Ketoreductase polypeptides for the production of (R)-3-hydroxythiolane

Patent metadata

Field	Value
Publication number	US-9394552-B2
Application number	US-201514597996-A
Country	US
Kind code	B2
Filing date	Jan 15, 2015
Priority date	Aug 24, 2007
Publication date	Jul 19, 2016
Grant date	Jul 19, 2016

Abstract

The present disclosure provides engineered ketoreductase enzymes having improved properties as compared to a naturally occurring wild-type ketoreductase enzyme. 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 chiral compounds.

First claim

What is claimed is:

1. A method for stereoselectively reducing substrate 3-ketothiolane- to product (R)-3-hydroxythiolane, which comprises contacting the 3-ketothiolane- with the ketoreductase polypeptide comprising an amino acid sequence that is at least 90% identical to the reference sequence SEQ ID NO:4 and which has a serine at the residue position corresponding to X147, under suitable reaction conditions for reducing or converting the 3-ketothiolase to (R)-3-hydroxythiolane, with a stereomeric excess of at least 70%.

2. The method of claim 1 , wherein 3-ketothiolane is reduced to (R)-3-hydroxythiolane with at least 90% steromeric excess.

3. The method of claim 1 , wherein 3-ketothiolane is reduced to (R)-3-hydroxythiolane with at least 98% steromeric excess.

4. The method of claim 1 , wherein the substrate is at least 100 g/L and at least 90% of the substrate is converted to product in less than 24 hrs, wherein the ketoreductase polypeptide is at least 0.8-1.0 g/L and comprises SEQ ID NO:143.

5. The method of claim 1 , wherein the substrate is at least 100 g/L and at least 90% of the substrate is converted to product in less than 20 hrs, wherein the ketoreductase polypeptide is at least about 0.8-1.0 g/L and is selected from the group consisting of SEQ ID NO:143.

6. The method of claim 1 , wherein the method is carried out at pH of about 7.5 or less.

7. The method of claim 1 , wherein the method is carried out at a pH of from about 5.0 to about 7.5.

8. The method of claim 1 , wherein the method is carried out with whole cells that express the ketoreductase enzyme, or an extract or lysate of such cells.

9. The method of claim 1 , wherein the ketoreductase is isolated or purified and the reduction reaction is carried out in the presence of a cofactor for the ketoreductase and optionally a regeneration system for the cofactor.

10. The method of claim 9 , wherein the cofactor regenerating system comprises glucose dehydrogenase and glucose; formate dehydrogenase and formate; or isopropanol and a secondary alcohol dehydrogenase.

11. The method of claim 9 , wherein the secondary alcohol dehydrogenase is the ketoreductase.

Assignees

• Codexis Inc

Inventors

• Liang Jack S
• Jenne Stephan
• Mundorff Emily
• Voladri Rama
• Lalonde James J
• Huisman Gjalt W

Classifications

• C12P17/167

  Heterorings having sulfur atoms as ring heteroatoms, e.g. vitamin B1, thiamine nucleus and open chain analogs · CPC title

• C12N9/0006

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

• C12P17/00 Primary

  Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms (C12P13/04 - C12P13/24 take precedence) · CPC title

• C12Y101/01184

  Carbonyl reductase (NADPH) (1.1.1.184) · CPC title

• Y02P20/52

  using catalysts, e.g. selective catalysts · CPC title