Ketoreductase polypeptides for the reduction of acetophenones

Patent metadata

Field	Value
Publication number	US-12415990-B2
Application number	US-202217932420-A
Country	US
Kind code	B2
Filing date	Sep 15, 2022
Priority date	Sep 13, 2007
Publication date	Sep 16, 2025
Grant date	Sep 16, 2025

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. A recombinant ketoreductase polypeptide capable of stereoselectively reducing acetophenone to (S)-1-phenethanol, which comprises an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 4 and comprises a substitution at the position corresponding to position 7 of the polypeptide of SEQ ID NO: 4.

2. The recombinant ketoreductase polypeptide of claim 1 , wherein the residue at the position corresponding to position 7 of the polypeptide of SEQ ID NO: 4 is an aromatic, non-polar, polar, histidine, proline, or basic residue.

3. The recombinant ketoreductase polypeptide of claim 1 , wherein said amino acid sequence of said engineered ketoreductase polypeptide further comprises at least one substitution at a position corresponding to a position in SEQ ID NO: 4, selected from the following substitutions:
the residue at the position corresponding to position 16 is a polar residue;
the residue at the position corresponding to position 43 is a nonpolar or polar residue;
the residue at the position corresponding to position 60 is an aromatic or non-polar, or aliphatic residue;
the residue at the position corresponding to position 94 is a cysteine, non-polar or an aliphatic residue;
the residue at the position corresponding to position 95 is a non-polar or aliphatic residue;
the residue at the position corresponding to position 96 is a polar or acidic residue;
the residue at the position corresponding to position 97 is a polar, non-polar, aliphatic, or basic residue;
the residue at the position corresponding to position 120 is an aromatic, non-polar or aliphatic residue;
the residue at the position corresponding to position 125 is a polar or non-polar residue;
the residue at the position corresponding to position 142 is a polar residue, serine or asparagine;
the residue at the position corresponding to position 147 is an aromatic, polar, non-polar, or aliphatic residue;
the residue at the position corresponding to position 149 is a non-polar or aromatic residue;
the residue at the position corresponding to position 150 is a constrained histidine, proline, or acidic residue;
the residue at the position corresponding to position 152 is a non-polar or polar residue;
the residue at the position corresponding to position 196 is an aliphatic, non-polar, or aromatic residue;
the residue at the position corresponding to position 202 is an aliphatic, aromatic, or a non-polar residue;
the residue at the position corresponding to position 205 is a basic, nonpolar or aliphatic residue; and
the residue at the position corresponding to position 206 is non-polar or aromatic residue.

4. The recombinant ketoreductase polypeptide of claim 1 , wherein the polypeptide is further capable of stereoselectively reducing the substrate 2′,6′-dichloro-3′-fluoroacetophenone to the product (S)-1-(2,6-dichloro-3-fluorophenyl) ethanol with a percent stereometric excess of at least 99%.

5. The recombinant ketoreductase polypeptide of claim 1 , wherein the polypeptide is further capable of reducing the substrate to the product at a rate greater than the rate capable by the ketoreductase polypeptide having the sequence of SEQ ID NO: 6 under the same conditions.

6. The recombinant ketoreductase polypeptide of claim 1 , wherein the polypeptide is further capable of reducing the substrate 2′,6′-dichloro-3′-fluoroacetophenone to the product (S)-1-(2,6-dichloro-3-fluorophenyl) ethanol at a rate that is at least 450% greater than the rate capable by the ketoreductase polypeptide having the sequence of SEQ ID NO: 6 under the same conditions.

7. The recombinant ketoreductase polypeptide of claim 1 , wherein the polypeptide is further capable of reducing the substrate 2′,6′-dichloro-3′-fluoroacetophenone to the product (S)-1-(2,6-dichloro-3-fluorophenyl) ethanol at a rate that is at least 1500% greater than the rate capable by the ketoreductase polypeptide having the sequence of SEQ ID NO: 6 under the same conditions.

8. The recombinant ketoreductase polypeptide of claim 1 , wherein the amino acid sequence of said recombinant ketoreductase polypeptide further comprises a proline at the position corresponding to position 190 of SEQ ID NO: 119.

Assignees

• Codexis Inc

Inventors

• Liang Jack
• Jenne Stephane J
• Mundorff Emily
• Ching Charlene
• Gruber John M
• Krebber Anke
• Huisman Gjalt W

Classifications

• C12N15/70

  Vectors or expression systems specially adapted for E. coli · CPC title

• C12Y101/01184

  Carbonyl reductase (NADPH) (1.1.1.184) · CPC title

• Y02P20/52

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

• C12Y101/01

  with NAD+ or NADP+ as acceptor (1.1.1) · CPC title

• C12P7/22

  aromatic · CPC title