Enzyme Variants With Improved Ester Synthase Properties

Abstract

The disclosure relates to enzyme variants with improved ester synthase properties for the production of fatty acid esters. Further contemplated are recombinant host cells that express such variants, cell cultures comprising the recombinant host cells and fatty acid ester compositions produced by such recombinant host cells.

1 .- 142 . (canceled) 
     
     
         143 . A variant thioesterase polypeptide comprising an amino acid sequence having at least about 90% sequence identity to SEQ ID NO: 51, wherein said variant Thioesterase polypeptide is genetically engineered to have at least one mutation at an amino acid position selected from the group consisting of amino acids 32, 33, 34, 38, 45, 46, 48, 49, 51, 52, 76, 81, 82, 84, 88, 112, 115, 116, 118, 119, 148, 149, 156, 159 and 164, wherein said variant thioesterase polypeptide has greater ester synthase activity compared to said corresponding wild type polypeptide. 
     
     
         144 . The variant thioesterase polypeptide of  claim 143 , wherein said polypeptide is a  Photobacterium profimdum  ( Ppro ) thioesterase polypeptide. 
     
     
         145 . The variant thioesterase polypeptide of  claim 143 , wherein expression of the variant thioesterase polypeptide in a recombinant host cell results in a higher titer of fatty ester compositions compared to a recombinant host cell expressing the corresponding wild type polypeptide. 
     
     
         146 . The variant ester synthase of  claim 145 , wherein said titer is at least about 5% or greater. 
     
     
         147 . The variant thioesterase polypeptide according to  claim 143 , comprising a mutation
 a) at amino acid position 32;   b) at amino acid position 33;   c) at amino acid position 34;   d) at amino acid position 45;   e) at amino acid position 48;   f) at amino acid position 46;   g) at amino acid position 49;   h) at amino acid position 51;   i) at amino acid position 52;   j) at amino acid position 76;   k) at amino acid position 82;   l) at amino acid position 84;   m) at amino acid position 112;   n) at amino acid position 115;   o) at amino acid position 116;   p) at amino acid position 148;   q) at amino acid position 149;   r) at amino acid position 156; or   s) at amino acid position 159.   
     
     
         148 . The variant thioesterase polypeptide according to  claim 143 , comprising at least one mutation selected from the group consisting of I45R, D48E, D48F, D48L, D48M, K32M, Q33A, Q33R, Q34R, I38Y, S46K, S46V, S46W, T49D, T49L, T49V, T49W, G51R, N52C, N52F, N52K, N52L, N52R, N76I, N76L, N76V, G81C, F82G, F82I, F82R, Q84A, Q84L, Q84V, R88H, V112L, N115F, N115W, N115Y, Y116H, Y116N, Y116P, Y116R, Y116S, Y116T, K118Q, R119C, I148Y, L149F, L149M, L149T, N156K, N156R, N156Y, L159K, L159M and D164T. 
     
     
         149 . The variant thioesterase polypeptide according to  claim 148 , wherein the thioesterase polypeptide comprises
 a) mutations S46V and Y116H;   b) mutations S46V, D48M, G51R, N76V and Y116H;   c) mutations S46V, D48M, T49L, G51R, N76V, Y116H and I148Y;   d) mutations S46V, D48M, T49L, G51R, N76V, N115F, Y116H and I148Y;   e) mutations Q33R, Q34R, S46V, D48M, T49L, G51R, N52R, N76V, V112L, N115W, Y116H and I148Y;   f) mutations Q33R, S46V, D48M, T49L, G51R, N52K, N76V, V112L, N115W, Y116H, I148Y and L149M; or   g) mutations S46V, D48M, T49L, G51R, N52R, N76V, Q84A, V112L, N115W, Y116H, I148Y and L149M.   
     
     
         150 . A recombinant host cell genetically engineered to express a variant thioesterase polypeptide according to  claim 143 . 
     
     
         151 . A cultured recombinant host cell comprising a polynucleotide sequence encoding a variant thioesterase polypeptide according to  claim 143 , wherein the recombinant host cell when expressing the variant thioesterase polypeptide produces a fatty acid ester composition with higher titer, higher yield and/or higher productivity of fatty acid esters compared to a host cell expressing the corresponding wild type thioesterase polypeptide, when cultured in medium containing a carbon source under conditions effective to express the variant thioesterase polypeptide, preferably wherein said titer is at least about 5% or greater. 
     
     
         152 . A cell culture comprising the recombinant host cell according to  claim 150  and a fatty ester composition. 
     
     
         153 . The cell culture of  claim 152 , wherein the fatty ester composition comprises
 a) one or more of a C 6 , C 8 , C 10 , C 12 , C 13 , C 14 , C 15 , C 16 , C 17  or C 18  fatty ester;   b) unsaturated fatty esters;   c) a fatty ester having a double bond at position 7 in the carbon chain between C 7  and C 8  from the reduced end of the fatty ester; or   d) branched chain fatty esters.   
     
     
         154 . The variant thioesterase polypeptide according to  claim 143 , wherein said variant thioesterase polypeptide comprises amino acids 73-81 of SEQ ID NO: 51 as follows LG[AG] [VIL]D[AG]LRG. 
     
     
         155 . A recombinant host cell comprising a polynucleotide encoding a variant polypeptide with increased ester synthase activity according to  claim 143 , wherein said recombinant host cell produces a fatty ester composition under conditions effective to express an ester synthase activity of the variant polypeptide. 
     
     
         156 . The variant polypeptide of according to  claim 155 , wherein said fatty ester composition comprises fatty acid methyl esters, fatty acid ethyl esters, fatty acid propyl esters, fatty acid isopropyl esters, fatty acid butyl esters, monoglycerides, fatty acid isobutyl esters, fatty acid 2-butyl esters, and/or fatty acid tert-butyl esters. 
     
     
         157 . The variant polypeptide of according to claim  1 , wherein said increased ester synthase activity results in properties selected from the group consisting of increased chain lengths of fatty acid esters, decreased chain lengths of fatty acid esters, and a change in fatty ester percentage of fatty acid species.