Ketoreductase-mediated stereoselective route to alpha chloroalcohols

Patent metadata

Field	Value
Publication number	US-2016160187-A1
Application number	US-201615046011-A
Country	US
Kind code	A1
Filing date	Feb 17, 2016
Priority date	Jun 22, 2009
Publication date	Jun 9, 2016
Grant date	—

Abstract

The present disclosure relates to engineered ketoreductase polypeptides and uses thereof for the preparation of α-chloroalcohols from α-chloroketones. Also provided are polynucleotides encoding the engineered ketoreductase polypeptides and host cells capable of expressing the engineered ketoreductase polypeptides

First claim

What is claimed is:

1 . An engineered ketoreductase polypeptide comprising an amino acid sequence at least 90% identical to SEQ ID NO:2 and that includes at least two of the following features:
residue corresponding to amino acid 2 of SEQ ID NO:2 is an aliphatic or nonpolar amino acid selected from alanine, leucine, valine, isoleucine, glycine and methionine; residue corresponding to amino acid 28 of SEQ ID NO:2 is an aliphatic or nonpolar amino acid selected from alanine, leucine, valine, isoleucine, glycine and methionine; residue corresponding to amino acid 34 of SEQ ID NO:2 is a polar amino acid selected from asparagine, glutamine, serine, and threonine; residue corresponding to amino acid 47 of SEQ ID NO:2 is an aliphatic or nonpolar amino acid selected from alanine, leucine, valine, isoleucine, glycine and methionine; residue corresponding to amino acid 50 of SEQ ID NO:2 is a basic amino acid selected from lysine and arginine; residue corresponding to amino acid 81 of SEQ ID NO:2 is a polar amino acid selected from among asparagine, glutamine, serine, and threonine; residue corresponding to amino acid 90 of SEQ ID NO:2 is an aliphatic or nonpolar amino acid selected from alanine, leucine, valine, isoleucine, glycine and methionine; residue corresponding to amino acid 91 of SEQ ID NO:2 is an aliphatic or nonpolar amino acid selected from alanine, leucine, valine, isoleucine, glycine and methionine, an aromatic amino acid selected from tyrosine, tryptophan, and phenylalanine, or a basic amino acid selected from lysine and arginine; residue corresponding to amino acid 94 of SEQ ID NO:2 is the basic amino acid, arginine; residue corresponding to amino acid 112 of SEQ ID NO:2 is an aromatic amino acid selected from tyrosine, tryptophan, and phenylalanine; residue corresponding to amino acid 117 of SEQ ID NO:2 is an acidic amino acid selected from aspartic acid and glutamic acid; residue corresponding to amino acid 143 of SEQ ID NO:2 is a basic amino acid selected from lysine and arginine; residue corresponding to amino acid 144 of SEQ ID NO:2 is a polar amino acid selected from asparagine, glutamine, serine, and threonine; residue corresponding to amino acid 145 of SEQ ID NO:2 is a nonpolar amino acid selected from alanine, leucine, valine, isoleucine, and methionine or an aliphatic amino acid selected from alanine, leucine, valine, isoleucine; residue corresponding to amino acid 148 of SEQ ID NO:2 is a constrained amino acid selected from proline and histidine; residue corresponding to amino acid 150 of SEQ ID NO:2 is a nonpolar or aliphatic amino acid selected from leucine, valine, isoleucine, glycine and methionine; residue corresponding to amino acid 152 of SEQ ID NO:2 is a nonpolar or aliphatic amino acid selected from alanine, leucine, valine, isoleucine, glycine and methionine; residue corresponding to amino acid 153 of SEQ ID NO:2 is a nonpolar or aliphatic amino acid selected from alanine, leucine, valine, isoleucine, glycine and methionine, or a constrained amino acid selected from histidine and proline; residue corresponding to amino acid 158 of SEQ ID NO:2 is a polar amino acid selected from asparagine, glutamine, and serine; residue corresponding to amino acid 190 of SEQ ID NO:2 is a nonpolar or an aliphatic amino acid selected from alanine, valine, leucine, isoleucine, glycine, and methionine; residue corresponding to amino acid 198 of SEQ ID NO:2 is a polar amino acid selected from asparagine, glutamine, and threonine; residue corresponding to amino acid 199 of SEQ ID NO:2 is an aliphatic or nonpolar amino acid selected from alanine, leucine, valine, glycine, and methionine, or a polar amino acid selected from asparagine, glutamine, serine, and threonine; residue corresponding to amino acid 200 of SEQ ID NO:2 is a nonpolar amino acid selected from alanine, leucine, valine, isoleucine, and glycine; residue corresponding to amino acid 217 of SEQ ID NO:2 is a polar amino acid selected from asparagine, glutamine, serine and threonine; residue corresponding to amino acid 225 of SEQ ID NO:2 is a nonpolar amino acid selected from valine, leucine, glycine, and methionine; residue corresponding to amino acid 231 of SEQ ID NO:2 is an aromatic amino acid selected from tyrosine, tryptophan, and phenylalanine; residue corresponding to amino acid 232 of SEQ ID NO:2 is a nonpolar amino acid selected from leucine, isoleucine, valine, glycine, and methionine; residue corresponding to amino acid 233 of SEQ ID NO:2 is a polar amino acid selected from asparagine, glutamine, serine, and threonine; residue corresponding to amino acid 244 of SEQ ID NO:2 is a nonpolar amino acid selected from alanine, leucine, isoleucine, valine, glycine, and methionine; residue corresponding to amino acid 260 of SEQ ID NO:2 is an aromatic amino acid selected from tyrosine and tryptophan; and residue corresponding to amino acid 261 of SEQ ID NO:2 is a polar amino acid selected from asparagine, glutamine, and threonine.

2 . The engineered ketoreductase polypeptide of claim 1 , wherein the amino acid sequence includes at least two of the following amino acid substitutions relative to SEQ ID NO:2: P2L; V28A; A34S; A47V; E50K; D81N; S90V; I91L; I91W; I91R; I91K; K94R; D112Y; G117D; S143R; V144T; G145A; R148H; A150G; F152L; N153G; N153V; N153H; T158S; G190A; S198N; I199M; I199L; I199N; M200I; A217T; I225V; P231F; A232V; E233Q; D244G; F260Y; and S261N.

3 . The engineered ketoreductase polypeptide of claim 1 , wherein the amino acid sequence includes one or more of the following features:
residue corresponding to amino acid 91 of SEQ ID NO:2 is selected from the group consisting of leucine, tryptophan, arginine, and lysine; residue corresponding to amino acid 144 of SEQ ID NO:2 is threonine; residue corresponding to amino acid 145 of SEQ ID NO:2 is alanine; residue corresponding to amino acid 150 of SEQ ID NO:2 is glycine; residue corresponding to amino acid 153 of SEQ ID NO:2 is selected from the group consisting of glycine, valine, and histidine; residue corresponding to amino acid 190 of SEQ ID NO:2 is alanine; residue corresponding to amino acid 199 of SEQ ID NO:2 is selected from the group consisting of methionine, leucine, and asparagine; and residue corresponding to amino acid 260 of SEQ ID NO:2 is tyrosine.

4 . The engineered ketoreductase polypeptide of claim 3 , wherein the amino acid sequence includes one or more of the following features:
residue corresponding to amino acid 91 of SEQ ID NO:2 is arginine; residue corresponding to amino acid 145 of SEQ ID NO:2 is alanine; residue corresponding to amino acid 153 of SEQ ID NO:2 is selected from the group consisting of glycine, and histidine; residue corresponding to amino acid 190 of SEQ ID NO:2 is alanine; and residue corresponding to amino acid 260 of SEQ ID NO:2 is tyrosine.

5 . The engineered ketoreductase polypeptide of claim 1 , wherein the polypeptide is capable of converting a reaction mixture comprising an initial concentration of at least 10 g/L (S)-tert-butyl 4-chloro-3-oxo-1-phenylbutan-2-ylcarbamate to tert-butyl (2S,3R)-4-chloro-3-hydroxy-1-phenylbutan-2-ylcarbamate with a conversion rate of at least 70% in 24 hours.

6 . The engineered ketoreductase polypeptide of claim 5 , wherein the polypeptide is capable of a conversion rate of at least 95% in 24 hours.

7 . The engineered ketoreductase polypeptide of claim 6 , wherein the polypeptide is capable of converting (S)-tert-butyl 4-chloro-3-oxo-1-phenylbutan-2-ylcarbamate to tert-butyl (2S,3R)-4-chloro-3-hydroxy-1-phenylbutan-2-ylcarbamate in at least 97% diastereomeric excess.

8 . The engineered ketoreductase polypeptide of claim 1 , wherein the amino acid sequence comprises at least one of the foll

Assignees

• Codexis Inc

Inventors

• Bong Yong Koy
• Vogel Michael
• Collier Steven J
• Mitchell Vesna
• Mavinahalli Jagadeesh

Classifications

• A61P7/04

  Antihaemorrhagics; Procoagulants; Haemostatic agents; Antifibrinolytic agents · CPC title

• 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

• C12P13/02

  Amides, e.g. chloramphenicol {or polyamides; Imides or polyimides; Urethanes, i.e. compounds comprising N-C=O structural element or polyurethanes (peptides C12P21/00 or C07K)} · CPC title

• C12P17/02

  Oxygen as only ring hetero atoms · CPC title