Methods for high taurine production in unicellular organisms

Abstract

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

1 . A cell comprising:
 (a) two units expressed as two monocistronic messages, wherein
 (i) a first exogenous DNA which comprises a single expression cassette, wherein the single expression cassette comprises a promoter operably linked to a polynucleotide which encodes a fused cysteine dioxygenase-like (CDOL) with a linker to a portion of the cysteine synthetase/PLP decarboxylase (partCS/PLP-DC) protein (CDOL-linker-SADL) and 
 (ii) a second expression unit that comprises a second promoter operably linked to a second polynucleotide which encodes a taurine-binding protein; or 
   (b) two units expressed as one polycistronic message, wherein
 (i) an exogenous DNA comprises a single expression cassette, wherein the single expression cassette comprises a promoter operably linked to a polynucleotide which encodes a fused cysteine dioxygenase-like (CDOL) with a linker to a portion of the cysteine synthetase/PLP decarboxylase (partCS/PLP-DC) protein (CDOL-linker-SADL) and 
 (ii) a second polynucleotide which encodes a taurine-binding protein, 
   
       wherein the expression units are expressed in the cell and wherein the cell produces taurine. 
     
     
         2 . The cell of  claim 1 , wherein CDOL comprises the nucleotide sequence SEQ ID NO:3. 
     
     
         3 . The cell of  claim 1 , wherein partCS/PLP-DC comprises nucleotides 1 through 3 plus 1414 through 2958 of the sequence SEQ ID NO:11. 
     
     
         4 . The cell of  claim 1 , wherein CDOL comprises the amino acid sequence SEQ ID NO:4. 
     
     
         5 . The cell of  claim 1 , wherein partCS/PLP-DC comprises amino acids 1 plus 472 through 985 of the sequence SEQ ID NO:12. 
     
     
         6 . The cell of  claim 1 , wherein the taurine-binding protein comprises the nucleotide sequence SEQ ID NO:16 or SEQ ID NO:18. 
     
     
         7 . The cell of  claim 1 , wherein the taurine binding protein comprises amino acid sequence SEQ ID NO:17 or SEQ ID NO:19. 
     
     
         8 . The cell of  claim 1  which is a prokaryotic cell. 
     
     
         9 . A method of producing hypotaurine or taurine, comprising growing the prokaryotic cell of  claim 1  under conditions which permit expression of the first and second polynucleotides of the two monocistronic genes or expression of the polynucleotide of the single polycistronic genes, thereby producing taurine. 
     
     
         10 . A pharmaceutical composition comprising a concentrate, extract or secretion of the prokaryotic cell of  claim 1 . 
     
     
         11 . A nutritional supplement comprising a concentrate, extract or secretion of the prokaryotic cell of  claim 1 . 
     
     
         12 . A food or food supplement comprising a concentrate, extract or secretion of the prokaryotic cell of  claim 1 . 
     
     
         13 . An animal feed or feed supplement comprising a concentrate, extract or secretion of the prokaryotic cell of  claim 1 . 
     
     
         14 . A feed of  claim 13 , wherein the feed is an aquafeed. 
     
     
         15 . A plant growth or yield enhancer comprising a concentrate, extract or secretion of the prokaryotic cell of  claim 1 . 
     
     
         16 . The bacterial cell of  claim 1  wherein the bacteria is selected from the group consisting of Proteobacteria, Alphaproteobacteria, Betaproteobacteria, Deltaproteobacteria, Epsilonproteobacteria, methanotrophs,  Bacillus, Salmonella, Lactococcus, Streptococcus, Brevibacterium , coryneform bacteria,  Bacillus subtilis, Brevibacterium ammoniagene, Corynebacterium crenatum, Corynebacterim pekinese, Corynebacterium glutamicumas, Erwinia citreus, Erwinia herbicola, Escherichia coli, Fusarium venenatum, Gluconobacter oxydans, Propionibacterium freudenreicheii , and  Propionibacterium denitrificans.    
     
     
         17 . A method of treating the cell of  claim 1  with an exogenous application of a sulfur-containing compound or sulfate thereby resulting in the production of taurine or increased hypotaurine or taurine in the cell. 
     
     
         18 . An animal feed or animal feed supplement comprising a concentrate, extract or secretion of the cell of  claim 17 , wherein the concentrate, extract or secretion contains hypotaurine or taurine. 
     
     
         19 . A bacterial cell line wherein the function of the TauD or cbl genes is disrupted by genetic knock out resulting in negligible expression of the gene products TDO or Cbl, identified herein as TauD/KO or cbl/KO. 
     
     
         20 . A TauD/KO or cbl/KO cell comprising:
 (a) two units expressed as two monocistronic messages, wherein
 (i) a first exogenous DNA which comprises a single expression cassette, wherein the single expression cassette comprises a promoter operably linked to a polynucleotide which encodes a fused cysteine dioxygenase-like (CDOL) with a linker to a portion of the cysteine synthetase/PLP decarboxylase (partCS/PLP-DC) protein (CDOL-linker-SADL) and 
 (ii) a second expression unit that comprises a second promoter operably linked to a second polynucleotide which encodes a taurine-binding protein; or 
   (b) two units expressed as one polycistronic message, wherein
 (i) an exogenous DNA comprises a single expression cassette, wherein the single expression cassette comprises a promoter operably linked to a polynucleotide which encodes a fused cysteine dioxygenase-like (CDOL) with a linker to a portion of the cysteine synthetase/PLP decarboxylase (partCS/PLP-DC) protein (CDOL-linker-SADL) and 
 (ii) a second polynucleotide which encodes a taurine-binding protein, 
   
       wherein the expression units are expressed in the cell and wherein the cell produces taurine. 
     
     
         21 . The cell of  claim 20 , wherein CDOL comprises the nucleotide sequence SEQ ID NO:3. 
     
     
         22 . The cell of  claim 20 , wherein partCS/PLP-DC comprises nucleotides 1 through 3 plus 1414 through 2958 of the sequence SEQ ID NO:11. 
     
     
         23 . The cell of  claim 20 , wherein CDOL comprises the amino acid sequence SEQ ID NO:4. 
     
     
         24 . The cell of  claim 20 , wherein partCS/PLP-DC comprises amino acids 1 plus 472 through 985 of the sequence SEQ ID NO:12. 
     
     
         25 . The cell of  claim 20 , wherein the taurine-binding protein comprises the nucleotide sequence SEQ ID NO:16 or SEQ ID NO:18. 
     
     
         26 . The cell of  claim 20 , wherein the taurine-binding protein comprises amino acid sequence SEQ ID NO:17 or SEQ ID NO:19. 
     
     
         27 . The cell of  claim 20 , which is a prokaryotic cell. 
     
     
         28 . A method of producing hypotaurine or taurine, comprising growing the prokaryotic cell of  claim 20  under conditions which permit expression of the first and second polynucleotides of the two monocistronic genes or expression of the polynucleotide of the single polycistronic genes, thereby producing taurine. 
     
     
         29 . A pharmaceutical composition comprising a concentrate, extract or secretion of the prokaryotic cell of  claim 20 . 
     
     
         30 . A nutritional supplement comprising a concentrate, extract or secretion of the prokaryotic cell of  claim 20 . 
     
     
         31 . A food or food supplement comprising a concentrate, extract or secretion of the prokaryotic cell of  claim 20 . 
     
     
         32 . An animal feed or feed supplement comprising a concentrate, extract or secretion of the prokaryotic cell of  claim 20 . 
     
     
         33 . A feed of  claim 32 , wherein the feed is an aquafeed. 
     
     
         34 . A plant growth or yield enhancer comprising a concentrate, extract or secretion of the prokaryotic cell of  claim 20 . 
     
     
         35 . The bacterial cell of  claim 20  wherein the bacteria is selected from the group consisting of Proteobacteria, Alphaproteobacteria, Betaproteobacteria, Deltaproteobacteria, Epsilonproteobacteria, methanotrophs,  Bacillus, Salmonella, Lactococcus, Streptococcus, Brevibacterium , coryneform bacteria,  Bacillus subtilis, Brevibacterium ammoniagene, Corynebacterium crenatum, Corynebacterim pekinese, Corynebacterium glutamicumas, Erwinia citreus, Erwinia herbicola, Escherichia coli, Fusarium venenatum, Gluconobacter oxydans, Propionibacterium freudenreicheii , and  Propionibacterium denitrificans.    
     
     
         36 . A method of treating the cell of  claim 20  with an exogenous application of a sulfur-containing compound or sulfate thereby resulting in the production of taurine or increased hypotaurine or taurine in the cell. 
     
     
         37 . An animal feed or animal feed supplement comprising a concentrate, extract or secretion of the cell of  claim 20 , wherein the concentrate, extract or secretion contains hypotaurine or taurine.