Ketoreductase polypeptides for the synthesis of chiral compounds

Patent metadata

Field	Value
Publication number	US-11236308-B2
Application number	US-202016874574-A
Country	US
Kind code	B2
Filing date	May 14, 2020
Priority date	Feb 10, 2015
Publication date	Feb 1, 2022
Grant date	Feb 1, 2022

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 polynucleotide encoding an engineered polypeptide comprising an amino acid sequence with at least 95% 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 polynucleotide of claim 1 , wherein the encoded engineered polypeptide further comprises 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 polynucleotide of claim 1 , wherein the encoded engineered polypeptide further comprises one of the following substitutions: 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/R/V, X213D/E/G/H/K/M/N/R, X217H/N, X233Q/T, X250G, and X252W.

4. The engineered polynucleotide of claim 1 , wherein the encoded engineered polypeptide further comprises a substitution at position X190.

5. The engineered polynucleotide of claim 1 , wherein the encoded engineered polypeptide further comprises a substitution at position X190, wherein the substitution is selected from P, A, T, or Q.

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

7. The engineered polynucleotide of claim 1 , in which the polynucleotide comprises a nucleic acid sequence selected from the group consisting of the odd-numbered sequence identifiers of SEQ ID NOS:7, 9, 11, and 19-235.

8. An expression vector comprising the polynucleotide of claim 1 .

9. A host cell comprising the polynucleotide of claim 1 .

10. A host cell comprising the expression vector of claim 8 .

11. The host cell of claim 9 , wherein said host cell is E. coli.

12. The host cell of claim 10 , wherein said host cell is E. coli.

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

• C12P17/12

  containing a six-membered hetero ring · CPC title

• C12Y101/01184

  Carbonyl reductase (NADPH) (1.1.1.184) · CPC title

• C12P41/002

  by oxidation/reduction reactions · CPC title

• C12N9/0006 Primary

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