Ketoreductase-mediated stereoselective route to alpha chloroalcohols

Patent metadata

Field	Value
Publication number	US-9404092-B2
Application number	US-201615046011-A
Country	US
Kind code	B2
Filing date	Feb 17, 2016
Priority date	Jun 22, 2009
Publication date	Aug 2, 2016
Grant date	Aug 2, 2016

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 polypeptide having ketoreductase activity which comprises an amino acid sequence at least 90% identical to SEQ ID NO: 2 and includes at least two features selected from the group consisting of:
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 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, and 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 polypeptide of claim 1 , wherein the amino acid sequence includes at least two amino acid substitutions relative to SEQ ID NO: 2 selected from the group consisting of 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 polypeptide of claim 1 , wherein the amino acid sequence includes one or more features selected from the group consisting of:
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 polypeptide of claim 3 , wherein the amino acid sequence includes one or more features selected from the group consisting of:
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 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 polypeptide of claim 5 , wherein the polypeptide is capable of a conversion rate of at least 95% in 24 hours.

7. The engineered 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 polypeptide of claim 1 , wherein the amino acid sequence comprises at

Assignees

• Codexis Inc

Inventors

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

Classifications

• C12N9/0008

  acting on the aldehyde or oxo group of donors (1.2) · CPC title

• C12N9/0006 Primary

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

• C12P17/02

  Oxygen as only ring hetero atoms · CPC title

• C12P7/28

  Acetone-containing products · 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