Designer cells for enantioselective reduction of ketones and use thereof in efficient production of enantioenriched alcohols

Patent metadata

Field	Value
Publication number	US-2016289713-A1
Application number	US-201414785286-A
Country	US
Kind code	A1
Filing date	Apr 17, 2014
Priority date	Apr 17, 2013
Publication date	Oct 6, 2016
Grant date	—

Abstract

The present invention is to provide a preparation of variant recombinant whole cell biocatalysts, referred herein as “designer cells” having significantly enhanced carbonyl reductase activity for use in the efficient production of variant industrially important enantiomerically enriched alcohols. More specifically, the alcohol is optically pure ethyl (S)-4-chloro-3-hydroxybutyrate, which is useful as chiral building block and an intermediate for the production of hydroxymethylglutaryl-CoA (HMG-CoA) reductase inhibitors.

First claim

1 . A designer cell that expresses a non-naturally occurring carbonyl reductase polypeptide of sequence selected from SEQ ID NO: 1, 3, 5 or 7 of the Sequence Listing on the surface of cell having 250-fold to 300-fold higher activity per unit mass of CRS polypeptide compared to the designer cell that expresses corresponding carbonyl reductase of SEQ ID NO: 13 or 15 or 17 or 19 of the sequence listing in cytoplasm of cell for conversion of ethyl 4-chloro-3-oxobutyrate of formula 1 to ethyl (S)-4-chloro-3-hydroxybutyrate of formula 2.

2 . (canceled)

3 . A designer cell as claimed in claim 1 that expresses a non-naturally occurring carbonyl reductase polypeptide of sequence selected from SEQ ID NO: 1, 3, 5 or 7 of the Sequence Listing on the surface of cell having 15-fold to 26-fold higher activity per unit cell mass compared to the designer cell that expresses corresponding carbonyl reductase of SEQ ID NO: 13 or 15 or 17 or 19 of the Sequence Listing in cytoplasm of cell for conversion of ethyl 4-chloro-3-oxobutyrate of formula 1 to ethyl (S)-4-chloro-3-hydroxybutyrate of formula 2.

4 . (canceled)

5 . A designer cell as claimed in claim 1 that expresses a non-naturally occurring carbonyl reductase polypeptide of SEQ ID NO: 1 of the Sequence Listing on the surface of cell having 50-fold to 275-fold higher activity per unit mass of CRS polypeptide compared to the designer cell that expresses corresponding carbonyl reductase of SEQ ID NO: 13 of the Sequence Listing in cytoplasm of cell for reduction of compound of formula 3

wherein R1=CH3, CH2X, (CH3)2CH, CF3 or CH3(CH2)n
R2=H, X or CH3(CH2)n;
R3=alkyl group such as CH3 or CH3(CH2)m;
X=Cl or Br;
n=1-4 and
m=1-8[M].

6 . A designer cell as claimed in claim 1 that expresses a non-naturally occurring carbonyl reductase polypeptide of SEQ ID NO: 1 of the Sequence Listing on the surface of cell having 50-fold to 180-fold higher activity per unit mass of CRS polypeptide compared to the designer cell that expresses corresponding carbonyl reductase of SEQ ID NO: 13 of the Sequence Listing in cytoplasm of cell for reduction of compound of formula 4

wherein R1=R2=R3=R4=R5=H, CH3, F, Cl, Br, I, CF3, NO2 or OCH3;
R6=alkyl group such as CH3 or CH3(CH2)n;
n=1 to 5.

7 . A designer cell as claimed in claim 1 that expresses a non-naturally occurring carbonyl reductase polypeptide of SEQ ID NO: 1 of the Sequence Listing on the surface of cell having about 3-fold to 15-fold higher activity per unit cell mass compared to the designer cell that expresses corresponding carbonyl reductase of SEQ ID NO: 13 in cytoplasm of cell for reduction of compound of formula 3 or compound of formula 4

wherein R1=CH3, CH2X, (CH3)2CH, CF3 or CH3(CH2)n
R2=H, X or CH3(CH2)n;
R3=alkyl group such as CH3 or CH3(CH2)m;
X=Cl or Br;
n=1-4 and m=1-8;

wherein R1=R2=R3=R4=R5=H, CH3, F, Cl, Br, I, CF3, NO2 or OCH3;
R6=alkyl group such as CH3 or CH3(CH2)n;
n=1 to 5.

8 . A designer cell that simultaneously expresses a non-naturally occurring CRS polypeptide of sequence selected from SEQ ID NO: 1, 3, 5 or 7 and a non-naturally occurring GDH polypeptide of sequence selected from SEQ ID NO: 9 or 11 of the Sequence Listing on the surface of cell that has 250-fold to 300-fold higher activity for conversion of ethyl 4-chloro-3-oxobutyrate of formula 1 to ethyl (S)-4-chloro-3-hydroxybutyrate of formula 2 per unit mass of CRS polypeptide and 200-fold to 250-fold enhanced activity for oxidation of glucose to gluconate per unit mass of GDH polypeptide compared to the designer cell that simultaneously expresses corresponding CRS of SEQ ID NO: 13 or 15 or 17 or 19 of the Sequence Listing and corresponding GDH of SEQ ID NO: 21 or 23 of the Sequence Listing in cytoplasm of cell.

9 . (canceled)

10 . A designer cell as claimed in claim 8 that simultaneously expresses a non-naturally occurring CRS polypeptide of sequence selected from SEQ ID NO: 1, 3, 5 or 7 and a non-naturally occurring GDH polypeptide of sequence selected from SEQ ID NO: 9, 11, 13 or 15 of the Sequence Listing on the surface of cell that has about 11-fold to 24-fold higher activity for conversion of ethyl 4-chloro-3-oxobutyrate of formula 1 to ethyl (S)-4-chloro-3-hydroxybutyrate of formula 2 per unit cell mass and 9-fold to 31-fold enhanced activity for oxidation of glucose to gluconate per unit cell mass compared to the designer cell that simultaneously expresses corresponding CRS of SEQ ID NO: 13 or 15 or 17 or 19 of the Sequence Listing and corresponding GDH of SEQ ID NO: 21 or 23 of the Sequence Listing in cytoplasm of cell.

11 . (canceled)

12 . A designer cell as claimed in claim 8 that simultaneously expresses a non-naturally occurring CRS polypeptide of SEQ ID NO: 1 and a non-naturally occurring GDH polypeptide of SEQ ID NO: 9 of the Sequence Listing on the surface of cell having about 55-fold to 270-fold higher activity per unit mass of CRS polypeptide compared to the designer cell that simultaneously expresses corresponding CRS of SEQ ID NO: 13 of the Sequence Listing and corresponding GDH of SEQ ID NO: 21 of the Sequence Listing in cytoplasm of cell for reduction of compound of formula 3

wherein R1=CH3, CH2X, (CH3)2CH, CF3 or CH3(CH2)n
R2=H, X or CH3(CH2)n;
R3=alkyl group such as CH3 or CH3(CH2)m;
X=Cl or Br;
n=1-4 and
m=1-8.

13 . A designer cell as claimed in claim 8 that simultaneously expresses a non-naturally occurring CRS polypeptide of SEQ ID NO: 1 and a non-naturally occurring GDH polypeptide of SEQ ID NO: 9 of the Sequence Listing on the surface of cell having about 40-fold to 156-fold higher activity per unit mass of CRS polypeptide compared to the designer cell that simultaneously expresses corresponding CRS of SEQ ID NO: 13 of the Sequence Listing and corresponding GDH of SEQ ID NO: 21 of the Sequence Listing in cytoplasm of cell for reduction of compound of formula 4

wherein R1=R2=R3=R4=R5=H, CH3, F, Cl, Br, I, CF3, NO2 or OCH3;
R6=alkyl group such as CH3 or CH3(CH2)n;
n=1 to 5.

14 . A designer cell as claimed in claim 8 that simultaneously expresses a non-naturally occurring CRS polypeptide of SEQ ID NO: 1 and a non-naturally occurring GDH polypeptide of SEQ ID NO: 9 of the Sequence Listing on the surface of cell having about 3 fold to 24-fold higher activity per unit cell mass compared to the designer cell that simultaneously expresses corresponding CRS of SEQ ID NO: 13 of the Sequence Listing and corresponding GDH of SEQ ID NO: 21 of the Sequence Listing in cytoplasm of cell for reduction of compound of compound of formula 3 or compound of formula 4

Assignees

• Council Scient Ind Res

Inventors

• Srivastava Gautam
• Kaur Suneet
• Jolly Ravinder Singh

Classifications

• C12P13/008

  containing a N-O bond, e.g. nitro (-NO2), nitroso (-NO) · CPC title

• C12P41/002

  by oxidation/reduction reactions · CPC title

• C12P7/62 Primary

  Carboxylic acid esters · CPC title

• C12P7/22

  aromatic · CPC title

• C12Y101/01184

  Carbonyl reductase (NADPH) (1.1.1.184) · CPC title