Taurine production by biosystems

Abstract

The present invention describes a method for fermentation production of taurine-containing products from unicellular organisms. More specifically, the present invention relates to genetic modification of taurine and / or substrate biosynthetic pathways in unicellular organisms, including bacteria, algae, microalgae, diatoms, yeast or fungi. The present invention also relates to fermentation and processing methods for the production of various taurine-containing products. The present invention also relates to the use of cells, fermentation broths or extracts containing taurine to produce products for use in food, feed, beverages, nutritional and health supplements, cosmetics, household products, pharmaceuticals or agricultural production.

Description

[Technical field]

[0001] Submitting sequences

[0001] This application is filed in electronic format together with a sequence listing. The sequence listing has the title Bio-based Taurine Production 2.txt, was created on January 30, 2022, and is 443 kb in size. The information in the electronic format of the sequence listing is incorporated herein by reference in its entirety.

[0002]

[0002] This invention is in the field of taurine production by unicellular organisms. [Background technology]

[0003]

[0003] Taurine, an essential nutrient for humans and animals

[0004] Taurine, a sulfonic acid, is an essential nutrient for humans and animals (1-6), required for cardiovascular, musculoskeletal, visual, and nervous system function (7), and has been associated with overall health and longevity in humans (1). Taurine is used as a required ingredient in many products, including infant formula, pet food, animal feed, energy drinks, nutritional functional foods, pharmaceuticals, household goods / cosmetics, and plant growth enhancers, in some cases by the FDA. Taurine occurs naturally in meat and other animal products (8), but as we move toward more plant-based foods and feeds, taurine must be added as an ingredient or taken as a dietary supplement (5, 9, 10).

[0004]

[0005] Currently, taurine for dietary supplement use is produced almost entirely through petroleum-based processes. (11) There is a real need for biologically synthesized sources of taurine that are safe, sustainable, and economically capable of being produced on a commercial scale.

[0005]

[0006] The present invention provides a cost-effective method for fermentative production of taurine by a single-cell organism. A method for optimizing taurine production by genetic improvement of the single-cell organism, growth and fermentation conditions, cost-effective nutrient medium, and downstream processing for taurine purification is provided.

[0006]

[0007] Taurine biosynthetic pathway

[0008] Several taurine biosynthetic pathways have been identified. Genes and corresponding gene products, as well as methods for using the genes and corresponding peptides to generate taurine in cells, have been described in the literature (12-20). Briefly, pathway 1: cysteine ​​and oxygen are converted to 3-sulfinoalanine by cysteine ​​dioxygenase (CDO) or a CDO homolog (21). 3-sulfinoalanine is converted to hypotaurine by sulfinoalanine decarboxylase (SAD), glutamic acid decarboxylase (GAD) (22, 23), or by part of cysteine ​​synthetase / PLP decarboxylase (partCS / PLP-DC) (16). Hypotaurine is converted to taurine by spontaneous conversion or by the activity of a yet to be identified hypotaurine dehydrogenase (HTDeHase). Pathway 2: Cysteamine and oxygen are converted to hypotaurine by cysteamine dioxygenase (ADO), which is then converted to taurine. Pathway 3: Cysteine ​​and sulfite are converted to cysteate and hydrogen sulfide by cysteine ​​lyase. Cysteate is converted to taurine by SAD (24) or cysteate sulfonic acid decarboxylase (CAD). Pathway 4: O-phosphoserine and sulfite are converted to cysteate by threonine synthase (TS) (25). Cysteate is then converted to taurine by either SAD or GAD. Pathway 5: Serine can be converted to 2-aminoacrylate by serine dehydratase (SDH) (26). 2-Aminoacrylate and 3'-phosphoadenosine-5'-phosphosulfate (PAPS) are then converted to cysteate by 3'-phosphoadenylylsulfate:2'-aminoacrylate C-sulfotransferase (PAPS-AS). Cysteate is converted to taurine by either SAD or GAD (26, 27). Pathway 6: Cysteine ​​synthetase / PLP decarboxylase (CS / PLP-DC) converts O-acetylserine and hydrogen sulfide or 2-aminoacrylate and PAPS to taurine.

[0007]

[0009] Genes and corresponding peptides involved in taurine synthesis in algal and microalgal species (28) include cysteine ​​dioxygenase (CDO), glutamic acid decarboxylase (GAD), sulfinoalanine decarboxylase (SAD), cysteate synthase (CS), cysteine ​​synthetase / PLP decarboxylase (CS / PLP-DC) or part of cysteine ​​synthetase / PLP decarboxylase (partCS / PLP-DC).

[0008]

[0010] Precursor Pathway

[0011] Similarities between the taurine biosynthetic pathways arise from the requirement for carbon, nitrogen, and sulfur in taurine production. Carbon and nitrogen are provided by components of the serine pathway. Sulfur (sulfate and thiosulfate) is supplied to the cell by a series of reactions involving uptake, reduction, and assimilation. Carbon from glucose enters the serine biosynthetic pathway by conversion of glycerate 1,3-bisphosphate to glycerate 3-phosphate by phosphoglycerate kinase, the product of the pgk gene (29). Glycerate 3-phosphate is converted to 3-phosphohydroxypyruvate by 3-phosphoglycerate dehydrogenase, the product of serA. The serA gene product is sensitive to feedback inhibition by serine, which can be achieved by deleting the last 197 amino acids (serA Δ197 ) (30). 3-Phosphohydroxypyruvate is converted to O-phospho-serine by phosphoserine aminotransferase, the product of serC, and O-phospho-serine is converted to serine by phosphoserine phosphatase, the product of serB. Serine and acetyl-CoA are converted to O-acetyl-serine by serine acetyltransferase, the product of cysE. The cysE gene product is sensitive to feedback inhibition by cysteine, but mutant cysE M201Ris insensitive to cysteine ​​inhibition (31). O-Acetyl-serine is converted to cysteine ​​by cysteine ​​synthase, the product of cysK. Cysteine ​​can be degraded by the product of tna (32). Other serine-based taurine precursors are derived from the compounds named above. The precursor 2-aminoacrylate is generated from serine by threonine dehydratase (28) or serine dehydratase (26), the products of ilvA. The ilvA gene product is sensitive to feedback inhibition by isoleucine, but mutant ilvA L447F is insensitive to isoleucine inhibition (33). 2-Aminoacrylate is converted to 2-ketobutyrate by 2-iminobutanoate / 2-iminopropanoate deaminase, the product of RidA or tdcF, or aminoacrylate peracid reductase, the product of rutC.

[0009]

[0012] Sulfur precursors for taurine biosynthesis are obtained from the sulfur (sulfate and thiosulfate) uptake and reduction pathways. The sulfate-thiosulfate uptake pathway is controlled by the products of sbp, cysP, cysU, cysW, and cysA. Sulfate and thiosulfate are bound by the products of sbp and cysP, respectively, and transported into the cell by the products of cysU, cysW, and cysA (34). Sulfate is converted to 3'-phosphoadenosine-5'-phosphosulfate (PAPS) by ATP sulfurylase and APS kinase, which are products of cysDNC. PAPS is converted to adenosine-3',5'-diphosphate (PAP) and sulfite by PAPS reductase, which is the product of cysH. PAP nucleotidase, which is the product of cysQ, is involved in PAPS regeneration. Sulfite is converted to sulfide by the product of cysIJ. O-acetyl-L-serine and sulfide are converted to cysteine ​​by CysK and CysM. CysM also synthesizes S-sulfocysteine ​​from O-acetyl-L-serine and thiosulfate (35). S-sulfocysteine ​​is converted to cysteine ​​by glutaredoxin (NrdH) or Grx.

[0010]

[0013] Decomposition of taurine and sulfonic acids

[0014] In the absence of sulfur, bacteria utilize the sulfonate uptake and degradation pathway or the taurine uptake and degradation pathway to mobilize carbon, nitrogen, or sulfur (36-39). The genes and corresponding peptides involved in taurine uptake and degradation are usually located on the same operon, tauABCD (40) and ssuEADCB (41), and are induced in the absence of nitrogen (42, 43) or sulfur (36) or in the presence of taurine (39, 44). In other bacteria, such as C. (Corynebacterium) glutamicum, the genes and corresponding peptides involved in sulfonate, taurine uptake and degradation are located on the ssuD1CBA and sueABCD2 operons (45).

[0011]

[0015] The degradative enzyme genes tauX and tauY encode taurine dehydrogenase (TDH) (43), tauD encodes taurine dioxygenase (TDO) (36), tpa encodes taurine-pyruvate aminotransferase (TPAT) (46), and ssuD and ssuE encode the two-component alkanesulfonate monooxygenase 2CASM (37).

[0012]

[0016] Transcriptional factors

[0017] In bacteria, there are several global regulators of sulfur metabolism. The cysB gene product is a LysR-type transcriptional activator of genes involved in sulfur uptake and reduction and cysteine ​​metabolism. cysB is highly conserved in Gram-negative bacteria (47). In Corynebacterium glutamicum, the transcriptional regulator methionine / cysteine ​​biosynthesis repressor (McbR) (48) represses the expression of genes involved in sulfur assimilation and cysteine ​​biosynthesis. The translational regulators Cbl and TauR control the expression and induction of the taurine degradation pathway in bacteria (36, 46). Cbl is a LysR-type transcriptional regulator of the sulfonate uptake and degradation pathway or the taurine uptake and degradation pathway in some bacteria (41, 49). The cbl genes are found in Proteobacteria, including members of the Alphaproteobacteria, Betaproteobacteria, and Gammaproteobacteria. Bacteria that lack the Cbl transcription regulator possess the McbR subfamily of activators, which includes TauR, which controls the taurine uptake and degradation system. TauR is found in the alphaproteobacteria Rhizobiales and Rhodobacterales, the betaproteobacteria Burkholderiaceae and Comamonadaceae, the gammaproteobacteria Enterobacteriales, Oceanospirillales, and Psychromonadales, and the alphaproteobacteria Rhizobiales and Rhodobacter.

[0013]

[0018] Taurine efflux transporter

[0019] Taurine can be exported outside the cell by the products of gadC, yhiM, or AAperm.

[0014]

[0020] Fermentation conditions and nutrient media

[0021] In the described invention, taurine is produced by fermentation. Methods for producing compounds by batch, fed-batch, continuous fermentation, or by tank or pond fermentation are well known to those skilled in the art (50-60).

[0015]

[0022] The culture medium used in the present invention depends on the requirements of the microorganism used for production. Descriptions of media defined for various microorganisms can be found in the literature (61-63). Carbon sources may be used individually or in combination and may include sugars and carbohydrates, such as glucose, sucrose, lactose, fructose, maltose, molasses, starch and cellulose, oils and fats, fatty acids, alcohols, and organic acids. Nitrogen sources may be used individually or as mixtures and may include organic nitrogen-containing compounds, such as peptone, tryptone, casein amino acids, yeast extract, meat extract, malt extract, corn steep liquor, soybean meal, and urea or inorganic compounds, such as ammonium sulfate, ammonium chloride, ammonium phosphate, ammonium carbonate, and ammonium nitrate. Potassium and phosphate sources may include potassium chloride, monopotassium phosphate, dipotassium phosphate, monosodium phosphate, and disodium phosphate. Magnesium sulfate or ferrous sulfate, micronutrients, amino acids, and vitamins are also required for growth.

[0016]

[0023] To adjust the pH of the culture, compounds such as sodium hydroxide, potassium hydroxide, ammonia, ammonium hydroxide, or acids such as phosphoric acid or sulfuric acid are used. To adjust the foaming, antifoaming agents are used. Aerobic conditions are maintained by mixing or introducing air or oxygen into the culture. Dissolved oxygen is 15%-40% depending on the growth phase and the microorganism. The temperature of the culture is 25°C-40°C, preferably 30°C-37°C, depending on the microorganism. Growth of the cell culture is maintained until the maximum taurine production is reached, typically within 10 hours to 100 hours, preferably within 15 hours to 30 hours.

[0017]

[0024] In the described invention, the fermentation broth contains taurine, the microbial cell mass, the organic by-products of the fermentation process, and any remaining medium components.

[0025] The concentration of synthesized taurine can be determined at various time points throughout the fermentation using thin layer chromatography (TLC), amino acid analyzer, high performance liquid chromatography (HPLC), mass spectrometry (MS), electrospray ionization mass spectrometry (ESI-MS), and liquid chromatography tandem mass spectrometry (LC-MS / MS).

[0018]

[0026] Downstream Processing: Separation and Purification

[0027] In the described invention, taurine is processed or purified into a product. The particular downstream processing used depends on several factors, including whether taurine is present in the cells (or biomass) or liquid, the form of the desired final taurine product, e.g., liquid or powder, and the desired purity and / or moisture level. In some product applications, the processing may include drying the cells and medium to an appropriate concentration and dryness. In some product applications, the processing may include purifying or partially purifying the taurine. To reduce costs and increase efficiency, the volume can be reduced at various points throughout the downstream processing by concentrating or removing water by evaporation, e.g., using falling film evaporators, reverse osmosis, or nanofiltration.

[0019]

[0028] If taurine is present in the fermentation broth liquid, the liquid may be separated from the biomass by centrifugation, filtration, decantation or a combination thereof. Further processing of the liquid containing taurine may include concentration or drying or purification steps for the production of a taurine product according to the invention. The purification steps may be selected from the group consisting of chromatographic techniques (54) or membrane-based processes (64), including ion exchange chromatography (64), ultrafiltration, precipitation, pH adjustment and nanofiltration (65), treatment with activated carbon (66) or crystallization. The purification steps or any combination thereof may be repeated until the taurine is purified to the desired specifications, for example for purity and water content.

[0020]

[0029] When taurine is present in cells of fermentation broth, the cells can be separated from the liquid by centrifugation, filtration, decantation or a combination thereof. The taurine-containing cells can be concentrated and used as product, or the cells can be broken by chemicals, pressure, mechanical force or ultrasonic treatment to release the contents. The broken cells with contents can be concentrated or dried and used as product, or the contents can be further processed to produce single cell protein, which can be concentrated or dried and used as product. Alternatively, the taurine in the broken cells can be separated from cell debris by centrifugation, filtration or decantation or a combination thereof, and then further purified as described above.

[0021]

[0030] If taurine is present in both the fermentation broth liquid and the cells, the liquid and cells can be separated and treated separately as described above or concentrated together. The taurine-containing concentrate can be used to produce products according to the invention or can be further processed by purification as described above.

[0022]

[0031] The taurine-containing product can be in various forms, such as a liquid, powder, paste, capsule or tablet. Summary of the Invention [Means for solving the problem]

[0023]

[0032] The present invention provides a method for the fermentative production of taurine-containing products in unicellular organisms. More specifically, the present invention encompasses the use of polynucleotides for taurine biosynthesis enzymes in combination with polynucleotides for serine biosynthesis and sulfur (sulfate or thiosulfate) uptake, reduction, and assimilation, and / or the use of peptide polynucleotides for degrading or transporting taurine to increase taurine in the cell or excrete taurine into the medium. The present invention also relates to fermentation and processing methods for the production of various products produced from cells, fermentation broths, or extracts containing taurine.

[0024]

[0033] For the purposes of promoting an understanding of the principles of the invention, reference will now be made to specific embodiments of the invention and specific language will be used to describe them. The materials, methods, and examples are illustrative only and not limiting.

[0025]

[0034] In some embodiments, the unicellular organism comprises one or more exogenous polynucleotides operably linked to a promoter. In other embodiments, expression of an endogenous polynucleotide of the unicellular organism is modified by an exogenous promoter.

[0026]

[0035] In one embodiment, the present invention provides a taurine biosynthetic pathway comprising exogenous polynucleotides CDO and SAD, and pgk, serA Δ197 The organism comprises a unicellular organism having a serine-based pathway modified to increase expression of cysPUWA, cysDNC, cysQ, cysH, and cysIJ, and a sulfur-based pathway modified to increase expression of cysPUWA, cysDNC, cysQ, cysH, and cysIJ, and to inhibit taurine degradation by knocking out tauD, ssuD, and ssuE, or to inhibit taurine degradation and reuptake of taurine into the cell by knocking out tauABCD, ssuEADCB, ssuD1CBA, or sueABCD2.

[0027]

[0036] In another embodiment, the present invention provides a taurine biosynthetic pathway comprising an exogenous polynucleotide, CS / PLP-DC, pgk, serA Δ197 The organism comprises a unicellular organism having a serine-based pathway modified to increase expression of serC, serD, and serB, and a sulfur-based pathway modified to increase expression of cysDNC and cysQ, and to inhibit taurine degradation by knocking out tauD, ssuD, and ssuE, or to inhibit taurine degradation and reuptake of taurine into the cell by knocking out tauABCD, ssuEADCB, ssuD1CBA, or sueABCD2.

[0028]

[0037] In another embodiment, the present invention provides a taurine biosynthetic pathway comprising an exogenous polynucleotide, CS / PLP-DC, and a serA Δ197 and a serine-based pathway modified by increasing expression of tauABCD, ssuEADCB, ssuD1CBA or sueABCD2 to inhibit the degradation of taurine and its reuptake into the cell.

[0029]

[0038] In another embodiment, the present invention provides a taurine biosynthetic pathway comprising exogenous polynucleotides TS and SAD, and pgk, serA Δ197 The organism comprises a unicellular organism with a serine-based pathway modified to increase expression of sbp, cysUWA, cysDNC, cysQ, and cysH, and a sulfur-based pathway modified to increase expression of sbp, cysUWA, cysDNC, cysQ, and cysH, and to inhibit taurine degradation by knocking out tauD, SsuD, and SsuE, and to inhibit cysteate degradation by knocking out cuyA.

[0030]

[0039] In another embodiment, the present invention provides a taurine biosynthetic pathway comprising exogenous polynucleotides ilvA and PAPS-AS, and serA Δ197The organism consists of a unicellular organism with a serine-based pathway modified to increase expression of serC, serB, and serC, and a sulfur-based pathway modified to increase expression of sbp, cysUWA, cysDNC, and cysQ, and to inhibit taurine degradation by knocking out tauD, ssuD, and ssuE.

[0031]

[0040] In another embodiment, the present invention provides an exogenous polynucleotide, ilvA L447F and the taurine biosynthetic pathway, including PAPS-AS, the taurine efflux transporter gadC, yhiM, and AAperm, and serA Δ197 The present invention is directed to a unicellular organism that has a serine-based pathway modified to increase expression of cysPUWA, cysDNC, and cysQ, and a sulfur-based pathway modified to increase expression of cysPUWA, cysDNC, and cysQ, inhibit taurine degradation by knocking out tauD, ssuD, and ssuE, and inhibit 2-aminoacrylate degradation by knocking out ridA, tdcF, and rutC.

[0032]

[0041] In certain embodiments, the invention comprises modified or mutated taurine-producing unicellular organisms, including bacteria, yeast, fungi, or unicellular algae, for use in food, feed, beverages, nutritional and health supplements, cosmetics, household products, pharmaceuticals, or agricultural production.

[0033]

[0042] In certain embodiments, the present invention also describes a method for growing cells by fermentation and a medium composition for growing the cells for the production of taurine or a taurine product, which may be a liquid, powder, paste, capsule or tablet.

[0034]

[0043] In certain embodiments, the unicellular organism is E. (Escherichia) coli and is grown in a medium comprising at least 5 g / L ammonium sulfate, at least 6 g / L dibasic potassium phosphate, at least 3 g / L monobasic sodium phosphate, at least 0.5 g / L magnesium sulfate, at least 6 g / L glucose, at least 0.1 g / L tryptone, at least 0.05 g / L yeast extract, and at least 0.25 mg / L pyridoxal 5'-phosphate (PLP).

[0035]

[0044] In certain embodiments, the present invention relates to methods of processing cells or media to grow cells and produce a wide range of products, including pure taurine or taurine-containing products. The methods may include isolating taurine to produce taurine with a purity level of greater than 10% pure, greater than 25% pure, greater than 50% pure, greater than 75% pure, or greater than 98% pure. [Brief description of the drawings]

[0036] [Figure 1]

[0045] FIG. 1 is a diagram showing pathways for taurine production in unicellular organisms (outer dotted rectangle). Genes are shown in bold text and molecules in normal text. The taurine pathway is shown by a thick line and the serine, cysteine, sulfur, and degradative pathways are shown by thin lines. Genes coding for taurine uptake and degradation are shown in squares. The natural conversion of hypotaurine to taurine is indicated by an *. [Diagram 2]

[0046] FIG. 2 is an HPLC chromatogram showing purified taurine. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0037]

[0047] The present invention provides a method for the production of taurine (2-aminoethanesulfonic acid) in unicellular organisms. In a preferred embodiment, the present invention provides a method for the genetic modification of unicellular organisms using genes encoding proteins in the taurine biosynthesis pathway, the serine biosynthesis pathway, and a method for increasing sulfur transport, reduction, and assimilation with silenced or knocked-out genes for taurine degradation, or knocked-out precursors or operons for taurine uptake and degradation. The present invention also provides a method for using unicellular organisms, including bacteria, microalgae, fungi, yeast, and algae, with increased levels of endogenous taurine or taurine derivatives, such as hypotaurine, for use in food, feed, beverages, nutritional and health supplements, cosmetics, daily necessities, pharmaceuticals, or agricultural production.

[0038]

[0048] In the present invention, group 1: CDO and SAD, GAD or partCS / PLP-DC; group 2: ADO; group 3: cysteine ​​lyase and SAD or GAD; group 4: TS and SAD or GAD; group 5: ilvA L447F and PAPS-AS and SAD; or Group 6: CS / PLP-DC.

[0039]

[0049] In the present invention, pgk, serA Δ197 , serC, serB, cysE M201R A method is presented for the modification of a unicellular organism to increase expression of one or more polynucleotides of a peptide in the serine or sulfate pathway, which are comprised of cysK, cysM, nrdH, sbp, cysUWA, cysPUWA, cysDNC, cysQ, cysH, and cysIJ.

[0040]

[0050] The present invention provides a method for the modification of unicellular organisms to block taurine uptake and degradation by silencing, mutating or knocking out one or more of the following operons: tauABC, ssuEADCB, ssuD1CBA or sueABCD2.

[0041]

[0051] The present invention provides a method for the modification of unicellular organisms to block taurine by silencing, mutating or knocking out one or more of the following genes: tauX, tauY, tauD, tpa, ssuD, ssuE or ssu1.

[0042]

[0052] The present invention presents a method for the modification of unicellular organisms to block the degradation of precursors by silencing, mutating or knocking out one or more of the following: genes for 2-aminoacrylate degrading enzymes: ridA, tdcF, and rutC, gene for cysteate degrading enzyme: cuyA, and genes for serine degrading enzymes: glyA, sdaA, and ilvA.

[0043]

[0053] The present invention provides a method for the modification of a unicellular organism to control the expression of one or more translational regulatory genes cbl, cysB, tauR or mcbR in the serine, sulfate or taurine pathway.

[0044]

[0054] The present invention provides a method for the modification of unicellular organisms by including one or more exogenous polynucleotides from the group consisting of the following genes for a peptide that transports taurine out of the cell: gadC, yhiM, and AAperm.

[0045]

[0055] Below is a list of suitable polynucleotides appropriate for each gene in certain embodiments. Other polynucleotides suitable for use with the present invention can be obtained by identifying polynucleotides by selectively hybridizing the polynucleotides to the named polynucleotides by hybridization under low stringency, medium stringency or high stringency conditions. Still other polynucleotides suitable for use with the present invention can be obtained by identifying similar polynucleotides that have substantial identity to the nucleic acid or encode polypeptides that have substantial identity to the amino acid sequence when used as a reference in sequence comparisons.

[0046]

[0056] Suitable polynucleotides for CDO are provided in SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, which encode peptides having the amino acid sequences of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, respectively.

[0047]

[0057] Suitable polynucleotides for SAD are provided in SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, which encode peptides having the amino acid sequences of SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, respectively.

[0048]

[0058] A suitable polynucleotide for GAD is provided in SEQ ID NO:15 and encodes a peptide having the amino acid sequence of SEQ ID NO:16.

[0059] Suitable polynucleotides for CS / PL_DC are provided in SEQ ID NO:17 and SEQ ID NO:78, which encode peptides having the amino acid sequences of SEQ ID NO:18 and SEQ ID NO:79, respectively.

[0049]

[0060] A suitable polynucleotide for ADO is provided in SEQ ID NO:19 and encodes a peptide having the amino acid sequence of SEQ ID NO:20.

[0061] Suitable polynucleotides for CL are provided in SEQ ID NO:21, SEQ ID NO:23, which encode peptides having the amino acid sequences of SEQ ID NO:22, SEQ ID NO:24, respectively.

[0050]

[0062] Suitable polynucleotides for TS are provided in SEQ ID NO:25 and SEQ ID NO:27, which encode peptides having the amino acid sequences of SEQ ID NO:26 and SEQ ID NO:28, respectively.

[0063] Suitable polynucleotides for ilvA are provided in SEQ ID NO:136 and SEQ ID NO:140, which encode peptides having the amino acid sequences of SEQ ID NO:137 and SEQ ID NO:141, respectively.

[0051]

[0064] ilvA L447F A suitable polynucleotide is provided in SEQ ID NO:29 and encodes a peptide having the amino acid sequence of SEQ ID NO:30.

[0065] Suitable polynucleotides for PAPS-AS are provided in SEQ ID NO:31 and SEQ ID NO:33, which encode peptides having the amino acid sequences of SEQ ID NO:32 and SEQ ID NO:34, respectively.

[0052]

[0066] A suitable polynucleotide for pgk is provided in SEQ ID NO:35 and encodes a peptide having the amino acid sequence of SEQ ID NO:36.

[0067] User A Δ197 A suitable polynucleotide is provided in SEQ ID NO:37 and encodes a peptide having the amino acid sequence of SEQ ID NO:38.

[0053]

[0068] A suitable polynucleotide for serB is provided in SEQ ID NO:39, and encodes a peptide having the amino acid sequence of SEQ ID NO:40.

[0069] A suitable polynucleotide for serC is provided in SEQ ID NO:41 and encodes a peptide having the amino acid sequence of SEQ ID NO:42.

[0054]

[0070] cysE M201R A suitable polynucleotide is provided in SEQ ID NO:43 and encodes a peptide having the amino acid sequence of SEQ ID NO:44.

[0071] Suitable polynucleotides for cysK are provided in SEQ ID NO:45 and SEQ ID NO:147, which encode peptides having the amino acid sequences of SEQ ID NO:46 and SEQ ID NO:148, respectively.

[0055]

[0072] A suitable polynucleotide for cysDNC is provided in SEQ ID NO:47 and encodes peptides having the amino acid sequences of SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50.

[0073] A suitable polynucleotide for cysQ is provided in SEQ ID NO:51 and encodes a peptide having the amino acid sequence of SEQ ID NO:52.

[0056]

[0074] A suitable polynucleotide for cysH is provided in SEQ ID NO:53 and encodes a peptide having the amino acid sequence of SEQ ID NO:54.

[0075] A suitable polynucleotide for cysIJ is provided in SEQ ID NO:55 and encodes a peptide having the amino acid sequence of SEQ ID NO:57, SEQ ID NO:56.

[0057]

[0076] A suitable polynucleotide for cysB is provided in SEQ ID NO:58 and encodes a peptide having the amino acid sequence of SEQ ID NO:59.

[0077] A suitable polynucleotide for tauX is provided in SEQ ID NO:60 and encodes a peptide having the amino acid sequence of SEQ ID NO:61.

[0058]

[0078] A suitable polynucleotide for tauY is provided in SEQ ID NO:62 and encodes a peptide having the amino acid sequence of SEQ ID NO:63.

[0079] A suitable polynucleotide for tauD is provided in SEQ ID NO:64 and encodes a peptide having the amino acid sequence of SEQ ID NO:65.

[0059]

[0080] A suitable polynucleotide for tpa is provided in SEQ ID NO:66 and encodes a peptide having the amino acid sequence of SEQ ID NO:67.

[0081] A suitable polynucleotide for tauABCD is provided in SEQ ID NO:68.

[0060]

[0082] A suitable polynucleotide for ssuEADCB is provided in SEQ ID NO:69.

[0083] Suitable polynucleotides for ssuD are provided in SEQ ID NO:70 and SEQ ID NO:72, which encode peptides having the amino acid sequences of SEQ ID NO:71 and SEQ ID NO:73, respectively.

[0061]

[0084] Suitable polynucleotides for ssuE are provided in SEQ ID NO:74 and SEQ ID NO:76, which encode peptides having the amino acid sequences of SEQ ID NO:75 and SEQ ID NO:77, respectively.

[0062]

[0085] Suitable polynucleotides for ridA are provided in SEQ ID NO:80, SEQ ID NO:149, SEQ ID NO:151 and encode peptides having the amino acid sequences of SEQ ID NO:81, SEQ ID NO:150, SEQ ID NO:152, respectively.

[0063]

[0086] A suitable polynucleotide for tdcF is provided in SEQ ID NO:82 and encodes a peptide having the amino acid sequence of SEQ ID NO:83.

[0087] A suitable polynucleotide for rutC is provided in SEQ ID NO:84 and encodes a peptide having the amino acid sequence of SEQ ID NO:85.

[0064]

[0088] A suitable polynucleotide for cuyA is provided in SEQ ID NO:86 and encodes a peptide having the amino acid sequence of SEQ ID NO:87.

[0089] Suitable polynucleotides for cbl are provided in SEQ ID NO:88 and SEQ ID NO:90, which encode peptides having the amino acid sequences of SEQ ID NO:89 and SEQ ID NO:91, respectively.

[0065]

[0090] Suitable polynucleotides for tauR are provided in SEQ ID NO:92 and SEQ ID NO:94, which encode peptides having the amino acid sequences of SEQ ID NO:93 and SEQ ID NO:95, respectively.

[0066]

[0091] A suitable polynucleotide for mcbR is provided in SEQ ID NO:96 and encodes a peptide having the amino acid sequence of SEQ ID NO:97.

[0092] A suitable polynucleotide for cysM is provided in SEQ ID NO:98 and encodes a peptide having the amino acid sequence of SEQ ID NO:99.

[0067]

[0093] Suitable polynucleotides for sdaA are provided in SEQ ID NO:100 and SEQ ID NO:102, which encode peptides having the amino acid sequences of SEQ ID NO:101 and SEQ ID NO:103, respectively.

[0068]

[0094] Suitable polynucleotides for glyA are provided in SEQ ID NO:104 and SEQ ID NO:106, which encode peptides having the amino acid sequences of SEQ ID NO:105 and SEQ ID NO:107, respectively.

[0069]

[0095] A suitable polynucleotide for tnaA is provided in SEQ ID NO:108 and encodes a peptide having the amino acid sequence of SEQ ID NO:109.

[0096] A suitable polynucleotide for cysPUWA is provided in SEQ ID NO:110 and encodes a peptide having the amino acid sequence of SEQ ID NO:111, SEQ ID NO:112, SEQ ID NO:113, SEQ ID NO:114.

[0070]

[0097] A suitable polynucleotide for nrdh is provided in SEQ ID NO:143 and encodes a peptide having the amino acid sequence of SEQ ID NO:144.

[0098] A suitable polynucleotide for sbp is provided in SEQ ID NO:160 and encodes a peptide having the amino acid sequence of SEQ ID NO:161.

[0071]

[0099] A suitable polynucleotide for ssuC is provided in SEQ ID NO:162 and encodes a peptide having the amino acid sequence of SEQ ID NO:163. [000100] A suitable polynucleotide for ssuB is provided in SEQ ID NO:164 and encodes a peptide having the amino acid sequence of SEQ ID NO:165.

[0072] [000101] A suitable polynucleotide for ssuA is provided in SEQ ID NO:166 and encodes a peptide having the amino acid sequence of SEQ ID NO:167. [000102] A suitable polynucleotide for ssuD1CBA is provided in SEQ ID NO:168.

[0073] [000103] A suitable polynucleotide for ssu1 is provided in SEQ ID NO:169 and encodes a peptide having the amino acid sequence of SEQ ID NO:170. [000104] A suitable polynucleotide for sueA is provided in SEQ ID NO:172 and encodes a peptide having the amino acid sequence of SEQ ID NO:173.

[0074] [000105] A suitable polynucleotide for sueB is provided in SEQ ID NO:174 and encodes a peptide having the amino acid sequence of SEQ ID NO:175. [000106] A suitable polynucleotide for sueC is provided in SEQ ID NO:176 and encodes a peptide having the amino acid sequence of SEQ ID NO:177.

[0075] [000107] A suitable polynucleotide for sueD2 is provided in SEQ ID NO:178 and encodes a peptide having the amino acid sequence of SEQ ID NO:179. [000108] A suitable polynucleotide for sueABCD2 is provided in SEQ ID NO:180.

[0076] [000109] Suitable polynucleotides for gadC are provided in SEQ ID NO:184, SEQ ID NO:186, SEQ ID NO:188, which encode peptides having the amino acid sequences of SEQ ID NO:185, SEQ ID NO:187, SEQ ID NO:189, respectively.

[0077] [000110] A suitable polynucleotide for yhiM is provided in SEQ ID NO:190 and encodes a peptide having the amino acid sequence of SEQ ID NO:191. [000111] Polynucleotides suitable for the amino acid permease AAperm are provided in SEQ ID NO:192, SEQ ID NO:194, SEQ ID NO:196, which encode peptides having the amino acid sequences of SEQ ID NO:193, SEQ ID NO:195, SEQ ID NO:197, respectively.

[0078] [000112] The present invention is not limited to the use of such amino acid sequences. The amino acid sequences, including the various enzymes and transcription factors listed, are considered to be within the scope of the present invention and to be substantially or sufficiently similar to the reference amino acid sequence. Although the present invention is not intended to be limited by any theory of how it achieves its advantageous results, it is believed that the identity between amino acid sequences required to maintain proper functionality is related to maintaining the tertiary structure of the polypeptide so that a particular interactive sequence is properly positioned and has the desired activity, and it is considered that a polypeptide containing such an interactive sequence under the appropriate spatial context has activity.

[0079] [000113] Another way in which similarity may exist between two amino acid sequences is when there are conservative substitutions between a group of given amino acids. The process of encoding a particular amino acid sequence may involve a DNA sequence having one or more base changes (i.e., insertions, deletions, substitutions) that do not result in a change in the encoded amino acid, or may involve base changes that may alter one or more amino acids but do not eliminate the functional properties of the polypeptide encoded by the DNA sequence.

[0080] [000114] One of skill in the art will recognize that a change in an amino acid sequence that alters, adds or deletes a single amino acid or a small number of amino acids in a coding sequence, e.g., a separate substitution, deletion or addition to a nucleic acid, peptide, polypeptide or protein sequence, is "sufficiently similar" if the change results in the replacement of an amino acid with a chemically similar amino acid.

[0081] [000115] Therefore, it is understood that the present invention does not merely encompass the specific polynucleotides encoding the proteins described herein. For example, modifications to sequences, such as deletions, insertions or substitutions in sequences that result in "silent" changes that do not substantially affect the functional properties of the resulting polypeptide, are expressly contemplated by the present invention. It is known to those skilled in the art that "universal" codes are not completely universal. Some mitochondrial and bacterial genomes deviate from the universal code, for example, some termination codons in the universal code specify amino acids in the mitochondrial or bacterial code. Thus, each silent variation of the nucleic acid encoding the polypeptide of the present invention is implicit in each polypeptide sequence described and is incorporated into the specification of the present invention.

[0082] [000116] It is understood that the alteration in nucleotide sequence reflects the degeneracy of genetic code or results in the production of chemically equivalent amino acids at a given site.Thus, the codon for the amino acid alanine, which is a hydrophobic amino acid, can be replaced by a codon that codes for another residue that is less hydrophobic, such as glycine, or a more hydrophobic residue, such as valine, leucine, or isoleucine.Similarly, the alteration that results in the replacement of one negatively charged residue with another, such as the replacement of aspartic acid with glutamic acid, or the replacement of one positively charged residue with another, such as the replacement of lysine with arginine, can also be expected to result in biologically equivalent products.

[0083] [000117] When preparing or synthetically modifying a nucleic acid, one of skill in the art can take into account the known codon preferences of the intended host in which the nucleic acid will be expressed. For example, the nucleic acid sequences of the invention can be expressed in a variety of species, but the sequences can be modified to take into account the particular codon and GC content preferences of the organism, as such preferences have been shown to vary (67-72).

[0084] [000118] Cloning technology [000119] Unless otherwise stated, the techniques utilized or discussed herein are standard methods well known to those skilled in the art. Certain terms utilized below and defined at the end of this section are used merely in a descriptive sense and not for purposes of limitation. The practice of the present invention utilizes, unless otherwise indicated, conventional techniques of botany, microbiology, mycology, phycology, tissue culture, molecular biology, chemistry, biochemistry, biotechnology, and recombinant DNA technology, which are within the skill of the art (73-80).

[0085] [000120] Polynucleotides suitable for use according to the invention can be obtained by cloning techniques using cDNA or genomic libraries, DNA, or cDNA from bacteria, algae, microalgae, diatoms, yeast or fungi, which are commercially available or can be constructed using standard methods known to those of skill in the art. Suitable nucleotide sequences can be isolated from DNA libraries obtained from a wide variety of species, by nucleic acid hybridization or amplification methods, such as polymerase chain reaction (PCR) procedures, using nucleotide sequences selected according to the invention as probes or primers.

[0086] [000121] Moreover, nucleic acid sequences can be constructed or amplified using chemical synthesis. The amplification product is called an amplicon. Moreover, if a particular nucleic acid sequence is of a length that makes full-length chemical synthesis impractical, the sequence can be divided into smaller segments that can be synthesized by methods known in the art and ligated together to form the desired full sequence. Alternatively, individual components or DNA fragments can be amplified by PCR, and adjacent fragments can be amplified together using fusion PCR (81), overlap PCR (82), or chemical (de novo) synthesis (83-87) by methods known in the art and using suppliers (e.g., DNA2.0, GElife technologies, GENEART, Gen9, GenScript).

[0087] [000122] The recombinant expression cassette or DNA construct comprises a promoter that directs transcription in a unicellular organism and is operably linked to the polynucleotide of the present invention described herein. A variety of different types of promoters have been described and used. As used herein, a polynucleotide is "operably linked" to a promoter or other nucleotide sequence when it is placed in a functional relationship with the promoter or other nucleotide sequence. The functional relationship between a promoter and a desired polynucleotide insert typically includes the polynucleotide and the promoter sequence contiguous to facilitate transcription of the polynucleotide sequence. Two nucleic acid sequences are further said to be operably linked if the nature of the binding between the two sequences (1) does not result in the introduction of a frameshift mutation, (2) does not interfere with the ability of the promoter region sequence to direct transcription of the desired nucleotide sequence, or (3) does not interfere with the ability of the desired nucleotide sequence to be transcribed by the promoter sequence region. Typically, the promoter element is generally located upstream (i.e., at the 5' end) of the nucleic acid insert coding sequence.

[0088] [000123] Although the promoter sequence may be ligated to the coding sequence prior to insertion into the vector, in other embodiments, a vector is selected that contains a promoter operable in the host cell into which the vector is inserted. In addition, certain preferred vectors have a region that codes for a ribosome binding site located between the promoter and the site into which the DNA sequence is inserted so as to be operably associated with the DNA sequence of the present invention and produce the desired polypeptide, i.e., the DNA sequence of the present invention, in frame.

[0089] [000124] A gene expression cassette may contain one or more polynucleotides (genes), each operably linked to a promoter and terminator to form a series of monocistronic mRNAs, or the genes may be arranged with one promoter and terminator to form a single polycistronic mRNA. A wide variety of operably cassettes are known to those of skill in the art.

[0090] [000125] Suitable promoters [000126] A wide variety of promoters are known to those of 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 (88-90). The characteristics (binding sites and regulatory elements) required for the identification and use of functional bacterial promoters are known to those of skill in the art (91-93). For purposes of describing the present invention, promoters are divided into two types: constitutive and non-constitutive (89, 94). Constitutive promoters are classified as those that provide a wide range of constitutive expression. There are also weak constitutive promoters and strong constitutive promoters (95). Other promoters are considered to be non-constitutive promoters (96-100).

[0091] [000127] Terminator [000128] In addition to the selection of a suitable promoter, the DNA construct requires a suitable transcription terminator to be bound downstream (3') after the stop codon (TGA, TAG or TAA) of the desired gene of the present invention for proper expression in unicellular organisms. Several such terminators are available and known to those skilled in the art. Terminators play an important role in RNA processing and stability as well as translation and can also control gene expression (101-110). The identification and use of terminators required for the expression of genes in unicellular organisms is known to those skilled in the art.

[0092] [000129] Selectability marker [000130] Selectable markers usually confer resistance to antibiotics, herbicides, or chemicals, or result in a color change that aids in the identification of transformed organisms. Vectors may also contain RNA stability signals, which are 3' regulatory sequence elements that enhance the stability of the transcribed RNA (111, 112).

[0093] [000131] Plastid transit peptide [000132] The present invention can target transformation to chloroplasts. Chloroplast-targeted transformation systems in algae are known to those of skill in the art (97, 99, 113-115).

[0094] [000133] A wide variety of plastid transit peptides that can be used in conjunction with the present invention are known to those of skill in the art. Suitable transit peptides that can be used to target any CDO, SAD, GAD, CS / PLP-DC, partCS / PLP-DC, TauA or TauK polypeptide to plastids include, but are not limited to, those described herein, in U.S. Pat. Nos. 8,779,237 (116), 8,674,180 (117), 8,420,888 (118), and 8,138,393 (119), and those described by Lee et al. (120) and von Heijne et al. (121). The identification and use of chloroplast plastid targeting sequences in algae is known to those of skill in the art (122-125). Cloning of a nucleic acid sequence encoding a transit peptide upstream and in frame with a nucleic acid sequence encoding a polypeptide involves standard molecular techniques known to those of skill in the art.

[0095] [000134] Suitable Vectors [000135] A wide variety of vectors can be utilized to transform unicellular organisms with constructs generated or selected according to the present invention, including high or low copy number plasmids, phage vectors, and cosmids. Vector systems, expression cassettes, culture methods, and transformation methods are known to those skilled in the art. Vectors can be selected such that the promoter and the polynucleotide encoding the desired polypeptide of the present invention that are operably linked are integrated into the genome of the unicellular organism. Other vectors that can be operably linked to the promoter and the polynucleotide encoding the polypeptide of the present invention are not integrated into the host genome, but the vector DNA with the cloned polynucleotide is replicated autonomously or semi-autonomously within the cell. A preferred embodiment of the present invention is expressed in unicellular organisms, but other embodiments can include expression in prokaryotes or unicellular eukaryotes, including but not limited to yeast, fungi, algae, microalgae, or microorganisms.

[0096] [000136] Those skilled in the art know that there are many expression systems available for expressing nucleic acids encoding the proteins of the invention. There are many commercially available recombinant vectors for transforming unicellular organisms. Standard molecular and cloning techniques (77, 80, 126) are available for generating recombinant expression cassettes that express polynucleotides encoding the desired polypeptides of the invention. No attempt will be made to describe in detail the various methods known for expressing proteins in prokaryotes or eukaryotes. Briefly, expression of isolated nucleic acids encoding the proteins of the invention is typically achieved by, for example, operably linking DNA or cDNA to a promoter and then incorporating it into an expression vector. The vectors may be suitable for replication and integration in either prokaryotes or eukaryotes. A typical expression vector contains transcription and translation terminators, initiation sequences, and promoters useful for regulating the expression of DNA encoding the proteins of the invention. To obtain high levels of expression of cloned genes, it is desirable to construct an expression vector that contains, at a minimum, a strong promoter to direct transcription, a ribosome binding site for translation initiation, and a transcription / translation terminator.

[0097] [000137] Expression in prokaryotes [000138] For use in prokaryotes, protocols for transformation and commonly used vectors (some with operators) with control sequences including promoters for transcription initiation along with ribosome binding site sequences are known to those of skill in the art. Those skilled in the art are familiar with molecular techniques and DNA vectors used in bacterial systems (127-131). In bacteria, one messenger RNA can code for one peptide (called monocistronic) or several independent peptides (called polycistronic). It is known to those skilled in the art that portions of polycistronic messenger RNA can be knocked out (132) or heterologous or exogenous genes can be expressed on monocistronic or polycistronic messenger RNA (130, 131). Genes can be expressed by modification of bacterial DNA (genome) through the use of knock-ins, gene insertions, or by allelic exchange (133-138). Specific gene targeting using PCR-based methods (139) and CRISPR / Cas (140-142) has been used in bacteria.

[0098] [000139] Expression in algae and microalgae [000140] Protocols for transformation and commonly used vectors with control sequences including a promoter for transcription initiation, optionally an operator, along with ribosome binding site sequences for use in algae and microalgae are known to those of skill in the art (89, 113, 143-153). Specific gene targeting systems including ZFNs (154) and transcription activator-like effector nucleases (TALENs) (155) have been used in algae.

[0099] [000141] Expression in non-plant eukaryotes [000142] Protocols for transformation and commonly used vectors are known to those skilled in the art. Control sequences, including promoters and ribosome binding site sequences for transcription initiation, for use in unicellular eukaryotes are also known to those skilled in the art. The present invention can be expressed in a variety of eukaryotic expression systems, such as yeast and protists. Vectors usually have expression control sequences, such as promoters, origins of replication, enhancer sequences, termination sequences, ribosome binding sites, RNA splice sites, polyadenylation sites, transcription terminator sequences, and selectable markers (156, 157). There are many vectors known to those skilled in the art that can be used with the present invention, including, but not limited to, pREP, pRIP, pD912, pD1201, pD1211, pD1221, pD1231, pYES2 / NT, pYSG-IBA, or pESC-TRP. Synthesis of heterologous proteins in yeast and fermentation of the products are known to those skilled in the art (158, 159). Protists that can be used include, but are not limited to, ciliates, amoebae, and flagellates. Yeasts and fungi that can be used with the present invention and molecular protocols for transformation, vectors required for expression of genes in such systems are known to those skilled in the art (160-165). A wide variety of vectors are available. Plasmid vectors are also available, which can be integrative, self-replicating high copy number vectors, or self-replicating low copy number vectors (166, 167). The most common vectors that complement chromosomal mutations in the host include functional genes, such as URA3, HIS3, LEU2, TRP1, and LYS2. In unicellular fungi, editing or targeting of specific genes has been used using PCR-based methods (168-170). zinc finger nucleases (ZFNs) ( 171 ), transcription activator-like effector nucleases (TALENs) ( 172 ), and clustered regularly interspaced short palindromic repeats / Cas (CRISPR / Cas) ( 173 , 174 ).

[0100] [000143] Those skilled in the art will recognize that modifications can be made to the protein of the present invention without reducing its biological activity. Some modifications can be made to facilitate cloning, expression, targeting, or to direct the location of the polypeptide in the host, or for purification. Such modifications are known to those skilled in the art and include, for example, the addition of methionine at the amino terminus to provide an initiation site, the addition of nucleic acid to insert a restriction site or a termination site.

[0101] [000144] In addition, the polynucleotide can be placed in a suitable vector that is used to transform unicellular organisms. The polypeptide can be expressed and then isolated from the transformed cells, or the metabolite can be synthesized 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.

[0102] [000145] The vector may contain another polynucleotide that encodes a signal polypeptide or signal sequence ("cellular location sequence") that directs the desired polypeptide within the host cell and accumulates the polypeptide in a specific cellular compartment, subcellular compartment, or membrane. Specific cellular compartments include the fungal or algal vacuole, chloroplast (absent in fungi), mitochondria, peroxisomes, secretory pathway, lysosomes, endoplasmic reticulum, nucleus, or Golgi apparatus. There are specific signal polypeptides or signal sequences that direct the transport of peptides to the periplasmic space in bacteria (175-177). Signal polypeptides or signal sequences are usually present at the amino terminus and are usually not present in the mature protein because proteases remove the signal peptide when the polypeptide reaches its final destination. Signal sequences can be primary sequences located at the N-terminus (121, 178-180), C-terminus (181, 182) or internally (183-185) or in tertiary structures (185). If the signal polypeptide or signal sequence that directs the polypeptide is not present on the vector, it is expected that the skilled person can incorporate the additional nucleotides required to code the signal polypeptide or signal sequence by ligation or PCR of appropriate nucleotides. The skilled person can identify the nucleotide sequence of the signal polypeptide or signal sequence using computer tools. There are many computer tools available for identifying target sequences or signal sequences. These include, but are not limited to, TargetP (186, 187), iPSORT (188), SignalP (189), PrediSi (190), ELSpred (191), HSLpred (192) and PSLpred (193), MultiLoc (194), SherLoc (195), ChloroP (196), MITOPROT (197), Predotar (198), 3D-PSSM (199), and PredAlgo (125). Further methods and protocols are discussed in the literature (194).

[0103] [000146] Transformation of host cells [000147] Transformation of unicellular organisms can be accomplished by a wide variety of methods within the purview of one of skill in the art (88, 90, 151, 200). One of skill in the art can use a variety of algae, diatom, fungal, yeast, and bacterial gene transfer techniques, including, but not limited to, Agrobacterium-mediated (201), glass beads and polyethylene glycol (PEG) (202, 203), electroporation (204-207), biolistics or ballistic particle acceleration (208-212), silicon carbide whisker technology (213, 214), viral infection (215, 216), or transposon / transposase complexes (217). Transformation can target organellular genomes (115). Other methods to edit, integrate or move genes into bacterial, fungal or 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).

[0104] [000148] Gene silencing by mutagenesis or recombinant techniques [000149] Genetic modification to silence or inactivate genes or corresponding gene products in unicellular organisms can be performed by radiation, chemical or UV-based mutagenesis followed by specific screening for biochemical traits or pathways (200, 218-222). Radiation-based mutations can silence or inactivate genes or corresponding gene products by DNA breakage and repair. Chemical or UV-based mutations usually result in a single DNA base pair change. Mutations can silence or inactivate genes or corresponding gene products by one of the following: (1) introducing frameshift mutations, (2) introducing premature stop codons, (3) interfering with the ability of the promoter region sequence to direct the transcription of the desired nucleotide sequence, (4) interfering with the ability of the desired nucleotide sequence to be transcribed by the promoter sequence region, or (5) introducing amino acid substitutions in the gene product to reduce or inhibit activity (enzymatic activity or binding) or disrupt the function of the gene product.

[0105] [000150] Targeted gene silencing or knockout can be achieved in unicellular organisms using phages or viruses (94, 223-227), transposons (217, 228-231), PCR-based targeting (168-170, 232), recombinases, or by allelic exchange (133-138). Targeted and random bacterial gene disruption can be achieved using group II introns (Targetrons) (233, 234), ZNFs (171), TALENs (172), CRISPER-Cas9, or clustered regularly interspaced short palindromic repeats interference (CRISPi) (140-142, 173, 174, 235, 236). In addition, RNA-mediated methods (237-242) or regulatory RNAs (243-245) have been used to silence or suppress gene expression in unicellular organisms, and such techniques and protocols are well known to those of skill in the art.

[0106] [000151] Suitable unicellular organisms [000152] A wide variety of unicellular host cells may be used in the present invention, including prokaryotic and unicellular eukaryotic host cells. These cells or organisms may include yeast, fungi, algae, microalgae, microorganisms, or unicellular photosynthetic organisms. Preferred host cells for the present invention are bacteria, including Archaea and Eubacteria. Members of the Proteobacteria, such as Alphaproteobacteria, Betaproteobacteria, Gammaproteobacteria, Deltaproteobacteria, and Epsilonproteobacteria, may serve as hosts for the present invention. Other bacteria may be used with the present invention, including Methanotrophs and Methylobacterium (246). Other bacterial genera that may serve as hosts for the present invention include, but are not limited to, Escherichia, Bacillus, Salmonella, Lactococcus, Lactobacillus, Streptococcus, Brevibacterium, and Coryneform bacteria.Some specific bacterial species that can be used in the present invention include Bacillus subtilis, Brevibacterium ammoniagenes, Corynebacterium crenatum, Corynebacterium pekinese, Corynebacterium glutamicum, Erwinia citreus, Erwinia herbicola, Escherichia coli, Fusarium venenatum, Gluconobacter oxydans, Propionibacterium freudenreicheii, Propionibacterium denitrificans, and the like. denitrificans, and Saccharomyces cerevisiae (50).

[0107] [000153] Cells of unicellular algae, unicellular photosynthetic organisms, and microscopic algae (microphytes or microalgae) may be used in the present invention. These include, but are not limited to, diatoms, green algae (Chlorophyta), and members of the phyla Euglenophyta, Dinoflagellata, Chrysophyta, Phaeophyta, red algae (Rhodophyta), Heterokontophyta, and Cyanobacteria. The present invention may also be used to increase taurine in algae that have been shown to synthesize taurine (28) or may have the ability to synthesize taurine (28) by binding taurine to taurine-binding proteins or by knocking out genes for taurine degradation.These include Coccomyxa spp., Chlorella spp., Trebouxia impressa, Tetraselmis spp., Chlamydomonas reinhardtii, Micromonas pusilla, Ostreococcus tauri, Navicula radiosa, Phaeodactylum tricornutum, Pseudo-nitzschia multiseries, Fragilariopsis cylindrus, Thalassiosira weissfrogii, and others. weissflogii, Nannochloropsis oceanica, Aureococcus anophagefferens, Saccharina japonica, Sargassum species, and Bigelowiella natans.

[0108] [000154] Protists that can be used in the present invention include, but are not limited to, ciliates, amoebae, and flagellates. Yeasts 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.

[0109] [000155] Once transformed, the unicellular organism can be treated with other "active substances" either before or during growth to further increase the production of taurine. "Active substances" as used herein refers to substances that have a beneficial effect on taurine production by the unicellular organism. Sulfur-containing compounds, such as sulfurous acid, sulfide, hydrogen sulfide, sulfate, taurine, hypotaurine, cysteate, 2-sulfacetaldehyde, homotaurine, homocysteine, cystathionine, N-acetylthiazolidine 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, can also be used to promote taurine production. Depending on the type of gene construct or recombinant expression cassette, other metabolites and nutrients can be used. These include, but are not limited to, sugars, carbohydrates, lipids, oligopeptides, monosaccharides (glucose, arabinose, fructose, xylose, and ribose), disaccharides (sucrose and trehalose), and polysaccharides, carboxylic acids (succinic acid, malic acid, and fumaric acid), vitamins, and nutrients such as phosphate, molybdate, or iron.

[0110] [000156] In some embodiments, the properties of the transgenic unicellular organism are modified using substances that increase intracellular sulfur concentration, such as sulfur, sulfite, sulfide, hydrogen sulfide, sulfate, taurine, hypotaurine, homotaurine, cysteate, 2-sulfacetaldehyde, N-acetylthiazolidine 4-carboxylic acid (ATCA), glutathione, and bile. In other embodiments, the substances increase nitrogen concentration. Either amino acids naturally occurring in proteins (e.g., cysteine, methionine, glutamic acid, glutamine, serine, alanine, or glycine) or amino acids not naturally occurring in proteins (e.g., GABA, citrulline, or ornithine), and / or polyamines, may be used for this purpose.

[0111] [000157] Pharmaceutical compositions [000158] The present invention provides pharmaceutical compositions comprising extracts of one or more of the modified unicellular organisms described above. Extracts containing hypotaurine or taurine can be used to synthesize or produce taurine derivatives (247, 248), taurine conjugates (249) or taurine polymers (250), which may have a wide range of commercial and medicinal applications (251). Some taurine derivatives can function as organogelators (252) or dyes (253) and can be used in the synthesis of nanosensors (254). Some taurine derivatives have anticonvulsant (247) or anticancer (255) properties. Other taurine derivatives are used in the treatment of alcoholism (256, 257). Taurine conjugate-carboxyethyl ester-polyrotaxanes increase anticoagulant activity (258). Taurine-containing polymers can improve wound healing (259, 260). Taurine-binding polymers, such as poly(gamma-glutamic acid sulfonate), are biodegradable and may have applications in the development of drug delivery systems, environmental materials, tissue engineering, and biomedical materials (261). Extracts of taurine-containing cells may be used in pharmaceutical or medicinal compositions to deliver taurine, hypotaurine, taurine conjugates, or taurine polymers for the treatment of congestive heart failure, hypertension, hepatitis, high cholesterol, fibrosis, epilepsy, autism, attention deficit hyperactivity disorder, retinal degeneration, diabetes, and alcoholism. It is also used as an antioxidant to improve mental function.

[0112] [000159] The pharma- ceutically acceptable solution of taurine, taurine derivatives, taurine conjugates or taurine polymers is in tablet, capsule, gel, ointment, film, patch, powder, or dissolved in liquid form.

[0113] [000160] Dietary supplements and feed [000161] Transgenic cells containing hypotaurine or taurine can be consumed or used to produce extracts for dietary supplements. Transgenic cells containing hypotaurine or taurine can be used for human consumption. Transgenic cells extracts containing hypotaurine or taurine can be used as dietary supplements, as antioxidants, or to improve physical or mental function. Extracts can be used in the form of liquid, powder, capsule or tablet.

[0114] [000162] Transgenic cells containing hypotaurine or taurine can be used as fish or animal feed or can be used to produce extracts for animal feed supplements. Transgenic cells containing hypotaurine or taurine can be used as animal or fish feed. Transgenic cell extracts containing taurine can be used as feed supplements in the form of liquid, powder, capsule or tablet.

[0115] [000163] Plant growth or yield enhancers [000164] Transgenic cells containing hypotaurine or taurine can be used as a plant growth or yield enhancer. Extracts of transgenic cells containing hypotaurine or taurine can be used as plant enhancers in the form of liquids, powders, capsules or tablets.

[0116] definition [000165] The term "polynucleotide" refers to a naturally occurring or synthetic linear and contiguous 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 serving a primary structural role. Unless otherwise indicated, nucleic acids or polynucleotides are written from left to right in a 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.

[0117] [000166] The terms "amplified" and "amplification" refer to the construction of multiple copies of a nucleic acid sequence or multiple copies complementary to a nucleic acid sequence using at least one of the nucleic acid sequences as a template. Amplification can be accomplished by chemical synthesis using either the solid-phase phosphoramidate technique or the polymerase chain reaction (PCR). Other amplification systems include the ligase chain reaction system, nucleic acid sequence-based amplification, the Q-beta replicase system, transcription-based amplification systems, and strand displacement amplification. The amplification product is called an amplicon.

[0118] [000167] As used herein, "promoter" includes reference to a DNA region upstream from the transcription start site and involved in the recognition and binding of either RNA polymerase I, II or III and other proteins to initiate transcription. A promoter includes necessary nucleic acid sequences near the transcription start site, e.g., in the case of a polymerase II type promoter, a TATA element. A promoter also optionally includes distal enhancer or repression elements, which may be located up to several thousand base pairs from the transcription start site. In bacteria, a promoter includes a Shine-Dalgarno or ribosome binding site, which may include the sequence AGGAGG (-35 box), and a Pribnow box or RNA polymerase binding site, which may include the sequence TATAAT (-10 box).

[0119] [000168] The term "algal promoter" refers to a promoter capable of initiating transcription in an algal cell. [000169] The term "exogenous promoter" refers to a promoter other than the native or natural promoter that drives transcription of a length of DNA of viral, bacterial or eukaryotic origin, including DNA from a microorganism, a plant, a plant virus, an invertebrate or a vertebrate.

[0120] [000170] The term "microbe" refers to any microorganism (including both eukaryotic and prokaryotic microorganisms), such as bacteria, fungi, yeast, bacteria, algae, and protists, as well as other unicellular organisms.

[0121] [000171] The term "constitutive" refers to a promoter that is active under most environmental and developmental conditions, such as, but not limited to, the CaMV35S promoter. [000172] The term "inducible promoter" refers to a promoter under chemical (including biomolecules, e.g., sugars, organic acids, or amino acids) or environmental control.

[0122] [000173] The terms "encoding" and "coding" refer to the process by which a polynucleotide conveys information to a cell, through the mechanisms of transcription and translation, so that a series of amino acids can be assembled into a specific amino acid sequence to produce a functional polypeptide, such as an active enzyme or ligand-binding protein.

[0123] [000174] The terms "polypeptide", "peptide", "protein" and "gene product" are used interchangeably herein to refer to a polymer of amino acid residues. The term applies to amino acid polymers in which one or more amino acid residues are artificial chemical analogs of the corresponding naturally occurring amino acids, as well as to naturally occurring amino acid polymers. Amino acids can be represented by their commonly known three letter or one letter symbols. Amino acid sequences are written from left to right in the amino to carboxy direction, respectively. Numeric ranges are inclusive of the numbers defining the range.

[0124] [000175] 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 or may include known analogs of natural amino acids that can function in a manner similar to the naturally occurring amino acids.

[0125] [000176] The term "degradation" with respect to "taurine degradative pathway," "taurine degradative enzyme," "taurine degradative system," and "taurine degradative protein" refers to the process of breakdown, catabolism or dissimilation of taurine.

[0126] [000177] The terms "cysteine ​​dioxygenase" and "CDO" refer to a protein that catalyzes the following reaction: Cysteine ​​+ oxygen = 3-sulfinoalanine 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 ​​bisulfite, or alanine 3-sulfinic acid.

[0127] [000178] The terms "sulfinoalanine decarboxylase" and "SAD" refer to a protein that catalyzes the following reaction: 3-Sulfinoalanine = Hypotaurine + CO2 NOTE: SAD is an alternative 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, cysteate decarboxylase (CAD), cysteic acid decarboxylase, and 3-sulfino-L-alanine carboxy-lyase. NOTE: The SAD reaction is also catalyzed by a subset of glutamic acid decarboxylases (GADs), enzymes that have been shown to catalyze the SAD reaction (22, 23).

[0128] [000179] Other names for hypotaurine are 2-aminoethanesulfinate, 2-aminoethylsulfinic acid, and 2-aminoethanesulfinic acid. [000180] Other names for taurine are 2-aminoethanesulfonic acid, aminoethanesulfonate, L-taurine, taurine ethyl ester, and taurine ketoisocaproic acid 2-aminoethanesulfinate.

[0129] [000181] The terms "threonine synthase" and "TS" refer to a protein that catalyzes the following reaction: O-phosphoserine and sulfite = cysteate Note: TS is another name for cysteate synthase.

[0130] [000182] The term "ilvA" or "ilvA gene product" refers to a protein that catalyzes the following reaction: Serine = 2-aminoacrylate NOTE: ilvA is another name for serine / threonine dehydratase, threonine dehydratase, Ser / Thr dehydratase, threonine deaminase, serine ammonia-lyase, serine dehydratase or SDH.

[0131] [000183] Other names for 2-aminoacrylate are 2-aminoacrylic acid, dehydroalanine, and 2-aminoprop-2-enoic acid. [000184] The term "3'-phosphoadenylylsulfate:2'aminoacrylate C-sulfotransferase" or "PAPS-AS" refers to a protein that catalyzes the following reaction: 2-Aminoacrylate + 3'-phosphoadenosine-5'-phosphosulfate = cysteate [000185] The terms "cysteamine dioxygenase" and "ADO" refer to a protein that catalyzes the following reaction: 2-Aminoethanethiol + O2 = Hypotaurine ADO is another name for 2-aminoethanethiol:oxygen oxidoreductase, persulfurase, cysteamine oxygenase, and cysteamine:oxygen oxidoreductase.

[0132] [000186] Other names for 2-aminoethanethiol are cysteamine or 2-aminoethane-1-thiol, b-mercaptoethylamine, 2-mercaptoethylamine, decarboxycysteine, and thioethanolamine.

[0133] [000187] The terms "cysteine ​​lyase" and "CL" refer to a protein that catalyzes the following reaction: Cysteine ​​+ Sulfite = Cysteate + Hydrogen Sulfide Other names for cysteine ​​lyase are cysteine ​​sulfite-lyase and cysteine ​​hydrogen-sulfide-lyase.

[0134] [000188] The terms "taurine-pyruvate aminotransferase" and "TPAT" refer to a protein that catalyzes the following reaction: Taurine + Pyruvic Acid = L-Alanine + 2-Sulfoacetaldehyde TPAT is another name for taurine transaminase or taurine transaminase aminotransferase. The term "Tpa" refers to the gene that codes for TPAT.

[0135] [000189] The terms "taurine dehydrogenase" and "TDH" refer to a protein that catalyzes the following reaction: Taurine + Water = Ammonia + 2-Sulfoacetaldehyde TDH is another name for taurine:oxidoreductase and taurine:ferricytochrome-c oxidoreductase.

[0136] [000190] The terms "tauX" or "tauY" refer to the genes encoding the small and large TDH subunits, respectively. [000191] The terms "taurine dioxygenase" and "TDO" refer to a protein that catalyzes the following reaction: Taurine + 2-oxoglutarate + O2 = sulfite + aminoacetaldehyde + succinate + CO2 TDO is another name for 2-aminoethanesulfonate dioxygenase, alpha-ketoglutarate-dependent taurine dioxygenase, and taurine or 2-oxoglutarate:O2 oxidoreductase.

[0137] [000192] The term "tauD" refers to the gene encoding TDO. [000193] The term "two-component alkanesulfonate monooxygenase" or "2CASM" refers to a protein that catalyzes the following reaction: Taurine + O2 + FMNH2 = Aminoacetaldehyde + SO3 2 +H2O+FMN or Taurine + O2 + Thioredoxin red = Aminoacetaldehyde + SO3 2 +H2O+Thioredoxin ox [000194] The terms "ssuDE," "ssuD," or "ssuE" refer to genes encoding two-component alkanesulfonate monooxygenases (2CASMs).

[0138] [000195] The terms "cysteine ​​synthetase / PLP decarboxylase" and "CS / PLP-DC" refer to a protein that catalyzes the following reaction: 2-Aminoacrylate + PAPS = Taurine O-phosphoserine + PAPS = taurine O-Acetyl-L-serine + Hydrogen Sulfide = Taurine [000196] The terms "part of cysteine ​​synthetase / PLP decarboxylase" and "partCS / PLP-DC" refer to proteins that catalyze decarboxylase reactions in which a carbon-carbon bond is cleaved to form subsequent substrates and end products, including, but not limited to: Cysteic acid = 2-aminoethanesulfonate + CO2 3-Sulfinoalanine = Hypotaurine + CO2 Glutamic acid = 4-aminobutanoic acid + CO2 [000197] Another name for 4-aminobutanoic acid is gamma-aminobutyric acid (GABA).

[0139] [000198] Other names for pyridoxal 5'-phosphate (PLP) are vitamin B6 and P-5-P. [000199] The term "recombinant" includes reference to a cell or vector that has been modified by the introduction of a heterologous nucleic acid. Recombinant cells may express genes not normally found in the cell, or express native genes that are otherwise abnormally expressed, under-expressed, or not expressed at all as a result of deliberate human intervention, or may reduce or eliminate expression of native genes as a result of deliberate human intervention.

[0140] [000200] The term "recombinant expression cassette" refers to a nucleic acid construct, produced recombinantly or synthetically, that has a set of specific nucleic acid elements that allow transcription of a specific 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 contains, among other sequences, the nucleic acid to be transcribed and a promoter.

[0141] [000201] The term "transgenic" includes reference to a unicellular organism that contains a heterologous polynucleotide within its genome. Generally, the heterologous polynucleotide is integrated into the genome in a manner that passes the polynucleotide 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 whose phenotype has been altered by the presence of heterologous nucleic acid, including cells altered or generated from an original transgenic cell by growth by budding or conjugation.

[0142] [000202] The term "vector" includes reference to a nucleic acid used in the transfection or transformation of a host cell into which a polynucleotide can be inserted. [000203] The term "selectively hybridizes" includes reference to hybridization of a nucleic acid sequence to a specific nucleic acid target sequence under stringent hybridization conditions that is detectably higher (e.g., at least twice background) than hybridization to non-target nucleic acid sequences, and to the substantial exclusion of non-target nucleic acids. Selectively hybridizing sequences typically have at least about 40% sequence identity to each other, preferably 60-90% sequence identity, and most preferably 100% sequence identity (i.e., complementarity).

[0143] [000204] The terms "stringent conditions" and "stringent hybridization conditions" include reference to conditions under which a probe hybridizes to a target sequence detectably more highly (e.g., at least twice as high as background) than other sequences. Stringent conditions are sequence-dependent and will be different in various circumstances. By controlling the stringency of hybridization and / or washing conditions, target sequences that may be up to 100% complementary to the probe can be identified (homologous probes). Alternatively, lower degrees of similarity can be detected by adjusting stringency conditions to allow some mismatches (heterologous probes). Optimally, the probe is approximately 500 nucleotides in length, but can vary widely in length from less than 500 nucleotides to as long as the entire length of the target sequence.

[0144] [000205] Typically, stringent conditions are those in which the salt concentration is less than about 1.5M Na ion, typically about 0.01-1.0M Na ion concentration (or other salt) at pH 7.0-8.3, and the temperature is at least about 30°C for short probes (e.g., 10-50 nucleotides) and at least about 60°C for long probes (e.g., more than 50 nucleotides). Stringent conditions can also be achieved by adding destabilizing substances, such as formamide or Denhardt's solution. Low stringency conditions include hybridization with a buffer solution of 30-35% formamide, 1M NaCl, 1% SDS (sodium dodecyl sulfate) at 37°C and washing with 1x-2x SSC (20x SSC = 3.0M NaCl / 0.3M trisodium citrate) at 50-55°C. Moderate stringency conditions include hybridization with 40-45% formamide, 1M NaCl, 1% SDS at 37°C and washing with 0.5x-1x SSC at 55-60°C. High stringency conditions include hybridization with 50% formamide, 1M NaCl, 1% SDS at 37°C and washing with 0.1x SSC at 60-65°C. Specificity is typically a function of post-hybridization washing, with the ionic strength being a determining factor, and the temperature of the final washing solution. For DNA-DNA hybrids, T m can be approximated (262), and T m = 81.5°C + 16.6(logM) + 0.41(%GC) - 0.61(%form) - 500 / L, where M is the molar concentration 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 hybrid length in base pairs. m is the temperature (under defined ionic strength and pH) at which 50% of a complementary target sequence hybridizes to a perfectly matched probe. m Since T drops by about 1°C for each 1% mismatch, mThe hybridization and / or washing conditions can be adjusted to hybridize to sequences of desired identity. For example, when searching for sequences with 90% or more identity, T m Generally, stringent conditions are those that achieve a thermal melting point (T) of a specific sequence and its complement at a defined ionic strength and pH. m ) is chosen to be approximately 5°C lower than the thermal melting point (T m ), whereas moderate stringency conditions may utilize hybridization and / or washing temperatures 1, 2, 3, or 4° C. lower than the thermal melting point (T m ), low stringency conditions may utilize hybridization and / or washing temperatures 6, 7, 8, 9, or 10° C. lower than the thermal melting point (T m ) can be used. m By using the above-mentioned methods, one skilled in the art will understand that variations in the stringency of the hybridization and / or washing solutions are essentially described. Extensive guides to nucleic acid hybridization can be found in the scientific literature (126, 263). Unless otherwise stated, in this application, high stringency is defined as hybridization with 4xSSC, 5xDenhardt's solution (5g Ficoll, 5g polyvinylpyrrolidone, 5g bovine serum albumin in 500ml water), 0.1mg / ml boiled salmon sperm DNA, and 25mM Na phosphate at 65°C, and washing with 0.1xSSC, 0.1% SDS at 65°C.

[0145] [000206] The following terms are used to describe 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."

[0146] [000207] The term "reference sequence" refers to a defined sequence used as a basis for sequence comparison. A reference sequence can be, for example, a segment of a full-length cDNA or gene sequence, or a subset or the entirety of a sequence that is specified as a complete cDNA or gene sequence.

[0147] [000208] The term "comparison window" includes reference to a particular contiguous segment of a polynucleotide sequence, where the polynucleotide sequence may be compared to a reference sequence, and a portion of the polynucleotide sequence in the comparison window may include additions or deletions (i.e., gaps) when compared to the reference sequence for optimal alignment. The comparison window is usually at least 20 contiguous nucleotides in length, and can optionally be 30, 40, 50, 100 nucleotides or longer. Those skilled in the art understand that the incorporation of gaps in a polynucleotide sequence alignment introduces gap penalties that are subtracted from the number of matches.

[0148] [000209] Alignment methods for comparison of nucleotide and amino acid sequences are well known to those of skill in the art. The local homology algorithm BESTFIT (264) can perform optimal alignment for sequence comparison using a homology alignment algorithm called GAP (265), and Tfasta and Fasta (266) can be used to search for similarity using computerized implementations of these algorithms that are widely available online or from various suppliers (Intelligenetics, Genetics Computer Group, Inc.). CLUSTAL allows alignment of multiple sequences (267-269), and the PileUp program can be used for optimal global alignment of multiple sequences (270). The BLAST family of programs can be used for similarity searches of nucleotide or protein databases. BLASTN searches nucleotide databases using a nucleotide query. BLASTP searches protein databases using a protein query. BLASTX searches protein databases using a translated nucleotide query, which is derived from a six-frame translation of the nucleotide query sequence (both strands). TBLASTN searches a translated nucleotide database using a back-translated protein query, whereas TBLASTX searches a translated nucleotide database using a translated nucleotide query.

[0149] [000210] GAP(265) maximizes the number of matches and minimizes the number of gaps in the alignment of two complete sequences. GAP considers all possible alignments and gap positions and produces an alignment with the greatest number of matched bases and the fewest gaps. It also calculates gap and gap extension penalties in units of matched bases. The default gap creation and gap extension penalty values ​​in version 10 of the Wisconsin Genetics Software Package are 8 and 2, respectively. Gap creation and gap extension penalties may be expressed as integers selected from the group of integers consisting of 0 to 100. GAP presents four figures of merit for alignments: quality, ratio, identity, and similarity. Quality is the metric that is maximized to align sequences. Ratio is the quality divided by the number of bases in the shorter segment. Percent identity is the percentage of symbols that actually match. Percent similarity is the percentage of symbols that are similar. Symbols opposite a gap are ignored. Similarity is scored if the scoring matrix value of a pair of symbols is equal to or greater than a similarity threshold of 0.50. The scoring matrix used in version 10 of the Wisconsin Genetics Software Package is BLOSUM62 (271).

[0150] [000211] Unless otherwise stated, sequence identity or similarity values ​​refer to values ​​obtained using the BLAST 2.0 suite of programs with default parameters (272). Those skilled in the art understand that BLAST searches can model proteins as random sequences and assume that proteins contain regions, called low-complexity regions, that are rich in non-random sequences, short repeats, or one or more amino acid residues. Such low-complexity regions may align between unrelated proteins even though other protein regions are quite different. Those skilled in the art may use low-complexity filter programs to reduce the number of low-complexity regions aligned in a search. Such filter programs include, but are not limited to, SEG (273, 274) and XNU (275).

[0151] [000212] The terms "sequence identity" and "identity" are used in the context of two nucleic acid or polypeptide sequences and include reference to residues in the two sequences that are identical when aligned for maximum correspondence over a specified comparison window. When using percentages of sequence identity with respect to proteins, it is recognized that non-identical residue positions often differ by conservative substitutions of amino acids, where the amino acid residue is replaced with another having similar chemical properties (e.g., charge or hydrophobicity) without altering the functional properties of the molecule. When sequences differ in conservative substitutions, the percent sequence identity may be adjusted upwards to correct for the conservative nature of the substitution. Sequences that differ by such conservative substitutions are said to have "sequence similarity" or "similarity." Scoring conservative substitutions allows for partial, rather than complete, mismatches (276), which increases the percentage sequence similarity.

[0152] [000213] The term "percentage of sequence identity" refers to a value determined by comparison over a comparison window of two optimally aligned sequences, where a portion of the polynucleotide sequence of the comparison window may contain gaps (additions or deletions) when compared to a reference sequence for optimal alignment. The percentage is calculated by determining the number of positions in both sequences where identical nucleic acid bases or amino acid residues occur to obtain the number of matching positions, dividing the number of matching positions by the total number of positions in the comparison window, and multiplying the result by 100 to obtain the percentage of sequence identity.

[0153] [000214] The term "substantial identity" of a polynucleotide sequence means that the polynucleotide comprises a sequence having 50-100% sequence identity, preferably at least 50% sequence identity, preferably at least 60%, preferably at least 70%, more preferably at least 80%, more preferably at least 90%, and most preferably at least 95% sequence identity, compared to a reference sequence using one of the alignment programs described and standard parameters. Those skilled in the art will recognize that such values ​​can be appropriately adjusted to determine the corresponding identity of proteins encoded by two nucleotide sequences by taking into account codon degeneracy, amino acid similarity, reading frame positioning, and the like. For such purposes, substantial identity of amino acid sequences usually means 50-100% sequence identity. Another indication that nucleotide sequences are substantially identical is whether two molecules hybridize to each other under low stringency, medium stringency, or high stringency conditions. Another indication that two nucleic acid sequences are substantially identical is whether the two polypeptides immunologically cross-react with the same antibody in a Western blot, immunoblot, or ELISA assay.

[0154] [000215] The term "substantial identity" in the context of a peptide indicates that the peptide comprises a sequence having 55-100% sequence identity to a reference sequence, preferably at least 55% sequence identity, preferably 60%, preferably 70%, more preferably 80%, and most preferably at least 90% or 95% sequence identity to the reference sequence over a specified comparison window. Preferably, optimal alignment is performed using a homology alignment algorithm (265). Thus, a peptide is substantially identical to a second peptide, e.g., the two peptides differ only by conservative substitutions. Another indication that amino acid sequences are substantially identical is whether the 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 the epitopes recognized by the antibodies differ by non-conservative changes when the epitopes are substantially identical.

[0155] [000216] The present invention provides isolated cells that contain DNA that does not express a functional taurine degrading enzyme, and some isolated cells of the present invention contain (i) exogenous DNA that disrupts the expression of the gene or makes the corresponding peptide of the degrading enzyme nonfunctional, (ii) base pair mutation that disrupts the expression of the gene or makes the corresponding peptide of the degrading enzyme nonfunctional, or (iii) deletion of the whole polynucleotide or part of the polynucleotide that disrupts the expression of the gene or makes the corresponding peptide of the degrading enzyme nonfunctional. Nonfunctional DNA can be due to changes in the promoter, part of the coding region or terminator to the polynucleotide that encodes the taurine degrading enzyme, including tauX, tauY, tauD, tpa, ssuD or ssuE, or changes in the gene that encodes the translational activator of the gene, including cbl or tauR, that render the gene product nonfunctional. The present invention also provides isolated cells that contain a nonfunctional gene or gene product of the taurine degrading enzyme due to the suppression or reduced accumulation of the corresponding RNA due to antisense RNA or RNA interference.

[0156] [000217] 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 illustrative purposes only and are not intended to limit the scope of the invention.

[0157] [000218] Publications and other materials used herein to clarify the background of the invention or to provide further details regarding the practice are incorporated by reference and are grouped together for convenience in their respective bibliographies.

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Platform engineering of Corynebacterium glutamicum with reduced pyruvate dehydrogenase complex activity for improved production of L-lysine, L-valine, and 2-ketoisovalerate. Appl Environ Microbiol. 2013;79(18):5566-75. EXAMPLES

[0159] Example 1 Creation of high taurine-producing microorganisms [000219] Step 1: Generate the ΔtauABCD polynucleotide (SEQ ID NO: 116) using chemical synthesis. The polynucleotide is cloned into the pTOF25 vector and transformed into E. coli K12 strain to knock out tauABCD (SEQ ID NO: 68) using the recombination method of Merlin et al. (277).

[0160] [000220] Step 2: Generate the ΔssuEADCB polynucleotide (SEQ ID NO:115) using chemical synthesis. The polynucleotide is cloned into the pTOF25 vector and transformed into the ΔtauABCD strain (from Example 1, Step 1) to knock out ssuEADCB (SEQ ID NO:69) using the recombination method of Merlin et al. (277).

[0161] [000221] Step 3: Generate the trcPUWA polynucleotide (SEQ ID NO:118) using chemical synthesis. The polynucleotide is cloned into the pTOF25 vector and transformed into the ΔtauABCD / ΔssuEADCB strain (from Example 1, Step 2) to knock in the constitutive promoter and replace the native promoter with cysPUWA (SEQ ID NO:110) using the recombination method of Merlin et al. (277).

[0162] [000222] Step 4: Generate the trcDNC polynucleotide (SEQ ID NO:117) using chemical synthesis. The polynucleotide is cloned into the pTOF25 vector and transformed into the ΔtauABCD / ΔssuEADCB / trcPUWA strain (from Example 1, step 3) to knock in the constitutive promoter and replace the native promoter with cysDNC (SEQ ID NO:47) using the recombination method of Merlin et al. (277).

[0163] [000223] Step 5: Use chemical synthesis to generate an operational polycistronic CDO / SAD / cysQ / cysH / cysIJ polynucleotide optimized for expression in the host cell line, as follows: a. The CDO gene is derived from SEQ ID NO:3 by removing nucleotides 4-159 (corresponding to the native transit peptide) and encodes the CDO peptide from Chlamydomonas reinhardtii (SEQ ID NO:4 minus amino acids 2-53). b. The SAD gene is taken from SEQ ID NO:9 and encodes the SAD peptide (SEQ ID NO:10) from zebrafish (Danio rerio). The c.cysQ gene is taken from SEQ ID NO:51 and encodes the cysQ peptide from E. coli (SEQ ID NO:52). The cysH gene is taken from SEQ ID NO:53 and encodes the cysH peptide from E. coli (SEQ ID NO:54). The e.cysIJ gene is taken from SEQ ID NO:55 and encodes the cysI peptide from E. coli (SEQ ID NO:57) and the cysJ peptide from E. coli (SEQ ID NO:56).

[0164] [000224] Step 6: The polynucleotide is cloned into a bacterial expression vector to make it functional. [000225] Step 7: Operable polycistronic pgk / serA using chemical synthesis Δ197 / serC / serB / cysE M201R To generate the / cysK / cysM polynucleotide. a. The pgk gene is obtained from SEQ ID NO:35 and encodes the pgk peptide (SEQ ID NO:36) from Corynebacterium glutamicum. b.serA Δ197 The gene is taken from SEQ ID NO: 37, serA from Corynebacterium glutamicum Δ 197 peptide (SEQ ID NO:38). The c.serC gene is obtained from SEQ ID NO:41 and encodes the serC peptide (SEQ ID NO:42) from Corynebacterium glutamicum. d. The serB gene is obtained from SEQ ID NO:39 and encodes the serB peptide from Corynebacterium glutamicum (SEQ ID NO:40). e.cysE M201R The gene is taken from SEQ ID NO: 43 and is cysE from E. coli. M201R It encodes the peptide (SEQ ID NO:44). f. The cysK gene is taken from SEQ ID NO:45 and encodes the cysK peptide from E. coli (SEQ ID NO:46). The g.cysM gene is taken from SEQ ID NO:98 and encodes the cysM peptide (SEQ ID NO:99) from E. coli.

[0165] [000226] Step 8: Polycistronic pgk / serA Δ197 / serC / serB / cysE M201R The / cysK / cysM polynucleotide is cloned into a bacterial expression vector having a selectable marker different from the vector of Example 1, step 6, to make it functional.

[0166] [000227] Step 9: CDO-SAD / cysQ / cysH / cysIJ construct (from Example 1, Step 6) and pgk / serA Δ197 / serC / serB / cysE M201R The vector carrying the / cysK construct (from Example 1, step 8) is co-transformed into the ΔtauABCD / ΔssuEADCB / trcPUWA / trcDNC strain (from Example 1, step 4) to verify the presence of both DNA constructs.

[0167] Example 2 Creation of another high taurine-producing microorganism [000228] Step 1: Generate ΔsdaA in the ΔtauABCD / ΔssuEADCB strain (from Example 1, Step 2) using a synthetic polynucleotide (SEQ ID NO:146) and the recombination method of Merlin et al. (277).

[0168] [000229] Step 2: Generate ΔglyA in the ΔtauABCD / ΔssuEADCB / ΔsdaA strain (from Example 2, Step 1) using a synthetic polynucleotide (SEQ ID NO:159) and the recombinant method of Merlin et al. (277).

[0169] [000230] Step 3: Generate trcDNC in the ΔtauABCD / ΔssuEADCB / ΔsdaA / ΔglyA strain (from Example 2, Step 2) using a synthetic polynucleotide (SEQ ID NO:117) and the recombination method of Merlin et al. (277).

[0170] [000231] Step 4: Generate trcUWA in strain ΔtauABCD / ΔssuEADCB / ΔsdaA / ΔglyA / trcDNC (from Example 2, Step 3) using a synthetic polynucleotide (SEQ ID NO:134) and the recombinant method of Merlin et al. (277).

[0171] [000232] Step 5: Generate ΔilvA in strain ΔtauABCD / ΔssuEADCB / ΔsdaA / ΔglyA / trcDNC / trcUWA (from Example 2, Step 4) using a synthetic polynucleotide (SEQ ID NO:135) and the recombination method of Merlin et al. (277).

[0172] [000233] Step 6: Use chemical synthesis to construct an operable polycistronic pgk / serA nucleotide sequence as described in Example 1, steps 7a-7f. Δ197 / serC / serB / cysE M201R / cysK polynucleotide is generated.

[0173] [000234] Step 7: The polynucleotide is cloned into a bacterial expression vector to make it functional. [000235] Step 8: Use chemical synthesis to generate an operational polycistronic CDO / SAD / cysQ / cysH / cysIJ / sbp polynucleotide optimized for expression in the host cell line, as follows: a. CDO, SAD, cysQ, cysH, and cysIJ are obtained as described in Example 1, steps 5a-5e. b.sbp is derived from SEQ ID NO:160 and encodes the sbp peptide from E. coli (SEQ ID NO:161).

[0174] [000236] Step 9: The polycistronic CDO / SAD / cysQ / cysH / cysIJ / sbp polynucleotide is cloned into a bacterial expression vector with a selectable marker different from the vector in Example 2, Step 7, to make it functional.

[0175] [000237] Step 10: CDO / SAD / cysQ / cysH / cysIJ / sbp construct (from Example 2, Step 9) and pgk / serA Δ197 / serC / serB / cysE M201R The vector carrying / cysK (from Example 2, step 7) is co-transformed into the ΔtauABCD / ΔssuEADCB / ΔsdaA / ΔglyA / trcDNC / trcUWA strain (from Example 2, step 5) to confirm the presence of both DNA constructs.

[0176] Example 3 Creation of another high taurine-producing microorganism [000238] Step 1: Generate ΔridA in strain ΔtauABCD / ΔssuEADCB / ΔsdaA / ΔglyA / trcDNC (from Example 2, Step 3) using a synthetic polynucleotide (SEQ ID NO:119) and the recombination method of Merlin et al. (277).

[0177] [000239] Step 2: Generate ΔtdcF in the ΔtauABCD / ΔssuEADCB / ΔsdaA / ΔglyA / trcDNC / ΔridA strain (from Example 3, Step 1) using a synthetic polynucleotide (SEQ ID NO:120) and the recombination method of Merlin et al. (277).

[0178] [000240] Step 3: Generate ΔrutC in strain ΔtauABCD / ΔssuEADCB / ΔsdaA / ΔglyA / trcDNC / ΔridA / ΔtdcF (from Example 3, Step 2) using a synthetic polynucleotide (SEQ ID NO:121) and the recombination method of Merlin et al. (277).

[0179] [000241] Step 4: Use chemical synthesis to construct an operable polycistronic pgk / serA nucleotide sequence as described in Example 1, steps 7a-7d. Δ197 / serC / serB polynucleotides are generated.

[0180] [000242] Step 5: Polycistronic pgk / serA Δ197 The / serC / serB polynucleotide is made functional by cloning into a bacterial expression vector. [000243] Step 6: Using chemical synthesis, produce an operational polycistronic CS / PLP-DC / IlvA optimized for expression in a host cell line, such as: L447F A polynucleotide is generated. The CS / PLP-DC gene is obtained from SEQ ID NO:17 and encodes the pgk peptide (SEQ ID NO:18) from Micromonas pusilla. b.IlvA L447F The gene is taken from SEQ ID NO: 29 and is IlvA from E. coli. L447F It encodes the peptide (SEQ ID NO:30).

[0181] [000244] Step 7: Polycistronic CS / PLP-DC / IlvA L447FThe polynucleotide is cloned into a bacterial expression vector having a selectable marker different from the vector in Example 3, step 5, to make it functional.

[0182] [000245] Step 8: Functionality pgk / serA Δ197 / serC / serB (from Example 3, step 5) and CS / PLP-DC / IlvA L447F The vector carrying the construct (from Example 3, step 7) is co-transformed into the ΔtauABCD / ΔssuEADCB / ΔsdaA / ΔglyA / trcDNC / ΔridA / ΔtdcF / ΔrutC strain (from Example 3, step 3) to verify the presence of both DNA constructs.

[0183] Example 4 Creation of another high taurine-producing microorganism [000246] Step 1: Use chemical synthesis to generate an operational polycistronic TS / partCS / PLP-DC polynucleotide optimized for expression in a host cell line, as follows: The TS gene is obtained from SEQ ID NO:27 and encodes the TS peptide (SEQ ID NO:28) from Euryarchaeota archaeon. b. The partCS / PLP-DC gene was derived from SEQ ID NO:17 by deleting nucleotides 4-1413 (removing the native transit peptide sequence and cysteine ​​synthetase peptide sequence but retaining the start codon) and encodes the partCS / PLP-DC peptide from Micromonas pusilla (SEQ ID NO:18 minus amino acids 2-471).

[0184] [000247] Step 2: The polycistronic TS / partCS / PLP-DC polynucleotide is cloned into a bacterial expression vector to make it functional. [000248] Step 3: Use chemical synthesis to construct an operable polycistronic pgk / serA nucleotide sequence as described in Example 1, steps 7a-7c. Δ197 / serC polynucleotide is produced.

[0185] [000249] Step 4: Polycistronic pgk / serA Δ197 The / serC polynucleotide is cloned into a bacterial expression vector having a selectable marker different from the vector of Example 4, step 2, to make it functional.

[0186] [000250] Step 5: Functionality pgk / serA Δ197 The vector carrying / serC (from Example 4, step 4) and the TS / partCS / PLP-DC construct (from Example 4, step 2) are co-transformed into the ΔtauABCD / ΔssuEADCB / ΔsdaA / ΔglyA / trcDNC strain (from Example 2, step 3) to confirm the presence of both DNA constructs.

[0187] Example 5 Creation of another high taurine-producing microorganism [000251] Step 1: A DNA fragment is generated using genomic DNA from Corynebacterium glutamicum and the primer pair SEQ ID NO:122 and SEQ ID NO:123. A second DNA fragment is generated using genomic DNA from Corynebacterium glutamicum and the primer pair SEQ ID NO:124 and SEQ ID NO:125. Each DNA fragment is purified and used in overlap PCR with primers SEQ ID NO:122 and SEQ ID NO:125 to generate a knockout fragment of ssuE (SEQ ID NO:76). The resulting fragment is cloned into pK19mobsacB vector and transformed into Corynebacterium glutamicum to replace ssuE with the ssuE knockout fragment by homologous recombination as described by Buchholz et al. (278).

[0188] [000252] Step 2: Generate ΔmcbR in the ΔssuE strain (from Example 5, Step 1) using the synthetic polynucleotide (SEQ ID NO:142) and the recombination method described by Buchholz et al. (278).

[0189] [000253] Step 3: Generate ΔilvA in the ΔssuE / ΔmcbR strain (from Example 5, Step 2) using the synthetic polynucleotide (SEQ ID NO:139) and the recombinant method described by Buchholz et al. (278).

[0190] [000254] Step 4: Generate ΔglyA in the ΔssuE / ΔmcbR / ΔilvA strain (from Example 5, Step 3) using the synthetic polynucleotide (SEQ ID NO:138) and the recombinant method described by Buchholz et al. (278).

[0191] [000255] Step 5: Polycistronic pgk / serA of Example 3 Step 5 Δ197 The / serC / serB polynucleotide is made functional by cloning into a bacterial expression vector.

[0192] [000256] Step 6: Use chemical synthesis to generate an operational polycistronic CDO / SAD / gadC polynucleotide optimized for expression in the host cell line, as follows: The CDO gene is taken from SEQ ID NO:1 and encodes the CDO peptide from zebrafish (SEQ ID NO:2). b. The SAD gene is taken from SEQ ID NO:9 and encodes the SAD peptide from zebrafish (SEQ ID NO:10). The c. gadC gene is taken from SEQ ID NO: 184 and encodes the GadC peptide from E. coli (SEQ ID NO: 185).

[0193] [000257] Step 7: The CDO / SAD / gadC polynucleotide is cloned into a bacterial expression vector with a selectable marker different from the vector in Example 5, Step 5, to make it functional.

[0194] [000258] Step 8: Functional CDO / SAD / gadC (from Example 5, Step 7) and pgk / serA Δ197The vector carrying / serC / serB (from Example 5, step 5) is co-transformed into the ΔssuE / ΔmcbR / ΔilvA / glyA strain (from Example 5, step 4) to confirm the presence of the DNA construct.

[0195] Example 6 A microbial fermentation process that produces at least 1.0 g / L of taurine in shake flasks [000259] Step 1: Grow a seed culture of taurine-producing bacteria (from Examples 1, 2, 3 or 4) in LB broth supplemented with the appropriate antibiotic on a rotary shaker at 37°C and 250 rpm for 12-20 hours.

[0196] [000260] Step 2: Inoculate the production medium with 1 / 50 volume of the seed culture. The production medium contains ammonium sulfate (5 g / L), dibasic potassium phosphate (6 g / L), monobasic sodium phosphate (3 g / L), magnesium sulfate (0.5 g / L), glucose (6 g / L), tryptone (0.1 g / L), yeast extract (0.05 g / L), and PLP (2.4 mg / L) at pH 7.0 with or without antibiotics. Grow the taurine-producing bacteria in the production medium in beveled flasks on a rotary shaker at 250 rpm and 30°C for 20-30 hours.

[0197] [000261] Step 3: Separate the cells from the broth by centrifugation. [000262] Step 4: Determine intracellular taurine concentration by HPLC and clarify broth.

[0198] Example 7 A microbial fermentation process that produces at least 25 g / L of taurine in a fermenter [000263] Step 1: Grow a seed culture of taurine-producing bacteria (from Examples 1, 2, 3 or 4) in LB broth supplemented with the appropriate antibiotic on a rotary shaker at 250 rpm and 37°C for 12-20 hours.

[0199] [000264] Step 2: Batch fermentation is performed in a 1.5 L bioreactor using the production medium from Example 6, Step 2, supplemented with antifoam. The pH is maintained at 7.0 with ammonium hydroxide, the temperature at 30°C, and the dissolved oxygen at >20% by adjusting the agitation rate and airflow.

[0200] [000265] Step 3: Separate the cells from the broth by centrifugation. [000266] Step 4: Determine intracellular taurine concentration by HPLC and clarify broth.

[0201] Example 8 Another microbial fermentation process that produces at least 1.0 g / L of taurine in shake flasks [000267] Step 1: Grow a seed culture of taurine producing bacteria (from Example 5) in LB broth supplemented with 0.5% glucose with appropriate antibiotics for 24 hours and on a rotary shaker at 200 rpm and 30°C for 48 hours.

[0202] [000268] Step 2: Inoculate the production medium with 1 / 10 volume of the seed culture. The production medium contained yeast extract (2 g / L), glucose (40 g / L), calcium carbonate (10 g / L), ammonium sulfate (15 g / L), dibasic potassium phosphate (1 g / L), monobasic potassium phosphate (1 g / L), sodium chloride (2 g / L), calcium chloride (80 mg / L), ferric chloride (3 mg / L), zinc sulfate heptahydrate (0.9 mg / L), copper sulfate (0.2 mg / L), manganese sulfate (0.4 mg / L), sodium molybdate (0.1 mg / L), sodium borate (0.3 mg / L), magnesium sulfate (1 g / L), thiamine hydrochloride (0.2 mg / L), biotin (0.2 mg / L), and PLP (2.4 mg / L) with or without antibiotics at pH 7.0. Taurine producing bacteria are grown in production medium in bevelled flasks on a rotary shaker at 250 rpm and 30° C. for 24 hours.

[0203] [000269] Step 3: Separate the cells from the broth by centrifugation. [000270] Step 4: Determine intracellular taurine concentration by HPLC and clarify broth.

[0204] Example 9 Another microbial fermentation process producing at least 25 g / L of taurine in a fermenter [000271] Step 1: A seed culture of taurine-producing bacteria (from Example 5) is grown in LB broth supplemented with appropriate antibiotics on a rotary shaker at 200 rpm and 30°C for 24 hours.

[0205] [000272] Step 2: Batch fermentation is carried out in a 1.5 L bioreactor using the production medium from Example 8, Step 2, supplemented with antifoam. The pH is maintained at 7.0 with potassium hydroxide and phosphoric acid, the temperature at 30°C, and the dissolved oxygen at >20% by adjusting the agitation rate and airflow.

[0206] [000273] Step 3: Separate the cells from the broth by centrifugation. [000274] Step 4: Determine intracellular taurine concentration by HPLC and clarify broth.

[0207] Example 10 Purification of at least 90% of taurine from clarified broth [000275] Step 1: Purify taurine from clarified broth (Examples 6-9, step 3) by cation exchange as follows. a. The solution is concentrated by ultrafiltration. b. Adjust the pH of the clarified broth solution to pH 4.0 with HCl. c. Add the solution to an activated cation exchange column. d. Wash the column with 0.1 N HCl. e. Elute taurine with deionized water.

[0208] [000276] Step 2: Dry the solution into a crystalline or powder form. [000277] Step 3: Determine taurine concentration by HPLC. Example 11 Purification of at least 90% taurine from fermentation cells [000278] Step 1: Suspend cells (from Examples 6, 7, 8 or 9, Step 3) in 0.1 N HCl.

[0209] [000279] Step 2: The cells are disrupted with chemicals, pressure, mechanical force or sonication to release their contents. [000280] Step 3: Separate cell debris from the supernatant by centrifugation.

[0210] [000281] Step 4: Purify taurine from the supernatant (Examples 6-9, Step 3) by cation exchange as described in Example 10, Steps 1a-1e. [000282] Step 5: Dry the solution into a crystalline or powder form.

[0211] [000283] Step 6: Determine taurine concentration by HPLC. [000284] The use of the terms "a," "an," and "the," and similar references in the context of the description of the present invention (particularly in the context of the claims which follow) should be construed to encompass both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. The terms "comprising," "having," "including," and "containing" should be construed as open-ended terms (i.e., meaning "including, but not limited to"), unless otherwise indicated herein. The recitation of ranges of values ​​herein is merely intended to serve as a shorthand method of individually referring to each separate value within the range, and each separate value is incorporated herein as if it were individually recited herein, unless otherwise indicated herein. All methods described herein can be performed in any suitable order, unless otherwise indicated herein or clearly contradicted by context. The use of any and all examples and exemplary language (e.g., "such as") provided herein is intended merely to better clarify the invention, unless otherwise stated, and does not pose a limitation on the scope of the invention. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention.

[0212] [000285] Embodiments of the invention are described herein, including the best way known to the inventors for carrying out the invention. Variations of such embodiments may become apparent to those skilled in the art upon reading the foregoing specification. The inventors anticipate that those skilled in the art will utilize such variations as appropriate, and the inventors intend that the invention be practiced other than as specifically described herein. Accordingly, the present invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto, as permitted by applicable law. Moreover, all combinations of the above-described elements in all possible variations are encompassed by the present invention, unless otherwise indicated herein or clearly contradicted by context. Embodiments of the invention are described herein, including the best way known to the inventors for carrying out the invention. Variations of such embodiments may become apparent to those skilled in the art upon reading the foregoing specification. The inventors anticipate that those skilled in the art will utilize such variations as appropriate, and the inventors intend that the invention be practiced other 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 clearly contradicted by context.

Claims

1. Growing the unicellular organism in a shake flask to produce 1 g / liter of taurine from the unicellular organism.

2. A method for producing taurine comprising:

2. Growing the unicellular organism in a fermentor or bioreactor to produce at least 20 g / liter of taurine from the unicellular organism.

2. A method for producing taurine comprising:

3. The unicellular organism expresses one or more exogenous taurine biosynthetic pathways, comprising: Deletion of at least one operon for taurine or sulfonate uptake and degradation; exogenous taurine efflux transporter, increased expression of genes in sulfate or thiosulfate transport or sulfur reduction or sulfur assimilation; Increased expression of genes in the serine biosynthetic pathway, Increased expression of genes in the cysteine ​​biosynthetic pathway, Increased expression of genes in the 2-aminoacrylate biosynthetic pathway; a deletion of at least one gene in the degradation of taurine, serine, cysteate, or 2-aminoacrylate; or Modulation of expression of genes for transcriptional regulators or activators of taurine, sulfur, or cysteine ​​metabolism The method of claim 1 or 2, comprising one or more of:

4. The unicellular organisms are Proteobacteria, Alphaproteobacteria, Betaproteobacteria, Deltaproteobacteria, Epsilonproteobacteria, Methanotrophs, and the like. ), Methylobacterium, Escherichia, Bacillus, Salmonella, Lactococcus, Streptococcus, Brevibacterium, Coryneform bacteria bacteria, Bacillus subtilis, Brevibacterium ammoniagenes, Corynebacterium crenatum, Corynebacterium pekinese, Corynebacterium glutamicum, Erwinia citreus, Erwinia herbicola, Escherichia coli, Fusarium venenatum 3. The method of claim 1 or 2, wherein the bacterium is selected from the group consisting of Propionibacterium venenatum, Gluconobacter oxydans, Propionibacterium freudenreichei, Propionibacterium denitrificans, and Saccharomyces cerevisiae.

5. Isolating the taurine to produce taurine having a purity level of greater than 10% pure, greater than 25% pure, greater than 50% pure, greater than 75% pure, or greater than 98% pure. The method of claim 1 or 2, further comprising:

6. 3. The method of claim 1 or 2, wherein the unicellular organism is Escherichia coli and the step of growing is carried out in a medium comprising at least 5 g / L ammonium sulfate, at least 6 g / L dibasic potassium phosphate, at least 3 g / L monobasic sodium phosphate, at least 0.5 g / L magnesium sulfate, at least 6 g / L glucose, at least 0.1 g / L tryptone, and at least 0.05 g / L yeast extract.

7. The method of claim 1 or 2, wherein the step of growing is carried out in a medium containing at least 0.25 mg / L pyridoxal 5'-phosphate (PLP).

8. 3. The method according to claim 1 or 2, wherein cells of unicellular organisms are chemically, physically or mechanically disrupted, dried and used in food, feed, beverages, nutritional and health supplements, cosmetics, daily necessities, pharmaceuticals or agricultural production.

9. A unicellular organism capable of producing at least 1 g / liter of taurine when grown in a shake flask or 20 g / liter of taurine when grown in a fermentor or bioreactor.

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