Methods of using ketoreductase polypeptides for reduction of acetophenones

Patent metadata

Field	Value
Publication number	US-9528131-B2
Application number	US-201414501416-A
Country	US
Kind code	B2
Filing date	Sep 30, 2014
Priority date	Sep 13, 2007
Publication date	Dec 27, 2016
Grant date	Dec 27, 2016

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 method for stereoselectively reducing 2,6-dichloro-3′-fluoroacetophenone substrate, optionally substituted at one or more positions selected from the group consisting of 4′ and 5′, to the corresponding substituted (S)-1-phenethanol, which comprises contacting the substrate with an engineered ketoreductase under reaction conditions suitable for stereoselectively reducing or converting the substrate to the corresponding substituted (S)-1-phenethanol product, wherein the engineered ketoreductase is derived from a wild-type Lactobacillus ketoreductase and wherein said engineered ketoreductase comprises an amino acid sequence at least 90% identical to the reference sequence of SEQ ID NO: 2, 4, or 98; and wherein said engineered ketoreductase is capable of stereoselectively reducing 2,6-dichloro-3′-fluoroacetophenone to (S)-1-(2,6-dichloro-3-fluorophenyl) ethanol.

2. The method of claim 1 , wherein the (S)-1-(2,6-dichloro-3-fluorophenyl) ethanol is formed in greater than 90% stereomeric excess.

3. The method of claim 1 , wherein the (S)-1-(2,6-dichloro-3-fluorophenyl) ethanol is formed in greater than 99% stereomeric excess.

4. The method of claim 1 , wherein at least 95% of the substrate is reduced to the product in less than 24 hours when the method is conducted with at least 200 g/L of substrate and with less than 2 g/L of the engineered ketoreductase.

5. The method of claim 1 , wherein the method is carried out with whole cells that express the engineered ketoreductase, or an extract or lysate of such cells.

6. The method of claim 1 , wherein the engineered ketoreductase is isolated and/or purified and the reduction reaction is carried out in the presence of a cofactor for the engineered ketoreductase.

7. The method of claim 6 , wherein the reduction reaction is carried out in the presence of a regeneration system for the cofactor.

8. The method of claim 7 , in which the cofactor regenerating system comprises glucose dehydrogenase and glucose; formate dehydrogenase and formate; or isopropanol and a secondary alcohol dehydrogenase.

9. The method of claim 8 , wherein the secondary alcohol dehydrogenase is the engineered ketoreductase.

10. The method of claim 1 , wherein the amino acid sequence of the engineered ketoreductase has a proline at a position corresponding to position 190 of SEQ ID NO: 2, 4, or 98.

11. The method of claim 9 , wherein said engineered ketoreductase comprises a residue at a position corresponding to a position in SEQ ID NO: 2, 4, or 98 selected from the group consisting of:
the residue at the position corresponding to position 7 is an aromatic, non-polar, polar, L-proline, L-histidine, or a basic residue;
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, 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 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 L-proline, L-histidine or an 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 nonpolar 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.

12. The method of claim 9 , wherein said engineered ketoreductase comprises a residue at a position corresponding to a position in SEQ ID NO: 2, 4, or 98 selected from the group consisting of:
the residue at the position corresponding to position 7 is threonine, proline, tryptophan, arginine, histidine, or asparagine;
the residue at the position corresponding to position 16 is serine;
the residue at the position corresponding to position 43 is isoleucine;
the residue at the position corresponding to position 60 is alanine;
the residue at the position corresponding to position 94 is alanine, valine or cysteine;
the residue at the position corresponding to position 95 is isoleucine or leucine;
the residue at the position corresponding to position 96 is serine, asparagine, threonine or glutamic acid;
the residue at the position corresponding to position 97 is lysine, threonine, valine, arginine, methionine, or isoleucine;
the residue at the position corresponding to position 120 is phenylalanine or valine;
the residue at the position corresponding to position 125 is glycine or serine;
the residue at the position corresponding to position 142 is asparagine;
the residue at the position corresponding to position 147 is phenylalanine, leucine, isoleucine, valine, or glutamine;
the residue at the position corresponding to position 149 is glycine or phenylalanine;
the residue at the position corresponding to position 150 is aspartic acid or histidine;
the residue at the position corresponding to position 152 is serine, threonine, or methionine;
the residue at the position corresponding to position 196 is valine, isoleucine, methionine, phenylalanine, or isoleucine;
the residue at the position corresponding to position 202 is alanine, tryptophan, tyrosine, or methionine;
the residue at the position corresponding to position 205 is arginine; and
the residue at the position corresponding to position 206 is methionine or tyrosine.

13. The method of claim 1 , wherein said engineered ketoreductase 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.

14. The method of claim 13 , wherein said engineered ketoreductase is further capable of reducing the substrate to the product 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.

15. The method of claim 1 , wherein said engineered ketoreductase is further capable of reducing the substrate to the product 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.

16. The method of claim 1 , wherein said engineered ketoreductase is capable of converting in less than 24 hours at least 95% of the 2′,6′-dichloro-3′-fluoroacetophenone substrate to (S)-1-(2,6-dichloro-3-fluorophenyl) ethanol when carried out with the engineered ketoreductase at an amount of less than 1% by weight with respect to the amount of the 2′,6′-dichloro-3′-fluoroacetophenone substrate.

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

• Y02E50/10

  Biofuels, e.g. bio-diesel · CPC title

• C12Y101/01

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

• C12P7/22 Primary

  aromatic · CPC title

• Y02P20/52

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