Microbiological production of short fatty acids and uses thereof

Abstract

The present invention relates to proteins involved in fatty acid synthesis, such as fatty acid synthases (FAS) variants, comprising one or more polypeptide chains, wherein said polypeptide chain(s) comprise one or more subunits comprising a malonyl/palmitoyl transferase domain (MPT domain), acetyl transferase domain (AT domain), and ketoacyl synthase domain (KS domain), and at least one amino acid substitution in the MPT domain at a position corresponding to R130, in the AT domain at a position corresponding to 1306, and/or in the KS domain, preferably in the acyl binding channel and/or at KS domain binding site to ACP, to modulate affinities of acyl intermediates, and optionally further amino acid substitution(s). The present invention relates to the respective polypeptide domains. The present invention further relates to nucleic acid molecules encoding the proteins (or the polypeptide domains) and to host cells containing said nucleic acid molecules. The present invention further relates to a method for the production of short fatty acids, CoA esters of short fatty acids, ethyl esters of short fatty acids, esters of short fatty acids with other metabolites, and/or enzyme bound short fatty acids (C 6 to C 12 ), comprising the expression of said nucleic acid molecules, preferably in said host cells. The present invention further relates to a method for the production of biofuels, flavoring compounds and/or fine chemicals, comprising the expression of said nucleic acid molecules, preferably in said host cells. The present invention also relates to the use of the proteins, nucleic acids molecule or host cells for the bulk production of short fatty acids (C 6 to C 12 ), the specific production of C 6 fatty acids and/or C 8 fatty acids, the bulk production of CoA esters of short fatty acids (C 6 to C 12 ), the specific production of C 6 -CoA esters and/or C 8 -CoA esters, the bulk production of ethyl esters of short fatty acids (C 6 to C 12 ), the specific production of C 6 fatty acid ethyl esters and/or C 8 fatty acid ethyl esters, the bulk production of esters of short fatty acids (C 6 to C 12 ) with other metabolites, the specific production of C 6 fatty acid esters with other metabolites and/or C 8 fatty acid esters with other metabolites, the bulk production of enzyme bound short fatty acids (C 6 to C 12 ), the specific production of enzyme bound C 6 fatty acids and/or enzyme bound C 8 fatty acids, the production of biofuels, fine chemicals and/or flavoring substances.

1 . A protein involved in fatty acid synthesis,
 said protein comprising one or more polypeptide chains, wherein said polypeptide chain(s) comprise
 (i) one or more subunits comprising the amino acid sequences of
 SEQ ID NO: 1 (malonyl/palmitoyl transferase domain, MPT domain); 
 SEQ ID NO: 2 (acetyl transferase domain, AT domain), and/or 
 SEQ ID NO: 3 (ketoacyl synthase domain, KS domain); 
 
 (ii) at least one amino acid substitution
 in the MPT domain at a position corresponding to R130 of the amino acid sequence of SEQ ID NO: 1; 
 in the AT domain at a position corresponding to 1306 of the amino acid sequence of SEQ ID NO: 2; 
 
 and/or
 in the KS domain, in the acyl binding channel; 
 
 wherein the amino acid sequence comprising the at least one amino acid substitution has at least 80% sequence identity to the respective amino acid sequence of SEQ ID NO: 1 and/or 2, and/or at least 80% sequence identity to the respective amino acid sequence of SEQ ID NO: 3, 
   provided that when the protein comprises an amino acid substitution G236S in the KS domain it comprises at least one additional amino acid substitution.   
     
     
         2 . The protein according to  claim 1 , comprising at least one further amino acid substitution in the KS domain selected from a position corresponding to Q193, N258 and D259 of the amino acid sequence of SEQ ID NO: 3. 
     
     
         3 . The protein according to  claim 1 , wherein the protein is type I FAS of  Saccharomyces cerevisiae.    
     
     
         4 . A polypeptide domain comprising
 (i) one or more subunits comprising the amino acid sequences of
 SEQ ID NO: 1 (malonyl/palmitoyl transferase domain, MPT domain); 
 SEQ ID NO: 2 (acetyl transferase domain, AT domain), or 
 SEQ ID NO: 3 (ketoacyl synthase domain, KS domain); 
   (ii) at least one amino acid substitution
 in the MPT domain at a position corresponding to R130 of the amino acid sequence of SEQ ID NO: 1; 
 in the AT domain at a position corresponding to 1306 of the amino acid sequence of SEQ ID NO: 2; 
   and/or
 in the KS domain in the acyl binding channel; 
   wherein the amino acid sequence comprising the at least one amino acid substitution has at least 80% sequence identity to the respective amino acid sequence of SEQ ID NO: 1 and/or 2, and/or at least 80% sequence identity to the respective amino acid sequence of SEQ ID NO: 3,   
       provided that when the polypeptide domain comprises an amino acid substitution G236S in the KS domain it comprises at least one additional amino acid substitution. 
     
     
         5 . The polypeptide domain according to  claim 4 , comprising at least one further amino acid substitution in the KS domain selected from a position corresponding to Q193, N258 and D259 of the amino acid sequence of SEQ ID NO: 3. 
     
     
         6 . The polypeptide domain according to  claim 1 , comprising amino acid substitution(s) R130K in the MPT domain (SEQ ID NO. 1), and/or I306A in the AT domain (SEQ ID NO: 2). 
     
     
         7 . The polypeptide domain according to  claim 1 , comprising amino acid substitution(s) in the acyl binding channel of the KS domain, wherein the amino acid substitutions comprise amino acid substitution(s) G236S, M237W and/or F265Y in the KS domain (SEQ ID NO: 3), and/or further comprising amino acid substitution(s) Q193A, Q193E, N258A, N258D and/or D259A in the KS domain (SEQ ID NO: 3). 
     
     
         8 . The protein according to  claim 1 , selected from the group of
 a protein comprising the amino acid substitutions I306A and G236S;   a protein comprising the amino acid substitutions I306A, R130K and F265Y;   a protein comprising the amino acid substitutions I306A, R130K and G236S;   a protein comprising the amino acid substitution R130K;   a protein comprising the amino acid substitutions I306A, R130K, G236S and M237W;   a protein comprising the amino acid substitutions I306A, G236S and M237W;   a protein comprising the amino acid substitutions G236S and M237W;   a protein comprising the amino acid substitutions I306A and R130K;   a protein comprising the amino acid substitutions R130K and G236S;   a protein comprising the amino acid substitutions I306A and F265Y;   a protein comprising the amino acid substitutions I306A, G236S and F265Y;   a protein comprising the amino acid substitutions R130K, G236S and M237W;   a protein comprising the amino acid substitutions G236S and F265Y;   a protein comprising the amino acid substitutions R130K, G236S and F265Y;   a protein comprising the amino acid substitutions I306A, R130K, G236S and F265Y;   a protein comprising the amino acid substitutions I306A, G236S, M237W and F265Y;   a protein comprising the amino acid substitution I306A;   a protein comprising the amino acid substitution M237W;   a protein comprising the amino acid substitution F265Y;   a protein comprising the amino acid substitutions I306A and M237W;   a protein comprising the amino acid substitutions R130K and M237W;   a protein comprising the amino acid substitutions R130K and F265Y;   a protein comprising the amino acid substitutions I306A, R130K and M237W;   a protein comprising the amino acid substitutions I306A, M237W and F265Y;   a protein comprising the amino acid substitutions R130K, M237W and F265Y;   a protein comprising the amino acid substitutions G236S, M237W and F265Y;   a protein comprising the amino acid substitutions M237W and F265Y;   a protein comprising the amino acid substitutions I306A, R130K, M237W and F265Y;   a protein comprising the amino acid substitutions R130K, G236S, M237W and F265Y;   a protein comprising the amino acid substitutions I306A, R130K, G236S, M237W and F265Y,   a protein comprising the amino acid substitutions I306A, R130K, G236S and D259A;   a protein comprising the amino acid substitutions I306A, R130K, G236S, M237W and D259A;   a protein comprising the amino acid substitutions I306A, R130K, G236S and N258A;   a protein comprising the amino acid substitutions I306A, R130K, G236S, M237W and N258A;   a protein comprising the amino acid substitutions I306A, R130K, G236S and N258D;   a protein comprising the amino acid substitutions I306A, R130K, G236S, M237W and N258D;   a protein comprising the amino acid substitutions I306A, R130K, G236S and Q193A;   a protein comprising the amino acid substitutions I306A, R130K, G236S, M237W and Q193A;   a protein comprising the amino acid substitutions I306A, R130K, G236S and Q193E; and   a protein comprising the amino acid substitutions I306A, R130K, G236S, M237W and Q193E;   wherein   R130K refers to R130K in the MPT domain of the amino acid sequence of SEQ ID NO: 1;   I306A refers to I306A in the AT domain of the amino acid sequence of SEQ ID NO: 2;   G236S refers to G236S in the KS domain of the amino acid sequence of SEQ ID NO: 3;   M237W refers to M237W in the KS domain of the amino acid sequence of SEQ ID NO: 3;   F265Y refers to F265Y in the KS domain of the amino acid sequence of SEQ ID NO: 3;   D259A refers to D259A in the KS domain of the amino acid sequence of SEQ ID NO: 3;   N258A refers to N258A in the KS domain of the amino acid sequence of SEQ ID NO: 3;   N258D refers to N258D in the KS domain of the amino acid sequence of SEQ ID NO: 3;   Q193A refers to Q193A in the KS domain of the amino acid sequence of SEQ ID NO: 3; and   Q193E refers to Q193E in the KS domain of the amino acid sequence of SEQ ID NO: 3.   
     
     
         9 . The protein according to  claim 1 , selected from
 variant I306A/G236S;   variant I306A/R130K/F265Y;   variant I306A/R130K/G236S;   variant R130K;   variant I306A/R130K/G236S/M237W;   variant I306A/G236S/M237W;   variant G236S/M237W;   variant I306A/R130K;   variant R130K/G236S;   variant I306A/F265Y;   variant I306A/G236S/F265Y;   variant R130K/G236S/M237W;   variant G236S/F265Y;   variant R130K/G236S/F265Y;   variant I306A/R130K/G236S/F265Y;   variant I306A/G236S/M237W/F265Y;   variant I306A;   variant M237W;   variant F265Y;   variant I306A/M237W;   variant R130K/M237W;   variant RI 30K/F265Y;   variant I306A/R130K/M237W;   variant I306A/M237W/F265Y;   variant R130K/M237W/F265Y;   variant G236S/M237W/F265Y;   variant M237W/F265Y;   variant I306A/R130K/M237W/F265Y;   variant R130K/G236S/M237W/F265Y;   variant I306A/R130K/G236S/M237W/F265Y;   variant I306A/RI 30K/G236S/D259A;   variant I306A/R130K/G236S/M237W/D259A;   variant I306A/R130K/G236S/N258A;   variant I306A/R130K/G236S/M237W/N258A;   variant I306A/R130K/G236S/N258D;   variant I306A/R130K/G236S/M237W/N258D;   variant I306A/R130K/G236S/Q193A;   variant I306A/R130K/G236S/M237W/Q193A;   variant I306A/R130K/G236S/Q193E; and   variant I306A/R130K/G236S/M237W/Q193E;   wherein   R130K refers to R130K in the MPT domain of the amino acid sequence of SEQ ID NO: 1;   I306A refers to I306A in the AT domain of the amino acid sequence of SEQ ID NO: 2;   G236S refers to G236S in the KS domain of the amino acid sequence of SEQ ID NO: 3;   M237W refers to M237W in the KS domain of the amino acid sequence of SEQ ID NO: 3; F265Y refers to F265Y in the KS domain of the amino acid sequence of SEQ ID NO: 3;   D259A refers to D259A in the KS domain of the amino acid sequence of SEQ ID NO: 3;   N258A refers to N258A in the KS domain of the amino acid sequence of SEQ ID NO: 3;   N258D refers to N258D in the KS domain of the amino acid sequence of SEQ ID NO: 3;   Q193A refers to Q193A in the KS domain of the amino acid sequence of SEQ ID NO: 3; and   Q193E refers to Q193E in the KS domain of the amino acid sequence of SEQ ID NO: 3.   
     
     
         10 . The protein according to  claim 1 , which causes elevated overall production of short fatty acids, CoA esters of short fatty acids, ethyl esters of short fatty acids, esters of short fatty acids with other metabolites, and/or enzyme bound short fatty acids (C 6  to C 12 ) compared to the wild type protein(s) or the protein(s) without such amino acid substitution(s). 
     
     
         11 . The protein according to  claim 1 , wherein the amino acid substitution(s) selected from
 I306A and G236S;   I306A and F265Y;   I306A, G236S and M237W;   I306A, R130K and G236S;   G236S and M237W;   I306A, R130K, G236S and M237W;   R130K;   I306A, G236S and F265Y;   I306A, R130K and F265Y;   I306A and R130K;   R130K and G236S;   R130K, G236S and M237W;   G236S and F265Y;   R130K, G236S and F265Y;   I306A, R130K, G236S and F265Y;   I306A, G236S, M237W and F265Y;   I306A;   M237W;   F265Y;   I306A and M237W;   R130K and M237W;   R130K and F265Y;   I306A, R130K and M237W;   I306A, M237W and F265Y;   R130K, M237W and F265Y;   G236S, M237W and F265Y;   M237W and F265Y;   I306A, R130K, M237W and F265Y;   R130K, G236S, M237W and F265Y;   I306A, R130K, G236S, M237W and F265Y;   I306A, R130K, G236S and N258A;   I306A, R130K, G236S and N258D;   I306A, R130K, G236S, M237W and N258A; and   I306A, R130K, G236S, M237W and N258D   wherein   R130K refers to R130K in the MPT domain of the amino acid sequence of SEQ ID NO: 1;   I306A refers to I306A in the AT domain of the amino acid sequence of SEQ ID NO: 2;   G236S refers to G236S in the KS domain of the amino acid sequence of SEQ ID NO: 3;   M237W refers to M237W in the KS domain of the amino acid sequence of SEQ ID NO: 3; F265Y refers to F265Y in the KS domain of the amino acid sequence of SEQ ID NO: 3;   N258A refers to N258A in the KS domain of the amino acid sequence of SEQ ID NO: 3;   N258D refers to N258D in the KS domain of the amino acid sequence of SEQ ID NO: 3;   increase(s) the selectivity for the production of C 6  fatty acids, C 6  fatty acid CoA esters, C 6  fatty acid ethyl esters, C 6  fatty acid esters with other metabolites, and/or enzyme bound C 6  fatty acids compared to wild type protein(s) or the protein without such amino acid substitution(s).   
     
     
         12 . The protein according to  claim 1 , wherein the amino acid substitution(s) selected from
 I306A, R130K and F265Y;   R130K;   I306A, R130K, G236S and M237W;   R130K and G236S;   I306A and R130K;   G236S and M237W;   I306A, R130K and G236S;   I306A, G236S and M237W;   R130K, G236S and M237W;   I306A and F265Y;   M237W;   I306A, G236S and F265Y;   G236S and F265Y;   I306A and G236S;   R130K, G236S and F265Y;   I306A, R130K, G236S and F265Y;   I306A, G236S, M237W and F265Y;   I306A;   F265Y;   I306A and M237W;   R130K and M237W;   R130K and F265Y;   I306A, R130K and M237W;   I306A, M237W and F265Y;   R130K, M237W and F265Y;   G236S, M237W and F265Y;   M237W and F265Y;   I306A, R130K, M237W and F265Y;   R130K, G236S, M237W and F265Y;   I306A, R130K, G236S, M237W and F265Y;   I306A, R130K, G236S and D259A,   I306A, R130K, G236S and N258A;   I306A, R130K, G236S and N258D;   I306A, R130K, G236S and Q193A;   I306A, R130K, G236S and Q193E;   I306A, R130K, G236S, M237W and D259A;   I306A, R130K, G236S, M237W and N258A;   I306A, R130K, G236S, M237W and N258D;   I306A, R130K, G236S, M237W and Q193A; and   I306A, R130K, G236S, M237W and Q193E   wherein   R130K refers to R130K in the MPT domain of the amino acid sequence of SEQ ID NO: 1;   I306A refers to I306A in the AT domain of the amino acid sequence of SEQ ID NO: 2;   G236S refers to G236S in the KS domain of the amino acid sequence of SEQ ID NO: 3;   M237W refers to M237W in the KS domain of the amino acid sequence of SEQ ID NO: 3; F265Y refers to F265Y in the KS domain of the amino acid sequence of SEQ ID NO: 3;   D259A refers to D259A in the KS domain of the amino acid sequence of SEQ ID NO: 3;   N258A refers to N258A in the KS domain of the amino acid sequence of SEQ ID NO: 3;   N258D refers to N258D in the KS domain of the amino acid sequence of SEQ ID NO: 3;   Q193A refers to Q193A in the KS domain of the amino acid sequence of SEQ ID NO: 3; and   Q193E refers to Q193E in the KS domain of the amino acid sequence of SEQ ID NO: 3;   increase(s) the selectivity for the production of C 8  fatty acids, C 8  fatty acid CoA esters, C 8  fatty acid ethyl esters, C 8  fatty acid esters with other metabolites, and/or enzyme bound C 8  fatty acids compared to wild type protein(s) or the protein without such amino acid substitution(s).   
     
     
         13 . The protein according to  claim 1 , wherein the amino acid substitution(s) selected from
 I306A, R130K, G236S and M237W;   G236S and M237W;   R130K;   I306A, G236S and F265Y;   I306A, R130K and G236S;   R130K and G236S;   I306A and R130K;   I306A, G236S and M237W;   I306A and F265Y;   M237W;   I306A, R130K and F265Y;   R130K, G236S and M237W;   G236S and F265Y;   I306A and G236S;   R130K, G236S and F265Y;   I306A, R130K, G236S and F265Y;   I306A, G236S, M237W and F265Y;   I306A;   F265Y;   I306A and M237W;   R130K and M237W;   R130K and F265Y;   I306A, R130K and M237W;   I306A, M237W and F265Y;   R130K, M237W and F265Y;   G236S, M237W and F265Y;   M237W and F265Y;   I306A, R130K, M237W and F265Y;   R130K, G236S, M237W and F265Y;   I306A, R130K, G236S, M237W and F265Y;   I306A, R130K, G236S and D259A,   I306A, R130K, G236S and N258A;   I306A, R130K, G236S and N258D;   I306A, R130K, G236S and Q193A;   I306A, R130K, G236S and Q193E;   I306A, R130K, G236S, M237W and D259A;   I306A, R130K, G236S, M237W and N258A;   I306A, R130K, G236S, M237W and N258D;   I306A, R130K, G236S, M237W and Q193A; and   I306A, R130K, G236S, M237W and Q193E   wherein   R130K refers to R130K in the MPT domain of the amino acid sequence of SEQ ID NO: 1;   I306A refers to I306A in the AT domain of the amino acid sequence of SEQ ID NO: 2;   G236S refers to G236S in the KS domain of the amino acid sequence of SEQ ID NO: 3;   M237W refers to M237W in the KS domain of the amino acid sequence of SEQ ID NO: 3; F265Y refers to F265Y in the KS domain of the amino acid sequence of SEQ ID NO: 3;   D259A refers to D259A in the KS domain of the amino acid sequence of SEQ ID NO: 3;   N258A refers to N258A in the KS domain of the amino acid sequence of SEQ ID NO: 3;   N258D refers to N258D in the KS domain of the amino acid sequence of SEQ ID NO: 3;   Q193A refers to Q193A in the KS domain of the amino acid sequence of SEQ ID NO: 3; and   Q193E refers to Q193E in the KS domain of the amino acid sequence of SEQ ID NO: 3;   increase(s) the selectivity for the production of C 10  to C 12  fatty acids, C 10  to C 12  fatty acid CoA esters, C 10  to C 12  fatty acid ethyl esters, C 10  to C 12  fatty acid esters with other metabolites, and/or enzyme bound C 10  to C 12  fatty acids compared to wild type protein(s) or the protein without such amino acid substitution(s).   
     
     
         14 . A nucleic acid molecule, encoding a a polypeptide domain according to  claim 4 . 
     
     
         15 . A host cell, containing a nucleic acid molecule according to  claim 14 . 
     
     
         16 . The host cell according to  claim 15 ,
 which has an elevated overall production of short fatty acids, CoA esters of short fatty acids, ethyl esters of short fatty acids, esters of short fatty acids with other metabolites, and/or enzyme bound short fatty acids (C 6  to C 12 ) compared to a cell not containing said nucleic acid molecule,
 or which has an increased yield of C 6  fatty acids, C 6  fatty acid CoA esters, C 6  fatty acid ethyl esters, C 6  fatty acid esters with other metabolites, and/or enzyme bound C 6  fatty acids compared to a cell not containing said nucleic acid molecule, 
 or which has an increased yield of C 8  fatty acids, C 8  fatty acid CoA esters, C 8  fatty acid ethyl esters, C 8  fatty acid esters with other metabolites, and/or enzyme bound C 8  fatty acids compared to a cell not containing said nucleic acid molecule, 
 or which has an increased yield of C 10  to C 12  fatty acids, C 10  to C 12  fatty acid CoA esters, C 10  to C 12  fatty acid ethyl esters, C 10  to C 12  fatty acid esters with other metabolites, and/or enzyme bound C 10  to C 12  fatty acids compared to a cell not containing said nucleic acid molecule. 
   
     
     
         17 . A method for the production of short fatty acids, CoA esters of short fatty acids, short fatty acid ethyl esters, short fatty acid esters with other metabolites, and/or enzyme bound short fatty acids (C 6  to C 12 ), comprising the expression of a nucleic acid molecule according to  claim 14 . 
     
     
         18 . A method for the production of a biofuel, a flavoring compound and/or a fine chemical (such as natural compounds), comprising the expression of a nucleic acid molecule according to  claim 14 . 
     
     
         19 . A method for:
 the bulk production of short fatty acids (C 6  to C 12 ),   the specific production of C 6  fatty acids,   the specific production of C 8  fatty acids,   the bulk production of CoA esters of short fatty acids (C 6  to C 12 ),   the specific production of C 6  fatty acid CoA esters,   the specific production of C 8  fatty acid CoA esters,   the bulk production of ethyl esters of short fatty acids (C 6  to C 12 ),   the specific production of C 6  fatty acid ethyl esters,   the specific production of C 8  fatty acid ethyl esters, or   the bulk production of short fatty acids (C 6  to C 12 ) esters with other metabolites,   the specific production of C 6  fatty acid esters with other metabolites,   the specific production of C 8  fatty acid esters with other metabolites,   the bulk production of enzyme bound short fatty acids (C 6  to C 12 ),   the specific production of enzyme bound C 6  fatty acids,   the specific production of enzyme bound C 8  fatty acids,   the production of biofuels,   the production of fine chemicals, where short fatty acids (C 6  to C 12 ) or their derivatives are used as a building block, or   the production of flavoring substances, wherein said method comprises the use of a nucleic acid molecule according to  claim 14 .   
     
     
         20 . The polypeptide domain, according to  claim 4 , having at least one amino acid substitution selected from G236, M237, and F265. 
     
     
         21 . The host cell, according to  claim 15 , wherein said host cell is a  Saccharomyces cerevisiae.