Ketoreductases and uses thereof

• US 8,415,127 B2
• Filed: 11/07/2011
• Issued: 04/09/2013
• Est. Priority Date: 02/08/2007
• Status: Active Grant

• Current Assignee: Codexis, Inc.
• Original Assignee: Codexis, Inc.
• Inventors: Ching, Charlene, Gruber, John M., Huisman, Gjalt W., Mundorff, Emily, Newman, Lisa M.
• Primary Examiner(s): PAK, YONG D
• Application Number:
  US13/290,773
• Publication Number:
  US 20120178142A1
• Time in Patent Office: 519 Days
• Field of Search: None
• US Class Current: 435/190
• CPC Class Codes: C12N 15/00 Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor mutants or genetically engineered microorganisms, per se C12N1/00, C12N5/00, C12N7/00; new plants per se A01H; plant reproduction by tissue culture techniques A01H4/00; new animals per se A01K67/00; use of medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases, gene therapy A61K48/00
• CPC Class Codes: C12N 15/70 Vectors or expression systems specially adapted for E. coli
• CPC Class Codes: C12N 9/0006 acting on CH-OH groups as donors (1.1)
• CPC Class Codes: C12Y 101/01184 Carbonyl reductase (NADPH) (1.1.1.184)

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.

Claims

1. An isolated or engineered polypeptide having ketoreductase activity which comprises an amino acid sequence with at least 90% sequence identity to SEQ ID NO:

2 and having an L at the amino acid residue corresponding to residue 147 of SEQ ID NO;

2.

2. The polypeptide of claim 1 in which the amino acid sequence further comprises one or more features selected from:

residue corresponding to residue 94 is an aromatic amino acid or G;

residue corresponding to residue 96 is any amino acid other than S/N;

residue corresponding to residue 153 is an aliphatic amino acid residue other than L;

residue corresponding to residue 199 is any amino acid residue other than L;

residue corresponding to residue 202 is G or an aliphatic amino acid residue other than A; and

residue corresponding to residue 206 is an aromatic amino acid residue.

3. The polypeptide of claim 2 in which the amino acid sequence further comprises one or more of features selected from:

residue corresponding to residue 94 is F, W, H, Y;

residue corresponding to residue 96 is G, I, C or an aromatic amino acid;

residue corresponding to residue 153 is G or A;

residue corresponding to residue 199 is K, I, N, R, V, Q, or W;

residue corresponding to residue 202 is I, L, or G; and

residue corresponding to residue 206 is F.

4. The polypeptide of claim 2 in which the amino acid sequence further comprises one or more of the features selected from:

residue 49 is a polar amino acid residue other than K;

residue 53 is an acidic amino acid residue;

residue 54 is a small or aliphatic amino acid residue other than T/P;

residue 60 is an aliphatic amino acid residue other than V;

residue 95 is an aliphatic amino acid other than V;

residue 97 is a small amino acid or G;

residue 109 is a basic amino acid residue other than K;

residue 165 is a hydroxyl or small amino acid residue;

residue 197 is a small amino acid residue or G;

residue 223 is an aliphatic amino acid residue other than L; and

residue 233 is a small amino acid residue or G.

5. The polypeptide of claim 4 in which the amino acid sequence comprises one or more features selected from:

residue 49 is R;

residue 53 is D;

residue 54 is A;

residue 60 is A;

residue 95 is L;

residue 97 is G;

residue 109 is R;

residue 165 is T;

residue 197 is G;

residue 223 is V; and

residue 233 is G.

6. The polypeptide of claim 1 which is capable of converting acetophenone to (R)-1-phenylethanol.

7. The polypeptide of claim 1 which comprises an amino acid sequence selected from the group consisting of SEQ ID NO:

6, 20, 22, 24, 26, 28, 30, 32, 34, 38, 40, 42, 44, 46, 48, 50, 52, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 110, 122, 124, 126, 136, and 138.