Ketoreductase polypeptides for the synthesis of chiral compounds

Patent metadata

Field	Value
Publication number	US-10696953-B2
Application number	US-201615543535-A
Country	US
Kind code	B2
Filing date	Feb 5, 2016
Priority date	Feb 10, 2015
Publication date	Jun 30, 2020
Grant date	Jun 30, 2020

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 a variety of chiral compounds.

First claim

What is claimed is:

1. An engineered polypeptide comprising an amino acid sequence with at least 90% sequence identity to SEQ ID NO:4 and an arginine or lysine at position X249 as compared to SEQ ID NO:4, and wherein said polypeptide has ketoreductase activity with at least 2-fold greater selectivity to the chiral compound 2a relative to chiral compound 2c as compared to SEQ ID NO:4.

2. The engineered polypeptide of claim 1 , wherein said amino acid sequence further comprises at least one of the following:
the residue corresponding to X68 is a non-polar, or aliphatic residue;
the residue corresponding to X102 is an acidic residue;
the residue corresponding to X110 is an acidic, or aromatic residue;
the residue corresponding to X114 is a non-polar residue;
the residue corresponding to X135 is a basic residue;
the residue corresponding to X144 is a non-polar, or aromatic residue;
the residue corresponding to X147 is a non-polar, or aliphatic residue;
the residue corresponding to X149 is a polar residue;
the residue corresponding to X153 is a polar, or aromatic residue;
the residue corresponding to X158 is a non-polar, or aliphatic residue;
the residue corresponding to X175 is a polar residue;
the residue corresponding to X190 is an aromatic, aliphatic, non-polar, or polar residue;
the residue corresponding to X196 is an aromatic, polar, basic, non-polar, or aliphatic residue;
the residue corresponding to X197 is an aromatic residue;
the residue corresponding to X198 is a non-polar, polar residue;
the residue corresponding to X201 is a polar residue;
the residue corresponding to X202 is an aromatic, or basic residue;
the residue corresponding to X203 is an aromatic, non-polar, or aliphatic residue;
the residue corresponding to X205 is a polar residue;
the residue corresponding to X206 is an aromatic residue;
the residue corresponding to X207 is an aromatic, acidic, non-polar, or aliphatic residue;
the residue corresponding to X209 is an aromatic, acidic, non-polar, polar, or aliphatic residue;
the residue corresponding to X210 is a non-polar, or aliphatic residue;
the residue corresponding to X212 is an aromatic, basic, non-polar, aliphatic, or acidic residue;
the residue corresponding to X213 is an aromatic, basic, acidic, polar, non-polar, or aliphatic residue;
the residue corresponding to X217 is a polar, or aromatic residue;
the residue corresponding to X233 is a polar residue;
the residue corresponding to X250 is a non-polar residue; and
the residue corresponding to X252 is an aromatic residue.

3. The engineered polypeptide of claim 1 , further comprising at least one substitution selected from X68V, X94G/Q, X102D, X110E/W, X114G, X135K, X144G/W, X147L, X149S, X150D/F/P/W, X153H/P/T/V/C, X158V, X175T, X190A/L/Q/T/W, X196H/I/K/N/Q, X1961, X196K/N/Q, X197Y, X198G/S, X201N, X202F/H/R/Y, X203G/L/W, X205T, X206H/W/Y, X207D/G/V/Y, X209E/F/G/I/M/T/V/W, X210L/P, X211H/I/P/S, X212A/E/G/H/N/P/RN, X213D/E/G/H/K/M/N/R, X217H/N, X233Q/T, X250G, and X252W.

4. The engineered polypeptide of claim 1 , further comprising a substitution at position X190.

5. The engineered polypeptide of claim 4 , comprising a substitution at position X190, wherein the substitution is selected from P, A, T, or Q.

6. The engineered polypeptide claim 1 , wherein the stereoselective activity of the polypeptide is increased at least 2-fold as compared to the corresponding activity of the reference polypeptide of SEQ ID NOS:4, 10, 26, and/or 42.

Assignees

• Codexis Inc

Inventors

• Alvizo Oscar
• Agard Nicholas J
• Xie Xinkai
• Entwistle David
• Kosjek Birgit

Classifications

• C12P7/40

  containing a carboxyl group {including Peroxycarboxylic acids} · CPC title

• C12N9/0006 Primary

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

• C12P17/12

  containing a six-membered hetero ring · CPC title

• C12Y101/01184

  Carbonyl reductase (NADPH) (1.1.1.184) · CPC title

• C12P7/02

  containing a hydroxy group · CPC title