,674,180 (119), 8,420,888 (120), and 8,138,393 (121) and in Lee et al. (122) and von Heijne et al, (123) Identification and use of chloroplast plastid targeting sequences for algae are known to those of ordinary skill in the art. (124-127) 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 known to those of ordinary skill in the art.

Plastid Transit Peptides

[0074] The invention can be targeted for transformation into the chloroplast. Chloroplast targeted transformation systems for algae are known by those of ordinary skill in the art. (113, 115, 128-130)

Suitable Vectors

[0075] A wide variety of vectors may be employed to transform a unicellular organism with a construct made or selected in accordance with the invention, including high- or low-copy number plasmids, phage vectors and cosmids. Vector systems, expression cassettes, culture methods, and transformation methods are known by those of ordinary skill in the art. 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 unicellular organism. Other vectors that can operably link promoter and polynucleotides that encode the polypeptide of the invention are incorporated are not incorporated into the host genome but the vector DNA with the clone polynucleotides are autonomously or semi autonomously replicated in the cell, Although the preferred embodiment of the invention is expressed in bacteria, other embodiments may include expression in prokaryotic or unicellular eukaryotic organisms including, but not limited to, yeast, fungi, algae, microalgae, or microbes.

[0076] 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 unicellular organism. Standard molecular and cloning techniques (85, 88, 131) are available to make a recombinant expression cassette that expresses the polynucleotide that encodes the desired polypeptide of the invention. No attempt will be made to describe in detail the various methods known for the expression of proteins in prokaryotes or eukaryotes. 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, to direct transcription, a ribosome-binding site for translational initiation, and a transcription/translation terminator.

Expression in Prokaryotes

[0077] Protocols for transformation as well as commonly used vectors with control sequences including promoters for transcription initiation (some with an operator), together with ribosome binding site sequences for use in prokaryotes are known to those of ordinary skill in the art. 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, (132) lactose, (132) and tryptophan (133) 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. (134-138) In bacteria one messenger RNA can encode for one peptide (referred to as monocistronic) or several independent peptides (referred to as polycistronic). It is known to those of ordinary skill in the art that a portion of a polycistronic messenger RNA can be knocked-out (139) or that heterologous or exogenous genes can be expressed on a monocistronic or polycistronic messenger RNA. (137, 138) Genes can be expressed by modification of bacterial DNA (genomic) through the use of knock-in, gene insertion, or by allelic exchange. (140-145) Specific gene targeting has been used in bacteria using PCR-based methods, (146) and CRISPR/Cas (147-149)

Expression in Algae and Microalgae

[0078] Protocols for transformation as well as commonly used vectors with control sequences include promoters for transcription initiation, optionally with an operator, together with ribosome binding site sequences for use in algae and microalgae are known to those of ordinary skill in the art. (105, 128, 150-160). Specific gene targeting systems have been used in algae including ZFNs (161) and transcription activator-like effector nucleases (TALENs). (162)

Expression in Non Plant Eukaryotes

[0079] Protocols for transformation, as well as commonly used vectors, are known to those of ordinary skill in the art. Also known to those of ordinary skill in the art are control sequences that include promoters for transcription initiation and ribosome binding site sequences for use in unicellular eukaryotes. The present invention can be expressed in a variety of eukaryotic expression systems such as yeast and protozoa. 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. (163, 164) There are numerous vectors that can be used with the invention that are known to those of ordinary skill in the art and include, but are not limited to, pREP, pRIP, pD912, pD1201, pD1211, pD1221, pD1231, pYES2/NT, pYSG-IBA, or pESC-TRP. Synthesis of heterologous proteins and fermentation of products in yeast is known to those of ordinary skill in the art. (165, 166) Protozoa that can be used include, but are not limited to, ciliates, amoebae and flagellates. Yeast and fungi that can be used with the invention and the molecular protocols for transformation, and the vectors required for expression of genes in these systems, are known to those of ordinary skill in the art. (167-172) A range of vectors is available. Also available are plasmid vectors, which may be integrative, autonomously replicating high copy-number vectors, or autonomously replicating low copy number vectors. (173, 174) The most common vectors that complement a chromosomal mutation in the host include functional genes such as URA3, HIS3, LEU2, TRP1 and LYS2. Specific gene editing or targeting has been used in unicellular fungi using PCR-based methods, (175-177) Zinc-finger nucleases (ZFNs), (178) transcription activator like effector nucleases (TALENs), (179) and clustered regularly interspaced short palindromic repeats/Cas (CRISPR/Cas). (180, 181)

[0080] One of ordinary skill in 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 known to those of ordinary 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.

[0081] In addition to the selection of a suitable promoter, the DNA constructs require an appropriate transcriptional terminator to be attached downstream of the desired gene of the invention for proper expression in unicellular organisms. Several such terminators are available and known to persons of ordinary skill in the art. These include, but are not limited to, the tml from CaMV and E9 from rbcS. A variety of available terminators known to function in unicellular organisms can be used in the present 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. Selectable markers usually confer resistance to an antibiotic, herbicide or chemical or provide color change, which aid the identification of transformed organisms. The vectors may also include a RNA stability signal, which are 3'-regulatory sequence elements that increase the stability of the transcribed RNA. (182, 183)

Terminators

[0082] 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 and may also control gene expression. (184-193) The identification and use of terminators that are required to express genes in unicellular organisms are known to those of ordinary skill in the art.

[0083] In addition, polynucleotides that encode a CDO, CDOL, SAD, SADL, partCS/PLP-DC or CS/PLP-DC can be placed in the appropriate vector used to transform unicellular organisms. The polypeptide can be expressed and then isolated from transformed cells, or metabolites can be synthetized and isolated from the transformed cells. Such transgenic organisms can be harvested, and subjected to large-scale protein or metabolite (taurine) extraction and purification techniques.

[0084] The vectors may include another polynucleotide insert that encodes a peptide or polypeptide and 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 PCR 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 CDO, CDOL, SAD, SADL, GADL1, CS/PLP-DC, partCS/PLP-DC, TauA, or TauK 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.

[0085] 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 vacuole, chloroplast (not in fungi), mitochondrion, peroxisomes, secretory pathway, lysosome, endoplasmic reticulum, nucleus or Golgi apparatus in fungi or algae. There are specific signal polypeptides or signal sequences to direct peptide transport to the periplasmic space in bacteria. (194-196) 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 (123, 197-199), C-terminus (200, 201) or internal (202-204) or tertiary structure. (204) 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 (205, 206), iPSORT (207), SignalP (208), PrediSi (209), ELSpred (210) HSLpred (211) and PSLpred (212), MultiLoc (213), SherLoc (214), ChloroP (215), MITOPROT (216), Predotar (217) 3D-PSSM (218) and PredAlgo. (127) Additional methods and protocols are discussed in the literature. (213)

Transformation of Host Cells

[0086] Transformation of an unicellular organism can be accomplished in a wide variety of ways within the scope of a person of ordinary skill in the art. (104, 106, 158, 219) Those of ordinary skill in the art can use different algal, diatom, fungal, yeast and bacteria gene transfer techniques that include, but not limited to, Agrobacterium-mediated (220) glass beads and polyethylene glycol (PEG), (221, 222) electroporation, (223-226) microprojectile bombardment or ballistic particle acceleration, (227-231) silicon carbide whisker methods, (232, 233), viral infection, (234, 235) or transposon/transposase complexes. (236) Transformation can be targeted to organellular genomes. (130) Other methods to edit, incorporate or move genes into bacteria, fungal algal genomes include, but are not limited to, Zinc-finger nucleases (ZFNs), transcription activator like effector nucleases (TALENs), or clustered regularly interspaced short palindromic repeats/Cas (CRISPR/Cas).

Gene Silencing by Mutagenesis or Using Recombinant Technologies

[0087] Genetic modification to silence or inactivate genes or their corresponding gene products of unicellular organisms can be conducted by radiation-, chemical- or UV-based mutagenesis followed by specific screening for biochemical traits or pathways. (219, 237-241) Radiation-based mutations can silence or inactive a gene or the corresponding gene product by DNA breakage and repair. Chemical- or UV-based mutations usually result in single DNA basepair changes. Mutations can silence or inactive a gene or the corresponding gene product by one of the following (1) result in the introduction of a frame-shift mutation; (2) result in the introduction of premature stop codon; (3) interfere with the ability of the promoter region sequence to direct the transcription of the desired nucleotide sequence, (4) interfere with the ability of the desired nucleotide sequence to be transcribed by the promoter sequence region or (5) introduce amino acid substitution in the gene product to reduce or inhibit activity (enzymatic activity or binding) or interfere with the function of the gene product.

[0088] Targeted gene silencing or knockouts can be made in unicellular organisms using phage or viruses, (110, 242-246) transposons, (236, 247-250) PCR-assisted targeting, (175-177, 251) recombinases or by allelic exchange. (140-145) targeted and random bacterial gene disruptions using a group II intron (Targetron), (252, 253) ZNFs, (178) TALENs, (179) CRISPER-Cas9 or clustered regularly interspaced short palindromic repeats interference (CRISPi). (147-149, 180, 181, 254, 255) In addition, RNA-mediated methods, (256-261) or regulatory RNAs (262-264) have been used to silence or suppress gene expression in unicellular organisms and these techniques and protocols are well known to one with ordinary skill in the art.

Suitable Unicellular Organisms

[0089] A wide variety of unicellular host cells may be used in the invention, including prokaryotic and unicellular eukaryotic host cells. These cells or organisms may include yeast, fungi, algae, microalgae, microbes, or unicellular photosynthetic organisms. Preferred host cells for this invention are bacteria including, archaebacteria and eubacteria. Proteobacteria such as members of Alphaproteobacteria, Betaproteobacteria, Gammaproteobacteria, Deltaproteobacteria, and Epsilonproteobacteria can host the invention. Other bacteria including methanotrophs (265) can be used with the invention. Other bacterial genera that can host the invention include, but are not limited to Bacillus, Salmonella, Lactococcus, Streptococcus, Brevibacterium and coryneform bacteria. Some specific bacterial species that can be used for the invention include, but are not limited to, Bacillus subtilis, Brevibacterium ammoniagene, Corynebacterium crenatum, Corynebacterium pekinese, Corynebacterium glutamicumas, Erwinia citreus, Erwinia herbicola, Escherichia coli, Fusarium venenatum, Gluconobacter oxydans, Propionibacterium freudenreicheii, and Propionibacterium denitrificans. (266).

[0090] Unicellular algae, unicellular photosynthetic organisms, and microscopic algae (microphytes or microalgae) cells may be used in the invention. These include, but are not limited to diatoms, green algae (Chlorophyta), and members of the Euglenophyta, Dinoflagellata, Chrysophyta, Phaeophyta, red algae (Rhodophyta), Heterokontophyta, and Cyanobacteria. The invention can also be used to increase the taurine by binding taurine with a taurine binding protein or knocking out genes for taurine degradation in algae that have been shown to synthesize taurine (55) or may have the capability to synthesize taurine. (55) These include but are not limited to Coccomyxa species, Chlorella species, Trebouxia impressa, Tetraselmis species, Chlamydomonas reinhardtii, Micromonas pusilla, Ostreococcus tauri, Navicula radiosa, Phaeodactylum tricornutum, Pseudo-nitzschia multiseries, Fragilariopsis cylindrus, Thalassiosira weissflogii, Nannochloropsis oceanica, Aureococcus anophagefferens, Saccharina japonica, Sargassum species and Bigelowiella natans.

[0091] Protozoa that may be used in the invention include, but are not limited, to ciliates, amoebae and flagellates. Yeast and unicellular 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 Saccharomyces pombe.

[0092] One embodiment of the invention (Embodiment number 1) is a method for the increased production of taurine in an unicellular organism by the following steps:

[0093] 1. operably link a promoter to the 5' end of a polynucleotide for a functional SAD (using SAD, SADl, GADL, partCS/PLP-DC, or CS/PLP-DC) operably linked to a terminator;

[0094] 2. insert the functional SAD construct (from step 1, Embodiment number 1) into a vector;

[0095] 3. operably link a promoter to the 5' end of the polynucleotide for a truncated functional Tau-binding protein (using TauA or TauK) operably linked to a teitninator;

[0096] 4. insert the taurine-binding protein polynucleotide construct (from step 3, Embodiment number 1) into a vector containing the functional SAD construct (from step 2, Embodiment number 1); and

[0097] 5. transform the vector containing the SAD and taurine-binding protein (from step 4, Embodiment number 1) constructs into a unicellular organism.

[0098] Another embodiment of the invention (Embodiment number 2) is a method for the increased production of taurine in a unicellular organism by the following steps:

[0099] 1. operably link a promoter to the 5' end of the polynucleotide for a functional CDO (using CDO or CDOL) operably linked to a terminator;

[0100] 2. insert the functional CDO polynucleotide construct (from step 1, Embodiment number 2) into a vector;

[0101] 3. insert the functional SAD construct (from step 1, Embodiment number 1) into a vector containing the functional CDO construct (from step 2, Embodiment number 2);

[0102] 4. insert the taurine-binding protein polynucleotide construct (from step 3, Embodiment number 1) into a vector containing the functional CDO and SAD constructs (from step 3, Embodiment number 2); and

[0103] 5. transform the vector containing the functional CDO, SAD, and Tau-binding protein constructs (from step 4, Embodiment number 2) constructs into a unicellular organism.

[0104] Another embodiment of the invention (Embodiment number 3) is a method for the increased production of taurine in a unicellular organism by the following steps:

[0105] 1. insert the taurine-binding protein polynucleotide construct (from step 3, Embodiment number 1) into a vector; and

[0106] 2. transform the vector containing the taurine-binding protein construct (from step 1, Embodiment number 3) into a unicellular organism.

[0107] Another embodiment of the invention (Embodiment number 4) is a method for the increased production of taurine in a unicellular organism by the following steps:

[0108] 1. operably link a promoter to the 5' end of the polynucleotide for a functional CDO (using either CDO or CDOL) that is linked in-frame, with no linker, with a polynucleotide for a functional SAD (using SAD, SADl, GADL, partCS/PLP-DC, or partCS/PLP-DC) operably linked to a terminator;

[0109] 2. insert the CDO/SAD construct (from step 1, Embodiment number 4) into a vector that contains the functional taurine-binding protein (from step 2, Embodiment number 3); and

[0110] 3. transform the vector containing the functional CDO/SAD and taurine-binding protein constructs (from step 2, Embodiment number 4) into a unicellular organism.

[0111] Another embodiment of the invention (Embodiment number 5) is a method for the increased production of taurine in a unicellular organism by the following steps:

[0112] 1. operably link a promoter to the 5' end of the polynucleotide for functional CDO (using CDO or CDOL) that is linked in-frame with a short, 3 to 66, polynucleotide (linker) to the 5' end of the polynucleotide for a functional SAD (using SAD, SADL, GADL1, partCS/PLP-DC, or CS/PLP-DC) operably linked to a terminator;

[0113] 2. insert the taurine-binding protein construct (from step 3 above, Embodiment number 1) into a vector containing the CDO/Linker/SAD construct (from step 1, Embodiment number 5); and

[0114] 3. transform the vector containing the functional CDO/Linker/SAD and Tau-binding protein constructs (from step 2, Embodiment number 5) into a unicellular organism.

[0115] Another embodiment of the invention (Embodiment number 6) is a method for the increased production of taurine in a unicellular organism by the following step:

[0116] 1. knockout the gene for a taurine degradation enzyme using chemical or genetic means by replacement or deletion of a promoter, a portion of the coding region, or terminator to one of the following genes, TauX, TauY, TauD, Tpa, SsuD, or SsuE genes using a pSC101.sub.ts-sacB, allelic exchange or .lamda.-red recombinase method in a unicellular organism; and

[0117] 2. transform the vector containing the SAD (from step 2, Embodiment number 1) constructs into the unicellular organism with the mutation or knocked-out TauX, TauY, TauD, Tpa, SsuD, or SsuE gene (from step 2, Embodiment number 6).

[0118] Another embodiment of the invention (Embodiment number 7) is a method for the increased production of taurine in a unicellular organism by the following step:

[0119] 1. transform the vector containing the SAD and taurine-binding protein construct (from step 4, Embodiment number 1) into the unicellular organism with the mutated or knocked-out TauX, TauY, TauD, Tpa, SsuD, or SsuE gene (from step 2, Embodiment number 6).

[0120] Another embodiment of the invention (Embodiment number 8) is a method for the increased production of taurine in a unicellular organism by the following step:

[0121] 1. transform the vector containing the functional CDO, SAD, and taurine-binding protein constructs (from step 5, Embodiment number 2) constructs into the unicellular organism with the mutated or knocked-out TauX, TauY, TauD, Tpa, SsuD, or SsuE gene (from step 2, Embodiment number 6).

[0122] Another embodiment of the invention (Embodiment number 9) is a method for the increased production of taurine in a unicellular organism by the following step:

[0123] 1. transform the vector containing the taurine-binding protein construct (from step 1, Embodiment number 3) into the unicellular organism with the mutated or knocked-out TauX, TauY, TauD, Tpa, SsuD, or SsuE gene (from step 2, Embodiment number 6).

[0124] Another embodiment of the invention (Embodiment number 10) is a method for the increased production of taurine in a unicellular organism by the following step:

[0125] 1. transform the vector containing the functional CDO/SAD construct (from step 2, Embodiment number 4) into the unicellular organism with the mutated or knocked-out TauX, TauY, TauD, Tpa, SsuD, or SsuE gene (from step 2, Embodiment number 6).

[0126] Another embodiment of the invention (Embodiment number 11) is a method for the increased production of taurine in a unicellular organism by the following step:

[0127] 1. transform the vector containing the CDO/SAD construct and taurine-binding protein (from step 3, Embodiment number 4) into the unicellular organism with the mutated or knocked-out TauX, TauY, TauD, Tpa, SsuD, or SsuE gene (from step 2, Embodiment number 6).

[0128] Another embodiment of the invention (Embodiment number 12) is a method for the increased production of taurine in a unicellular organism by the following step:

[0129] 1. transform the vector containing the CDO/Linker/SAD construct (from step 1, Embodiment number 5) into the unicellular organism with the mutated or knocked-out TauX, TauY, TauD, Tpa, SsuD, or SsuE gene (from step 2, Embodiment number 6).

[0130] Another embodiment of the invention (Embodiment number 13) is a method for the increased production of taurine in a unicellular organism by the following step:

[0131] 1. transform the vector containing the CDO/Linker/SAD and taurine-binding protein constructs (from step 2, Embodiment number 5) into the unicellular organism with the mutated or knocked-out TauX, TauY, TauD, Tpa, SsuD, or SsuE gene (from step 2, Embodiment number 6).

[0132] Another embodiment of the invention (Embodiment number 14) is a method for the increased production of taurine in a unicellular organism by the following steps:

[0133] 1. introduce a mutation or knock out the gene for the transcription regulator of the taurine degradation pathways using chemical or genetic means by replacement or deletion of a promoter, a portion of the coding region, or terminator to one of the following genes, cbl, or TauR genes using a pSC101.sub.ts-sacB, allelic exchange or -red recombinase method and select the mutant or knocked-out unicellular organism; and

[0134] 2. transform the vector containing the SAD (from step 2, Embodiment number 1) construct into the unicellular organism with the mutated or knocked-out cbl or TauR gene (from step 2, Embodiment number 14).

[0135] Another embodiment of the invention (Embodiment number 15) is a method for the increased production of taurine in a unicellular organism by the following step:

[0136] 1. transform the vector containing the SAD and taurine-binding protein construct (from step 4, Embodiment number 1) into the unicellular organism with the mutated or knocked-out cbl or TauR gene (from step 2, Embodiment number 14).

[0137] Another embodiment of the invention (Embodiment number 16) is a method for the increased production of taurine in a unicellular organism by the following step:

[0138] 1. transform the vector containing the functional CDO and SAD constructs (from step 3, Embodiment number 2) constructs into the unicellular organism with mutated or knocked-out cbl or TauR gene (from step 2, Embodiment number 14).

[0139] Another embodiment of the invention (Embodiment number 17) is a method for the increased production of taurine in a unicellular organism by the following step:

[0140] 1. transform the vector containing the functional CDO, SAD, and taurine-binding protein constructs (from step 5, Embodiment number 2) constructs into the unicellular organism with mutated or knocked-out cbl or TauR gene (from step 2, Embodiment number 14).

[0141] Another embodiment of the invention (Embodiment number 18) is a method for the increased production of taurine in a unicellular organism by the following step:

[0142] 1. transform the vector containing the taurine-binding protein construct (from step 1, Embodiment number 3) into an unicellular organism into the unicellular organism with the mutated or knocked-out cbl or TauR gene (from step 2, Embodiment number 14)

[0143] Another embodiment of the invention (Embodiment number 19) is a method for the increased production of taurine in a unicellular organism by the following step:

[0144] 1. transform the vector containing the functional CDO/SAD construct (from step 2, Embodiment number 4) into the unicellular organism with mutated or knocked-out cbl or TauR gene (from step 2, Embodiment number 14).

[0145] Another embodiment of the invention (Embodiment number 20) is a method for the increased production of taurine in a unicellular organism by the following step:

[0146] 1. transform the vector containing the CDO/SAD construct and taurine-binding protein (from step 3, Embodiment number 4) into the unicellular organism with the mutated or knocked-out cbl or TauR gene (from step 2, Embodiment number 14).

[0147] Another embodiment of the invention (Embodiment number 21) is a method for the increased production of taurine in a unicellular organism by the following step:

[0148] 1. transform the vector containing the CDO/Linker/SAD construct (from step 1, Embodiment number 5) into the unicellular organism with the mutated or knocked-out cbl or TauR gene (from step 2, Embodiment number 14).

[0149] Another embodiment of the invention (Embodiment number 22) is a method for the increased production of taurine in a unicellular organism by the following step:

[0150] 1. transform the vector containing the CDO/Linker/SAD and Tau-binding protein constructs (from step 2, Embodiment number 5) into the unicellular organism with the mutated or knocked-out chi or TauR gene (from step 2, Embodiment number 14).

[0151] Once transformed, the unicellular organism may be treated with other "active agents" either prior to or during the growth to further increase production of taurine. "Active agent," as used herein, refers to an agent that has a beneficial effect on the taurine or amino acid production by the unicellular organism. Some of these agents may be precursors of end products for the reaction catalyzed by CDO, CDOL, SAD, SADL, GADL1, 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 sulfite, sulfide, hydrogen sulfide, sulfate, taurine, hypotaurine, 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 CDO, CDOL, SAD, SADL, GADL1, 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 CDO, CDOL, SAD, SADL, GADL1, 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.

[0152] In some embodiments properties of a transgenic unicellular organism are altered using an agent which increases sulfur concentration in the cell, such as sulfur, sulfite, sulfide, hydrogen 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 not naturally occur in proteins (e.g., GABA, citrulline, or ornithine) and/or polyamines can be used for this purpose.

Pharmaceutical Compositions

[0153] The invention provides pharmaceutical compositions that comprise extracts of one or more transgenic organisms described above. Extracts containing hypotaurine or taurine can be used to synthesize or manufacture taurine derivatives, (267, 268) taurine-conjugates (269) or taurine-polymers (270) that may have a wide range of commercial and medicinal applications. (271) Some taurine derivatives can function as organogelators (272) or dyes (273) and can be used in nanosensor synthesis. (274) Some taurine derivatives have anticonvulsant (267) or anti-cancer (275) properties. Other taurine derivatives are used in the treatment of alcoholism. (276, 277) Taurine-conjugated carboxyethylester-polyrotaxanes increase anticoagulant activity. (278) Taurine-containing polymers may increase wound healing. (279, 280) 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. (281) Extracts from taurine-containing cells 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.

[0154] 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

[0155] Transgenic cells containing hypotaurine or taurine may be consumed or used to make extracts for nutritional supplements. Transgenic cells that contain hypotaurine or taurine may be used for human consumption. Extracts from transgenic cells 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.

[0156] Transgenic cells containing hypotaurine or taurine may be used as fish or animal feed or used to make extracts for the supplementation of animal feed. Transgenic cells that contain hypotaurine or taurine may be used as animal or fish feed. Extracts from transgenic cells containing taurine may be used as feed supplements in the form of a liquid, powder, capsule or tablet.

Enhancer of Plant Growth or Yield

[0157] Transgenic cells that contain hypotaurine or taurine may be used as an enhancer for plant growth or yield. Extracts from transgenic cells containing hypotaurine or taurine may be used as plant enhancers in the form of a liquid, powder, capsule or tablet.

Fermentation and Taurine Purification

[0158] 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. (266, 282-292)

[0159] 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. (283, 285, 286, 293-296)

Definitions

[0160] 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.

[0161] 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.

[0162] 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. In bacteria the promoter includes a Shine-Dalgarno or ribosomal binding site that can include the sequence AGGAGG (-35 box) and a Pribnow box or RNA polymerase binding site that can include the sequence TATAAT (-10 box).

[0163] The term "algal promoter" refers to a promoter capable of initiating transcription in algal cells.

[0164] 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.

[0165] The term "microbe" refers to any microorganism (including both eukaryotic and prokaryotic microorganisms), such as bacteria, fungi, yeast, bacteria, algae and protozoa, as well as other unicellular organisms.

[0166] 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.

[0167] The term "inducible promoter" refers to a promoter that is under chemical (including biomolecules such as sugars, organic acids or amino acids) or environmental control.

[0168] 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.

[0169] 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.

[0170] 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.

[0171] The term "degradation" in reference to the "taurine degradation pathway", "taurine degradation enzymes", "taurine degradation system", and "taurine degradation proteins" refers to the process of breakdown, catabolism, or dissimilation of taurine.

[0172] The term's "cysteine dioxygenase" and "CDO" refer to the protein (EC:1.13.11.20) that catalyzes the following reaction:

cysteine+oxygen=3-sulfinoalanine

[0173] 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.

[0174] The terms "sulfinoalanine decarboxylase" and "SAD" refer to the protein (4.1.1.29) that catalyzes the following reaction:

3-sulfinoalanine=hypotaurine+CO.sub.2

[0175] 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 carboxy-lyase.

[0176] NOTE: the SAD reaction is also catalyzed by GADL1 (4.1.1.15) (glutamic acid decarboxylase like 1). Although called GADL1 the enzyme has been shown to catalyze the SAD reaction. (52, 53)

[0177] Other names for hypotaurine are 2-aminoethane sulfinate, 2-aminoethylsulfinic acid, and 2-aminoethanesulfinic acid.

[0178] Other names for taurine are 2-aminoethane sulfonic acid, aminoethanesulfonate, L-taurine, taurine ethyl ester, and taurine ketoisocaproic acid 2-aminoethane sulfinate.

[0179] The terms "cysteamine dioxygenase" and "ADO" refer to the protein (EC 1.13.11.19) that catalyzes the following reaction:

2-aminoethanethiol+O.sub.2=hypotaurine

[0180] ADO is another name for 2-aminoethanethiol:oxygen oxidoreductase, persulfurase, cysteamine oxygenase, and cysteamine:oxygen oxidoreductase,

[0181] Other names for 2-aminoethanethiol are cysteamine or 2-aminoethane-1-thiol, b-mercaptoethylamine, 2-mercaptoethylamine, decarboxycysteine, and thioethanolamine.

[0182] The terms "taurine-pyruvate aminotransferase" and "TPAT" refer to the protein (EC 2.6.1.77) that catalyzes the following reaction:

taurine+pyruvate=L-alanine+2-sulfoacetaldehyde

[0183] TPAT is another name for taurine transaminase or taurine transaminase aminotransferase

[0184] The term "Tpa" refers to the gene that encodes TPAT.

[0185] 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

[0186] SA is another name for acetyl-phosphate:sulfite S-acetyltransferase or Xsc.

[0187] The terms "taurine dehydrogenase" and "TDH" refer to the protein (EC:1.4.2.-) that catalyzes the following reaction:

taurine+water=ammonia+2-sulfoacetaldehyde

[0188] TDH is another name for taurine:oxidoreductase, taurine:ferricytochrome-c oxidoreductase,

[0189] The term "TauX" or "Taut" refers to the genes that encode for the small and large subunits of TDH, respectively.

[0190] The terms "taurine dioxygenase" and "TDO" refer to the protein (EC:1.14.11.17) that catalyzes the following reaction:

taurine+2-oxoglutarate+O.sub.2=sulfite+aminoacetaldehyde+succinate+CO.su- b.2

[0191] TDO is another name for 2-aminoethanesulfonate dioxygenase, alpha-ketoglutarate-dependent taurine dioxygenase, taurine, or 2-oxoglutarate:O.sub.2 oxidoreductase.

[0192] 2-oxoglutarate is another name for alpha-ketoglutarate.

[0193] The term "TauD" refers to the gene that encodes TDO.

[0194] The term "two-component alkanesulfonate monooxygenase" or "2CASM" that catalyzes the following reaction:

taurine+O.sub.2+FMNH.sub.2=Aminoacetaldehyde+SO.sub.3.sup.2+H.sub.2O+FMN

or

taurine+O.sub.2+Thioredoxin.sub.red=Aminoacetaldehyde+SO.sub.3.sup.2+H.s- ub.2O+Thioredoxin.sub.ox

[0195] The term "SssuDE", "SsuD" or "SsuE" refers to the genes that encode the two-component alkanesulfonate monooxygenase (2CASM).

[0196] The term "functional" with reference to CDO, CDOL, SAD, SADL, GADL1, partCS/PLP-DC, or CS/PLP-DC refers to peptides, proteins or enzymes that catalyze the CDOL, SADL, ADO, TPAT, or CS/PLP-DC reactions, respectively.

[0197] 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

[0198] The terms "portion of the cysteine synthetase/PLP decarboxylase" and "partCS/PLP-DC" refers to the protein that catalyzes a decarboxylyase reaction which cleaves carbon-carbon bonds and includes, but is not limited to, the following substrate and end-products:

Aspartate beta-alanine CO.sub.2

Glutamate 4-aminobutanoate+CO.sub.2

Cysteic acid=2-aminoethane sulfonate CO.sub.2

[0199] Note: another name for 4-aminobutanoate is gamma-aminobutyric acid (GABA).

[0200] 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.

[0201] 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.

[0202] The term "transgenic" includes reference to a unicellular, 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 the genotype of which has been altered by the presence of heterologous nucleic acid including those cell altered or created by budding or conjugation propagation from the initial transgenic cell.

[0203] 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.

[0204] 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.

[0205] 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.

[0206] 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.degree. C. for short probes (e.g., 10 to 50 nucleotides) and at least about 60.degree. 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.degree. C., and a wash in 1.times. to 2.times.SSC (20.times.SSC=3.0 M NaCl/0.3 M trisodium citrate) at 50 to 55.degree. C. Moderate stringency conditions include hybridization in 40 to 45% formamide, 1 M NaCl, 1% SDS at 37.degree. C., and a wash in 0.5.times. to 1.times.SSC at 55 to 60.degree. C. High stringency conditions include hybridization in 50% formamide, 1 M NaCl, 1% SDS at 37.degree. C., and a wash in 0.1.times.SSC at 60 to 65.degree. 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.sub.m can be approximated (297), where the T.sub.m=81.5.degree. 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.sub.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.sub.m is reduced by about 1.degree. C. for each 1% of mismatching; thus, T.sub.m, hybridization and/or wash conditions can be adjusted to hybridize to sequences of the desired identity. For example, if sequences with .gtoreq.90% identity are sought, the T.sub.m can be decreased 10.degree. C. Generally, stringent conditions are selected to be about 5.degree. C. lower than the thermal melting point (T.sub.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.degree. C. lower than the thermal melting point (T.sub.m); moderately stringent conditions can utilize a hybridization and/or wash at 6, 7, 8, 9 or 10.degree. C. lower than the thermal melting point (T.sub.m); low stringency conditions can utilize a hybridization and/or wash at 11, 12, 13, 14, 15 or 20.degree. C. lower than the thermal melting point (T.sub.m). Using the equation, hybridization and wash compositions, and desired T.sub.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. (131, 298) Unless otherwise stated, in the present application high stringency is defined as hybridization in 4.times.SSC, 5.times.Denhardt solution (5 g Ficoll, 5 g polyvinylpyrrolidone, 5 g bovine serum albumin in 500 ml of water), 0.1 mg/ml boiled salmon sperm DNA, and 25 mM Na phosphate at 65.degree. C., and a wash in 0.1.times.SSC, 0.1%.COPYRGT. SDS at 65.degree. C.

[0207] 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."

[0208] 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.

[0209] 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.

[0210] 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, BESTFIT, (299) can perform an optimal alignment of sequences for comparison using a homology alignment algorithm called GAP, (300) search for similarity using Tfasta and Fasta, (301) 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 (302-304) and program PileUp can be used for optimal global alignment of multiple sequences. (305) 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.

[0211] GAP (300) 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. (306)

[0212] Unless otherwise stated, sequence identity or similarity values refer to the value obtained using the BLAST 2.0 suite of programs using default parameters. (307) 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 (308, 309) and XNU. (310)

[0213] 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, (311) thereby increasing the percentage sequence similarity.

[0214] 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.

[0215] The term "substantial identity" of polynucleotide sequences means that a polynucleotide comprises a sequence that has between 50-100%.COPYRGT. 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.

[0216] 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%.COPYRGT. sequence identity to the reference sequence over a specified comparison window. Preferably, optimal alignment is conducted using the homology alignment algorithm (300). 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 ELISA 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.

[0217] 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

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Example 1

Development of a Transgenic Bacterium with a TauD Knockout that Expresses CDOL without Transit Peptide Fused with a Linker to partCS/PLP-DC Using Chemical Synthesis

[0532] Step 1: Use PCR to amplify the TauD (SEQ ID NO:20) using 500 ng of DNA from E. coli strain K12 and the primers for SEQ ID NO:44 and SEQ ID NO:45. Use the PCR-amplified fragment to knockout TauD with X Red-mediated recombination as described by Datsenko and Wanner (251) and Baba et al. (139)

[0533] Step 2: Use chemical synthesis to make a DNA construct that contains a CDOL gene (SEQ ID NO:3) without the transit peptide, linker (SEQ ID NO:15), partCS/PLP-DC gene (SEQ ID NO:11) all in frame. Clone the CDOL/linker/partCS/PLP-DC fragment into a bacterial expression vector, such as pET11, pKK223-3, or pSF-Tac, so it is functional.

[0534] The CDOL gene is as follows:

[0535] a. Derived from SEQ ID NO:3 by removing nucleotides 4 through 159 (corresponding to the native transit peptide) and without the stop codon, optimized for expression in E. coli and encoding a CDOL peptide from Chlamydomonas reinhardtii (SEQ ID NO:4 minus amino acids 2 through 53); and

[0536] The partCS/PIP-DC gene is as follows:

[0537] a. Derived from SEQ ID NO:11 by removing nucleotides 1 through 1413 (corresponding to the native transit and cysteine synthetase peptides), optimized for expression in E. coli and encoding a partCS/PLP-DC peptide from Micromonas pusilla (SEQ ID NO:12 minus amino acids 1 through 471); and

[0538] Step 3: Transform the vector with the functional CDOL/linker/partCS/PLP-DC construct (from Step 2, EXAMPLE 1) into the TauD knockout E. coli strain (from Step 1, EXAMPLE 1) and confirm the presence of the DNA construct.

Example 2

[0539] Development of a Transgenic Bacterium with a TauD Knockout that Expresses CDOL without Transit Peptide Fused with a Linker to partCS/PLP-DC and a Taurine Binding Peptide Using Chemical Synthesis

[0540] Step 1: Use chemical synthesis to make a DNA construct that contains a taurine binding protein (SEQ ID NO:16 or SEQ ID NO:18) without the transit peptide. Clone the taurine binding protein into a bacterial expression vector, such as pET11, pKK223-3, or pSF-Tac, so it is functional.

[0541] The taurine-binding protein gene is as follows:

[0542] a. Derived from SEQ ID NO:16 by removing nucleotides 4 through 66 (corresponding to the periplasmic transit peptide) and encoding a truncated taurine-binding peptide from E. coli (SEQ ID NO:17 minus amino acids 2 through 22); or

[0543] b. Derived from SEQ ID NO:18, by removing nucleotides 4 through 93, (corresponding to the periplasmic transit peptide), optimized for expression in. E. coli and encoding a truncated taurine-binding peptide from Roseobacter denitrificans (SEQ ID NO:19 minus amino acids 2 through 31);

[0544] Step 2: Transform the DNA vector with the taurine-binding protein (from Step 1, EXAMPLE 2) into the TauD knockout E. coli strain that contains the vector with the CDOL/linker/partCS/PLP-DC (from Step 3, EXAMPLE 1). Select for antibiotic resistance, and confirm the presence of the DNA constructs.

Example 3

Development of a Transgenic Bacterium with a Cbl Knockout that Expresses CDOL without Transit Peptide Fused with a Linker to partCS/PLP-DC Using Chemical Synthesis

[0545] Step 1: Use PCR to amplify the cbl (SEQ ID NO:36) using 500 ng of DNA from E. coli strain K12 and the primers for SEQ ID NO:46 and SEQ ID NO:47. Use the PCR-amplified fragment to knockout cbl with X Red-mediated recombination as described by Datsenko and Wanner (251) and Baba et al. (139)

[0546] Step 2: Transform the vector with the functional CDOL/linker/partCS/PLP-DC construct (from Step 2, EXAMPLE 1) into the cbl knockout E. coli strain (from Step 1, EXAMPLE 3) and confirm the presence of the DNA construct.

Example 4

Development of a Transgenic Bacterium with a Cbl Knockout that Expresses CDOL without Transit Peptide Fused with a Linker to partCS/PLP-DC and a Taurine Binding Peptide Using Chemical Synthesis

[0547] Step 1: Transform the DNA vector with the taurine-binding protein (from Step 1, EXAMPLE 2) into the cbl knockout E. coli strain that contains the vector with the CDOL/linker/partCS/PLP-DC (from Step 2, EXAMPLE 3) and confirm the presence of the DNA constructs.

Example 5

Development of a Transgenic Bacterium with a SsuD Knockout that Expresses CDOL without Transit Peptide Fused with a Linker to partCS/PLP-DC Using Chemical Synthesis

[0548] Step 1: Use PCR to amplify the SsuD (SEQ ID NO:22) using 500 ng of DNA from E. coli strain K12 and the primers for SEQ ID NO:48 and SEQ ID NO:49. Use the PCR-amplified fragment to knockout SsuD with .lamda. Red-mediated recombination as described by Datsenko and Wanner (251) and Baba et al. (139)

[0549] Step 2: Transform the vector with the functional CDOL/linker/partCS/PLP-DC construct (from Step 2, EXAMPLE 1) into the SsuD knockout E. coli strain (from Step 1, EXAMPLE 5) and confirm the presence of the DNA construct.

Example 6

Development of a Transgenic Bacterium with a SsuD Knockout that Expresses CDOL without Transit Peptide Fused with a Linker to partCS/PLP-DC and a Taurine Binding Peptide Using Chemical Synthesis

[0550] Step 1: Transform the DNA vector with the taurine-binding protein (from Step 1, EXAMPLE 2) into the SsuD knockout E. coli strain that contains the vector with the CDOL/linker/partCS/PLP-DC (from Step 2, EXAMPLE 5) and confirm the presence of the DNA constructs.

Example 7

Development of a Transgenic Bacterium with a SsuE Knockout that Expresses CDOL without Transit Peptide Fused with a Linker to partCS/PLP-DC Using Chemical Synthesis

[0551] Step 1: Use PCR to amplify the SsuE (SEQ ID NO:24) using 500 ng of DNA from E. coli strain K12 and the primers for SEQ ID NO:50 and SEQ ID NO:51. Use the PCR-amplified fragment to knockout SsuE with X, Red-mediated recombination as described by Datsenko and Wanner (251) and Baba et al. (139)

[0552] Step 2: Transform the vector with the functional CDOL/Linker/partCS/PLP-DC construct (from Step 2, EXAMPLE 1) into the SsuE knockout E. coli strain (from Step 1, EXAMPLE 7) and confirm the presence of the DNA construct.

Example 8

Development of a Transgenic Bacterium with a SsuE Knockout that Expresses CDOL without Transit Peptide Fused with a Linker to partCS/PLP-DC and a Taurine Binding Peptide Using Chemical Synthesis

[0553] Step 1: Transform the DNA vector with the taurine-binding protein (from Step 1, EXAMPLE 2) into the SsuE knockout E coli strain that contains the vector with the CDOL/linker/partCS/PLP-DC (from Step 2, EXAMPLE 7) and confirm the presence of the DNA constructs.

Example 9

Development of Another Type of Transgenic Bacterium with a SsuD Knockout that Expresses CDOL without Transit Peptide Fused with a Linker to partCS/PLP-DC Using Chemical Synthesis

[0554] Step 1: Use overlap PCR to amplify a knockout fragment for SsuD (SEQ ID NO:26) using genome DNA from Corynebacterium glutamicum and the pK19mobsacB vector as described by Buchholz et al. (312) Generate independent DNA fragments using the primer pairs SEQ ID NO:52 and SEQ ID NO:53 and genome DNA from C. glutamicum and SEQ ID NO:54 and SEQ ID NO:55 and genome DNA from C. glutamicum. Purify each DNA fragment and mix in equal amounts in an overlap PCR using primers SEQ ID NO:52 and SEQ ID NO:55. Clone the resulting fusion product containing the SsuD gene with an internal deletion of 875 bp (SsuD knockout fragment) into pK19mobsacB. Replace the SsuD1 gene with the SsuD knockout fragment by homologous recombination. (312)

[0555] Step 2: Use chemical synthesis to make a DNA construct that contains a CDOL gene (SEQ ID NO:3) without the transit peptide, linker (SEQ ID NO:15), partCS/PLP-DC gene (SEQ ID NO:11) all in frame. Clone the CDOL/linker/partCS/PLP-DC fragment into a bacterial expression vector, such as pET11, pKK223-3, or pSF-Tac, so it is functional.

[0556] The CDOL gene is as follows:

[0557] a. Derived from SEQ ID NO:3 by removing nucleotides 4 through 159 (corresponding to the native transit peptide) and without the stop codon, optimized for expression in C. glutamicum and encoding a CDOL peptide from Chlamydomonas reinhardtii (SEQ ID NO:4 minus amino acids 2 through 53); and

[0558] The partCS/PLP-DC gene is as follows:

[0559] a. Derived from SEQ ID NO:11 by removing nucleotides 1 through 1413 (corresponding to the native transit and cysteine synthetase peptides), optimized for expression in in C. glutamicum and encoding a partCS/PLP-DC peptide from Micromonas pusilla (SEQ ID NO:12 minus amino acids 1 through 471); and

[0560] Step 3: Transform the vector with the functional CDOL/linker/partCS/PLP-DC construct (from Step 2, EXAMPLE 9) into the SsuD knockout C. glutamicum strain (from Step 1, EXAMPLE 9) and confirm the presence of the DNA construct.

Example 10

Development of Another Type of Transgenic Bacterium with a SsuD Knockout that Expresses CDOL without Transit Peptide Fused with a Linker to partCS/PLP-DC and a Taurine Binding Peptide Using Chemical Synthesis

[0561] Step 1: Use chemical synthesis to make a DNA construct that contains a taurine binding protein (SEQ ID NO:16 or SEQ ID NO:18) without the transit peptide. Clone the taurine binding protein into a bacterial expression vector, such as pET11, pKK223-3, or pSF-Tac, so it is functional.

[0562] The taurine binding protein gene is as follows:

[0563] a. Derived from SEQ ID NO:16 by removing nucleotides 4 through 66 (corresponding to the periplasmic transit peptide) and encoding a truncated taurine-binding peptide from C. glutamicum (SEQ ID NO:17 minus amino acids 2 through 22); or

[0564] b. Derived from SEQ ID NO:18 by removing nucleotides 4 through 93, (corresponding to the periplasmic transit peptide), optimized for expression in C. glutamicum and encoding a truncated taurine-binding peptide from Roseobacter denitrificans (SEQ ID NO:19 minus amino acids 2 through 31);

[0565] Step 2: Transform the DNA vector with the taurine-binding protein (from Step 1, EXAMPLE 10) into the SsuD knockout C. glutamicum strain (from Step 1, EXAMPLE 9) and confirm the presence of the DNA construct.

Example 11

Development of Another Type of Transgenic Bacterium with a SsuE Knockout that Expresses CDOL without Transit Peptide Fused with a Linker to partCS/PLP-DC Using Chemical Synthesis

[0566] Step 1: Use overlap PCR to amplify a knockout fragment for SsuE (SEQ ID NO:28) using genome DNA from Corynebacterium glutamicum and the pK19mobsacB vector as described by Buchholz et al. (312) Generate independent DNA fragments using the primer pairs SEQ ID NO:56 and SEQ ID NO:57 and genome DNA from C. glutamicum and SEQ ID NO:58 and SEQ ID NO:59 and genome DNA from C. glutamicum. Purify each DNA fragment and mix in equal amounts in an overlap PCR using primers SEQ ID NO:56 and SEQ ID NO:59. Clone the resulting fusion product, containing the SsuE gene with an internal deletion of 735 bp (SsuE knockout fragment), into pK19mobsacB. Replace the SsuE gene with the SsuE knockout fragment by homologous recombination. (312)

[0567] Step 2: Transform the vector with the functional CDOL/linker/partCS/PLP-DC construct (from Step 2, EXAMPLE 9) into the SsuE knockout C. glutamicum strain (from Step 1, EXAMPLE 11) and confirm the presence of the DNA construct.

Example 12

Development of Another Transgenic Bacterium with a SsuE Knockout that Expresses CDOL without Transit Peptide Fused with a Linker to partCS/PLP-DC and a Taurine Binding Peptide Using Chemical Synthesis

[0568] Step 1: Transform the DNA vector with the taurine-binding protein (from Step 1, EXAMPLE 10) into the SsuE knockout C. glutamicum strain (from Step 1, EXAMPLE 11) and confirm the presence of the DNA construct.

Example 13

Development of Another Transgenic Bacterium with a Cbl Knockout that Expresses CDOL without Transit Peptide Fused with a Linker to partCS/PLP-DC Using Chemical Synthesis

[0569] Step 1: Use overlap PCR to amplify a knockout fragment for cbl (SEQ ID NO:38) using genome DNA from Corynebacterium glutamicum, and the pK19mobsacB vector as described by Buchholz et al. (312) Generate independent DNA fragments using the primer pairs SEQ ID NO:60 and SEQ ID NO:61 and genome DNA from C. glutamicum and SEQ ID NO:62 and SEQ ID NO:63 and genome DNA from C. glutamicum. Purify each DNA fragment and mix in equal amounts in an overlap PCR using primers SEQ ID NO:60 and SEQ ID NO:63. Clone the resulting fusion product, containing the cbl gene with an internal deletion of 563 bp (cbl knockout fragment) into pK19mobsacB. Replace the cbl gene with the cbl knockout fragment by homologous recombination. (312)

[0570] Step 2: Transform the vector with the functional CDOL/linker/partCS/PLP-DC construct (from Step 2, EXAMPLE 9) into the cbl knockout C. glutamicum strain (from Step 1, EXAMPLE 13) and confirm the presence of the DNA construct.

Example 14

Development of Another Transgenic Bacterium with a Cbl Knockout that Expresses CDOL without Transit Peptide Fused with a Linker to partCS/PLP-DC and a Taurine Binding Peptide Using Chemical Synthesis

[0571] Step 1: Transform the DNA vector with the taurine binding protein (from Step 1, EXAMPLE 10) into the cbl knockout C. glutamicum strain (from Step 1, EXAMPLE 13) and confirm the presence of the DNA construct.

Example 15

Development of a Transgenic Bacterium with a TauR Knockout that Expresses CDOL without Transit Peptide Fused with a Linker to partCS/PLP-DC Using Chemical Synthesis

[0572] Step 1: Use overlap PCR to amplify a knockout fragment for TauR (SEQ ID NO:40) using genome DNA from Corynebacterium glutamicum and the pK19mobsacB vector as described by Buchholz et al. (312) Generate independent DNA fragments using the primer pairs SEQ ID NO:64 and SEQ ID NO:65 and genome DNA from C. glutamicum and SEQ ID NO:66 and SEQ ID NO:67 and genome DNA from C. glutamicum. Purify each DNA fragment and mix in equal amounts in an overlap PCR using primers SEQ ID NO:64 and SEQ ID NO:67. Clone the resulting fusion product, containing the TauR gene with an internal deletion of 1052 bp (TauR knockout fragment) into pK19mobsacB. Replace the TauR gene with the TauR knockout fragment by homologous recombination. (312)

[0573] Step 2: Transform the vector with the functional CDOL/linker/partCS/PLP-DC construct (from Step 2, EXAMPLE 9) into the TauR knockout C. glutamicum strain (from Step 1, EXAMPLE 15) and confirm the presence of the DNA construct.

Example 16

Development of a Transgenic Bacterium with a TauR Knockout that Expresses CDOL without Transit Peptide Fused with a Linker to partCS/PLP-DC and a Taurine Binding Peptide Using Chemical Synthesis

[0574] Step 1: Transform the DNA vector with the taurine-binding protein (from Step 1, EXAMPLE 10) into the TauR knockout C. glutamicum strain (from Step 1, EXAMPLE 15) and confirm the presence of the DNA construct.

Example 17

Development of a Transgenic Alga with a Native Taurine Biosynthetic Pathway that Expresses a Taurine Binding Protein Using Chemical Synthesis

[0575] Step 1: Use chemical synthesis to make a DNA construct that contains a taurine binding protein (SEQ ID NO:16 or SEQ ID NO:18) without the transit peptide. Clone the taurine binding protein into an algal expression vector, such as pCB740 or pD1-Kan, so it is functional.

[0576] The taurine binding protein gene is as follows:

[0577] a. Derived from SEQ ID NO:16 by removing nucleotides 4 through 66 (corresponding to the periplasmic transit peptide), optimized for expression in Chlamydomonas reinhardtii or Ostreococcus tauri and encoding a truncated taurine-binding peptide from E. coli (SEQ ID NO:17 minus amino acids 2 through 22); or

[0578] b. Derived from SEQ ID NO:18 by removing nucleotides 4 through 93, (corresponding to the periplasmic transit peptide), optimized for expression in Chlamydomonas reinhardtii or Ostreococcus tauri and encoding a truncated taurine-binding protein from Roseobacter denitrificans (SEQ ID NO:19 minus amino acids 2 through 31);

[0579] Step 2: Transform the DNA vector with the taurine-binding protein (from Step 1, EXAMPLE 17) into Chlamydomonas reinhardtii or Ostreococcus tauri and confirm the presence of the DNA constructs.

Example 18

Development of a Transgenic Alga with a Native Taurine Biosynthetic Pathway that Expresses a Taurine Binding Protein with a Chloroplast Transit Peptide Using Chemical Synthesis

[0580] Step 1: Use chemical synthesis to make a DNA construct that contains a taurine binding protein (SEQ ID NO:16 or SEQ ID NO:18) with the plastid transit peptide (SEQ ID NO:13). Clone the taurine-binding protein into an algal expression vector, such as pCB740 or pD1-Kan, so it is functional.

[0581] The nucleotide sequence for the plastid transit peptide (SEQ ID NO:13) encodes the peptide SEQ ID NO:14.

[0582] The taurine binding protein gene is as follows:

[0583] a. Derived from SEQ ID NO:16 by removing nucleotides 1 through 66 (corresponding to the periplasmic transit peptide), optimized for expression in Chlamydomonas reinhardtii or Ostreococcus tauri and encoding a truncated taurine-binding peptide from E. coli (SEQ ID NO:17 minus amino acids 1 through 22); or

[0584] b. Derived from SEQ ID NO:18 by removing nucleotides 4 through 93, (corresponding to the periplasmic transit peptide), optimized for expression in Chlamydomonas reinhardtii or Ostreococcus tauri and encoding a truncated taurine-binding peptide from Roseobacter denitrificans (SEQ ID NO:19 minus amino acids 2 through 31);

[0585] Step 2: Transform the DNA vector with the taurine-binding protein (from Step 1, EXAMPLE 18) into Chlamydomonas reinhardtii or Ostreococcus tauri and confirm the presence of the DNA constructs.

Example 19

Development of a Transgenic Alga with a Native Taurine Biosynthetic Pathway that Expresses a Taurine Binding Protein in the Chloroplast Via Chloroplast Transformation Using Chemical Synthesis

[0586] Step 1: Make the following construct: an atpA promoter-59UTR (untranslated region) operably linked to taurine binding protein and the atpA terminator (TatpA). Use the chloroplast destination expression for Chlamydomonas reinhardtii as described by Oey et al (115) Use chemical synthesis to make a DNA construct that contains a taurine binding protein (SEQ ID NO:16 or SEQ ID NO:18) without a transit peptide with XbaI at the 5' end and a NcoI site at the 3end. Clone the taurine-binding protein into the XbaI/NcoI site (remove the GFP fragment) of the Entry vector. Recombine the atpA/taurine binding protein/atpA cassette from the Entry vector into the Destination vector, pC-Dest/psbA.

[0587] The taurine binding protein gene is as follows:

[0588] a. Derived from SEQ ID NO:16 by removing nucleotides 4 through 66 (corresponding to the periplasmic transit peptide), optimized for expression in Chlamydomonas reinhardtii and encoding a truncated taurine-binding peptide from E. coli (SEQ ID NO:17 minus amino acids 2 through 22) with an XbaI site 5' of the start codon and a NcoI site 3' of the stop codon; or

[0589] b. Derived from SEQ ID NO:18 by removing nucleotides 4 through 93, (corresponding to the periplasmic transit peptide), optimized for expression in Chlamydomonas reinhardtii and encoding a truncated taurine-binding peptide from Roseobacter denitrificans (SEQ ID NO:19 minus amino acids 2 through 31) with an XbaI site 5' of the start codon and a NcoI site 3' of the stop codon;

[0590] Step 2: Transform the DNA vector with the Destination vector containing the atpA promoter/taurine binding protein/TatpA (from. Step 1, EXAMPLE 19) into Chlamydomonas reinhardtii and confirm the presence of the DNA construct.

Example 20

Development of a Transgenic Fungus that Expresses CDOL without the Transit Peptide Fused with a Linker to partCS/PLP-DC and a Taurine Binding Protein Using Chemical Synthesis

[0591] Step 1: Use chemical synthesis to make a DNA construct that contains a CDOL gene (SEQ ID NO:3) without the transit peptide, linker (SEQ ID NO:15), partCS/PLP-DC gene (SEQ ID NO:11) all in frame. Clone the CDOL/linker/partCS/PLP-DC fragment into a fungal expression vector so it is functional.

[0592] The CDOL gene is as follows:

[0593] a. Derived from SEQ ID NO:3 by removing nucleotides 4 through 159 (corresponding to the native transit peptide) and without the stop codon, optimized for expression in yeast and encoding a CDOL peptide from Chlamydomonas reinhardtii (SEQ ID NO:4 minus amino acids 2 through 53); and

[0594] The partCS/PLP-DC gene is as follows:

[0595] a. Derived from SEQ ID NO:11 by removing nucleotides 1 through 1413 (corresponding to the native transit and cysteine synthetase peptides), optimized for expression in yeast and encoding a partCS/PLP-DC peptide from Micromonas pusilla (SEQ ID NO:12 minus amino acids 1 through 471); and

[0596] Step 3: Transform the vector with the functional CDOL/linker/partCS/PLP-DC construct (from Step 2, EXAMPLE 20) into yeast and confirm the presence of the DNA construct.

[0597] Step 4: Use chemical synthesis to make a DNA construct that contains a taurine-binding protein (SEQ ID NO:16 or SEQ ID NO:18) without the transit peptide. Clone the taurine-binding protein into a fungal expression vector, such as pESC-TRP, pYES2/NT, or pYSG-IBA, so it is functional.

[0598] The taurine-binding protein gene is as follows:

[0599] a. Derived from SEQ ID NO:16 by removing nucleotides 4 through 66 (corresponding to the periplasmic transit peptide), optimized for expression in yeast, and encoding a truncated taurine-binding protein (SEQ ID NO:17 minus amino acids 2 through 22); or

[0600] b. Derived from SEQ ID NO:18, by removing nucleotides 4 through 93, (corresponding to the periplasmic transit peptide), optimized for expression in yeast and encoding a truncated taurine-binding protein from Roseobacter denitrificans (SEQ ID NO:19 minus amino acids 2 through 31);

[0601] Step 4: Transform the DNA vector with the taurine-binding protein (from Step 3, EXAMPLE 20) into the yeast strain that contains the vector with the CDOL/linker/partCS/PLP-DC (from Step 3, EXAMPLE 20) and confirm the presence of the DNA constructs.

Example 21

Development of a Transgenic Fungus that Expresses CS/PLP-DC without the Transit Peptide and a Taurine Binding Protein Using Chemical Synthesis

[0602] Step 1: Use chemical synthesis to make a DNA construct that contains a CS/PLP-DC gene (SEQ ID NO:11) without the transit peptide. Clone the CS/PLP-DC fragment into a fungal expression vector, such as pESC-TRP, pYES2/NT, or pYSG-IBA vector, so it is functional.

[0603] The CS/PLP-DC gene is as follows:

[0604] a. Derived from SEQ ID NO:11, by removing nucleotides 4 through 234, (corresponding to the native transit peptide) optimized for expression in yeast, and encoding a CS/PLP-DC peptide from Micromonas pusilla (SEQ ID NO:12 minus amino acids 2 through 78); and

[0605] Step 2: Transform the vector with the functional CS/PLP-DC construct (from Step 1, EXAMPLE 21) into yeast and confirm the presence of the DNA construct.

[0606] Step 3: Use chemical synthesis to make a DNA construct that contains a taurine-binding protein (SEQ ID NO:16 or SEQ ID NO:18) without the transit peptide. Clone the taurine-binding protein into a fungal expression vector, such as pESC-TRP, pYES2/NT, or pYSG-IBA, so it is functional.

[0607] The taurine-binding protein gene is as follows:

[0608] a. Derived from SEQ ID NO:16 by removing nucleotides 4 through 66 (corresponding to the periplasmic transit peptide), optimized for expression in yeast, and encoding a truncated taurine-binding peptide (SEQ ID NO:17 minus amino acids 2 through 22); or

[0609] b. Derived from SEQ ID NO:18, by removing nucleotides 4 through 93, (corresponding to the periplasmic transit peptide), optimized for expression in yeast, and encoding a truncated taurine-binding peptide from Roseobacter denitrificans (SEQ ID NO:19 minus amino acids 2 through 31);

[0610] Step 4: Transform the DNA vector with the taurine-binding protein (from Step 3, EXAMPLE 21) into the yeast strain that contains the vector with the CS/PLP-DC (from Step 2, EXAMPLE 21) and confirm the presence of the DNA constructs.

Example 22

Development of a Transgenic Fungus with that Expresses CDO without the Transit Peptide Fused with a Linker SAD and a Taurine Binding Protein Using Chemical Synthesis

[0611] Step 1: Use chemical synthesis to make a DNA construct that contains a CDO gene (SEQ ID NO:1) without the transit peptide, linker (SEQ ID NO:15), SAD gene (SEQ ID NO:5) all in frame. Clone the CDO/linker/SAD fragment into a fungal expression vector so it is functional.

[0612] The CDO gene is as follows:

[0613] a. Derived from SEQ ID NO:1 without the stop codon, optimized for expression in yeast and encoding a CDO peptide from Danio rerio (SEQ ID NO:2); and

[0614] The SAD gene is as follows:

[0615] a. Derived from SEQ ID NO:5 optimized for expression in yeast and encoding a SAD peptide from Danio rerio (SEQ ID NO:6); and

[0616] Step 3: Transform the vector with the functional CDO/linker/SAD construct (from Step 1, EXAMPLE 22) into yeast and confirm the presence of the DNA construct.

[0617] Step 4: Use chemical synthesis to make a DNA construct that contains a taurine-binding protein (SEQ ID NO:16 or SEQ ID NO:18) without the transit peptide. Clone the taurine-binding protein into a fungal expression vector, such as pESC-TRP, pYES2/NT, or pYSG-IBA, so it is functional.

[0618] The taurine-binding protein gene is as follows:

[0619] a. Derived from SEQ ID NO:16 by removing nucleotides 4 through 66 (corresponding to the periplasmic transit peptide), optimized for expression in yeast, and encoding a truncated taurine-binding protein (SEQ ID NO:17 minus amino acids 2 through 22); or

[0620] b. Derived from SEQ ID NO:18, by removing nucleotides 4 through 93, (corresponding to the periplasmic transit peptide), optimized for expression in yeast, and encoding a truncated taurine-binding protein from Roseobacter denitrificans (SEQ ID NO:19 minus amino acids 2 through 31);

[0621] Step 4: Transform the DNA vector with the taurine-binding protein (from Step 3, EXAMPLE 20) into the yeast strain that contains the vector with the CDO/linker/SAD (from Step 3, EXAMPLE 22) and confirm the presence of the DNA constructs.

Example 23

Development of a Transgenic Fungus that Expresses CDOL without the Transit Peptide Fused with a Linker GADL1 and a Taurine Binding Protein Using Chemical Synthesis

[0622] Step 1: Use chemical synthesis to make a DNA construct that contains a CDOL gene (SEQ ID NO:3) without the transit peptide, linker (SEQ ID NO:15), GADL1 gene (SEQ ID NO:9) all in-frame. Clone the CDOL/linker/GADL1 fragment into a fungal expression vector so it is functional.

[0623] The CDOL gene is as follows:

[0624] a. Derived from SEQ ID NO:3 by removing nucleotides 4 through 159 (corresponding to the native transit peptide) and without the stop codon, optimized for expression in E. coli and encoding a CDOL peptide from Chlamydomonas reinhardtii (SEQ ID NO:4 minus amino acids 2 through 53); and

[0625] The GADL1 gene is as follows:

[0626] a. Derived from SEQ ID NO:9 optimized for expression in yeast and encoding a GADL1 peptide from Dania rerio (SEQ ID NO:10); and

[0627] Step 3: Transform the vector with the functional CDOL/linker/GADL1 construct (from Step 1, EXAMPLE 23) into yeast and confirm the presence of the DNA construct.

[0628] Step 4: Use chemical synthesis to make a DNA construct that contains a taurine-binding protein (SEQ ID NO:16 or SEQ ID NO:18) without the transit peptide. Clone the taurine-binding protein into a fungal expression vector, such as pESC-TRP, pYES2/NT, or pYSG-IBA, so it is functional.

[0629] The taurine-binding protein gene is as follows:

[0630] a. Derived from SEQ ID NO:16 by removing nucleotides 4 through 66 (corresponding to the periplasmic transit peptide), optimized for expression in yeast, and encoding a truncated taurine-binding protein (SEQ ID NO:17 minus amino acids 2 through 22); or

[0631] b. Derived from SEQ ID NO:18, by removing nucleotides 4 through 93, (corresponding to the periplasmic transit peptide), optimized for expression in yeast, and encoding a truncated taurine-binding protein from Roseobacter denitrificans (SEQ ID NO:19 minus amino acids 2 through 31);

[0632] Step 4: Transform the DNA vector with the taurine-binding protein (from Step 3, EXAMPLE 23) into the yeast strain that contains the vector with the CDO/linker/GADL1 (from Step 3, EXAMPLE 22) and confirm the presence of the DNA constructs.

Example 24

Development of a Transgenic Bacterium with TauX Suppressed that Expresses CDOL without the Transit Peptide Fused with a Linker to partCS/PLP-DC Using Chemical Synthesis

[0633] Step 1: Use chemical synthesis to make an antisense construct to silence or suppress TauX (SEQ ID NO: 30) and clone into the pBAD vector as described by Stefan et al. (313)

[0634] The TauX antisense is as follows:

[0635] a. Fuse the polynucleotides for SEQ ID NO:68 to polynucleotides 1 through 360 of SEQ ID NO:30. Clone the TauX antisense fragment into the bacterial expression vector, pBAD so TauX antisense fragment can be expressed.

[0636] Step 2: Transform the vector with the TauX antisense construct (from Step 1, EXAMPLE 24) into Roseobacter denitrificans and confirm the presence of the DNA construct.

[0637] Step 3: Use chemical synthesis to make a DNA construct that contains a CDOL gene (SEQ ID NO:3) without the transit peptide, linker (SEQ ID NO:15), partCS/PLP-DC gene (SEQ ID NO:11) all in frame. Clone the CDOL/linker/partCS/PLP-DC fragment into a bacterial expression vector, such as pET11, pKK223-3, or pSF-Tac, so it is functional.

[0638] The CDOL gene is as follows:

[0639] a. Derived from SEQ ID NO:3 by removing nucleotides 4 through 159 (corresponding to the native transit peptide) and without the stop codon, optimized for expression in. Roseobacter denitrificans and encoding a CDOL peptide from Chlamydomonas reinhardtii (SEQ ID NO:4 minus amino acids 2 through 53); and

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

[0641] a. Derived from SEQ ID NO:11 by removing nucleotides 1 through 1413 (corresponding to the native transit and cysteine synthetase peptides), optimized for expression in Roseobacter denitrificans and encoding a partCS/PLP-DC peptide from Micromonas pusilla (SEQ ID NO:12 minus amino acids 1 through 471); and

[0642] Step 4: Transform the vector with the functional CDOL/linker/partCS/PLP-DC construct (from Step 3, EXAMPLE 24) into the TauX knockdown Roseobacter denitrificans strain (from Step 1, EXAMPLE 24) and confirm the presence of the DNA construct.

Example 25

Development of a Transgenic Bacterium with a TauY Suppressed that Expresses CDOL without the Transit Peptide Fused with a Linker to partCS/PLP-DC Using Chemical Synthesis

[0643] Step 1: Use chemical synthesis to make an antisense construct to silence or suppress TauY (SEQ ID NO: 32) and clone into the pBAD vector as described by Stefan et al. (313)

[0644] The TauY antisense is as follows:

[0645] a. Fuse the polynucleotides for SEQ ID NO:68 to polynucleotides 1 through 360 of SEQ ID NO:32. Clone the TauY antisense fragment into a bacterial expression vector, pBAD, so the TauY antisense fragment can be expressed.

[0646] Step 2: Transform the vector with the TauY antisense construct (from Step 1, EXAMPLE 25) into Roseobacter denitrificans and confirm the presence of the DNA construct

[0647] Step 3: Transform the vector with the functional CDOL/linker/partCS/PLP-DC construct (from Step 3, EXAMPLE 24) into the TauY knockdown Roseobacter denitrificans strain (from Step 1, EXAMPLE 25) and confirm the presence of the DNA construct.

Example 26

[0648] Development of a Transgenic Bacterium with a Tpa Suppressed that Expresses CDOL without the Transit Peptide Fused with a Linker to partCS/PLP-DC Using Chemical Synthesis

[0649] Step 1: Use chemical synthesis to make an antisense construct to silence or suppress Tpa (SEQ ID NO: 34) and clone into the pBAD vector as described by Stefan et al. (313)

[0650] The TauY antisense is as follows:

[0651] a. Fuse the polynucleotides for SEQ ID NO:68 to polynucleotides 1 through 360 of SEQ ID NO:34. Clone the Tpa antisense fragment into the bacterial expression vector, pBAD, so Tpa antisense fragment can be expressed.

[0652] Step 2: Transform the vector with the Tpa antisense construct (from Step 1, EXAMPLE 26) into Roseobacter denitrificans and confirm the presence of the DNA construct.

[0653] Step 3: Transform the vector with the functional CDOL/linker/partCS/PLP-DC construct (from Step 3, EXAMPLE 24) into the Tpa knockdown Roseobacter denitrificans strain (from Step 1, EXAMPLE 26) and confirm the presence of the DNA construct.

Example 27

Develop Bacteria with Taurine

[0654] Grow bacteria with CS/PLP-DC (such as from EXAMPLE 1) and induce gene expression with the appropriate inducer associated with the vector. Collect the cells and confirm that the cells express the CS/PLP-DC peptide (.about.96.6 kDa) using western blot analysis and that have increased taurine using HPLC analysis.

Example 28

Develop Aquafeed Using Bacterial Cells with Taurine

[0655] Grow bacteria with CS/PLP-DC (such as from EXAMPLE 1) and induce gene expression with the appropriate inducer associated with the vector. Collect the cells and process for use as an additive to feed.

Example 28

Develop an E. coli Strain that Produces Taurine

[0656] This example demonstrates the use of a TauD knockout that expresses a CDOL fused to SADL with a linker (CDOL-linker-partCS/PLP-DC) (such as from EXAMPLE 1) to produce taurine in an E. coli. Transformed E. coli were confirmed by selection and PCR analysis. E. coli were grown in ZYP media (314) and induced using autoinduction with an 8:1 lactose to glucose ratio. Free amino acids were extracted from 2 hr culture after the addition of cysteine (200 uM) to determine the level of taurine using high-performance liquid chromatography (HPLC). The bacteria were separated from the supernatant by centrifugation and the level of taurine was determined in the pellet and supernatant. The taurine levels were 0.26% and 1.0% of total extracted free amino acids for the pellet and supernatant, respectively.

[0657] 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.

[0658] 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.

Sequence CWU 1

1

681606DNADanio rerio 1atggagcaga ctgaagtcat gaagcccgag actctggagg atctgatcaa aactctgcat 60cagatcttcc agagcgactc catcaatgtg gaggaggtgc agaacctgat ggagtcctac 120cagagcaacc cgcaggactg gatgaagttc gccaagttcg accagtacag gtacaccagg 180aacctcgtgg atgaaggaaa cggaaagttc aacctgatga tcctgtgctg gggtgaagga 240cacggcagca gcatccatga ccacacagac tcgcactgct tcctgaagct gctgcagggt 300cagctgaagg agacgctgtt cgactggccc gaccgcaagc tgcagagcgg catgaagccc 360cgcggccaga gcgtgctgca ggagaaccag tgcgcgtaca tcaacgactc tctgggactc 420caccgtgtgg agaatgtgag ccacacagag ccggccgtga gtctgcacct ttacagtcct 480ccgttccaga gctgccgcac gtttgaccag cgcaccggac accacaacac cgtcaagatg 540accttctgga gcaaatatgg cgagaggacg ccctatgagc tgagcgtctc gcaggagaat 600aactga 6062201PRTDanio rerio 2Met Glu Gln Thr Glu Val Met Lys Pro Glu Thr Leu Glu Asp Leu Ile 1 5 10 15 Lys Thr Leu His Gln Ile Phe Gln Ser Asp Ser Ile Asn Val Glu Glu 20 25 30 Val Gln Asn Leu Met Glu Ser Tyr Gln Ser Asn Pro Gln Asp Trp Met 35 40 45 Lys Phe Ala Lys Phe Asp Gln Tyr Arg Tyr Thr Arg Asn Leu Val Asp 50 55 60 Glu Gly Asn Gly Lys Phe Asn Leu Met Ile Leu Cys Trp Gly Glu Gly 65 70 75 80 His Gly Ser Ser Ile His Asp His Thr Asp Ser His Cys Phe Leu Lys 85 90 95 Leu Leu Gln Gly Gln Leu Lys Glu Thr Leu Phe Asp Trp Pro Asp Arg 100 105 110 Lys Leu Gln Ser Gly Met Lys Pro Arg Gly Gln Ser Val Leu Gln Glu 115 120 125 Asn Gln Cys Ala Tyr Ile Asn Asp Ser Leu Gly Leu His Arg Val Glu 130 135 140 Asn Val Ser His Thr Glu Pro Ala Val Ser Leu His Leu Tyr Ser Pro 145 150 155 160 Pro Phe Gln Ser Cys Arg Thr Phe Asp Gln Arg Thr Gly His His Asn 165 170 175 Thr Val Lys Met Thr Phe Trp Ser Lys Tyr Gly Glu Arg Thr Pro Tyr 180 185 190 Glu Leu Ser Val Ser Gln Glu Asn Asn 195 200 3810DNAChlamydomonas reinhardtii 3atgtcttcta tcatcgctat gcctatcaac gaggacggtg tcgttgtggt cgaccgcaag 60ctgctgggca acgaggtcga gagcaaggcc cgctgcgcgg acaccgcctg caccgcggct 120gcgcccgccc cgcccgccac ggcggccgcg cccacctcca tgccggagct gttgcaggcg 180ttgcagcgcg ccattgacga ggagaaggcc actggccagg tcgccatcaa cgctgtggac 240cagacgcccg agtccgctgc gcggctgagc gcccgcgtgc aggctctact ctcggcctac 300accagctcca actcgggcga ctggcgacgc tacgccatgt tcaacgacat ccactacgtg 360cgcaacctgg tggatgccaa tgaggacttt gaactaattg ttctttgttg gaagcgcggg 420caagtcagcc gcgtgcacaa ccacgccaac gcgcactgct ggctggcggt gctggacggc 480gagatgcgcg agacgcagtt ccagcgcgcg tccgcgccgc ccggctgccc cgcgcccgcg 540gcctcggagc acgatggcag cactgtgtac gtggagccca cacaggtgtc cgacatgcga 600gtgggtgacg ccggctacat caacgactcc atggcgctgc acaacgtggg gtgttgcatg 660cccgccctgg ccgctggcga ggagggcccc gagggcgggg tgacgctgca ctgctacgcc 720cccccgattc gccgcgtcaa gatctatgag gacagcaagg tcacggagcg cgtgcccggc 780tactactcca agggcggagt gcgcgtttga 8104269PRTChlamydomonas reinhardtii 4Met Ser Ser Ile Ile Ala Met Pro Ile Asn Glu Asp Gly Val Val Val 1 5 10 15 Val Asp Arg Lys Leu Leu Gly Asn Glu Val Glu Ser Lys Ala Arg Cys 20 25 30 Ala Asp Thr Ala Cys Thr Ala Ala Ala Pro Ala Pro Pro Ala Thr Ala 35 40 45 Ala Ala Pro Thr Ser Met Pro Glu Leu Leu Gln Ala Leu Gln Arg Ala 50 55 60 Ile Asp Glu Glu Lys Ala Thr Gly Gln Val Ala Ile Asn Ala Val Asp 65 70 75 80 Gln Thr Pro Glu Ser Ala Ala Arg Leu Ser Ala Arg Val Gln Ala Leu 85 90 95 Leu Ser Ala Tyr Thr Ser Ser Asn Ser Gly Asp Trp Arg Arg Tyr Ala 100 105 110 Met Phe Asn Asp Ile His Tyr Val Arg Asn Leu Val Asp Ala Asn Glu 115 120 125 Asp Phe Glu Leu Ile Val Leu Cys Trp Lys Arg Gly Gln Val Ser Arg 130 135 140 Val His Asn His Ala Asn Ala His Cys Trp Leu Ala Val Leu Asp Gly 145 150 155 160 Glu Met Arg Glu Thr Gln Phe Gln Arg Ala Ser Ala Pro Pro Gly Cys 165 170 175 Pro Ala Pro Ala Ala Ser Glu His Asp Gly Ser Thr Val Tyr Val Glu 180 185 190 Pro Thr Gln Val Ser Asp Met Arg Val Gly Asp Ala Gly Tyr Ile Asn 195 200 205 Asp Ser Met Ala Leu His Asn Val Gly Cys Cys Met Pro Ala Leu Ala 210 215 220 Ala Gly Glu Glu Gly Pro Glu Gly Gly Val Thr Leu His Cys Tyr Ala 225 230 235 240 Pro Pro Ile Arg Arg Val Lys Ile Tyr Glu Asp Ser Lys Val Thr Glu 245 250 255 Arg Val Pro Gly Tyr Tyr Ser Lys Gly Gly Val Arg Val 260 265 51449DNADanio rerio 5atggacgagt ctgatgggaa gctgttcctt actgaggctt tcaacataat catggaagaa 60attcttaaca aaggaaggga cttgaaggag aaggtttgtg agtggaaaga tccagatcag 120ctgagatctc tcctggacct cgaacttcgg gatcatggag aatgtcatga gaagctgctg 180cagagggttc gagatgtggc caaatacagc gtaaaaactt gtcatcctcg gttcttcaat 240cagctgtttg ctggcgtgga ctatcatgca ctgacaggac ggctcatcac tgaaaccctc 300aataccagcc aatacaccta tgaagtggct ccagtgtttg tcctgatgga ggaggaagtg 360atcagtaagc ttcgctctct ggttggctgg tcagaaggag atgggatctt ttgtcctgga 420ggatccatgt ctaacatgta tgccattaac gtcgctcggt actgggcttt tcctcaagtg 480aagacaaaag gcttgtgggc cgcaccacgg atggctatat ttacatcaca acagagtcat 540tactccgtga aaaaaggagc tgcgtttctt ggtattggaa cagaaaatgt tttcattgtg 600caagtggatg agagcggcag catgatacca gaagacctgg aggcaaaaat tgtgcaggca 660aaatcccaag acgctgttcc gtttttcgta aacgccacag ccggaaccac agtgcaggga 720gcctttgacc ctctgaagcg catagctgac atatgtgaaa gaaacggcat gtggatgcat 780gttgacgccg catggggagg aagcgtgctg ttttccaaaa agcacagaca tctggttgca 840ggaatagaaa gagcaaactc ggtgacttgg aatcctcaca aaatgcttct gacgggactg 900cagtgctctg tgattttgtt cagagatact acgaatttgc tcatgcactg tcacagtgcc 960aaagccacat acttgttcca gcaagacaag ttctacgaca caagtctgga cacgggcgac 1020aaatccatcc agtgtggccg gaaggtggat tgcctcaagc tctggctcat gtggaaggca 1080atcggagcta gtggtctttc acagcgtgtc gataaggcct ttgccctcac taggtattta 1140gttgaagaaa tggagaaacg ggagaatttc cagctggtct gtaaggggcc gtttgtgaac 1200gtttgcttct ggtttattcc acccagtctg aaaggaaagg agaacagccc agattaccag 1260gaaagactat ccaaggtggc gccagtcatt aaagagagga tgatgaagcg aggaacgatg 1320atggtgggat atcagccaat ggatgaacac gtcaacttct tccgcatggt ggttgtttct 1380ccacagctca caaccaaaga catggatttc ttccttgatg agatggagaa actcgggaag 1440gatctatga 14496482PRTDanio rerio 6Met Asp Glu Ser Asp Gly Lys Leu Phe Leu Thr Glu Ala Phe Asn Ile 1 5 10 15 Ile Met Glu Glu Ile Leu Asn Lys Gly Arg Asp Leu Lys Glu Lys Val 20 25 30 Cys Glu Trp Lys Asp Pro Asp Gln Leu Arg Ser Leu Leu Asp Leu Glu 35 40 45 Leu Arg Asp His Gly Glu Cys His Glu Lys Leu Leu Gln Arg Val Arg 50 55 60 Asp Val Ala Lys Tyr Ser Val Lys Thr Cys His Pro Arg Phe Phe Asn 65 70 75 80 Gln Leu Phe Ala Gly Val Asp Tyr His Ala Leu Thr Gly Arg Leu Ile 85 90 95 Thr Glu Thr Leu Asn Thr Ser Gln Tyr Thr Tyr Glu Val Ala Pro Val 100 105 110 Phe Val Leu Met Glu Glu Glu Val Ile Ser Lys Leu Arg Ser Leu Val 115 120 125 Gly Trp Ser Glu Gly Asp Gly Ile Phe Cys Pro Gly Gly Ser Met Ser 130 135 140 Asn Met Tyr Ala Ile Asn Val Ala Arg Tyr Trp Ala Phe Pro Gln Val 145 150 155 160 Lys Thr Lys Gly Leu Trp Ala Ala Pro Arg Met Ala Ile Phe Thr Ser 165 170 175 Gln Gln Ser His Tyr Ser Val Lys Lys Gly Ala Ala Phe Leu Gly Ile 180 185 190 Gly Thr Glu Asn Val Phe Ile Val Gln Val Asp Glu Ser Gly Ser Met 195 200 205 Ile Pro Glu Asp Leu Glu Ala Lys Ile Val Gln Ala Lys Ser Gln Asp 210 215 220 Ala Val Pro Phe Phe Val Asn Ala Thr Ala Gly Thr Thr Val Gln Gly 225 230 235 240 Ala Phe Asp Pro Leu Lys Arg Ile Ala Asp Ile Cys Glu Arg Asn Gly 245 250 255 Met Trp Met His Val Asp Ala Ala Trp Gly Gly Ser Val Leu Phe Ser 260 265 270 Lys Lys His Arg His Leu Val Ala Gly Ile Glu Arg Ala Asn Ser Val 275 280 285 Thr Trp Asn Pro His Lys Met Leu Leu Thr Gly Leu Gln Cys Ser Val 290 295 300 Ile Leu Phe Arg Asp Thr Thr Asn Leu Leu Met His Cys His Ser Ala 305 310 315 320 Lys Ala Thr Tyr Leu Phe Gln Gln Asp Lys Phe Tyr Asp Thr Ser Leu 325 330 335 Asp Thr Gly Asp Lys Ser Ile Gln Cys Gly Arg Lys Val Asp Cys Leu 340 345 350 Lys Leu Trp Leu Met Trp Lys Ala Ile Gly Ala Ser Gly Leu Ser Gln 355 360 365 Arg Val Asp Lys Ala Phe Ala Leu Thr Arg Tyr Leu Val Glu Glu Met 370 375 380 Glu Lys Arg Glu Asn Phe Gln Leu Val Cys Lys Gly Pro Phe Val Asn 385 390 395 400 Val Cys Phe Trp Phe Ile Pro Pro Ser Leu Lys Gly Lys Glu Asn Ser 405 410 415 Pro Asp Tyr Gln Glu Arg Leu Ser Lys Val Ala Pro Val Ile Lys Glu 420 425 430 Arg Met Met Lys Arg Gly Thr Met Met Val Gly Tyr Gln Pro Met Asp 435 440 445 Glu His Val Asn Phe Phe Arg Met Val Val Val Ser Pro Gln Leu Thr 450 455 460 Thr Lys Asp Met Asp Phe Phe Leu Asp Glu Met Glu Lys Leu Gly Lys 465 470 475 480 Asp Leu 71629DNAGuillardia theta 7atggtgcccc ccgccttgca tgaagggttc tgcagccctc gaggcaggac ttgttgctct 60caggtgggac acgtggagtt gttggagagc tgggaaacgc aggggaacaa gctgagatgc 120gagcaagacc tcctgctggc caaggttccc tctcgcttcc accaccttga ggaagtggcc 180gagctggatg atatcttcag ggaggtgtat cctctgatcc ggcagtacga gacggagaac 240gcgctagcag acgagcacaa ggtgctggag ttcaggacgc cagcggagct gaaggaggag 300gtggacgtgg ggctgcctga ggagggatct gtggagaaat ttgtcgaggg atgcagaagc 360tctatgaagt acagcgtccg aacgagtcac ccgcgcttca tgaaccagct ctatgctggc 420agcgacccgg cagggcaggt ggcagagctg ctcagtgctg tgctgaacac caccatccac 480acgtacgggg cagctccctt cttctccgtg ctggagcggc aggtgatcga gaagctgggg 540aggatgctgg ggtttcagga gcatgtcgac ggcgtctttg cccccggagg ctcgtacgcg 600aacatggtgg cgctgatagt tgcgaggaac cagcacttcc ctcatgtgcg ggagcatggc 660tggaggagcg acgacaaacc tgttatcttc acttcttccc atgctcacta ctctgtcgcc 720aaggctgcca tgatcacggg gatggggtcg aatcaagtgg tcgctgtgcc tacggacgag 780cagggaagaa tgcagcctgc agcgctggag gaggagatta tgcgagcaaa ggagagcgga 840cggaagcctt tctacgtgag ctgcacggca gggacgacag tgactggggc gtttgacccg 900attgacgaga tctgtcagat atgtagaagg catgagatgt ggctgcacac ggatggcgcg 960tggggaggag ctgcaatatt ctcggaggag cacagaaatc ttctacgagg agttgagggc 1020gtcgatagct tctgcttgaa tccgcacaag atgctggggg tcccgatgca gtgctccgtg 1080ctcatcctca acaaccacga ggggcgctcg agaggagcaa cagaggaaga gagcttggat 1140ctcgggcaga agtcgctgca gtgcggaagg aaacctgatt gcctaaagct ctggctctgc 1200tggaagcgac atggaacccg cgggtttgca aggagggtag atcgcgcgta taccttctcg 1260cagaagttcg cagaaatggt cagaagggac cccaggttct acctgctgat ggacccgatc 1320tcctgcaacg tctgcttctt ctacctccct ccctccctcc ggcagcagct ggtggacaga 1380aacctcaacg acttggaaaa ggaggaggcg cagcggcagc tcaaggagtt ccatgctcga 1440ctcggtcagg ttactcagat catctacagg aggatgcaga aagacggcaa gatgctcatc 1500aacttcagcc ctcttaaaga cagagatctg cctcacttct tccgagccgt catgatccag 1560cagagagtaa cggaagacga tcttgttttc atcctcgatc attttgaaca tctgggaaag 1620gacctctag 16298542PRTGuillardia theta 8Met Val Pro Pro Ala Leu His Glu Gly Phe Cys Ser Pro Arg Gly Arg 1 5 10 15 Thr Cys Cys Ser Gln Val Gly His Val Glu Leu Leu Glu Ser Trp Glu 20 25 30 Thr Gln Gly Asn Lys Leu Arg Cys Glu Gln Asp Leu Leu Leu Ala Lys 35 40 45 Val Pro Ser Arg Phe His His Leu Glu Glu Val Ala Glu Leu Asp Asp 50 55 60 Ile Phe Arg Glu Val Tyr Pro Leu Ile Arg Gln Tyr Glu Thr Glu Asn 65 70 75 80 Ala Leu Ala Asp Glu His Lys Val Leu Glu Phe Arg Thr Pro Ala Glu 85 90 95 Leu Lys Glu Glu Val Asp Val Gly Leu Pro Glu Glu Gly Ser Val Glu 100 105 110 Lys Phe Val Glu Gly Cys Arg Ser Ser Met Lys Tyr Ser Val Arg Thr 115 120 125 Ser His Pro Arg Phe Met Asn Gln Leu Tyr Ala Gly Ser Asp Pro Ala 130 135 140 Gly Gln Val Ala Glu Leu Leu Ser Ala Val Leu Asn Thr Thr Ile His 145 150 155 160 Thr Tyr Gly Ala Ala Pro Phe Phe Ser Val Leu Glu Arg Gln Val Ile 165 170 175 Glu Lys Leu Gly Arg Met Leu Gly Phe Gln Glu His Val Asp Gly Val 180 185 190 Phe Ala Pro Gly Gly Ser Tyr Ala Asn Met Val Ala Leu Ile Val Ala 195 200 205 Arg Asn Gln His Phe Pro His Val Arg Glu His Gly Trp Arg Ser Asp 210 215 220 Asp Lys Pro Val Ile Phe Thr Ser Ser His Ala His Tyr Ser Val Ala 225 230 235 240 Lys Ala Ala Met Ile Thr Gly Met Gly Ser Asn Gln Val Val Ala Val 245 250 255 Pro Thr Asp Glu Gln Gly Arg Met Gln Pro Ala Ala Leu Glu Glu Glu 260 265 270 Ile Met Arg Ala Lys Glu Ser Gly Arg Lys Pro Phe Tyr Val Ser Cys 275 280 285 Thr Ala Gly Thr Thr Val Thr Gly Ala Phe Asp Pro Ile Asp Glu Ile 290 295 300 Cys Gln Ile Cys Arg Arg His Glu Met Trp Leu His Thr Asp Gly Ala 305 310 315 320 Trp Gly Gly Ala Ala Ile Phe Ser Glu Glu His Arg Asn Leu Leu Arg 325 330 335 Gly Val Glu Gly Val Asp Ser Phe Cys Leu Asn Pro His Lys Met Leu 340 345 350 Gly Val Pro Met Gln Cys Ser Val Leu Ile Leu Asn Asn His Glu Gly 355 360 365 Arg Ser Arg Gly Ala Thr Glu Glu Glu Ser Leu Asp Leu Gly Gln Lys 370 375 380 Ser Leu Gln Cys Gly Arg Lys Pro Asp Cys Leu Lys Leu Trp Leu Cys 385 390 395 400 Trp Lys Arg His Gly Thr Arg Gly Phe Ala Arg Arg Val Asp Arg Ala 405 410 415 Tyr Thr Phe Ser Gln Lys Phe Ala Glu Met Val Arg Arg Asp Pro Arg 420 425 430 Phe Tyr Leu Leu Met Asp Pro Ile Ser Cys Asn Val Cys Phe Phe Tyr 435 440 445 Leu Pro Pro Ser Leu Arg Gln Gln Leu Val Asp Arg Asn Leu Asn Asp 450 455 460 Leu Glu Lys Glu Glu Ala Gln Arg Gln Leu Lys Glu Phe His Ala Arg 465 470 475 480 Leu Gly Gln Val Thr Gln Ile Ile Tyr Arg Arg Met Gln Lys Asp Gly 485 490 495 Lys Met Leu Ile Asn Phe Ser Pro Leu Lys Asp Arg Asp Leu Pro His 500 505 510 Phe Phe Arg Ala Val Met Ile Gln Gln Arg Val Thr Glu Asp Asp Leu 515 520 525 Val Phe Ile Leu Asp His Phe Glu His Leu Gly Lys Asp Leu 530 535 540 91776DNADanio rerio 9atggcggcgt ctgcaccctc ctcctcctct tctggcggcg ttccggatcc caactcgaca 60aatttacagc caccttcctc aaactacgac tggagtggag tcgctcatgg atgtacaagg 120aagcttggaa tgaagatatg tgggttcttg cagaagaaca ataatgttga cgacaagggt 180cgaattgtcg ggttgtttaa cgaccagcag cccaggagta ttttaacccg ggacaacgag 240cgagattccc gcttcagacg cacagagacg gacttctcca atctgtatgc aagagatctg 300cttcctgcta aaaatggcga ggagtacacc atgcagttcc tgctggaggt ggtggagatc 360ctcactaact acgtgcgcaa gaccttcgac agatccacca aagtgctgga cttccaccat 420ccacaccagc

tgctggaagg catggagggc ttcaacctgg agctgtgtga ccagcccgag 480agtctggagc agatcctggt ggactgcagg gacactctca aatatggagt ccggacaggt 540cacccaaggt tttttaacca gctgtcttca ggactagaca tcatcggttt agctggagaa 600tggttgacct ccactgccaa caccaacatg ttcacgtatg agattgcgcc agtgtttgtc 660ctgatggagc agctcacact gaagaagatg cgagagattg tcggctggcc gaacggagaa 720ggagatggca ttttctcacc aggaggagcc atctccaaca tgtacagcgt gatggtggct 780cgatataaac actatcctga gattaaaatc aaaggcatgg cggcggctcc cagactggtg 840ctgttcacct cagaacacag tcactactct ataaagaagg ccagtgcagt gttgggtttc 900ggcacagaga atctgatcct gctgagaacg gatgaaagag gtcgagttat tccagctgat 960ttggaggcca aagtcattga cgccaagcag aagggctttg tgccgatgtt tgtgaacgca 1020acggctggat ctacagtgta cggagccttc gacccaatca atgagatcgc cgacatctgt 1080gagaaataca acatgtggct tcacgtagat ggagcgtggg gtggaggttt gctgatgtct 1140agaaaacaca aacacaagct cagtggcatt gagagagcaa actccgtcac ctggaaccca 1200cacaagatga tgggtgttcc tctacagtgc tccgccattc tggtccggga gaagggtctt 1260ctacagggct gtaattccat gtgcgctgga tatctctttc agccggataa gcagtatgac 1320gtcacctatg acacggggga caaggccata cagtgtggcc gtcatgtaga catcttcaaa 1380ttctggctca tgtggaagtc aaagggcact actggttttg agaagcacat tgacaggtgt 1440ctggagctgt cggagtatct ctaccacaag atcaagaaca gagaaggata tgagatggtg 1500tttcaagggg agccacagca cacaaatgta tgtttctggt acattcctcc aagcctgcgg 1560cttctgccag atggagagga gaaacgacat cggcttcata aggtcgcccc aaagatcaag 1620gcactgatga tggagtgcgg gacaacaatg gtgggctacc agcctcaggg tgagaaggtt 1680aacttcttca ggatggtggt ctccaatccg gcggttacca ggtctgacat tgacttcctg 1740atcgatgaga tagaaagact gggacaggat ttatag 177610591PRTDanio rerio 10Met Ala Ala Ser Ala Pro Ser Ser Ser Ser Ser Gly Gly Val Pro Asp 1 5 10 15 Pro Asn Ser Thr Asn Leu Gln Pro Pro Ser Ser Asn Tyr Asp Trp Ser 20 25 30 Gly Val Ala His Gly Cys Thr Arg Lys Leu Gly Met Lys Ile Cys Gly 35 40 45 Phe Leu Gln Lys Asn Asn Asn Val Asp Asp Lys Gly Arg Ile Val Gly 50 55 60 Leu Phe Asn Asp Gln Gln Pro Arg Ser Ile Leu Thr Arg Asp Asn Glu 65 70 75 80 Arg Asp Ser Arg Phe Arg Arg Thr Glu Thr Asp Phe Ser Asn Leu Tyr 85 90 95 Ala Arg Asp Leu Leu Pro Ala Lys Asn Gly Glu Glu Tyr Thr Met Gln 100 105 110 Phe Leu Leu Glu Val Val Glu Ile Leu Thr Asn Tyr Val Arg Lys Thr 115 120 125 Phe Asp Arg Ser Thr Lys Val Leu Asp Phe His His Pro His Gln Leu 130 135 140 Leu Glu Gly Met Glu Gly Phe Asn Leu Glu Leu Cys Asp Gln Pro Glu 145 150 155 160 Ser Leu Glu Gln Ile Leu Val Asp Cys Arg Asp Thr Leu Lys Tyr Gly 165 170 175 Val Arg Thr Gly His Pro Arg Phe Phe Asn Gln Leu Ser Ser Gly Leu 180 185 190 Asp Ile Ile Gly Leu Ala Gly Glu Trp Leu Thr Ser Thr Ala Asn Thr 195 200 205 Asn Met Phe Thr Tyr Glu Ile Ala Pro Val Phe Val Leu Met Glu Gln 210 215 220 Leu Thr Leu Lys Lys Met Arg Glu Ile Val Gly Trp Pro Asn Gly Glu 225 230 235 240 Gly Asp Gly Ile Phe Ser Pro Gly Gly Ala Ile Ser Asn Met Tyr Ser 245 250 255 Val Met Val Ala Arg Tyr Lys His Tyr Pro Glu Ile Lys Ile Lys Gly 260 265 270 Met Ala Ala Ala Pro Arg Leu Val Leu Phe Thr Ser Glu His Ser His 275 280 285 Tyr Ser Ile Lys Lys Ala Ser Ala Val Leu Gly Phe Gly Thr Glu Asn 290 295 300 Leu Ile Leu Leu Arg Thr Asp Glu Arg Gly Arg Val Ile Pro Ala Asp 305 310 315 320 Leu Glu Ala Lys Val Ile Asp Ala Lys Gln Lys Gly Phe Val Pro Met 325 330 335 Phe Val Asn Ala Thr Ala Gly Ser Thr Val Tyr Gly Ala Phe Asp Pro 340 345 350 Ile Asn Glu Ile Ala Asp Ile Cys Glu Lys Tyr Asn Met Trp Leu His 355 360 365 Val Asp Gly Ala Trp Gly Gly Gly Leu Leu Met Ser Arg Lys His Lys 370 375 380 His Lys Leu Ser Gly Ile Glu Arg Ala Asn Ser Val Thr Trp Asn Pro 385 390 395 400 His Lys Met Met Gly Val Pro Leu Gln Cys Ser Ala Ile Leu Val Arg 405 410 415 Glu Lys Gly Leu Leu Gln Gly Cys Asn Ser Met Cys Ala Gly Tyr Leu 420 425 430 Phe Gln Pro Asp Lys Gln Tyr Asp Val Thr Tyr Asp Thr Gly Asp Lys 435 440 445 Ala Ile Gln Cys Gly Arg His Val Asp Ile Phe Lys Phe Trp Leu Met 450 455 460 Trp Lys Ser Lys Gly Thr Thr Gly Phe Glu Lys His Ile Asp Arg Cys 465 470 475 480 Leu Glu Leu Ser Glu Tyr Leu Tyr His Lys Ile Lys Asn Arg Glu Gly 485 490 495 Tyr Glu Met Val Phe Gln Gly Glu Pro Gln His Thr Asn Val Cys Phe 500 505 510 Trp Tyr Ile Pro Pro Ser Leu Arg Leu Leu Pro Asp Gly Glu Glu Lys 515 520 525 Arg His Arg Leu His Lys Val Ala Pro Lys Ile Lys Ala Leu Met Met 530 535 540 Glu Cys Gly Thr Thr Met Val Gly Tyr Gln Pro Gln Gly Glu Lys Val 545 550 555 560 Asn Phe Phe Arg Met Val Val Ser Asn Pro Ala Val Thr Arg Ser Asp 565 570 575 Ile Asp Phe Leu Ile Asp Glu Ile Glu Arg Leu Gly Gln Asp Leu 580 585 590 112958DNAMicromonas pusilla 11atgtccgcgg cgacgggatc attatcccta cccctactcg ggcatctcgc gacctcgcgt 60aacgcacgcg cgcgtcggaa ccgcgccgcc gcggccatcc ccggcgtctc cctcgggaaa 120tcgacctcgg ttttcactcc gcgaggtcct aagcgcatcg cgcgcgtcgt cacctcgaag 180gcgggcccgc attcgaaccc tccgagggcg atatcgaccg tcgacgacgt cctcgcgttc 240accgtgccca ccgacgagcc cgcggccgag accgcctccc ccgccgacag cgactgcgaa 300ggcgagttct gcgacatgaa ggagagctcg tgcacgacga gggacctcat cggcagcacg 360ccgctgctcg atctgagcgc gtactccctg aaccccaccg tgaagatcct cgcgaagtgc 420gagtacctca acccgtccgg gtccatcaaa gaccgcatcg cgacgcacat cctggacaag 480gcgatcaaga gcggcgatct caagcccggg atgaccgtcg tcgcggcgac gtccgggaac 540accggcgccg cgatcgcgat ggcgtgcgcg ttgcgcgggt acgactacat cgtcatcacc 600aacgagaaga cgtccaagga gaaggtggac gcgatgagag cgtacggcgg cgaggtgatc 660gtctccccgt ccggggtgtc cccggacgac ccacagcact accagaacat cgagaacaag 720ctgtgcgagg agaaccccgg gacgtactac ggcgtggatc agtataacaa cccgtacaac 780gcggacgcgt acgaggcgac gctcgggccg gagatttggc gtcagagcgt gggcgcggtg 840acgcacttca tcgtcggcgg cagcaccggc ggcacggtca gcggcacggg gaggtacttg 900aagcaagaga acccggacgt gaggatcgtc ctcgcggacc cgagagggag cgtgttctgg 960gaccacgtcg tcaacggcgt cgccgccgac gacgtcaagg tgtccaagtc gtgggagacg 1020gagggcgtcg gcaaggattc catccccggg tgcctcgacg tctcgatcgt ggacgggatg 1080gtgcgcgcga cggacgagca ggcgttcggc gtgtgccgcg agctcgcgag cagcgacggc 1140ctcctcgtcg gcggcagcag cggtctgaac ctccacgcct cgcgcgtgtt atccggcgac 1200gtcgcggacg acagcgtcat cgtcacggtg ttcccggaca acggcgtgaa gtacctgtcg 1260aagatttaca acgacgactg gctcgactcg aagaagatgg gcggcgcaaa gaactcggac 1320gggaacgcgg agagagccgc ggagtgcgag gtgtactggc gcccggacgc gctctcgttc 1380gcggagcgaa aggcggcggc ggacgccgcc gccgccgccg ccgtcgaggg cgacaacctc 1440tggcccgagg acgagaccga gcgcgagctc aagttcctgg aggaactcgc gccgaagctg 1500acgcagtacc acagagactc catcaagggc gacgagcgcg tgcacagcaa gctccagtcc 1560ccggaggagc tcgcggcgac gttcgccgcc gcgggggcgc ccatcgacct cgcggagggc 1620gacgcccccg cgacggagga gcaactcgcg ctcgcggtgc aggcggtcat ggacaactcg 1680gtccgctcct cgcacccgat gttcttgaac cagctgtacg ccggcgtcga cgtcgtcgcg 1740ctcgcggggg agtggaccgc gagcgcgttg aacgccaacg tgcacacgtt tgaagtcgcg 1800ccggtgctca cggagattga gaaagccgtc ctcgcgaaaa ccgcgcggat gtggctgaac 1860aagcccgggt ctaagacgac gccgccgcac gacggtctgc tcgtccccgg cgggtccctg 1920gcgaacatgt actcgatgat cctcgcgcgc gatcgcgcgg agccggaggc gaagaccaag 1980ggcgcgagcg gcaacctcgt cgcgttttgc tcggagcagt cgcactactc gtacaaaaag 2040tccgcgatgg tcatgggcct cgggatggac aacatgatca aggtgaagtg cgaccagtcc 2100ggcgcgatga tcccggcgga gctcgagaag gcggttcagg aggccaagtc ccggggcaag 2160gtgccgttct acgtcggcac caccgcgggg tccaccgtgc tcggcgcctt tgacgactac 2220gaaggctgcg cggacgtctg cgaaaagcac gacatgtgga tgcacgtcga cggcgcgtgg 2280ggcggcgccg cggcgctgtc cccgacgaga aggcacaatc tccagggcgc gaacagagcg 2340gactcgttct gctggaaccc gcacaagatg ctcgggttgc cgctccagtg ctccatcttc 2400gtgacgaagc aacccggggc gctgtccaag gcgaacgccg cgcaggcgga ctacttgttc 2460cagccggaca agaacaacgc cgccgcggac ctcggcgacc gcacgattca gtgcggacgc 2520aaggcggacg ccctcaagat ctggctcgcg tggaaggcgc gcggagacga aggctgggcg 2580aatctcgtgg accgctcctt tggcctcgcg gagtacgtcg aggcgtcggt gcgcgagcgg 2640tgcgaaaaag acggctcgtt cgtcctcgcc gcgcccgcgc agtgcgcgaa catcgggttc 2700tggtacgtgc ccccgcgcct gaggccgttc gatgtcgagt ccgcgaccgc ggaccagctc 2760acggagattg ggttcgtcgc cccgaagctg aaggaccgga tgcaacggac cggggacgcg 2820atgatcgggt tccagccgat cgactcgatg aaccttccaa actttttccg actcgtgctt 2880ccaaactcga ggcacctgtc gaagaacgcg ctcgacgcta tgctcgatcg catggacgac 2940atgggcaaag acctgtga 295812985PRTMicromonas pusilla 12Met Ser Ala Ala Thr Gly Ser Leu Ser Leu Pro Leu Leu Gly His Leu 1 5 10 15 Ala Thr Ser Arg Asn Ala Arg Ala Arg Arg Asn Arg Ala Ala Ala Ala 20 25 30 Ile Pro Gly Val Ser Leu Gly Lys Ser Thr Ser Val Phe Thr Pro Arg 35 40 45 Gly Pro Lys Arg Ile Ala Arg Val Val Thr Ser Lys Ala Gly Pro His 50 55 60 Ser Asn Pro Pro Arg Ala Ile Ser Thr Val Asp Asp Val Leu Ala Phe 65 70 75 80 Thr Val Pro Thr Asp Glu Pro Ala Ala Glu Thr Ala Ser Pro Ala Asp 85 90 95 Ser Asp Cys Glu Gly Glu Phe Cys Asp Met Lys Glu Ser Ser Cys Thr 100 105 110 Thr Arg Asp Leu Ile Gly Ser Thr Pro Leu Leu Asp Leu Ser Ala Tyr 115 120 125 Ser Leu Asn Pro Thr Val Lys Ile Leu Ala Lys Cys Glu Tyr Leu Asn 130 135 140 Pro Ser Gly Ser Ile Lys Asp Arg Ile Ala Thr His Ile Leu Asp Lys 145 150 155 160 Ala Ile Lys Ser Gly Asp Leu Lys Pro Gly Met Thr Val Val Ala Ala 165 170 175 Thr Ser Gly Asn Thr Gly Ala Ala Ile Ala Met Ala Cys Ala Leu Arg 180 185 190 Gly Tyr Asp Tyr Ile Val Ile Thr Asn Glu Lys Thr Ser Lys Glu Lys 195 200 205 Val Asp Ala Met Arg Ala Tyr Gly Gly Glu Val Ile Val Ser Pro Ser 210 215 220 Gly Val Ser Pro Asp Asp Pro Gln His Tyr Gln Asn Ile Glu Asn Lys 225 230 235 240 Leu Cys Glu Glu Asn Pro Gly Thr Tyr Tyr Gly Val Asp Gln Tyr Asn 245 250 255 Asn Pro Tyr Asn Ala Asp Ala Tyr Glu Ala Thr Leu Gly Pro Glu Ile 260 265 270 Trp Arg Gln Ser Val Gly Ala Val Thr His Phe Ile Val Gly Gly Ser 275 280 285 Thr Gly Gly Thr Val Ser Gly Thr Gly Arg Tyr Leu Lys Gln Glu Asn 290 295 300 Pro Asp Val Arg Ile Val Leu Ala Asp Pro Arg Gly Ser Val Phe Trp 305 310 315 320 Asp His Val Val Asn Gly Val Ala Ala Asp Asp Val Lys Val Ser Lys 325 330 335 Ser Trp Glu Thr Glu Gly Val Gly Lys Asp Ser Ile Pro Gly Cys Leu 340 345 350 Asp Val Ser Ile Val Asp Gly Met Val Arg Ala Thr Asp Glu Gln Ala 355 360 365 Phe Gly Val Cys Arg Glu Leu Ala Ser Ser Asp Gly Leu Leu Val Gly 370 375 380 Gly Ser Ser Gly Leu Asn Leu His Ala Ser Arg Val Leu Ser Gly Asp 385 390 395 400 Val Ala Asp Asp Ser Val Ile Val Thr Val Phe Pro Asp Asn Gly Val 405 410 415 Lys Tyr Leu Ser Lys Ile Tyr Asn Asp Asp Trp Leu Asp Ser Lys Lys 420 425 430 Met Gly Gly Ala Lys Asn Ser Asp Gly Asn Ala Glu Arg Ala Ala Glu 435 440 445 Cys Glu Val Tyr Trp Arg Pro Asp Ala Leu Ser Phe Ala Glu Arg Lys 450 455 460 Ala Ala Ala Asp Ala Ala Ala Ala Ala Ala Val Glu Gly Asp Asn Leu 465 470 475 480 Trp Pro Glu Asp Glu Thr Glu Arg Glu Leu Lys Phe Leu Glu Glu Leu 485 490 495 Ala Pro Lys Leu Thr Gln Tyr His Arg Asp Ser Ile Lys Gly Asp Glu 500 505 510 Arg Val His Ser Lys Leu Gln Ser Pro Glu Glu Leu Ala Ala Thr Phe 515 520 525 Ala Ala Ala Gly Ala Pro Ile Asp Leu Ala Glu Gly Asp Ala Pro Ala 530 535 540 Thr Glu Glu Gln Leu Ala Leu Ala Val Gln Ala Val Met Asp Asn Ser 545 550 555 560 Val Arg Ser Ser His Pro Met Phe Leu Asn Gln Leu Tyr Ala Gly Val 565 570 575 Asp Val Val Ala Leu Ala Gly Glu Trp Thr Ala Ser Ala Leu Asn Ala 580 585 590 Asn Val His Thr Phe Glu Val Ala Pro Val Leu Thr Glu Ile Glu Lys 595 600 605 Ala Val Leu Ala Lys Thr Ala Arg Met Trp Leu Asn Lys Pro Gly Ser 610 615 620 Lys Thr Thr Pro Pro His Asp Gly Leu Leu Val Pro Gly Gly Ser Leu 625 630 635 640 Ala Asn Met Tyr Ser Met Ile Leu Ala Arg Asp Arg Ala Glu Pro Glu 645 650 655 Ala Lys Thr Lys Gly Ala Ser Gly Asn Leu Val Ala Phe Cys Ser Glu 660 665 670 Gln Ser His Tyr Ser Tyr Lys Lys Ser Ala Met Val Met Gly Leu Gly 675 680 685 Met Asp Asn Met Ile Lys Val Lys Cys Asp Gln Ser Gly Ala Met Ile 690 695 700 Pro Ala Glu Leu Glu Lys Ala Val Gln Glu Ala Lys Ser Arg Gly Lys 705 710 715 720 Val Pro Phe Tyr Val Gly Thr Thr Ala Gly Ser Thr Val Leu Gly Ala 725 730 735 Phe Asp Asp Tyr Glu Gly Cys Ala Asp Val Cys Glu Lys His Asp Met 740 745 750 Trp Met His Val Asp Gly Ala Trp Gly Gly Ala Ala Ala Leu Ser Pro 755 760 765 Thr Arg Arg His Asn Leu Gln Gly Ala Asn Arg Ala Asp Ser Phe Cys 770 775 780 Trp Asn Pro His Lys Met Leu Gly Leu Pro Leu Gln Cys Ser Ile Phe 785 790 795 800 Val Thr Lys Gln Pro Gly Ala Leu Ser Lys Ala Asn Ala Ala Gln Ala 805 810 815 Asp Tyr Leu Phe Gln Pro Asp Lys Asn Asn Ala Ala Ala Asp Leu Gly 820 825 830 Asp Arg Thr Ile Gln Cys Gly Arg Lys Ala Asp Ala Leu Lys Ile Trp 835 840 845 Leu Ala Trp Lys Ala Arg Gly Asp Glu Gly Trp Ala Asn Leu Val Asp 850 855 860 Arg Ser Phe Gly Leu Ala Glu Tyr Val Glu Ala Ser Val Arg Glu Arg 865 870 875 880 Cys Glu Lys Asp Gly Ser Phe Val Leu Ala Ala Pro Ala Gln Cys Ala 885 890 895 Asn Ile Gly Phe Trp Tyr Val Pro Pro Arg Leu Arg Pro Phe Asp Val 900 905 910 Glu Ser Ala Thr Ala Asp Gln Leu Thr Glu Ile Gly Phe Val Ala Pro 915 920 925 Lys Leu Lys Asp Arg Met Gln Arg Thr Gly Asp Ala Met Ile Gly Phe 930 935 940 Gln Pro Ile Asp Ser Met Asn Leu Pro Asn Phe Phe Arg Leu Val Leu 945 950 955 960 Pro Asn Ser Arg His Leu Ser Lys Asn Ala Leu Asp Ala Met Leu Asp 965 970 975 Arg Met Asp Asp Met Gly Lys Asp Leu 980 985 13177DNAArabidopsis thaliana 13atggctgctt atggtcaaat ctcctcggga atgactgtag atcctcaggt tctctcttcc 60tccagaaaca ttggagtttc cctatcacct ctccggagaa cactaatcgg cgccggagtt 120aggtctacta gtatctctct ccgtcaatgt tctctctccg ttagatcgat taaaatc 1771459PRTArabidopsis thaliana 14Met Ala Ala Tyr Gly Gln Ile Ser Ser Gly Met Thr Val Asp Pro Gln 1 5 10

15 Val Leu Ser Ser Ser Arg Asn Ile Gly Val Ser Leu Ser Pro Leu Arg 20 25 30 Arg Thr Leu Ile Gly Ala Gly Val Arg Ser Thr Ser Ile Ser Leu Arg 35 40 45 Gln Cys Ser Leu Ser Val Arg Ser Ile Lys Ile 50 55 1539DNAArtificial SequenceSynthetic sequence 15agtactgaag gcgaagttaa cgcggaagaa gaaggcttt 39161020DNAEscherichia coli 16atgaatgttt ttattcctga atactgctcc cataacaaga caggggagca gacaatcatg 60gcaatttcat cgcgtaacac acttcttgcc gcactggcat tcatcgcttt tcaggcacag 120gcggtgaacg tcaccgtggc gtatcaaacc tcagccgaac cggcgaaagt ggctcaggcc 180gacaacacct ttgctaaaga aagcggagca accgtggact ggcgtaagtt tgacagcgga 240gccagcatcg tgcgggcgct ggcttcaggc gacgtgcaaa tcggcaacct cggttccagc 300ccgttagcgg ttgcagccag ccaacaggtg ccgattgaag tcttcttgct ggcgtcaaaa 360ctgggtaact ccgaagcgct ggtggtaaag aaaactatca gcaaaccgga agatctgatt 420ggcaaacgca tcgccgtacc gtttatctcc accacccact acagcctgct ggcggcactg 480aaacactggg gcattaaacc cgggcaagtg gagattgtga acctgcagcc gcccgcgatt 540atcgctgcct ggcagcgggg agatattgat ggtgcttatg tctgggcacc ggcggttaac 600gccctggaaa aagacggcaa ggtgttgacc gattctgaac aggtcgggca gtggggcgcg 660ccaacgctgg acgtctgggt ggtgcgcaaa gattttgccg agaaacatcc tgaggtcgtg 720aaagcgttcg ctaaaagcgc catcgatgct cagcaaccgt acattgctaa cccagacgtg 780tggctgaaac agccggaaaa catcagcaaa ctggcgcgtt taagcggcgt gcctgaaggt 840gacgttccgg ggctggtgaa ggggaatacc tatctgacgc cgcagcaaca aacggcagaa 900ctgaccggac cggtgaacaa agcgatcatc gacaccgcgc agtttttgaa agagcagggc 960aaggtcccgg ctgtagcgaa tgattacagc cagtacgtta cctcgcgctt cgtgcaataa 102017320PRTEscherichia coli 17Met Ala Ile Ser Ser Arg Asn Thr Leu Leu Ala Ala Leu Ala Phe Ile 1 5 10 15 Ala Phe Gln Ala Gln Ala Val Asn Val Thr Val Ala Tyr Gln Thr Ser 20 25 30 Ala Glu Pro Ala Lys Val Ala Gln Ala Asp Asn Thr Phe Ala Lys Glu 35 40 45 Ser Gly Ala Thr Val Asp Trp Arg Lys Phe Asp Ser Gly Ala Ser Ile 50 55 60 Val Arg Ala Leu Ala Ser Gly Asp Val Gln Ile Gly Asn Leu Gly Ser 65 70 75 80 Ser Pro Leu Ala Val Ala Ala Ser Gln Gln Val Pro Ile Glu Val Phe 85 90 95 Leu Leu Ala Ser Lys Leu Gly Asn Ser Glu Ala Leu Val Val Lys Lys 100 105 110 Thr Ile Ser Lys Pro Glu Asp Leu Ile Gly Lys Arg Ile Ala Val Pro 115 120 125 Phe Ile Ser Thr Thr His Tyr Ser Leu Leu Ala Ala Leu Lys His Trp 130 135 140 Gly Ile Lys Pro Gly Gln Val Glu Ile Val Asn Leu Gln Pro Pro Ala 145 150 155 160 Ile Ile Ala Ala Trp Gln Arg Gly Asp Ile Asp Gly Ala Tyr Val Trp 165 170 175 Ala Pro Ala Val Asn Ala Leu Glu Lys Asp Gly Lys Val Leu Thr Asp 180 185 190 Ser Glu Gln Val Gly Gln Trp Gly Ala Pro Thr Leu Asp Val Trp Val 195 200 205 Val Arg Lys Asp Phe Ala Glu Lys His Pro Glu Val Val Lys Ala Phe 210 215 220 Ala Lys Ser Ala Ile Asp Ala Gln Gln Pro Tyr Ile Ala Asn Pro Asp 225 230 235 240 Val Trp Leu Lys Gln Pro Glu Asn Ile Ser Lys Leu Ala Arg Leu Ser 245 250 255 Gly Val Pro Glu Gly Asp Val Pro Gly Leu Val Lys Gly Asn Thr Tyr 260 265 270 Leu Thr Pro Gln Gln Gln Thr Ala Glu Leu Thr Gly Pro Val Asn Lys 275 280 285 Ala Ile Ile Asp Thr Ala Gln Phe Leu Lys Glu Gln Gly Lys Val Pro 290 295 300 Ala Val Ala Asn Asp Tyr Ser Gln Tyr Val Thr Ser Arg Phe Val Gln 305 310 315 320 181017DNARoseobacter denitrificans 18atgacatttc tttcacggat cacgtccggc acagcgattg ccctgacggc gaccatcatg 60agcatcggcg cggctgatgc caaaaacttc aagatcgccg tgggcgacag cggcggcagc 120agccaggaag ccaccggttt ggctttcatc gaagcccttg aggagctttc gggcggcgag 180cacactgcaa cgctgtttct gaacggacag ttggggtccg agcaagacac agtcaacgac 240gcggccatcg gctcgctcga catgtcgatc ctggcgatca acaacgtgac accgttctcg 300ccaactgttg gcgtcttctc gcttccatac gtgatcctga gcctcgaaga tgctgaaaag 360ctgacccagg gcccgatcgg tcaggaactg acagaaaaca caatcgaaga cgcaggcgtt 420cgtatcgtgg cctggaccta cacgggtttc cgccgcctga ccaattccaa aaagccggtc 480acatccgttg ccgatctgca aggtctcgtc attcgcgttc ccaagaacga aatcatgatc 540gacacctaca aggcctgggg catcagccca acgccgatgg catggtcgga aacctttgcg 600ggcctgcaaa ccggcgttgt cgacggtcag gacaacccct acaccaccat caacgcgatg 660aagttctacg aagtacaaaa gtacgtcacg aacatccgct acatcttctc catcgaacct 720ctgatcgtgt ccgagcaggt gtttcaggag ctttccgctg aagatcagga aatcattctg 780gaagcaggca agcgcgcgac ggccgcgtct gcacagttcc tgcgcgacaa ggaagcagag 840atcaaggaac tgctggtcga aaaaggcatg cagatcgacg acccggtcaa caatgagcag 900gagttcattg atctggcgac agcagctgtc tggccgaagt tctacgacag catcggcggc 960atcgaaaaga tgaacgctgt tctggctgaa atcggccgcg agccggtctc cgaataa 101719338PRTRoseobacter denitrificans 19Met Thr Phe Leu Ser Arg Ile Thr Ser Gly Thr Ala Ile Ala Leu Thr 1 5 10 15 Ala Thr Ile Met Ser Ile Gly Ala Ala Asp Ala Lys Asn Phe Lys Ile 20 25 30 Ala Val Gly Asp Ser Gly Gly Ser Ser Gln Glu Ala Thr Gly Leu Ala 35 40 45 Phe Ile Glu Ala Leu Glu Glu Leu Ser Gly Gly Glu His Thr Ala Thr 50 55 60 Leu Phe Leu Asn Gly Gln Leu Gly Ser Glu Gln Asp Thr Val Asn Asp 65 70 75 80 Ala Ala Ile Gly Ser Leu Asp Met Ser Ile Leu Ala Ile Asn Asn Val 85 90 95 Thr Pro Phe Ser Pro Thr Val Gly Val Phe Ser Leu Pro Tyr Val Ile 100 105 110 Leu Ser Leu Glu Asp Ala Glu Lys Leu Thr Gln Gly Pro Ile Gly Gln 115 120 125 Glu Leu Thr Glu Asn Thr Ile Glu Asp Ala Gly Val Arg Ile Val Ala 130 135 140 Trp Thr Tyr Thr Gly Phe Arg Arg Leu Thr Asn Ser Lys Lys Pro Val 145 150 155 160 Thr Ser Val Ala Asp Leu Gln Gly Leu Val Ile Arg Val Pro Lys Asn 165 170 175 Glu Ile Met Ile Asp Thr Tyr Lys Ala Trp Gly Ile Ser Pro Thr Pro 180 185 190 Met Ala Trp Ser Glu Thr Phe Ala Gly Leu Gln Thr Gly Val Val Asp 195 200 205 Gly Gln Asp Asn Pro Tyr Thr Thr Ile Asn Ala Met Lys Phe Tyr Glu 210 215 220 Val Gln Lys Tyr Val Thr Asn Ile Arg Tyr Ile Phe Ser Ile Glu Pro 225 230 235 240 Leu Ile Val Ser Glu Gln Val Phe Gln Glu Leu Ser Ala Glu Asp Gln 245 250 255 Glu Ile Ile Leu Glu Ala Gly Lys Arg Ala Thr Ala Ala Ser Ala Gln 260 265 270 Phe Leu Arg Asp Lys Glu Ala Glu Ile Lys Glu Leu Leu Val Glu Lys 275 280 285 Gly Met Gln Ile Asp Asp Pro Val Asn Asn Glu Gln Glu Phe Ile Asp 290 295 300 Leu Ala Thr Ala Ala Val Trp Pro Lys Phe Tyr Asp Ser Ile Gly Gly 305 310 315 320 Ile Glu Lys Met Asn Ala Val Leu Ala Glu Ile Gly Arg Glu Pro Val 325 330 335 Ser Glu 20852DNAEscherichia coli 20atgagtgaac gtctgagcat taccccgctg gggccgtata tcggcgcaca aatttcgggt 60gccgacctga cgcgcccgtt aagcgataat cagtttgaac agctttacca tgcggtgctg 120cgccatcagg tggtgtttct acgcgatcaa gctattacgc cgcagcagca acgcgcgctg 180gcccagcgtt ttggcgaatt gcatattcac cctgtttacc cgcatgccga aggggttgac 240gagatcatcg tgctggatac ccataacgat aatccgccag ataacgacaa ctggcatacc 300gatgtgacat ttattgaaac gccacccgca ggggcgattc tggcagctaa agagttacct 360tcgaccggcg gtgatacgct ctggaccagc ggtattgcgg cctatgaggc gctctctgtt 420cccttccgcc agctgctgag tgggctgcgt gcggagcatg atttccgtaa atcgttcccg 480gaatacaaat accgcaaaac cgaggaggaa catcaacgct ggcgcgaggc ggtcgcgaaa 540aacccgccgt tgctacatcc ggtggtgcga acgcatccgg tgagcggtaa acaggcgctg 600tttgtgaatg aaggctttac tacgcgaatt gttgatgtga gcgagaaaga gagcgaagcc 660ttgttaagtt ttttgtttgc ccatatcacc aaaccggagt ttcaggtgcg ctggcgctgg 720caaccaaatg atattgcgat ttgggataac cgcgtgaccc agcactatgc caatgccgat 780tacctgccac agcgacggat aatgcatcgg gcgacgatcc ttggggataa accgttttat 840cgggcggggt aa 85221283PRTEscherichia coli 21Met Ser Glu Arg Leu Ser Ile Thr Pro Leu Gly Pro Tyr Ile Gly Ala 1 5 10 15 Gln Ile Ser Gly Ala Asp Leu Thr Arg Pro Leu Ser Asp Asn Gln Phe 20 25 30 Glu Gln Leu Tyr His Ala Val Leu Arg His Gln Val Val Phe Leu Arg 35 40 45 Asp Gln Ala Ile Thr Pro Gln Gln Gln Arg Ala Leu Ala Gln Arg Phe 50 55 60 Gly Glu Leu His Ile His Pro Val Tyr Pro His Ala Glu Gly Val Asp 65 70 75 80 Glu Ile Ile Val Leu Asp Thr His Asn Asp Asn Pro Pro Asp Asn Asp 85 90 95 Asn Trp His Thr Asp Val Thr Phe Ile Glu Thr Pro Pro Ala Gly Ala 100 105 110 Ile Leu Ala Ala Lys Glu Leu Pro Ser Thr Gly Gly Asp Thr Leu Trp 115 120 125 Thr Ser Gly Ile Ala Ala Tyr Glu Ala Leu Ser Val Pro Phe Arg Gln 130 135 140 Leu Leu Ser Gly Leu Arg Ala Glu His Asp Phe Arg Lys Ser Phe Pro 145 150 155 160 Glu Tyr Lys Tyr Arg Lys Thr Glu Glu Glu His Gln Arg Trp Arg Glu 165 170 175 Ala Val Ala Lys Asn Pro Pro Leu Leu His Pro Val Val Arg Thr His 180 185 190 Pro Val Ser Gly Lys Gln Ala Leu Phe Val Asn Glu Gly Phe Thr Thr 195 200 205 Arg Ile Val Asp Val Ser Glu Lys Glu Ser Glu Ala Leu Leu Ser Phe 210 215 220 Leu Phe Ala His Ile Thr Lys Pro Glu Phe Gln Val Arg Trp Arg Trp 225 230 235 240 Gln Pro Asn Asp Ile Ala Ile Trp Asp Asn Arg Val Thr Gln His Tyr 245 250 255 Ala Asn Ala Asp Tyr Leu Pro Gln Arg Arg Ile Met His Arg Ala Thr 260 265 270 Ile Leu Gly Asp Lys Pro Phe Tyr Arg Ala Gly 275 280 22 1146DNAEscherichia coli 22atgagtctga atatgttctg gtttttaccg acccacggtg acgggcatta tctgggaacg 60gaagaaggtt cacgcccggt tgatcacggt tatctgcaac aaattgcgca agcggcggat 120cgtcttggct ataccggtgt gctaattcca acggggcgct cctgcgaaga tgcgtggctg 180gttgccgcat cgatgatccc ggtgacgcag cggctgaagt ttcttgtcgc cctgcgtccc 240agcgtaacct cacctaccgt tgccgcccgc caggccgcca cgcttgaccg tctctcaaat 300ggacgtgcgt tgtttaacct ggtcacaggc agcgatccac aagagctggc aggcgacgga 360gtgttccttg atcatagcga gcgctacgaa gcctcggcgg aatttaccca ggtctggcgg 420cgtttattgc agagagaaac cgtcgatttc aacggtaaac atattcatgt gcgcggagca 480aaactgctct tcccggcgat tcaacagccg tatccgccac tttactttgg cggatcgtca 540gatgtcgccc aggagctggc ggcagaacag gttgatctct acctcacctg gggcgaaccg 600ccggaactgg ttaaagagaa aatcgaacaa gtgcgggcga aagctgccgc gcatggacgc 660aaaattcgtt tcggtattcg tctgcatgtg attgttcgtg aaactaacga cgaagcgtgg 720caggccgccg agcggttaat ctcgcatctt gatgatgaaa ctatcgccaa agcacaggcc 780gcattcgccc ggacggattc cgtagggcaa cagcgaatgg cggcgttaca taacggcaag 840cgcgacaatc tggagatcag ccccaattta tgggcgggcg ttggcttagt gcgcggcggt 900gccgggacgg cgctggtggg cgatggtcct acggtcgctg cgcgaatcaa cgaatatgcc 960gcgcttggca tcgacagttt tgtgctttcg ggctatccgc atctggaaga agcgtatcgg 1020gttggcgagt tgctgttccc gcttctggat gtcgccatcc cggaaattcc ccagccgcag 1080ccgctgaatc cgcaaggcga agcggtggcg aatgatttta tcccccgtaa agtcgcgcaa 1140agctaa 114623381PRTEscherichia coli 23Met Ser Leu Asn Met Phe Trp Phe Leu Pro Thr His Gly Asp Gly His 1 5 10 15 Tyr Leu Gly Thr Glu Glu Gly Ser Arg Pro Val Asp His Gly Tyr Leu 20 25 30 Gln Gln Ile Ala Gln Ala Ala Asp Arg Leu Gly Tyr Thr Gly Val Leu 35 40 45 Ile Pro Thr Gly Arg Ser Cys Glu Asp Ala Trp Leu Val Ala Ala Ser 50 55 60 Met Ile Pro Val Thr Gln Arg Leu Lys Phe Leu Val Ala Leu Arg Pro 65 70 75 80 Ser Val Thr Ser Pro Thr Val Ala Ala Arg Gln Ala Ala Thr Leu Asp 85 90 95 Arg Leu Ser Asn Gly Arg Ala Leu Phe Asn Leu Val Thr Gly Ser Asp 100 105 110 Pro Gln Glu Leu Ala Gly Asp Gly Val Phe Leu Asp His Ser Glu Arg 115 120 125 Tyr Glu Ala Ser Ala Glu Phe Thr Gln Val Trp Arg Arg Leu Leu Gln 130 135 140 Arg Glu Thr Val Asp Phe Asn Gly Lys His Ile His Val Arg Gly Ala 145 150 155 160 Lys Leu Leu Phe Pro Ala Ile Gln Gln Pro Tyr Pro Pro Leu Tyr Phe 165 170 175 Gly Gly Ser Ser Asp Val Ala Gln Glu Leu Ala Ala Glu Gln Val Asp 180 185 190 Leu Tyr Leu Thr Trp Gly Glu Pro Pro Glu Leu Val Lys Glu Lys Ile 195 200 205 Glu Gln Val Arg Ala Lys Ala Ala Ala His Gly Arg Lys Ile Arg Phe 210 215 220 Gly Ile Arg Leu His Val Ile Val Arg Glu Thr Asn Asp Glu Ala Trp 225 230 235 240 Gln Ala Ala Glu Arg Leu Ile Ser His Leu Asp Asp Glu Thr Ile Ala 245 250 255 Lys Ala Gln Ala Ala Phe Ala Arg Thr Asp Ser Val Gly Gln Gln Arg 260 265 270 Met Ala Ala Leu His Asn Gly Lys Arg Asp Asn Leu Glu Ile Ser Pro 275 280 285 Asn Leu Trp Ala Gly Val Gly Leu Val Arg Gly Gly Ala Gly Thr Ala 290 295 300 Leu Val Gly Asp Gly Pro Thr Val Ala Ala Arg Ile Asn Glu Tyr Ala 305 310 315 320 Ala Leu Gly Ile Asp Ser Phe Val Leu Ser Gly Tyr Pro His Leu Glu 325 330 335 Glu Ala Tyr Arg Val Gly Glu Leu Leu Phe Pro Leu Leu Asp Val Ala 340 345 350 Ile Pro Glu Ile Pro Gln Pro Gln Pro Leu Asn Pro Gln Gly Glu Ala 355 360 365 Val Ala Asn Asp Phe Ile Pro Arg Lys Val Ala Gln Ser 370 375 380 24576DNAEscherichia coli 24atgcgtgtca tcaccctggc gggtagtcct cgctttcctt ctcgctccag ctccttgctg 60gaatatgcgc gggaaaaact aaatggcctg gatgtagagg tttatcactg gaatctgcaa 120aacttcgccc cggaagatct actttatgct cgtttcgata gtccggcact caagaccttc 180accgaacagc tgcaacaggc cgatgggctg attgtcgcca cgcctgtgta taaagccgcc 240tattccggtg cgttgaaaac cctgctcgac ctgctgccag aacgcgcttt gcaaggcaaa 300gtggtgctac cgctggcgac gggcggtacc gtggcccatc tgctggcggt cgattatgcc 360cttaaaccag ttttaagcgc actgaaagct caggagatcc tgcacggcgt gtttgccgat 420gactcacaag taattgatta ccatcacaga ccccagttca cgccaaatct gcaaacccgt 480cttgataccg cgctagaaac tttctggcag gcattgcacc gccgcgatgt tcaggttcct 540gaccttctgt ctctgcgagg taatgcccat gcgtaa 57625191PRTEscherichia coli 25Met Arg Val Ile Thr Leu Ala Gly Ser Pro Arg Phe Pro Ser Arg Ser 1 5 10 15 Ser Ser Leu Leu Glu Tyr Ala Arg Glu Lys Leu Asn Gly Leu Asp Val 20 25 30 Glu Val Tyr His Trp Asn Leu Gln Asn Phe Ala Pro Glu Asp Leu Leu 35 40 45 Tyr Ala Arg Phe Asp Ser Pro Ala Leu Lys Thr Phe Thr Glu Gln Leu 50 55 60 Gln Gln Ala Asp Gly Leu Ile Val Ala Thr Pro Val Tyr Lys Ala Ala 65 70 75 80 Tyr Ser Gly Ala Leu Lys Thr Leu Leu Asp Leu Leu Pro Glu Arg Ala 85 90 95 Leu Gln Gly Lys Val Val Leu Pro Leu Ala Thr Gly Gly Thr Val Ala 100 105 110 His Leu Leu Ala Val Asp Tyr Ala Leu Lys Pro Val Leu Ser Ala Leu 115 120 125 Lys Ala Gln Glu Ile Leu His Gly Val Phe Ala Asp Asp Ser Gln Val 130 135 140 Ile Asp Tyr His His Arg Pro Gln Phe Thr Pro Asn Leu Gln Thr Arg 145 150

155 160 Leu Asp Thr Ala Leu Glu Thr Phe Trp Gln Ala Leu His Arg Arg Asp 165 170 175 Val Gln Val Pro Asp Leu Leu Ser Leu Arg Gly Asn Ala His Ala 180 185 190 26 1146DNACorynebacterium glutamicum 26atgacattaa ctttccattg gttcctatcc acttcaggcg attcccgcgg catcatcggc 60ggcggtcacg gtgcagaaaa atccggcacc tcccgcgaat tgagccacag ctacctcaag 120cagttggcgc tagctgccga gaccaacggt tttgaatctg tcctgacacc aacgggcacg 180tggtgcgaag atgcgtggat tactgacgct tctttgattg aggcgacaaa acgcttgaag 240ttcctcgttg cgcttcgccc tgggcagatt ggacctacgc tgtctgctca aatggcttct 300actttccagc gtctgtctgg caaccgtttg ctgatcaatg tggtcaccgg tggggaagat 360gcggagcagc gtgcgtttgg tgatttcttg aacaaggagg agcgctacgc ccgtaccgga 420gaattcttgg atatcgtgag ccgcttgtgg cgaggcgaaa ccgtcacgca ccacggtgaa 480cacctgcagg tggagcaagc tagccttgcg catccgccag agattattcc ggagattctt 540tttggtggat cgtcgccagc tgcaggtgag gtggctgcac gttatgcgga cacctatctc 600acgtggggtg aaactcccga tcaggtggcg cagaaaatca actggatcaa cgagctagca 660gcacagcgcg gccgggaact gcgccatgga atccgcttcc atgtgatcac ccgcgatacg 720tctgaagaag catgggtggt ggcagagaag ttgattagcg gggtcactcc agaacaggtc 780gctaaggctc aagccgggtt tgcaacgtct aagtcggagg ggcagcgccg gatggctgag 840ctgcacagca agggtcgtgc ctttactagt ggctcaactg ctcgtgatct ggaggtgtat 900cccaatgtgt gggcaggcgt cggtttgctt cgcggaggtg caggaacagc ccttgtgggc 960tcgcatgaag aggtcgccga tcgcatcgaa gaatacgcag cactcggctt ggatcagttt 1020gtactgtcgg gttatccaaa cttggaggag gccttccact tcggtgaggg tgtgattccg 1080gagctgctgc gccgcggtgt ggatatcaaa aatcaagaat cacgagtttt ggaacctgtt 1140gggtaa 114627381PRTCorynebacterium glutamicum 27Met Thr Leu Thr Phe His Trp Phe Leu Ser Thr Ser Gly Asp Ser Arg 1 5 10 15 Gly Ile Ile Gly Gly Gly His Gly Ala Glu Lys Ser Gly Thr Ser Arg 20 25 30 Glu Leu Ser His Ser Tyr Leu Lys Gln Leu Ala Leu Ala Ala Glu Thr 35 40 45 Asn Gly Phe Glu Ser Val Leu Thr Pro Thr Gly Thr Trp Cys Glu Asp 50 55 60 Ala Trp Ile Thr Asp Ala Ser Leu Ile Glu Ala Thr Lys Arg Leu Lys 65 70 75 80 Phe Leu Val Ala Leu Arg Pro Gly Gln Ile Gly Pro Thr Leu Ser Ala 85 90 95 Gln Met Ala Ser Thr Phe Gln Arg Leu Ser Gly Asn Arg Leu Leu Ile 100 105 110 Asn Val Val Thr Gly Gly Glu Asp Ala Glu Gln Arg Ala Phe Gly Asp 115 120 125 Phe Leu Asn Lys Glu Glu Arg Tyr Ala Arg Thr Gly Glu Phe Leu Asp 130 135 140 Ile Val Ser Arg Leu Trp Arg Gly Glu Thr Val Thr His His Gly Glu 145 150 155 160 His Leu Gln Val Glu Gln Ala Ser Leu Ala His Pro Pro Glu Ile Ile 165 170 175 Pro Glu Ile Leu Phe Gly Gly Ser Ser Pro Ala Ala Gly Glu Val Ala 180 185 190 Ala Arg Tyr Ala Asp Thr Tyr Leu Thr Trp Gly Glu Thr Pro Asp Gln 195 200 205 Val Ala Gln Lys Ile Asn Trp Ile Asn Glu Leu Ala Ala Gln Arg Gly 210 215 220 Arg Glu Leu Arg His Gly Ile Arg Phe His Val Ile Thr Arg Asp Thr 225 230 235 240 Ser Glu Glu Ala Trp Val Val Ala Glu Lys Leu Ile Ser Gly Val Thr 245 250 255 Pro Glu Gln Val Ala Lys Ala Gln Ala Gly Phe Ala Thr Ser Lys Ser 260 265 270 Glu Gly Gln Arg Arg Met Ala Glu Leu His Ser Lys Gly Arg Ala Phe 275 280 285 Thr Ser Gly Ser Thr Ala Arg Asp Leu Glu Val Tyr Pro Asn Val Trp 290 295 300 Ala Gly Val Gly Leu Leu Arg Gly Gly Ala Gly Thr Ala Leu Val Gly 305 310 315 320 Ser His Glu Glu Val Ala Asp Arg Ile Glu Glu Tyr Ala Ala Leu Gly 325 330 335 Leu Asp Gln Phe Val Leu Ser Gly Tyr Pro Asn Leu Glu Glu Ala Phe 340 345 350 His Phe Gly Glu Gly Val Ile Pro Glu Leu Leu Arg Arg Gly Val Asp 355 360 365 Ile Lys Asn Gln Glu Ser Arg Val Leu Glu Pro Val Gly 370 375 380 28924DNACorynebacterium glutamicum 28atgacgtccc cgcataattt tgtcagtggt gctattgatc tgggtgaggt gaaagcgcgt 60gcggatgcgc gccagaaggc ccatgagcag gggccggtaa ctcagggcat tgctagttcc 120cttgatgtga ccatggagaa cctggagaat gaggtgctgc gtcgttccac gcaggttccg 180gtgattgttc tcgtgggtac cccgcgcagc cctgattcgg agcagttgaa gtcggatctg 240accacgcttg ctgctgaaag tggcaggaag ttcattttcg gttatgtcaa tgctgatacc 300gatgctgatg tggcccaggt gtttggggtg cagggcttgc cgtcggtgat tgctgtggca 360gcgggacgcc ctctggctga tttccagggc ggacagccag cggatgcact aaagcagtgg 420actgatcagg tggttcaggc tgtgggtgga cagctggaag gactgccaga ggaggccaca 480gacggcgaac aagaagacgc tcctgtggaa gacccccgct tcgatgctgc cactgatgct 540ctaaaccgtg gcgctttcga tgaggcgatt gcggtttatg agtccatttt ggcgcaggag 600ccaaacaacg ctgatgcgaa gcaggcacgc gataccgcaa agctgttggg ccggcttgcc 660acggtggatc cttcggtgga tgttgtcgct gctgcagatg ctgatccaac aaacgttgat 720ctggcctaca cagcagctga cgcggctgtt gttgcgggtg atcctgaggc tgcctttgat 780cgtttaattg ctctgctgac catcagcgct ggcgatcaga agaatcaggt gaaggaacgt 840ttgctggagc tgtttggcat gtttgagacc gccgatcccc gtgtgctgca ggcgcgagga 900aagatggcca gcgcgctgtt ctaa 92429307PRTCorynebacterium glutamicum 29Met Thr Ser Pro His Asn Phe Val Ser Gly Ala Ile Asp Leu Gly Glu 1 5 10 15 Val Lys Ala Arg Ala Asp Ala Arg Gln Lys Ala His Glu Gln Gly Pro 20 25 30 Val Thr Gln Gly Ile Ala Ser Ser Leu Asp Val Thr Met Glu Asn Leu 35 40 45 Glu Asn Glu Val Leu Arg Arg Ser Thr Gln Val Pro Val Ile Val Leu 50 55 60 Val Gly Thr Pro Arg Ser Pro Asp Ser Glu Gln Leu Lys Ser Asp Leu 65 70 75 80 Thr Thr Leu Ala Ala Glu Ser Gly Arg Lys Phe Ile Phe Gly Tyr Val 85 90 95 Asn Ala Asp Thr Asp Ala Asp Val Ala Gln Val Phe Gly Val Gln Gly 100 105 110 Leu Pro Ser Val Ile Ala Val Ala Ala Gly Arg Pro Leu Ala Asp Phe 115 120 125 Gln Gly Gly Gln Pro Ala Asp Ala Leu Lys Gln Trp Thr Asp Gln Val 130 135 140 Val Gln Ala Val Gly Gly Gln Leu Glu Gly Leu Pro Glu Glu Ala Thr 145 150 155 160 Asp Gly Glu Gln Glu Asp Ala Pro Val Glu Asp Pro Arg Phe Asp Ala 165 170 175 Ala Thr Asp Ala Leu Asn Arg Gly Ala Phe Asp Glu Ala Ile Ala Val 180 185 190 Tyr Glu Ser Ile Leu Ala Gln Glu Pro Asn Asn Ala Asp Ala Lys Gln 195 200 205 Ala Arg Asp Thr Ala Lys Leu Leu Gly Arg Leu Ala Thr Val Asp Pro 210 215 220 Ser Val Asp Val Val Ala Ala Ala Asp Ala Asp Pro Thr Asn Val Asp 225 230 235 240 Leu Ala Tyr Thr Ala Ala Asp Ala Ala Val Val Ala Gly Asp Pro Glu 245 250 255 Ala Ala Phe Asp Arg Leu Ile Ala Leu Leu Thr Ile Ser Ala Gly Asp 260 265 270 Gln Lys Asn Gln Val Lys Glu Arg Leu Leu Glu Leu Phe Gly Met Phe 275 280 285 Glu Thr Ala Asp Pro Arg Val Leu Gln Ala Arg Gly Lys Met Ala Ser 290 295 300 Ala Leu Phe 305 30387DNARoseobacter denitrificans 30atgaccaaaa cactgacagc tcaggacttg tccgacacct ttgacgcctt caatcgccat 60gacgttgatg gcgtcatgac acatttcgcc gatgattgcg tgttctacac cgtgggcggg 120gatgaagcct atggcgccaa agtcgaaggc gcagaagcga ttgccaaagc attctctgcc 180gtctgggcgg gcatgaagga cgcccattgg gatcatcaca gccactttgt gcatggggat 240cgcgccgtat ccgaatggac gttctccgga actggcgcgg acggcatgcg catcgaagca 300cagggcgctg acctctttac cctgcgcgac ggcaagatca tcgtgaaaca ggccctgcgc 360aaatcccgcc cgcccttcaa ggcttaa 38731128PRTRoseobacter denitrificans 31Met Thr Lys Thr Leu Thr Ala Gln Asp Leu Ser Asp Thr Phe Asp Ala 1 5 10 15 Phe Asn Arg His Asp Val Asp Gly Val Met Thr His Phe Ala Asp Asp 20 25 30 Cys Val Phe Tyr Thr Val Gly Gly Asp Glu Ala Tyr Gly Ala Lys Val 35 40 45 Glu Gly Ala Glu Ala Ile Ala Lys Ala Phe Ser Ala Val Trp Ala Gly 50 55 60 Met Lys Asp Ala His Trp Asp His His Ser His Phe Val His Gly Asp 65 70 75 80 Arg Ala Val Ser Glu Trp Thr Phe Ser Gly Thr Gly Ala Asp Gly Met 85 90 95 Arg Ile Glu Ala Gln Gly Ala Asp Leu Phe Thr Leu Arg Asp Gly Lys 100 105 110 Ile Ile Val Lys Gln Ala Leu Arg Lys Ser Arg Pro Pro Phe Lys Ala 115 120 125 321395DNARoseobacter denitrificans 32atgccacata gaccaaagca ctggcccaag gccagctacg atcccaaata cgatcctatc 60gtcgacgcgg gtcccggtca caaccgggac cacgcaccga cctattggat tggtacggcg 120gggacgccac ctgaagatga cgggccggtg tcgggtgaca tcgatgcgga tgtcgtcgtt 180gtcggctctg gctatacagg tctgtctacc gcaatccacc tggcgaagga ccacggcatc 240aaggcgcatg tccttgaagc caacacagtc gcctggggct gttccacccg caatggcggg 300caggcacaga tttcttccgg tcgtctcaag cggtcggagt ggatcaagcg gtggggcgtg 360gatgtcgcca aaggcatgca cgccgaggtc tgtgaagcct tcgaactgtt caatgatctg 420atcgggtcag atgacattga ttgcgacccg caaaccgggg gccatttcta tattgcccac 480cgcgaaaagg tcatggcgaa gctggaaaag gaatgtgccg tcctgaacga cacgtttggc 540tatggctctc gcattctgtc gcgcgacgaa ctacacgaaa aatacgtgcg ggatcaggaa 600gcacacggtg ccctttggga accggacggg acctcgatcc acgcggcaaa actggccttc 660agctacgtgc gtcttgcgcg caaactcggc gccaagatcc acacggccag cccggtcatg 720gggtggaaga ccgtgaacgg tgtgcatcac ctcaccacgc ccggtggcac ggtgcgcgca 780cgtgccgtgg ccttggcgac agcgggctac acaccgccgg ggctgaacga aaagaccaag 840caccggctca tgccgatcct gtcaaactcc atcgtgacgc gtccgctgag cgatgaggaa 900aaggcgggat gcggttttca ggtgaaatct ccgctgactg acacgcgcac cttgcggcac 960tactaccgct atctgcccga cggacgggtc cagatcggca gccgcagtgc gattacaggt 1020cgagacgcag agaaccccag acatctggag cttctgcaga aaggtctcta tcgcaagttc 1080cccgtgctcg aaggcattga actggattac tcctggtggg gatgggtgga tgtcagccat 1140gacatgatgc cacgcatttt ccagccaaac ccgaagcaaa caatctttta tgcgatgggc 1200tacggcggca acggggtgat gtattccgca caggccggca agcgcatggc gcaaatggtt 1260gcgggcgaag gcaaggacct caaacttccg atcttcacct cgcaactgcc aagccacggt 1320gttctgacac ccttccgcag gttgggccag cgcatggcct acccctacta ctaccttcgc 1380gatgaaattc tctga 139533464PRTRoseobacter denitrificans 33Met Pro His Arg Pro Lys His Trp Pro Lys Ala Ser Tyr Asp Pro Lys 1 5 10 15 Tyr Asp Pro Ile Val Asp Ala Gly Pro Gly His Asn Arg Asp His Ala 20 25 30 Pro Thr Tyr Trp Ile Gly Thr Ala Gly Thr Pro Pro Glu Asp Asp Gly 35 40 45 Pro Val Ser Gly Asp Ile Asp Ala Asp Val Val Val Val Gly Ser Gly 50 55 60 Tyr Thr Gly Leu Ser Thr Ala Ile His Leu Ala Lys Asp His Gly Ile 65 70 75 80 Lys Ala His Val Leu Glu Ala Asn Thr Val Ala Trp Gly Cys Ser Thr 85 90 95 Arg Asn Gly Gly Gln Ala Gln Ile Ser Ser Gly Arg Leu Lys Arg Ser 100 105 110 Glu Trp Ile Lys Arg Trp Gly Val Asp Val Ala Lys Gly Met His Ala 115 120 125 Glu Val Cys Glu Ala Phe Glu Leu Phe Asn Asp Leu Ile Gly Ser Asp 130 135 140 Asp Ile Asp Cys Asp Pro Gln Thr Gly Gly His Phe Tyr Ile Ala His 145 150 155 160 Arg Glu Lys Val Met Ala Lys Leu Glu Lys Glu Cys Ala Val Leu Asn 165 170 175 Asp Thr Phe Gly Tyr Gly Ser Arg Ile Leu Ser Arg Asp Glu Leu His 180 185 190 Glu Lys Tyr Val Arg Asp Gln Glu Ala His Gly Ala Leu Trp Glu Pro 195 200 205 Asp Gly Thr Ser Ile His Ala Ala Lys Leu Ala Phe Ser Tyr Val Arg 210 215 220 Leu Ala Arg Lys Leu Gly Ala Lys Ile His Thr Ala Ser Pro Val Met 225 230 235 240 Gly Trp Lys Thr Val Asn Gly Val His His Leu Thr Thr Pro Gly Gly 245 250 255 Thr Val Arg Ala Arg Ala Val Ala Leu Ala Thr Ala Gly Tyr Thr Pro 260 265 270 Pro Gly Leu Asn Glu Lys Thr Lys His Arg Leu Met Pro Ile Leu Ser 275 280 285 Asn Ser Ile Val Thr Arg Pro Leu Ser Asp Glu Glu Lys Ala Gly Cys 290 295 300 Gly Phe Gln Val Lys Ser Pro Leu Thr Asp Thr Arg Thr Leu Arg His 305 310 315 320 Tyr Tyr Arg Tyr Leu Pro Asp Gly Arg Val Gln Ile Gly Ser Arg Ser 325 330 335 Ala Ile Thr Gly Arg Asp Ala Glu Asn Pro Arg His Leu Glu Leu Leu 340 345 350 Gln Lys Gly Leu Tyr Arg Lys Phe Pro Val Leu Glu Gly Ile Glu Leu 355 360 365 Asp Tyr Ser Trp Trp Gly Trp Val Asp Val Ser His Asp Met Met Pro 370 375 380 Arg Ile Phe Gln Pro Asn Pro Lys Gln Thr Ile Phe Tyr Ala Met Gly 385 390 395 400 Tyr Gly Gly Asn Gly Val Met Tyr Ser Ala Gln Ala Gly Lys Arg Met 405 410 415 Ala Gln Met Val Ala Gly Glu Gly Lys Asp Leu Lys Leu Pro Ile Phe 420 425 430 Thr Ser Gln Leu Pro Ser His Gly Val Leu Thr Pro Phe Arg Arg Leu 435 440 445 Gly Gln Arg Met Ala Tyr Pro Tyr Tyr Tyr Leu Arg Asp Glu Ile Leu 450 455 460 341392DNARoseobacter denitrificans 34atggacggca atttcaatga aaatgatatc tcccgcgtcg tcgaagcaga ccgcgcgcat 60atctggcacc atctgagcca gcacaaacct tacgagacaa cagacccgcg catcattgtc 120gaaggcaagg gcatgaaggt ttgggaccag aagggcaaag agcatcttga tgccgtctcc 180ggtggggtct ggaccgtcaa tgtcggctat ggccgcgaac gcatcgccaa cgccgtgcgg 240gaccagttgg tcaagttgaa ctatttcgcc ggctccgcag gctccatccc cggtgccatg 300ttcgccgagc gtctgatcga gaagatgccg gggctgagcc gcgtttatta ctgcaattcc 360ggctccgagg cgaatgaaaa agccttcaag atggtccgcc agatcgcgca caaacgctat 420ggcggcaaaa agcacaaggt gctttatcgc gagcgtgact atcacggcac caccatttcc 480gccctttccg caggcgggca ggacgaacgg aacgcacaat atggcccctt cacgcccggt 540ttcgtgcgcg tgccccattg ccttgaatac cgcgcctttg aacaggaagg ggcgccacag 600gaaaactacg gtgtctgggc ggcggatcag atcgaaaagg taatcctcgc cgaagggccc 660gataccgtgg gcggcctgtg ccttgaaccg gtcactgcag gtggcggggt gatcacgccc 720cccgatggct actgggagcg tgtgcaggaa atctgccaca aatacgacat cctgctgcat 780atcgacgagg tcgtatgcgg cgtcggtcgg accggcacat ggttcggcta tcagcactac 840ggcatccagc cggatatggt cacgatggcc aagggtgtcg cgtccggtta cgcggcgatc 900gcctgccttg tgaccaatga aaaagtcttc gacatgttca aggatgacgc ctcggatccg 960ctgaactact tccgcgacat ctcgaccttt gggggctgca cggcgggtcc ggcagctgcg 1020ctggaaaacc tgtcgatcat cgaagaagaa ggcctgctgg acaacaccac ggaacagggg 1080gcctatatgc tcgactgtct gggcggcttg atggacaagc acaagatcat cggccaggtg 1140cgcggcaagg ggctgttcct cggtgccgaa ctggtcgagg atcgcgacac gcgcaaaccg 1200gttgacgaaa ggctcgcgca agcggtggtc gcggactgca tgcaacaggg tgtgatcatc 1260ggcgtgacca accgctctct gccgggcaag aacaacacgc tgtgtttctc gcccgccctg 1320atcgccagca aggatgacat tgaccacatc tgcgacgcgg tggacggtgc gctgtcgcgc 1380gttttcggct aa 139235463PRTRoseobacter denitrificans 35Met Asp Gly Asn Phe Asn Glu Asn Asp Ile Ser Arg Val Val Glu Ala 1 5 10 15 Asp Arg Ala His Ile Trp His His Leu Ser Gln His Lys Pro Tyr Glu 20 25 30 Thr Thr Asp Pro Arg Ile Ile Val Glu Gly Lys Gly Met Lys Val Trp 35 40 45 Asp Gln Lys Gly Lys Glu His Leu Asp Ala Val Ser Gly Gly Val Trp 50 55 60 Thr Val Asn Val Gly Tyr Gly Arg Glu Arg Ile Ala Asn Ala Val Arg 65 70 75 80 Asp Gln Leu Val Lys Leu Asn Tyr Phe Ala Gly Ser Ala Gly Ser Ile 85 90 95 Pro Gly Ala Met Phe Ala Glu Arg Leu Ile Glu Lys Met Pro Gly Leu

100 105 110 Ser Arg Val Tyr Tyr Cys Asn Ser Gly Ser Glu Ala Asn Glu Lys Ala 115 120 125 Phe Lys Met Val Arg Gln Ile Ala His Lys Arg Tyr Gly Gly Lys Lys 130 135 140 His Lys Val Leu Tyr Arg Glu Arg Asp Tyr His Gly Thr Thr Ile Ser 145 150 155 160 Ala Leu Ser Ala Gly Gly Gln Asp Glu Arg Asn Ala Gln Tyr Gly Pro 165 170 175 Phe Thr Pro Gly Phe Val Arg Val Pro His Cys Leu Glu Tyr Arg Ala 180 185 190 Phe Glu Gln Glu Gly Ala Pro Gln Glu Asn Tyr Gly Val Trp Ala Ala 195 200 205 Asp Gln Ile Glu Lys Val Ile Leu Ala Glu Gly Pro Asp Thr Val Gly 210 215 220 Gly Leu Cys Leu Glu Pro Val Thr Ala Gly Gly Gly Val Ile Thr Pro 225 230 235 240 Pro Asp Gly Tyr Trp Glu Arg Val Gln Glu Ile Cys His Lys Tyr Asp 245 250 255 Ile Leu Leu His Ile Asp Glu Val Val Cys Gly Val Gly Arg Thr Gly 260 265 270 Thr Trp Phe Gly Tyr Gln His Tyr Gly Ile Gln Pro Asp Met Val Thr 275 280 285 Met Ala Lys Gly Val Ala Ser Gly Tyr Ala Ala Ile Ala Cys Leu Val 290 295 300 Thr Asn Glu Lys Val Phe Asp Met Phe Lys Asp Asp Ala Ser Asp Pro 305 310 315 320 Leu Asn Tyr Phe Arg Asp Ile Ser Thr Phe Gly Gly Cys Thr Ala Gly 325 330 335 Pro Ala Ala Ala Leu Glu Asn Leu Ser Ile Ile Glu Glu Glu Gly Leu 340 345 350 Leu Asp Asn Thr Thr Glu Gln Gly Ala Tyr Met Leu Asp Cys Leu Gly 355 360 365 Gly Leu Met Asp Lys His Lys Ile Ile Gly Gln Val Arg Gly Lys Gly 370 375 380 Leu Phe Leu Gly Ala Glu Leu Val Glu Asp Arg Asp Thr Arg Lys Pro 385 390 395 400 Val Asp Glu Arg Leu Ala Gln Ala Val Val Ala Asp Cys Met Gln Gln 405 410 415 Gly Val Ile Ile Gly Val Thr Asn Arg Ser Leu Pro Gly Lys Asn Asn 420 425 430 Thr Leu Cys Phe Ser Pro Ala Leu Ile Ala Ser Lys Asp Asp Ile Asp 435 440 445 His Ile Cys Asp Ala Val Asp Gly Ala Leu Ser Arg Val Phe Gly 450 455 460 36951DNAEscherichia coli 36atgaatttcc aacaactaaa gataatccgc gaggctgcac gtcaggatta caacctgaca 60gaggttgcga atatgctttt tacctcacag tcaggcgtca gccgtcatat tcgggaactg 120gaggatgaac ttggcatcga aatatttgtt cgacgaggta agcgactgct gggcatgact 180gaaccgggca aagcattact ggtcattgca gaacgtattc tgaatgaagc cagtaatgtt 240cgtcggcttg cagacctgtt taccaacgat acgtctggcg ttctcactat tgcaacgacg 300catactcagg cacgttatag cttgccagag gtcattaaag cttttcgcga acttttcccg 360gaggttcggc tcgagctaat ccaggggacg ccacaggaaa ttgcgacatt gttgcaaaat 420ggcgaagctg atattggtat cgccagcgag cgtttgagta atgacccgca gctcgtcgcc 480ttcccgtggt ttcgttggca ccatagtttg cttgttccac acgatcatcc cttgacgcaa 540atttcaccat tgacgctgga atcaatagcg aagtggccgt taatcactta ccgacagggg 600attacggggc gctcacgtat tgatgacgca tttgcccgca aaggtttgct ggcagatatt 660gtattaagtg cgcaggattc tgatgtcatt aaaacctatg ttgctcttgg gcttgggatc 720ggattagttg ccgagcaatc cagtggcgaa caagaggaag agaatttaat ccgcctggat 780acgcggcatc tttttgatgc taatactgtc tggttgggac tgaagcgagg acaacttcag 840cgtaactatg tctggcgctt tctggaactt tgtaatgcag gactgtcagt tgaggatatc 900aagcggcagg tgatggaaag cagtgaagag gaaattgatt atcagatata g 95137316PRTEscherichia coli 37Met Asn Phe Gln Gln Leu Lys Ile Ile Arg Glu Ala Ala Arg Gln Asp 1 5 10 15 Tyr Asn Leu Thr Glu Val Ala Asn Met Leu Phe Thr Ser Gln Ser Gly 20 25 30 Val Ser Arg His Ile Arg Glu Leu Glu Asp Glu Leu Gly Ile Glu Ile 35 40 45 Phe Val Arg Arg Gly Lys Arg Leu Leu Gly Met Thr Glu Pro Gly Lys 50 55 60 Ala Leu Leu Val Ile Ala Glu Arg Ile Leu Asn Glu Ala Ser Asn Val 65 70 75 80 Arg Arg Leu Ala Asp Leu Phe Thr Asn Asp Thr Ser Gly Val Leu Thr 85 90 95 Ile Ala Thr Thr His Thr Gln Ala Arg Tyr Ser Leu Pro Glu Val Ile 100 105 110 Lys Ala Phe Arg Glu Leu Phe Pro Glu Val Arg Leu Glu Leu Ile Gln 115 120 125 Gly Thr Pro Gln Glu Ile Ala Thr Leu Leu Gln Asn Gly Glu Ala Asp 130 135 140 Ile Gly Ile Ala Ser Glu Arg Leu Ser Asn Asp Pro Gln Leu Val Ala 145 150 155 160 Phe Pro Trp Phe Arg Trp His His Ser Leu Leu Val Pro His Asp His 165 170 175 Pro Leu Thr Gln Ile Ser Pro Leu Thr Leu Glu Ser Ile Ala Lys Trp 180 185 190 Pro Leu Ile Thr Tyr Arg Gln Gly Ile Thr Gly Arg Ser Arg Ile Asp 195 200 205 Asp Ala Phe Ala Arg Lys Gly Leu Leu Ala Asp Ile Val Leu Ser Ala 210 215 220 Gln Asp Ser Asp Val Ile Lys Thr Tyr Val Ala Leu Gly Leu Gly Ile 225 230 235 240 Gly Leu Val Ala Glu Gln Ser Ser Gly Glu Gln Glu Glu Glu Asn Leu 245 250 255 Ile Arg Leu Asp Thr Arg His Leu Phe Asp Ala Asn Thr Val Trp Leu 260 265 270 Gly Leu Lys Arg Gly Gln Leu Gln Arg Asn Tyr Val Trp Arg Phe Leu 275 280 285 Glu Leu Cys Asn Ala Gly Leu Ser Val Glu Asp Ile Lys Arg Gln Val 290 295 300 Met Glu Ser Ser Glu Glu Glu Ile Asp Tyr Gln Ile 305 310 315 38927DNACorynebacterium glutamicum 38atggacaacg acggcggaga catgcgaatc gacgacctac gcagcttcat ttcagtcgcc 60caatcaggcc acctcaccga aaccgccgaa agattaggca tcccgcagcc cacactttcc 120agacgaatca gccgagtgga aaaacacgca ggcaccccac ttttcgaccg cgccggccgc 180aaactcgtcc tcaaccaacg aggccacgcc ttcctcaacc acgccagcgc catcgtcgca 240gaattcaact ccgccgcaac tgaaatcaaa cgcctcatgg acccagaaaa aggcacaatc 300cgactggact tcatgcattc cttgggcact tggatggtcc ccgaacttat ccgaacattc 360cgcgccgaac accccaatgt agaattccaa ctccaccaag cggcagcaat gctcctggta 420gatcgtgttt tggctgatga aactgacctc gcattagttg gccccaaacc tgccgaggtt 480ggtacctctt tagggtgggc gccactgctt cgtcaacgac ttgccctagc tgttcccgca 540gatcaccggc ttgcctcttt ttctggccaa ggagaattgc cgttgattag tgcgacggaa 600gaacctttcg tggcgatgcg agcaggtttc ggcacccgac tcctcatgga tgcattagcc 660gaagaagccg gttttgttcc caatgtggtt ttcgaatcca tggagctcac caccgtcgca 720gggcttgtca gcgcaggtct cggcgttggt gtggttccga tggatgatcc gtaccttccc 780acagtgggaa tcgtgcaacg cccacttagt ccacccgcat atagggaact cggtctggta 840tggaggctta acgcgggacc tgcaccggcc gtggataact tccggaagtt cgtggcggga 900tcgagatatg cattagaaga gggctga 92739308PRTCorynebacterium glutamicum 39Met Asp Asn Asp Gly Gly Asp Met Arg Ile Asp Asp Leu Arg Ser Phe 1 5 10 15 Ile Ser Val Ala Gln Ser Gly His Leu Thr Glu Thr Ala Glu Arg Leu 20 25 30 Gly Ile Pro Gln Pro Thr Leu Ser Arg Arg Ile Ser Arg Val Glu Lys 35 40 45 His Ala Gly Thr Pro Leu Phe Asp Arg Ala Gly Arg Lys Leu Val Leu 50 55 60 Asn Gln Arg Gly His Ala Phe Leu Asn His Ala Ser Ala Ile Val Ala 65 70 75 80 Glu Phe Asn Ser Ala Ala Thr Glu Ile Lys Arg Leu Met Asp Pro Glu 85 90 95 Lys Gly Thr Ile Arg Leu Asp Phe Met His Ser Leu Gly Thr Trp Met 100 105 110 Val Pro Glu Leu Ile Arg Thr Phe Arg Ala Glu His Pro Asn Val Glu 115 120 125 Phe Gln Leu His Gln Ala Ala Ala Met Leu Leu Val Asp Arg Val Leu 130 135 140 Ala Asp Glu Thr Asp Leu Ala Leu Val Gly Pro Lys Pro Ala Glu Val 145 150 155 160 Gly Thr Ser Leu Gly Trp Ala Pro Leu Leu Arg Gln Arg Leu Ala Leu 165 170 175 Ala Val Pro Ala Asp His Arg Leu Ala Ser Phe Ser Gly Gln Gly Glu 180 185 190 Leu Pro Leu Ile Ser Ala Thr Glu Glu Pro Phe Val Ala Met Arg Ala 195 200 205 Gly Phe Gly Thr Arg Leu Leu Met Asp Ala Leu Ala Glu Glu Ala Gly 210 215 220 Phe Val Pro Asn Val Val Phe Glu Ser Met Glu Leu Thr Thr Val Ala 225 230 235 240 Gly Leu Val Ser Ala Gly Leu Gly Val Gly Val Val Pro Met Asp Asp 245 250 255 Pro Tyr Leu Pro Thr Val Gly Ile Val Gln Arg Pro Leu Ser Pro Pro 260 265 270 Ala Tyr Arg Glu Leu Gly Leu Val Trp Arg Leu Asn Ala Gly Pro Ala 275 280 285 Pro Ala Val Asp Asn Phe Arg Lys Phe Val Ala Gly Ser Arg Tyr Ala 290 295 300 Leu Glu Glu Gly 305 401362DNACorynebacterium glutamicum 40atgcttgccg accttcccat cgccttaaac ccacacgaac caacatccat ccccacgcag 60ctcacagaac agatccgtcg tctcgtggcg aggggaattc tcaccccagg agacccgctt 120cccagcagtc gctcactatc cacccaattg ggggtatccc gcggcagtgt ggtgaccgct 180tatgaccaat tggccggtga aggctacctc agcaccgccc gcggttccgg tacaacgatc 240aacccagatc tgcatttgtt gaagcctgtg gaaattgaga agaaggagac gtcgagaagc 300gtcccgcccc cgctgctcaa cctgagcccc ggcgtgcccg ataccgcgac gctcgccgat 360tccgcatggc gcgctgcgtg gcgcgaagcc tgcgccaagc cacccacgca ctcccctgag 420cagggacttt tgaggctgcg gatcgagatc gccgaccacc tgcgccagat gcgtggcctc 480atggtcgagc cggagcagat catcgtcacc gccggcgcgc gcgaggggct gagtctgctg 540ctgcgcacca tggatgcgcc tgcccgcatc ggcgtcgaat cgcccggcta ccccagcctg 600cgccgcatcc cgcaggtgct tggccatgag acgatcgatg tgccgaccga cgaatccggc 660ctcgtacccc gcgcgctgcc ccacgacctc aacgcgctac tggtaacccc tagccatcaa 720tatccctacg gcggctcgct gcccgccgat cgccgcaccg cgctagtcgc gtgggctgag 780gcaaacgatg cgttgcttat tgaagacgac ttcgattctg agctgcgcta cgtcggtatg 840ccgcttccgc cgctgcgtgc gctggcgccc gatcgcacga ttctgctcgg cacgttttcc 900tccgtgatca caccacaagt cgcctgcgga tacctcatcg cgccgacgcc ccaggcgcgc 960gtgctcgcca cgcttcgcgg gattctcggc cagccagtcg gcgccatcac ccaacacgcg 1020ctcgcgtcct acctcgcctc aggcgcttta cgacgccgca cccaacgttt gcggcgcctt 1080taccgacacc gccgctccat cgtccaagac accctcggtg acctcccgaa tacgcagctt 1140cgccccatca acggtggcct ccacgcagtt ctcctttgcg acaaacccca agacctcgtc 1200gtcaccacac tcgcctcccg aggccttaac gtcaccgcgc tttcccacta ctggggcggc 1260accggcgcag acaacggcat cgtcttcggc ttcggctccc acgacgaaga caccctcaga 1320tgggtgcttg ctgagatcag cgatgcggtg tctctaggct aa 136241453PRTCorynebacterium glutamicum 41Met Leu Ala Asp Leu Pro Ile Ala Leu Asn Pro His Glu Pro Thr Ser 1 5 10 15 Ile Pro Thr Gln Leu Thr Glu Gln Ile Arg Arg Leu Val Ala Arg Gly 20 25 30 Ile Leu Thr Pro Gly Asp Pro Leu Pro Ser Ser Arg Ser Leu Ser Thr 35 40 45 Gln Leu Gly Val Ser Arg Gly Ser Val Val Thr Ala Tyr Asp Gln Leu 50 55 60 Ala Gly Glu Gly Tyr Leu Ser Thr Ala Arg Gly Ser Gly Thr Thr Ile 65 70 75 80 Asn Pro Asp Leu His Leu Leu Lys Pro Val Glu Ile Glu Lys Lys Glu 85 90 95 Thr Ser Arg Ser Val Pro Pro Pro Leu Leu Asn Leu Ser Pro Gly Val 100 105 110 Pro Asp Thr Ala Thr Leu Ala Asp Ser Ala Trp Arg Ala Ala Trp Arg 115 120 125 Glu Ala Cys Ala Lys Pro Pro Thr His Ser Pro Glu Gln Gly Leu Leu 130 135 140 Arg Leu Arg Ile Glu Ile Ala Asp His Leu Arg Gln Met Arg Gly Leu 145 150 155 160 Met Val Glu Pro Glu Gln Ile Ile Val Thr Ala Gly Ala Arg Glu Gly 165 170 175 Leu Ser Leu Leu Leu Arg Thr Met Asp Ala Pro Ala Arg Ile Gly Val 180 185 190 Glu Ser Pro Gly Tyr Pro Ser Leu Arg Arg Ile Pro Gln Val Leu Gly 195 200 205 His Glu Thr Ile Asp Val Pro Thr Asp Glu Ser Gly Leu Val Pro Arg 210 215 220 Ala Leu Pro His Asp Leu Asn Ala Leu Leu Val Thr Pro Ser His Gln 225 230 235 240 Tyr Pro Tyr Gly Gly Ser Leu Pro Ala Asp Arg Arg Thr Ala Leu Val 245 250 255 Ala Trp Ala Glu Ala Asn Asp Ala Leu Leu Ile Glu Asp Asp Phe Asp 260 265 270 Ser Glu Leu Arg Tyr Val Gly Met Pro Leu Pro Pro Leu Arg Ala Leu 275 280 285 Ala Pro Asp Arg Thr Ile Leu Leu Gly Thr Phe Ser Ser Val Ile Thr 290 295 300 Pro Gln Val Ala Cys Gly Tyr Leu Ile Ala Pro Thr Pro Gln Ala Arg 305 310 315 320 Val Leu Ala Thr Leu Arg Gly Ile Leu Gly Gln Pro Val Gly Ala Ile 325 330 335 Thr Gln His Ala Leu Ala Ser Tyr Leu Ala Ser Gly Ala Leu Arg Arg 340 345 350 Arg Thr Gln Arg Leu Arg Arg Leu Tyr Arg His Arg Arg Ser Ile Val 355 360 365 Gln Asp Thr Leu Gly Asp Leu Pro Asn Thr Gln Leu Arg Pro Ile Asn 370 375 380 Gly Gly Leu His Ala Val Leu Leu Cys Asp Lys Pro Gln Asp Leu Val 385 390 395 400 Val Thr Thr Leu Ala Ser Arg Gly Leu Asn Val Thr Ala Leu Ser His 405 410 415 Tyr Trp Gly Gly Thr Gly Ala Asp Asn Gly Ile Val Phe Gly Phe Gly 420 425 430 Ser His Asp Glu Asp Thr Leu Arg Trp Val Leu Ala Glu Ile Ser Asp 435 440 445 Ala Val Ser Leu Gly 450 421464DNAArtificial SequenceSynthetic construct 42atggcgattt cccccgaaac cttctttctc gcctccgaag ccgagggcac gctccagacc 60cggatccgcc agatggtggc cgaggggatc ctgaccggcc gcttccgccc gggcgagaaa 120ctgccctcct cgcgcaagct cgccgcgcat ctgggcgtca gccggatcac cgtgacactc 180gcctataccg aacttcaggc cgacgattac atcacctcgc gcggccggtc gggctattac 240gtgtccgaca acgcgcccga accgccgtcc tttcccgcgc gcgcacccgg gcagtcgtcg 300gtcgactggt cgcgcgccat cggccagcgg tttcgcggca ccgaaccgca ttcgaaaccc 360ggcaactggg ccgatttccg ctatccgttc atctatggcc aggccgatcc gaccctgttc 420gacgcggcca attggcggct ctgtgccctc caggccctgg ggcgcaagga tttcgccgcg 480ctgaccaccg attacaacga cagcgacgat cccgaattgc tggatttcat cgcccgccag 540atcctgcccc gccgcggcat cctggccggg ccggacgaaa tcctgctgac gctgggcgcc 600cagaacgcgc tgtggctgac cgcccaggtg ctgctgaccc agcgccggac cgcggcgatc 660gaggatccct gctacccggc cctgcgcggc atcctgaccc aggcgcgctg ccacctgcac 720gcggtcccgg tggatcgcga cgggttgccg cccgaggcga tccccgacgg caccaacgtg 780gtgttctgca cccccagcca ccaatgcccg accaccgcga ccatgccgat gtcgcgccgc 840catgccctgc tggaacgcgc cgaggccgag gatttcctga tcgtcgagga tgattacgaa 900ttcgagatgt cgttcctcaa atccccctcg ccggcgctga aatcgctcga ccggcacggg 960cgggtgatct acgtcggctc tttctccaaa tcgctgtttc cgggcctgcg cctgggctat 1020ctggtcggcc ccgagccgtt catccgggag gcccgcgccc tgcgtgccag cgtgctgcgc 1080cacccgccgg gccatatcca gcgcaccgtc acctatttcc tgtctcttgg ccattacgac 1140gccctgatcc gccgcatggg ccgggcctat cacgaccggc gccggatcat ggaccgcgcc 1200ctgcacgatc acgggctgac cgtcgccgga tcgggctcct tcggcggctc gtccttctgg 1260atgcgcgcgc ccgcgggcgt cgatacggcc gagctggccc gccgcctgtc cgccgacagc 1320gtgctgatcg aaccgggcca gccgttcttt gccggcaccg cgccgcccgg gcggttctac 1380cgcctggcct atagttcgat ctctggcccg cgcatccccg acgggatcgc gcggatcgcc 1440gccgcgctgg agaactggtc ctag 146443487PRTArtificial SequenceSynthetic construct 43Met Ala Ile Ser Pro Glu Thr Phe Phe Leu Ala Ser Glu Ala Glu Gly 1 5 10 15 Thr Leu Gln Thr Arg Ile Arg Gln Met Val Ala Glu Gly Ile Leu Thr 20 25 30 Gly Arg Phe Arg Pro Gly Glu Lys Leu Pro Ser Ser Arg Lys Leu Ala 35 40 45 Ala His Leu Gly Val Ser Arg Ile Thr Val Thr Leu Ala Tyr Thr Glu 50 55 60 Leu Gln Ala Asp Asp Tyr Ile Thr Ser Arg Gly Arg Ser Gly Tyr Tyr 65 70 75 80 Val Ser Asp Asn Ala Pro Glu Pro Pro Ser Phe Pro Ala Arg Ala Pro 85 90

95 Gly Gln Ser Ser Val Asp Trp Ser Arg Ala Ile Gly Gln Arg Phe Arg 100 105 110 Gly Thr Glu Pro His Ser Lys Pro Gly Asn Trp Ala Asp Phe Arg Tyr 115 120 125 Pro Phe Ile Tyr Gly Gln Ala Asp Pro Thr Leu Phe Asp Ala Ala Asn 130 135 140 Trp Arg Leu Cys Ala Leu Gln Ala Leu Gly Arg Lys Asp Phe Ala Ala 145 150 155 160 Leu Thr Thr Asp Tyr Asn Asp Ser Asp Asp Pro Glu Leu Leu Asp Phe 165 170 175 Ile Ala Arg Gln Ile Leu Pro Arg Arg Gly Ile Leu Ala Gly Pro Asp 180 185 190 Glu Ile Leu Leu Thr Leu Gly Ala Gln Asn Ala Leu Trp Leu Thr Ala 195 200 205 Gln Val Leu Leu Thr Gln Arg Arg Thr Ala Ala Ile Glu Asp Pro Cys 210 215 220 Tyr Pro Ala Leu Arg Gly Ile Leu Thr Gln Ala Arg Cys His Leu His 225 230 235 240 Ala Val Pro Val Asp Arg Asp Gly Leu Pro Pro Glu Ala Ile Pro Asp 245 250 255 Gly Thr Asn Val Val Phe Cys Thr Pro Ser His Gln Cys Pro Thr Thr 260 265 270 Ala Thr Met Pro Met Ser Arg Arg His Ala Leu Leu Glu Arg Ala Glu 275 280 285 Ala Glu Asp Phe Leu Ile Val Glu Asp Asp Tyr Glu Phe Glu Met Ser 290 295 300 Phe Leu Lys Ser Pro Ser Pro Ala Leu Lys Ser Leu Asp Arg His Gly 305 310 315 320 Arg Val Ile Tyr Val Gly Ser Phe Ser Lys Ser Leu Phe Pro Gly Leu 325 330 335 Arg Leu Gly Tyr Leu Val Gly Pro Glu Pro Phe Ile Arg Glu Ala Arg 340 345 350 Ala Leu Arg Ala Ser Val Leu Arg His Pro Pro Gly His Ile Gln Arg 355 360 365 Thr Val Thr Tyr Phe Leu Ser Leu Gly His Tyr Asp Ala Leu Ile Arg 370 375 380 Arg Met Gly Arg Ala Tyr His Asp Arg Arg Arg Ile Met Asp Arg Ala 385 390 395 400 Leu His Asp His Gly Leu Thr Val Ala Gly Ser Gly Ser Phe Gly Gly 405 410 415 Ser Ser Phe Trp Met Arg Ala Pro Ala Gly Val Asp Thr Ala Glu Leu 420 425 430 Ala Arg Arg Leu Ser Ala Asp Ser Val Leu Ile Glu Pro Gly Gln Pro 435 440 445 Phe Phe Ala Gly Thr Ala Pro Pro Gly Arg Phe Tyr Arg Leu Ala Tyr 450 455 460 Ser Ser Ile Ser Gly Pro Arg Ile Pro Asp Gly Ile Ala Arg Ile Ala 465 470 475 480 Ala Ala Leu Glu Asn Trp Ser 485 4470DNAEscherichia coli 44ctgcgcgcgt tacagcgccg cctgacgccc tggcatggag aagtacaatg attccgggga 60tccgtcgacc 704570DNAEscherichia coli 45agggggcgag gggaccgtcc actctcgtat taccccgccc gataaaacgg tgtaggctgg 60agctgcttcg 704670DNAEscherichia coli 46attagacttt aacaataacg ggaaatctga actgcccgga gtttaccgtg attccgggga 60tccgtcgacc 704770DNAEscherichia coli 47aaaagcccgc ttttatagcg ggatttttgc tatatctgat aatcaatttc tgtaggctgg 60agctgcttcg 704870DNAEscherichia coli 48attcgccagc gcatctggca gcccactcaa ctggaaggaa aacaattatg attccgggga 60tccgtcgacc 704970DNAEscherichia coli 49tcttcactgg cgttgccatt atttcttcct tagctttgcg cgactttacg tgtaggctgg 60agctgcttcg 705070DNAEscherichia coli 50gttatcaatg ttaacaaaaa aagaacaatt ggttataagg agagagtatg attccgggga 60tccgtcgacc 705170DNAEscherichia coli 51gcaatcccgc cagcgccagt ttaatgatgt tacgcatggg cattacctcg tgtaggctgg 60agctgcttcg 705229DNACorynebacterium glutamicum 52cgcggatccc tttccattgg ttcctatcc 295341DNACorynebacterium glutamicum 53cttcgatgcg atcggcgacc tcgggaggtg ccggattttt c 415441DNACorynebacterium glutamicum 54gaaaaatccg gcacctcccg aggtcgccga tcgcatcgaa g 415529DNACorynebacterium glutamicum 55cccaagcttc ccaacaggtt ccaaaactc 295629DNACorynebacterium glutamicum 56cgcggatccg cataattttg tcagtggtg 295740DNACorynebacterium glutamicum 57acctgattct tctgatcgcc ctggcgcgca tccgcacgcg 405841DNACorynebacterium glutamicum 58cgcgtgcgga tgcgcgccag ggcgatcaga agaatcaggt g 415930DNACorynebacterium glutamicum 59cccaagcttt agaacagcgc gctggccatc 306029DNACorynebacterium glutamicum 60cgcggatccc aacgacggcg gagacatgc 296143DNACorynebacterium glutamicum 61ccatcggaac cacaccaacg ccggccggcg cggtcgaaaa gtg 436243DNACorynebacterium glutamicum 62cacttttcga ccgcgccggc cggcgttggt gtggttccga tgg 436330DNACorynebacterium glutamicum 63cccaagcttg gccggcgcgg tcgaaaagtg 306430DNACorynebacterium glutamicum 64cgcggatccc cttcccatcg ccttaaaccc 306541DNACorynebacterium glutamicum 65cgaggtcttg gggtttgtcg cctgggaagc gggtctcctg g 416641DNACorynebacterium glutamicum 66ccaggagacc cgcttcccag gcgacaaacc ccaagacctc g 416731DNACorynebacterium glutamicum 67cccaagcttg cctagagaca ccgcatcgct g 3168102DNAArtificial SequenceSynthetic construct 68acccgttttt tgggctaacg ggaggaatta accatggtgt agatgggcgc atcgtaaccg 60tgcatctgcc agtttgaggg gacgacgaca gtatcggcct ca 102

Claims

1. A cell comprising: (a) two units expressed as two monocistronic messages, wherein (i) a first exogenous DNA which comprises a single expression cassette, wherein the single expression cassette comprises a promoter operably linked to a polynucleotide which encodes a fused cysteine dioxygenase-like (CDOL) with a linker to a portion of the cysteine synthetase/PLP decarboxylase (partCS/PLP-DC) protein (CDOL-linker-SADL) and (ii) a second expression unit that comprises a second promoter operably linked to a second polynucleotide which encodes a taurine-binding protein; or (b) two units expressed as one polycistronic message, wherein (i) an exogenous DNA comprises a single expression cassette, wherein the single expression cassette comprises a promoter operably linked to a polynucleotide which encodes a fused cysteine dioxygenase-like (CDOL) with a linker to a portion of the cysteine synthetase/PLP decarboxylase (partCS/PLP-DC) protein (CDOL-linker-SADL) and (ii) a second polynucleotide which encodes a taurine-binding protein, wherein the expression units are 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:3.

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:11.

4. The cell of claim 1, wherein CDOL comprises the amino acid sequence SEQ ID NO:4.

5. The cell of claim 1, wherein partCS/PLP-DC comprises amino acids 1 plus 472 through 985 of the sequence SEQ ID NO:12.

6. The cell of claim 1, wherein the taurine-binding protein comprises the nucleotide sequence SEQ ID NO:16 or SEQ ID NO:18.

7. The cell of claim 1, wherein the taurine binding protein comprises amino acid sequence SEQ ID NO:17 or SEQ ID NO:19.

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

9. A method of producing hypotaurine or taurine, comprising growing the prokaryotic cell of claim 1 under conditions which permit expression of the first and second polynucleotides of the two monocistronic genes or expression of the polynucleotide of the single polycistronic genes, 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 prokaryotic cell of claim 1.

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

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

14. A feed of claim 13, wherein the feed is an aquafeed.

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

16. The bacterial cell of claim 1 wherein the bacteria is selected from the group consisting of Proteobacteria, Alphaproteobacteria, Betaproteobacteria, Deltaproteobacteria, Epsilonproteobacteria, methanotrophs, Bacillus, Salmonella, Lactococcus, Streptococcus, Brevibacterium, coryneform bacteria, 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.

17. A method of treating the cell of claim 1 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 cell.

18. An animal feed or animal feed supplement comprising a concentrate, extract or secretion of the cell of claim 17, wherein the concentrate, extract or secretion contains hypotaurine or taurine.

19. A bacterial cell line wherein the function of the TauD or cbl genes is disrupted by genetic knock out resulting in negligible expression of the gene products TDO or Cbl, identified herein as TauD/KO or cbl/KO.

20. A TauD/KO or cbl/KO cell comprising: (a) two units expressed as two monocistronic messages, wherein (i) a first exogenous DNA which comprises a single expression cassette, wherein the single expression cassette comprises a promoter operably linked to a polynucleotide which encodes a fused cysteine dioxygenase-like (CDOL) with a linker to a portion of the cysteine synthetase/PLP decarboxylase (partCS/PLP-DC) protein (CDOL-linker-SADL) and (ii) a second expression unit that comprises a second promoter operably linked to a second polynucleotide which encodes a taurine-binding protein; or (b) two units expressed as one polycistronic message, wherein (i) an exogenous DNA comprises a single expression cassette, wherein the single expression cassette comprises a promoter operably linked to a polynucleotide which encodes a fused cysteine dioxygenase-like (CDOL) with a linker to a portion of the cysteine synthetase/PLP decarboxylase (partCS/PLP-DC) protein (CDOL-linker-SADL) and (ii) a second polynucleotide which encodes a taurine-binding protein, wherein the expression units are expressed in the cell and wherein the cell produces taurine.

21. The cell of claim 20, wherein CDOL comprises the nucleotide sequence SEQ ID NO:3.

22. The cell of claim 20, wherein partCS/PLP-DC comprises nucleotides 1 through 3 plus 1414 through 2958 of the sequence SEQ ID NO:11.

23. The cell of claim 20, wherein CDOL comprises the amino acid sequence SEQ ID NO:4.

24. The cell of claim 20, wherein partCS/PLP-DC comprises amino acids 1 plus 472 through 985 of the sequence SEQ ID NO:12.

25. The cell of claim 20, wherein the taurine-binding protein comprises the nucleotide sequence SEQ ID NO:16 or SEQ ID NO:18.

26. The cell of claim 20, wherein the taurine-binding protein comprises amino acid sequence SEQ ID NO:17 or SEQ ID NO:19.

27. The cell of claim 20, which is a prokaryotic cell.

28. A method of producing hypotaurine or taurine, comprising growing the prokaryotic cell of claim 20 under conditions which permit expression of the first and second polynucleotides of the two monocistronic genes or expression of the polynucleotide of the single polycistronic genes, thereby producing taurine.

29. A pharmaceutical composition comprising a concentrate, extract or secretion of the prokaryotic cell of claim 20.

30. A nutritional supplement comprising a concentrate, extract or secretion of the prokaryotic cell of claim 20.

31. A food or food supplement comprising a concentrate, extract or secretion of the prokaryotic cell of claim 20.

32. An animal feed or feed supplement comprising a concentrate, extract or secretion of the prokaryotic cell of claim 20.

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

34. A plant growth or yield enhancer comprising a concentrate, extract or secretion of the prokaryotic cell of claim 20.

35. The bacterial cell of claim 20 wherein the bacteria is selected from the group consisting of Proteobacteria, Alphaproteobacteria, Betaproteobacteria, Deltaproteobacteria, Epsilonproteobacteria, methanotrophs, Bacillus, Salmonella, Lactococcus, Streptococcus, Brevibacterium, coryneform bacteria, 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.

36. A method of treating the cell of claim 20 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 cell.

37. An animal feed or animal feed supplement comprising a concentrate, extract or secretion of the cell of claim 20, wherein the concentrate, extract or secretion contains hypotaurine or taurine.