Designer cells for enantioselective reduction of ketones and use thereof in efficient production of enantioenriched alcohols
AssigneeCouncil of Scientific and Industrial Research
InventorsGautam Srivastava, Suneet Kaur, Ravinder Singh Jolly
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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.
Claims17
A designer cell that expresses a non-naturally occurring carbonyl reductase polypeptide of sequence SEQ ID NO:
- 1 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.
A designer cell that simultaneously expresses a non-naturally occurring CRS polypeptide of sequence SEQ ID NO:
- 1 and a non-naturally occurring GDH polypeptide of sequence SEQ ID NO:
- 9 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.
A process for production of optically enriched aryl alcohols comprising steps of:
- a. providing a ketone having formula 1, formula 3 or formula 4 b. contacting said ketone with the designer cell of claim 1 and 0.005 to 0.02 mol % nicotinamide adenine dinucleotide phosphate, reduced (NADPH) and 100 to 500 units glucose dehydrogenase and a buffer solution of pH 5.0 to 9.0 to form a reaction mixture;
- c. adding to the reaction mixture obtained in step (b) an organic solvent such as ethyl acetate, butyl acetate, diethylether, methyl n-butyl ether or di-n-butyl ether in the ratio ranging between 10:1 to 1:1;
- d. energetically mixing the reaction mixture on magnetic stirrer at constant temperature of 20 to 40° C.;
- e. extracting the product obtained in step (d) in ethyl acetate followed by isolating the product having formula 2, formula 5 or formula 6 respectively wherein Formula 2 is in about 100% enantiomeric excess,
- 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;
- or
- 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.
Dependencies are inferred from the claim text.