Ketoreductase polypeptides and polynucleotides

Patent metadata

Field	Value
Publication number	US-11021729-B2
Application number	US-201816605576-A
Country	US
Kind code	B2
Filing date	Apr 13, 2018
Priority date	Apr 27, 2017
Publication date	Jun 1, 2021
Grant date	Jun 1, 2021

Abstract

The present invention provides engineered ketoreductase and phosphite dehydrogenase enzymes having improved properties as compared to a naturally occurring wild-type ketoreductase and phosphite dehydrogenase enzymes, as well as polynucleotides encoding the engineered ketoreductase and phosphite dehydrogenase enzymes, host cells capable of expressing the engineered ketoreductase and phosphite dehydrogenase enzymes, and methods of using the engineered ketoreductase and phosphite dehydrogenase enzymes to synthesize a chiral catalyst used in the synthesis of antiviral compounds, such as nucleoside inhibitors. The present invention further provides methods of using the engineered enzymes to deracemize a chiral alcohol in a one-pot, multi-enzyme system.

First claim

What is claimed is:

1. An engineered ketoreductase variant having at least 95% or more sequence identity to SEQ ID NO: 138, wherein the residue at position 95 is not valine or the residue at position 135 is not valine or the residue at position 139 is not isoleucine and wherein said positions are numbered with reference to SEQ ID NO: 138.

2. The engineered ketoreductase variant of claim 1 having at least 95% or more sequence identity to SEQ ID NO:112.

3. The engineered ketoreductase variant of claim 1 having at least 95% or more sequence identity to SEQ ID NO:124, and at least an additional substitution at position 207, wherein said positions are numbered with reference to SEQ ID NO:124.

4. The engineered ketoreductase variant of claim 1 having at least one additional substitution or substitution set at positions selected from 19, 24/43/47/49/67/68/70/91/220, 24/68/91/218/220, 67, 72, 74/75/78/108, 75/78/99/108/215/224, 78/107, 95, 96, and 114, wherein said positions are numbered with reference to SEQ ID NO:138.

5. The engineered ketoreductase variant of claim 1 , wherein said engineered ketoreductase comprises a polypeptide sequence selected from the even-numbered sequences set forth in SEQ ID NOS: 114 to 170.

6. A composition comprising at least one engineered ketoreductase variant provided in claim 1 .

7. A method for deracemization of a chiral alcohol comprising at least one engineered ketoreductase variant of claim 1 , comprising at least one engineered phosphite dehydrogenase variant, at least one chiral alcohol, and at least one co-factor, under conditions such that said chiral alcohol is deracemized.

8. The method of claim 7 , wherein said method is conducted in one pot reaction.

Assignees

• Codexis Inc

Inventors

• Moore Jeffrey C
• Liang Jack
• Penfield Jonathan
• Nazor Jovana
• Dellas Nikki
• Mitchell Vesna
• Duan Da
• Farasat Iman
• Rodriguez-Granillo Agustina
• Murphy Grant
• Marshall Nicholas

Classifications

• C12Y120/01001

  Phosphonate dehydrogenase (1.20.1.1) · CPC title

• C12P41/002 Primary

  by oxidation/reduction reactions · CPC title

• C12Y101/01002

  Alcohol dehydrogenase (NADP+) (1.1.1.2), i.e. aldehyde reductase · CPC title

• C12N9/0004 Primary

  Oxidoreductases (1.) · CPC title

• C12P7/02

  containing a hydroxy group · CPC title