Lov-D acyltransferase mediated acylation

Patent metadata

Field	Value
Publication number	US-9909156-B2
Application number	US-201615199095-A
Country	US
Kind code	B2
Filing date	Jun 30, 2016
Priority date	Sep 30, 2009
Publication date	Mar 6, 2018
Grant date	Mar 6, 2018

Abstract

Methods for the improved acylation of chemical substrates using LovD acyltransferases, thioesters having acyl groups, and (i) thiol scavengers and/or (ii) precipitating agents are presented. An improved method for the production of simvastatin using (i) activated charcoal as a thiol scavenger and/or (ii) ammonium hydroxide as a precipitating agent is also presented.

First claim

What is claimed is:

1. A method of making a statin compound, comprising contacting a LovD acyltransferase substrate comprising monacolin J, with a LovD acyltransferase, wherein said LovD acyltransferase is a recombinant variant comprising SEQ ID NO:2 comprising a substitution at position 157, relative to SEQ ID NO:2, in the presence of a thioester donor comprising alpha-dimethylbutyryl thioester, and an agent selected from a thiol scavenger, a precipitating agent and combinations thereof, under conditions which yield a statin compound.

2. The method of claim 1 , in which the monacolin J is a monacolin J hydroxy acid salt.

3. The method of claim 1 , in which the monacolin J is monacolin J lactone.

4. The method of claim 1 , in which the LovD acyltransferase substrate is 6-hydroxy-6-des-methyl-monacolin-J.

5. The method of claim 4 , in which the 6-hydroxyl-6-desmethyl-monacolin J is a 6-hydroxy-6-des-methyl monacolin J hydroxy acid salt.

6. The method of claim 4 , in which the 6-hydroxy-6-desmethyl monacolin J is 6-hydroxyl-6-des-methyl monacolin J lactone.

7. The method of claim 1 , in which the thioester donor is an alpha-dimethylbutyryl thioester.

8. The method of claim 7 , in which the alpha-dimethylbutyryl thioester is selected from the group consisting of α-dimethylbutyryl-S-methyl-mercaptopropionate (DMB-S-MMP), dimethylbutyryl-S-ethyl mercaptopropionate (DMB-S-EMP), dimethylbutyryl-S-methyl thioglycolate (DMB-S-MTG), dimethylbutyryl-S-methyl mercaptobutyrate (DMB-S-MMB), S-2-acetamidoethyl 2,2-dimethylbutanethioate, S-acetamidomethyl 2,2-dimethylbutanethioate and methyl 2-(2,2-dimethylbutanoylthio)acetate.

9. The method of claim 8 , in which the alpha-dimethylbutyryl thioester is DMB-S-MMP.

10. The method of claim 1 , which is carried out in an aqueous medium at a pH in the range of about pH 8 to pH 9.5.

11. The method of claim 1 , which is carried out in an aqueous buffer having an initial pH of about pH 9.

12. The method of claim 1 , in which the agent is a thiol scavenging agent.

13. The method of claim 12 , in which the thiol scavenging agent is activated charcoal.

14. The method of claim 1 , in which the agent is a precipitating agent.

15. The method of claim 14 , in which the precipitating agent is ammonium hydroxide.

16. The method of claim 1 , in which the LovD acyltransferase is a LovD variant comprising the amino acid sequence of SEQ ID NO: 16.

17. A method of making simvastatin hydroxy acid ammonium salt comprising contacting monacolin J or lovastatin with the LovD acyltransferase disclosed in the claim 1 in the presence of an alpha-dimethylbutyryl thioester donor and ammonium hydroxide under conditions which yield simvastatin hydroxy acid ammonium salt.

18. The method of claim 17 , which is carried out in aqueous solution at a pH in the range of about pH 8 to pH 9.5.

19. The method of claim 17 , which is carried out in an aqueous buffer having an initial pH of about pH 9.

Assignees

• Codexis Inc
• Codexis Inc

Inventors

• Collier Steven J
• Teo Ee Ling
• Sukumaran Joly
• Wilson Robert J
• Xu Junye

Classifications

• C12P7/62

  Carboxylic acid esters · CPC title

• C12N15/52

  Genes encoding for enzymes or proenzymes · CPC title

• C12P17/06 Primary

  containing a six-membered hetero ring, e.g. fluorescein · CPC title

• C12N9/1025

  Acyltransferases (2.3) · CPC title

• C12Y203/01

  transferring groups other than amino-acyl groups (2.3.1) · CPC title