Polynucleotides encoding ketoreductase polypeptides for reduction of acetophenones

Patent metadata

Field	Value
Publication number	US-9873863-B2
Application number	US-201615353165-A
Country	US
Kind code	B2
Filing date	Nov 16, 2016
Priority date	Sep 13, 2007
Publication date	Jan 23, 2018
Grant date	Jan 23, 2018

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 non-naturally occurring polynucleotide encoding an engineered ketoreductase polypeptide derived from a wild-type Lactobacillus ketoreductase having ketoreductase activity, wherein said engineered ketoreductase polypeptide 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 product when carried out at an amount of less than 1% by weight with respect to the amount of the 2′,6′-dichloro-3′-fluoroacetophenone substrate, and wherein said engineered ketoreductase polypeptide comprises an amino acid sequence at least 90% identical to SEQ ID NO: 2, 4, or 98, and further wherein said amino acid sequence of said engineered ketoreductase polypeptide comprises at least one substitution at a position corresponding to a position in SEQ ID NO: 2, 4, or 98, selected from the following substitutions:
the residue at the position corresponding to position 7 is an aromatic, non-polar, polar, constrained, or 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 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 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 a non-polar or aromatic residue.

2. The non-naturally occurring polynucleotide of claim 1 , wherein the amino acid sequence of said engineered ketoreductase polypeptide comprises a proline at a position corresponding to position 190 of SEQ ID NO: 2, 4, or 98.

3. The non-naturally occurring polynucleotide of claim 1 , wherein the amino acid sequence of said engineered ketoreductase polypeptide comprises an amino acid sequence having at least one substitution, at a position corresponding to a position in SEQ ID NO:2, 4, or 98, selected from:
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, or phenylalanine;
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; and
wherein optionally the amino acid sequence has one or more residue differences at other amino acid residues as compared to SEQ ID NO:2, 4, or 98.

4. The non-naturally occurring polynucleotide of claim 1 , wherein said engineered ketoreductase is 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.

5. The non-naturally occurring polynucleotide of claim 1 , wherein said engineered ketoreductase polypeptide 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.

6. An expression vector comprising the non-naturally occurring polynucleotide of claim 1 .

7. A host cell comprising the expression vector of claim 6 .

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

• C12P7/22

  aromatic · CPC title

• C12Y101/01184

  Carbonyl reductase (NADPH) (1.1.1.184) · CPC title

• C12Y101/01

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