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

• US 9,394,552 B2
• Filed: 01/15/2015
• Issued: 07/19/2016
• Est. Priority Date: 08/24/2007
• Status: Active Grant

• Current Assignee: Codexis, Inc.
• Original Assignee: Codexis, Inc.
• Inventors: Liang, Jack S, Jenne, Stephan, Mundorff, Emily, Voladri, Rama, Lalonde, James J., Huisman, Gjalt W.
• Primary Examiner(s): RAGHU, GANAPATHIRAM
• Application Number:
  US14/597,996
• Publication Number:
  US 20150125910A1
• Time in Patent Office: 551 Days
• Field of Search: 435/117, 435/189, 435/190, 435/69.1, 536/23.2, 530/350
• US Class Current: 1/1
• CPC Class Codes: C12N 9/0006 acting on CH-OH groups as donors (1.1)
• CPC Class Codes: C12P 17/00 Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms C12P13/04 - C12P13/24 take precedence
• CPC Class Codes: C12P 17/167 Heterorings having sulfur atoms as ring heteroatoms, e.g. vitamin B1, thiamine nucleus and open chain analogs
• CPC Class Codes: C12Y 101/01184 Carbonyl reductase (NADPH) (1.1.1.184)
• CPC Class Codes: Y02P 20/52 using catalysts, e.g. selective catalysts

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.

Claims

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.