Biocatalysts for the preparation of hydroxy substituted carbamates

Patent metadata

Field	Value
Publication number	US-2019177702-A1
Application number	US-201916284235-A
Country	US
Kind code	A1
Filing date	Feb 25, 2019
Priority date	Nov 18, 2011
Publication date	Jun 13, 2019
Grant date	—

Abstract

The present disclosure relates to engineered ketoreductase polypeptides for the preparation of hydroxyl substituted carbamate compounds, and polynucleotides, vectors, host cells, and methods of making and using the ketoreductase polypeptides.

First claim

What is claimed is:

1 . An engineered ketoreductase polypeptide capable of converting substrate compound (2), tert-butyl(1-oxo-1-phenylhex-5-yn-2-yl)carbamate, to product compound (1), tert-butyl(1-hydroxy-1-phenylhex-5-yn-2-yl)carbamate,

wherein the ketoreductase polypeptide comprises an amino acid sequence having at least 80% sequence identity to SEQ ID NO:4 and comprises a substitution at position 206, further comprising one or more residue differences as compared to SEQ ID NO:4 selected from: X7S; X17M; X17Q; X17R; X23V; X27L; X29G; X40R; X60I; X64V; X71P; X87L; X94A; X94P; X94S; X95M; X96L; X96Y; X105G; X113I; X122A; X127R; X131S; X144V; X145L; X147I; X147L; X147Q; X150Y; X152G; X153G; X157C; X173L; X195A; X195G; X196M; X198S; X199H; X208R; X216R; X221S; X243S; X245I; X249F; X249G; and X249Y.

2 . The engineered ketoreductase polypeptide of claim 1 , wherein the amino acid sequence of said ketoreductase polypeptide comprises the substitution X206F/L and one or more residue differences as compared to SEQ ID NO:4 selected from: X7S; X17M; X17Q; X17R; X23V; X27L; X29G; X40R; X60I; X64V; X71P; X87L; X94A; X94P; X94S; X95M; X105G; X113I; X122A; X127R; X131S; X144V; X145L; X147I; X147L; X147Q; X150Y; X152G; X153G; X157C; X173L; X196M; X198S; X208R; X216R; X221S; X243S; X245I; X249F; X249G; and X249Y.

3 . The engineered ketoreductase polypeptide of claim 1 , wherein the amino acid sequence of said ketoreductase polypeptide comprises X206F/L, and at least one or more residue differences as compared to SEQ ID NO:4 selected from: X17Q/R/M; X40R; X64V; X94P; X96L/Y; X144V; X147Q/I/L; X157C; X195A/G; X196M; and X199H.

4 . The engineered ketoreductase polypeptide of claim 1 , wherein the ketoreductase polypeptide is capable of converting the substrate compound (2) to the product compound (1) with at least 10 fold the activity of the reference polypeptide of SEQ ID NO:4, wherein the amino acid sequence comprises the substitution X206F/L, and one or more residue differences as compared to SEQ ID NO:4 selected from: X40R; X60I; X71P; X94P; X94A; X95M; X96L; X96Y; X127R; X144V; X1451; X150Y; X152G; X153G; X157C; X195A; X195G; X196M; X198S; X199H; X216R, X245I, X245F; X249Y; and X249F.

5 . The engineered ketoreductase polypeptide of claim 1 , wherein the ketoreductase polypeptide has increased thermal stability as compared to the reference polypeptide of SEQ ID NO:4 or 32, wherein the amino acid sequence comprises the substitution X206F/L, and one or more residue differences as compared to SEQ ID NO:4 selected from: X17Q; X17R; X17W; X40R; X64V; X71P; X87L; X94S; X94P; X147Q; X147I; X147L; X157C, X198S; X249Y; and X249F.

6 . The engineered ketoreductase polypeptide of claim 1 , wherein the ketoreductase polypeptide is capable of forming product syn-tert-butyl(1-hydroxy-1-phenylhex-5-yn-2-yl)carbamate compounds (1a) and (1c)

in diastereomeric excess of anti-tert-butyl(1-hydroxy-1-phenylhex-5-yn-2-yl)carbamate compounds (1b) and (1d).

7 . The engineered ketoreductase polypeptide of claim 6 , wherein the amino acid sequence of the ketoreductase polypeptide further comprises or more residue differences as compared to SEQ ID NO:4 selected from: X17Q; X64V; X71P; X87L; X96L/Y; X147I; X157C; X195A/G; X196M; X199H; and X249F.

8 . The engineered ketoreductase polypeptide of claim 7 , wherein said ketoreductase polypeptide is capable of forming product compounds (1a) and (1c) in a diastereomeric ratio of at least 50 over compounds (1b) and (1d).

9 . A polynucleotide encoding the engineered ketoreductase polypeptide of claim 1 .

10 . An expression vector comprising the polynucleotide of claim 9 .

11 . The expression vector of claim 10 , comprising at least one control sequence.

12 . The expression vector of claim 11 , in which the control sequence is a promoter.

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

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

15 . The host cell of claim 13 , wherein said host cell is E. coli.

16 . The host cell of claim 14 , wherein said host cell is E. coli.

17 . A method of preparing an engineered polypeptide comprising culturing the host cell of claim 13 , under conditions suitable for expression of the polypeptide, optionally further comprising isolating the engineered polypeptide.

18 . A method of preparing an engineered polypeptide comprising culturing the host cell of claim 14 , under conditions suitable for expression of the polypeptide, optionally further comprising isolating the engineered polypeptide.

19 . A method of preparing an engineered polypeptide comprising culturing the host cell of claim 15 , under conditions suitable for expression of the polypeptide, optionally further comprising isolating the engineered polypeptide.

20 . A method of preparing an engineered polypeptide comprising culturing the host cell of claim 16 , under conditions suitable for expression of the polypeptide, optionally further comprising isolating the engineered polypeptide.

Assignees

• Codexis Inc

Inventors

• Cabirol Fabien Louis
• Chen Haibin
• Gohel Anupam
• Collier Steven J
• Smith Derek J
• Kosjek Birgit
• Janey Jacob

Classifications

• C12N15/70

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

• Y02P20/52

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

• C12P13/02

  Amides, e.g. chloramphenicol {or polyamides; Imides or polyimides; Urethanes, i.e. compounds comprising N-C=O structural element or polyurethanes (peptides C12P21/00 or C07K)} · CPC title

• C12Y101/01184

  Carbonyl reductase (NADPH) (1.1.1.184) · CPC title

• C12N9/0006 Primary

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