US2015133698A1PendingUtilityA1

Production of fatty alcohols from engineered microorganisms

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Assignee: CODEXIS INCPriority: Apr 20, 2012Filed: Apr 19, 2013Published: May 14, 2015
Est. expiryApr 20, 2032(~5.7 yrs left)· nominal 20-yr term from priority
Inventors:Kaman ChanFernando ValleYoram BarakLouis ClarkKristian Karlshoej
C12P 5/02C07C 31/125C12P 7/6436A61K 2800/10C12P 7/04A61K 8/342C11D 3/2031A61Q 19/00C12Y 301/02C12Y 602/01003Y02E50/10C12N 9/0008C12N 9/1029C12Y 102/0105C12N 9/16C12N 15/70C12N 9/93C12Y 301/02014C12Y 203/01086
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Claims

Abstract

The invention generally relates to the production of a fatty alcohol composition from recombinant microbial cells. The fatty alcohols are produced by expressing a gene encoding a heterologous fatty alcohol forming acyl-CoA reductase (“FAR”); a gene encoding a heterologous thioesterase (“TE”) gene and a gene encoding an acyl-CoA synthetase (“ACS”).

Claims

exact text as granted — not AI-modified
1 . A recombinant bacterial microbial culture that produces a composition of fatty alcohols said fatty alcohol composition comprising fatty alcohols having a carbon chain length of at least 60% of C12, C14 and C16 fatty alcohols, the culture comprising a recombinant bacterial microorganism comprising (a) a gene encoding a heterologous thioesterase (“TE”); (b) a gene encoding a heterologous fatty alcohol forming acyl-CoA reductase (“FAR”) and (c) an over-expressed acyl-CoA synthetase (“ACS”). 
     
     
         2 . The recombinant bacterial culture of  claim 1 , wherein the recombinant bacterial microorganism is  E. coli , the heterologous TE has an amino acid sequence comprising at least 90% sequence identity to SEQ ID NO: 10 or SEQ ID NO: 35; the heterologous FAR has an amino acid sequence comprising at least 90% sequence identity to SEQ ID NOs: 2, 4, 6, 37 or 39; and the over-expressed ACS has an amino acid sequence comprising at least 90% sequence identity to SEQ ID NO: 8. 
     
     
         3 . The recombinant bacterial culture of  claim 1 , wherein said fatty alcohol composition comprises fatty alcohols having a carbon chain length of at least 20% of C12 fatty alcohols. 
     
     
         4 . The recombinant bacterial culture of  claim 1 , wherein the fatty alcohol composition comprises at least 80% by weight of C12, C14 or C16 fatty alcohols. 
     
     
         5 . The recombinant bacterial culture of  claim 1 , wherein a) the heterologous TE has an amino acid sequence comprising at least 95% sequence identity to SEQ ID NO: 35; b) the heterologous FAR has an amino acid sequence comprising at least 95% sequence identity to SEQ ID NO: 39; c) the over-expressed ACS has an amino acid sequence comprising at least 95% sequence identity to SEQ ID NO: 8 and d) said fatty alcohol composition comprises fatty alcohols having a carbon chain length of at least 30% of C12 fatty alcohols. 
     
     
         6 . The recombinant bacterial culture of  claim 1 , wherein the heterologous FAR has 98% sequence identity to SEQ ID NO: 39 and said fatty alcohol composition comprises fatty alcohols having a carbon chain length of at least 50% of C12 fatty alcohols. 
     
     
         7 . The recombinant bacterial culture of  claim 1 , wherein the fatty alcohol composition is recovered from the culture. 
     
     
         8 . A recombinant bacterial microorganism comprising (a) a gene encoding a heterologous thioesterase (“TE”); (b) a gene encoding a heterologous fatty alcohol forming acyl-CoA reductase (“FAR”) and (c) an over-expressed acyl-CoA synthetase (“ACS”); wherein the recombinant bacterial microorganism when cultured in the presence of a carbon source under suitable culture conditions produces a fatty alcohol composition. 
     
     
         9 . The recombinant bacterial microorganism of  claim 8 , wherein the microorganism is  E. coli.   
     
     
         10 . The recombinant bacterial microorganism of  claim 8 , wherein the TE is encoded by a fatB gene. 
     
     
         11 . The recombinant bacterial microorganism of  claim 10 , wherein the TE comprises at least 85% (88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to SEQ ID NO: 10 or SEQ ID NO: 35. 
     
     
         12 . The recombinant bacterial microorganism of  claim 8 , wherein the FAR is encoded by a gene derived from the following organisms  Marinobacter algicola, Marinobacter aquaeolei, Oceanobacter  sp.,  Mus musculus , or  Hahella chejuensis.   
     
     
         13 . The recombinant bacterial microorganism of  claim 8 , wherein the FAR has an amino acid sequence comprising at least 75% (80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%) sequence identity to SEQ ID NOs: 2, 4, 6, 37 or 39. 
     
     
         14 . The recombinant bacterial microorganism of  claim 8 , wherein the FAR is a variant FAR having at least 90% (91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to SEQ ID NOs: 2, 4, 6, 37 or 39. 
     
     
         15 . The recombinant bacterial microorganism of  claim 14 , wherein the gene encoding the FAR comprises a nucleic acid sequence having at least 90% (91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%) sequence identity to SEQ ID NOs: 3, 5, 7, 36 or 38. 
     
     
         16 . The recombinant bacterial microorganism of  claim 8 , wherein the ACS has an amino acid sequence comprising at least 85% (88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to SEQ ID NO: 8. 
     
     
         17 . The recombinant bacterial microorganism of  claim 16 , wherein the gene encoding the ACS comprises a nucleic acid sequence having at least 90% (91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to SEQ ID NO: 7. 
     
     
         18 . The recombinant bacterial microorganism of  claim 8 , further comprising
 a) an inactivated fadR gene, said fadR gene comprising a nucleic acid sequence having at least 90% (91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to SEQ ID NO: 11 and/or   b) an inactivated fadE gene, said fadE gene comprising a nucleic acid sequence having at least 90% (91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to SEQ ID NO: 13.   
     
     
         19 . The fatty alcohol composition produced by the cultured recombinant bacterial microorganism of  claim 8 . 
     
     
         20 . The fatty alcohol composition produced by the recombinant microorganism of  claim 8 , wherein the fatty alcohol composition comprises at least 60% by weight of C12, C14, or C16 fatty alcohols or any combination thereof. 
     
     
         21 . The fatty alcohol composition produced according to  claim 20 , wherein the fatty alcohol composition comprises at least 80% by weight of C12, C14 or C16 fatty alcohols or any combination thereof. 
     
     
         22 . A method of producing a fatty alcohol composition, the method comprising culturing a recombinant bacterial microorganism of  claim 8 , under suitable culture conditions with a suitable carbon source to produce a fatty alcohol composition and producing said fatty alcohol composition. 
     
     
         23 . The method according to  claim 22 , wherein at least 50% of the fatty alcohols produced by the recombinant bacterial cells are secreted into the culture. 
     
     
         24 . The method according to  claim 22 , further comprising recovering the produced fatty alcohol composition. 
     
     
         25 . The method according to  claim 24  further comprising reducing the recovered fatty alcohols to produce alkanes. 
     
     
         26 . The method according to  claim 24  further comprising esterifying the recovered fatty alcohols to produce fatty esters. 
     
     
         27 . The method according to  claim 22 , wherein the fatty alcohol composition comprises at least 60% by weight of C12, C14, or C16 fatty alcohols or any combination thereof. 
     
     
         28 . The method according to  claim 22 , wherein the fatty alcohol composition comprises at least 0.5 g/L of fatty alcohols. 
     
     
         29 . The method according to  claim 28 , wherein the fatty alcohol composition comprises at least 5.0 g/L of fatty alcohols. 
     
     
         30 . The method according to  claim 22 , wherein the microorganism is cultured at a temperature in the range of 20° C. to 45° C.; a pH in the range of pH 5 to pH 7; and for a time in the range of from 16 hours to 144 hours. 
     
     
         31 . The method according to  claim 22 , wherein the microorganism is cultured in the presence of fermentable sugars obtained from a cellulosic feedstock. 
     
     
         32 . A fatty alcohol composition comprising the fatty alcohols produced according to the method of  claim 22 . 
     
     
         33 . The fatty alcohol composition of  claim 32 , wherein said composition is used in a detergent composition. 
     
     
         34 . The fatty alcohol composition of  claim 32 , wherein said composition is used in a personal care composition. 
     
     
         35 . The fatty alcohol composition of  claim 32 , wherein said composition is used in a cleaning composition.

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