Engineered acyltransferases having altered substrate specificity
AssigneeKez.Biosolutions GmbH
InventorsAlexander Rittner, Jan Degen, Mirk Joppe
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Abstract
The present invention relates to the fields of fatty acid- and polyketide synthesis and protein engineering. In particular, it relates to the provision of engineered acyltransferases having altered substrate specificity as well as means and methods of using acyltransferases of the present invention for polyketide synthesis.
Claims20
An acyltransferase having altered substrate specificity, wherein the acyltransferase comprises one or more amino acid exchange(s) at reference position(s)
- (i) T161 according to SEQ ID NO:
- 29, or an analogous threonine, serine or asparagine residue at said reference position as defined in Table 1, wherein the amino acid is exchanged to alanine, glycine, valine, leucine, isoleucine, tyrosine, phenylalanine, tryptophan, aspartate or glutamate;
- and/or
- (ii) F184 according to SEQ ID NO:
- 29, or an analogous phenylalanine residue at said reference position as defined in Table 1, wherein the amino acid is exchanged to alanine, glycine, valine, leucine, isoleucine, methionine, aspartate or glutamate;
- and/or
- (iii) R286 according to SEQ ID NO:
- 29, or an analogous arginine residue at said reference position as defined in Table 1, wherein the amino acid is exchanged to alanine, glycine, valine, leucine, isoleucine, methionine, aspartate or glutamate;
- and/or
- (iv) K186 according to SEQ ID NO:
- 29, or an analogous lysine or arginine residue at said reference position as defined in Table 1, wherein the amino acid is exchanged to a negatively charged amino acid or to glycine, alanine, valine, leucine, isoleucine or methionine;
- and/or
- (v) K285 according to SEQ ID NO:
- 29, or an analogous lysine or arginine residue at said reference position as defined in Table 1, wherein the amino acid is exchanged to a negatively charged amino acid;
- and/or
- (vi) R300 according to SEQ ID NO:
- 29, or an analogous lysine or arginine residue at said reference position as defined in Table 1, wherein the amino acid is exchanged to a negatively charged amino acid, and/or
- (vii) A282 according to SEQ ID NO:
- 29, or an analogous alanine residue at said reference position as defined in Table 1, wherein the amino acid is exchanged to valine, leucine, isoleucine, phenylalanine or tyrosine;
- and/or
- (viii) G137 according to SEQ ID NO:
- 29, or an analogous glycine residue at said reference position as defined in Table 1, wherein the amino acid is exchanged to valine, leucine, isoleucine, phenylalanine or tyrosine
The acyltransferase of any of the preceding claims, wherein the acyltransferase is an isolated acyltransferase originating from a fatty acid synthase.
The acyltransferase of any of the proceeding claims, wherein the acyltransferase comprises amino acid exchanges to glutamate at reference positions (iv) and (vi), or amino acid exchanges to aspartate at reference positions (iv) and (vi), or an amino acid exchange to glutamate at reference position (iv) and an amino acid exchange to aspartate at reference position (vi), or an amino acid exchange to aspartate at reference position (iv) and an amino acid exchange to glutamate at reference position (vi).
The acyltransferase of any of the preceding claims, wherein the acyltransferase comprises an amino acid exchange to glutamate or aspartate at reference position (iii) and optionally further comprises an amino acid exchange at reference position:
- (ix) D160 according to SEQ ID NO:
- 29, or an analogous aspartate or glutamate residue at said reference position as defined in Table 1, wherein the amino acid is exchanged to arginine, histidine, lysine, glutamine, or asparagine.
The acyltransferase of any of the preceding claims, wherein the acyltransferase originates from a fatty acid synthase of an organism selected from vertebrata, including aves and mammalia, including artiodactyla, perissodactyla, carnivora, primates and rodentia, optionally wherein the acyltransferase originates from a fatty acid synthase comprising or consisting of a wild type amino acid sequence selected from any one of SEQ ID NO:
- 1 to 28, or a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% sequence identity thereto.
The acyltransferase of any of the preceding claims, wherein the acyltransferase comprises or consists of an amino acid sequence of any of SEQ ID NO:
- 57 to 82 or 111 to 133, or a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% sequence identity thereto.
A fusion protein comprising at least one acyltransferase of any one of the preceding claims, preferably wherein the fusion protein further comprises at least one linker domain and optionally at least one ketoacyl synthase domain.
Dependencies are inferred from the claim text.