Methods And Compositions For Improved Production Of Fatty Acids And Derivatives Thereof
The invention relates to compositions and methods, including polynucleotide sequences, amino acid sequences, and engineered host cells for producing fatty acids and derivates of fatty acids such as acyl-CoA, terminal olefins, fatty aldehydes, fatty alcohols, alkanes, alkenes, wax esters, ketones and internal olefins through altered expression of the transcription factor, fadR.
1 .- 46 . (canceled)
47 . A recombinant host cell which is genetically engineered to have an increased level of expression of a FadR polypeptide as compared to the level of expression of the FadR polypeptide in a corresponding wild-type host cell,
wherein one or more of the titer, yield, or productivity of a fatty acid or a derivative thereof produced by the recombinant host cell is increased relative to that of a corresponding wild-type host cell when the recombinant host cell is cultured in a culture medium under conditions permissive for the production of the fatty acid or derivative thereof.
48 . The recombinant host cell of claim 47 , wherein:
the fatty acid or derivative thereof is an acyl-ACP, an acyl-CoA, a fatty acid, a fatty aldehyde, a short chain alcohol, a long chain alcohol, a fatty alcohol, a wax ester, a fatty ester, a fatty acid methyl ester (FAME), a fatty acid ethyl ester (FAEE), a hydrocarbon, a ketone, a terminal olefin, or an internal olefin;
the fatty acid or derivative thereof is a C 6 , C 7 , C 8 , C 9 , C 10 , C 11 , C 12 , C 13 , C 14 , C 15 , C 16 , C 17 , C 18 , C 19 , C 20 , C 21 , C 22 , C 23 , C 24 , C 25 , or a C 26 fatty acid or derivative thereof;
the fatty acid or derivative thereof is saturated or unsaturated; and
the fatty acid or derivative thereof is branched or unbranched.
49 . The recombinant host cell of claim 47 , wherein the FadR polypeptide is encoded by a fadR gene obtained from Escherichia, Salmonella, Citrobacter, Enterobacter, Klebsiella, Cronobacter, Yersinia, Serratia, Erwinia, Pectobacterium, Photorhabdus, Edwardsiella, Shewanella , or Vibrio.
50 . The recombinant host cell of claim 47 , wherein the FadR polypeptide is a wild-type FadR polypeptide or a mutant FadR polypeptide.
51 . The recombinant host cell of claim 47 , wherein the FadR polypeptide comprises an amino acid sequence having at least 80% sequence identity to the amino acid sequence of SEQ ID NO: 1.
52 . The recombinant host cell of claim 50 , wherein the mutant FadR polypeptide comprises an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 1 and comprises a substitution mutation at position 219 of SEQ ID NO: 1, wherein the amino acid residue corresponding to amino acid 219 of SEQ ID NO: 1 is substituted with an asparagine residue.
53 . The recombinant host cell of claim 47 , wherein the level of expression of the FadR polypeptide in the recombinant host cell is regulated by an expression control sequence operably linked to a polynucleotide sequence which encodes the FadR polypeptide.
54 . The recombinant host cell of claim 53 , wherein the expression control sequence is a promoter or a ribosome binding site.
55 . The recombinant host cell of claim 47 , wherein the recombinant host cell further comprises an altered level of expression of one or more genes corresponding to fabA, fabB, iclR, fadA, fadB, fadD, fadE, fadl, fadJ, fadL, fadM, uspA, aceA, aceB, and aceK, as compared to the level of expression of the gene(s) in a corresponding wild-type host cell.
56 . The recombinant host cell of claim 47 , wherein the recombinant host cell further expresses or overexpresses one or more of a thioesterase, a carboxylic acid reductase (CAR), an alcohol dehydrogenase, an ester synthase, a decarboxylase, an aldehyde decarbonylase, a fatty alcohol forming acyl-CoA reductase (FAR), an acyl-ACP reductase, an acyl-CoA reductase, OleA, OleCD, OleBCD, an acetyl-CoA carboxylase complex, a biotin protein ligase, or a phosphopantetheinyl transferase (PPTase).
57 . The recombinant host cell of claim 56 , wherein the acetyl-CoA carboxylase complex is encoded by one or more acetyl-CoA carboxylase subunit genes obtained from one or more of Corynebacterium glutamicum, Escherichia coli, Lactococcus lactis, Kineococcus radiotolerans, Desulfovibrio desulfuricans, Erwinia amylovora, Rhodospirillum rubrum, Vibrio furnissii, Stenotrophomonas maltophilia, Synechocystis sp. PCC6803, or Synechococcus elongatus.
58 . The recombinant host cell of claim 56 , wherein:
(a) a thioesterase is expressed or overexpressed, and the recombinant host cell produces a fatty acid;
(b) a thioesterase and a carboxylic acid reductase are expressed or overexpressed, and the recombinant host cell produces a fatty aldehyde or a fatty alcohol or a combination thereof;
(c) a thioesterase, a carboxylic acid reductase, and an alcohol dehydrogenase are expressed or overexpressed, and the recombinant host cell produces a fatty alcohol;
(d) a thioesterase, a carboxylic acid reductase, and a phosphopantetheinyl transferase (PPTase) are expressed or overexpressed, and the recombinant host cell produces a fatty alcohol;
(e) a thioesterase, a carboxylic acid reductase, an alcohol dehydrogenase, and a phosphopantetheinyl transferase (PPTase) are expressed or overexpressed, and the recombinant host cell produces a fatty alcohol; or
(f) an ester synthase is expressed or overexpressed, and the recombinant host cell produces a fatty ester or a wax ester.
59 . A method of producing a fatty acid or a derivative thereof, the method comprising culturing the recombinant host cell of claim 47 in a culture medium under conditions permissive for the production of the fatty acid or derivative thereof.
60 . The method of claim 59 , further comprising isolating the fatty acid or derivative thereof.
61 . The method of claim 59 , wherein the FadR polypeptide comprises an amino acid sequence having at least 80% sequence identity to the amino acid sequence of SEQ ID NO: 1.
62 . The method of claim 59 , wherein the FadR polypeptide comprises an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 1 and comprises a substitution mutation at position 219 of SEQ ID NO: 1, wherein the amino acid residue corresponding to amino acid 219 of SEQ ID NO: 1 is substituted with an asparagine residue.
63 . The method of claim 59 , wherein:
(a) the FadR polypeptide is a wild-type FadR polypeptide;
(b) the FadR polypeptide is a mutant FadR polypeptide; or
(c) the FadR polypeptide is encoded by a fadR gene obtained from Escherichia, Salmonella, Citrobacter, Enterobacter, Klebsiella, Cronobacter, Yersinia, Serratia, Erwinia, Pectobacterium, Photorhabdus, Edwardsiella, Shewanella , or Vibrio.
64 . The method of claim 59 , wherein the level of expression of the FadR polypeptide in the recombinant host cell is regulated by an expression control sequence operably linked to a polynucleotide sequence which encodes the FadR polypeptide.
65 . The method of claim 64 , wherein the expression control sequence is a promoter or a ribosome binding site.
66 . The method of claim 59 , wherein the recombinant host cell further expresses or overexpresses one or more of a thioesterase, a carboxylic acid reductase (CAR), an alcohol dehydrogenase, an ester synthase, a decarboxylase, an aldehyde decarbonylase, a fatty alcohol forming acyl-CoA reductase (FAR), an acyl-ACP reductase, an acyl-CoA reductase, OleA, OleCD, OleBCD, one or more acetyl-CoA carboxylase complex subunit genes, a biotin protein ligase, or a phosphopantetheinyl transferase (PPTase).
67 . The method of claim 59 , wherein:
the fatty acid or derivative thereof is an acyl-ACP, an acyl-CoA, a fatty acid, a fatty aldehyde, a short chain alcohol, a long chain alcohol, a fatty alcohol, a wax ester, a fatty ester, a fatty acid methyl ester (FAME), a fatty acid ethyl ester (FAEE), a hydrocarbon, a ketone, a terminal olefin, or an internal olefin;
the fatty acid or derivative thereof is a C 6 , C 7 , C 8 , C 9 , C 10 , C 11 , C 12 , C 13 , C 14 , C 15 , C 16 , C 17 , C 18 , C 19 , C 20 , C 21 , C 22 , C 23 , C 24 , C 25 , or a C 26 fatty acid or derivative thereof;
the fatty acid or derivative thereof is saturated or unsaturated; and
the fatty acid or derivative thereof is branched or unbranched.
68 . The method of claim 59 , wherein the recombinant host cell further comprises an altered level of expression of one or more genes corresponding to fabA, fabB, icIR, fadA, fadB, fadD, fadE, fadI, fadJ, fadL, fadM, uspA, aceA, aceB, and aceK, as compared to the level of expression of the gene(s) in a corresponding wild-type host cell.