Ketoreductase polypeptides

Patent metadata

Field	Value
Publication number	US-2019161740-A1
Application number	US-201916272507-A
Country	US
Kind code	A1
Filing date	Feb 11, 2019
Priority date	Aug 29, 2008
Publication date	May 30, 2019
Grant date	—

Abstract

The present disclosure provides engineered ketoreductase enzymes having improved properties as compared to a naturally occurring wild-type ketoreductase enzyme including the capability of reducing 5-((4S)-2-oxo-4-phenyl (1,3-oxazolidin-3-yl))-1-(4-fluorophenyl) pentane-1,5-dione to (4S)-3-[(5S)-5-(4-fluorophenyl)-5-hydroxypentanoyl]-4-phenyl-1,3-oxazolidin-2-one. Also provided are polynucleotides encoding the engineered ketoreductase enzymes, host cells capable of expressing the engineered ketoreductase enzymes, and methods of using the engineered ketoreductase enzymes to synthesize the intermediate (4S)-3-[(5S)-5-(4-fluorophenyl)-5-hydroxypentanoyl]-4-phenyl-1,3-oxazolidin-2-one in a process for making Ezetimibe.

First claim

What is claimed is:

1 . An engineered polynucleotide encoding an engineered polypeptide having ketoreductase activity, wherein said engineered polypeptide comprises a region with an amino acid sequence having at least 90% sequence identity to residues 90 to 211 of SEQ ID NO: 130, comprising a substitution at position X190, and further comprising one or more of the following features selected from:
residue corresponding to X94 is asparagine, glycine, serine, or a polar residue; residue corresponding to X95 is an aliphatic residue; residue corresponding to X96 is glutamine, asparagine, or threonine; residue corresponding to X101 is an acidic, non-polar, or a polar residue; residue corresponding to X105 is an acidic or non-polar residue; residue corresponding to X108 is a hydrophilic, polar or constrained residue; residue corresponding to X111 is a non-polar or aliphatic residue; residue corresponding to X112 is an acidic or polar residue; residue corresponding to X113 is a non-polar or aliphatic residue; residue corresponding to X117 is a non-polar or a polar residue; residue corresponding to X127 is a basic residue; residue corresponding to X147 is a non-polar, aliphatic, aromatic, or hydrophobic residue; residue corresponding to X152 is a non-polar, basic, or hydrophilic residue; residue corresponding to X157 is a polar residue; residue corresponding to X163 is a non-polar or aliphatic residue; residue corresponding to X176 is a non-polar or aliphatic residue; residue corresponding to X194 is a constrained, basic, or polar residue; residue corresponding to X197 is a hydrophilic, acidic, basic, aliphatic or a non-polar residue; residue corresponding to X198 is an acidic, basic, hydrophilic, or non-polar residue; residue corresponding to X199 is an acidic, aliphatic, or non-polar residue; residue corresponding to X200 is an acidic or constrained residue; residue corresponding to X202 is a non-polar residue; residue corresponding to X206 is a non-polar, aromatic, or hydrophobic residue; residue corresponding to X211 is a basic residue, and wherein the amino acid sequence can optionally have one or more differences at other amino acid residues in the domain as compared to the reference sequence.

2 . An engineered polynucleotide encoding an engineered polypeptide having ketoreductase activity, wherein said engineered polypeptide comprises a region with an amino acid sequence having at least 90% sequence identity to residues-90 to 211 of SEQ ID NO: 130, comprising a substitution at position X190, and further comprising one or more of the following features selected from:
residue corresponding to X94 is asparagine, glycine, or serine; residue corresponding to X95 is leucine or methionine; residue corresponding to X96 is glutamine, asparagine, or threonine; residue corresponding to X101 is glycine or asparagine; residue corresponding to X105 is glycine; residue corresponding to X108 is histidine or serine; residue corresponding to X112 is aspartic acid; residue corresponding to X113 is alanine; residue corresponding to X117 is serine; residue corresponding to X127 is arginine; residue corresponding to X147 is leucine; residue corresponding to X152 is methionine or lysine; residue corresponding to X157 is threonine; residue corresponding to X163 is isoleucine; residue corresponding to X176 is valine; residue corresponding to X194 is arginine or glutamine; residue corresponding to X197 is valine or glutamic acid; residue corresponding to X198 is glycine, glutamic acid, or lysine; residue corresponding to X199 is aspartic acid; residue corresponding to X200 is proline; residue corresponding to X202 is glycine; residue corresponding to X206 is a glycine; residue corresponding to X211 is a arginine or lysine; and wherein the amino acid sequence can optionally have one or more differences at other amino acid residues in the domain as compared to the reference sequence.

3 . An expression vector comprising the polynucleotide of claim 1 , operably linked to one or more control sequences suitable for directing expression of the encoded polypeptide in a host cell.

4 . The expression vector of claim 3 , wherein the control sequence comprises a promoter.

5 . The expression vector of claim 4 , wherein the promoter comprises an E. coli promoter.

6 . The expression vector of claim 3 , wherein the control sequence comprises a secretion signal.

7 . A host cell comprising the expression vector of claim 3 .

8 . The host cell of claim 7 , wherein said host cell is E. coli.

Assignees

• Codexis Inc

Inventors

• Mundorff Emily
• De Vries Erik Jan

Classifications

• C12Y101/01184

  Carbonyl reductase (NADPH) (1.1.1.184) · CPC title

• C12N9/0006 Primary

  acting on CH-OH groups as donors (1.1) · CPC title

• C12N15/00

  Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor (mutants or genetically engineered microorganisms, per se C12N1/00, C12N5/00, C12N7/00; new plants per se A01H; plant reproduction by tissue culture techniques A01H4/00; new animals per se A01K67/00; use of medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases, gene therapy A61K48/00) · CPC title

• C12P7/02

  containing a hydroxy group · CPC title

• C12P17/14

  Nitrogen or oxygen as hetero atom and at least one other diverse hetero ring atom in the same ring · CPC title