Lov-D acyltransferase mediated acylation

Patent metadata

Field	Value
Publication number	US-10858680-B2
Application number	US-201916543817-A
Country	US
Kind code	B2
Filing date	Aug 19, 2019
Priority date	Sep 30, 2009
Publication date	Dec 8, 2020
Grant date	Dec 8, 2020

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 non-naturally occurring polynucleotide encoding an isolated or recombinant variant LovD polypeptide having at least two-fold greater acyltransferase activity than the wild-type Aspergillus terreus acyltransferase of SEQ ID NO:2, which comprises the amino acid sequence of SEQ ID NO:2 that includes the mutations L174F and A178L and from 1 to 30 additional mutations.

2. The non-naturally occurring polynucleotide encoding the variant LovD polypeptide of claim 1 , which has at least 10-fold greater acyltransferase activity than the wild-type A. terrus acyltransferase of SEQ ID NO:2.

3. The non-naturally occurring polynucleotide encoding the variant LovD polypeptide of claim 1 , that includes the following additional mutations: A123P, N191S/G, A247S and L361M.

4. The non-naturally occurring polynucleotide encoding the variant LovD polypeptide of claim 3 , further comprising one or more additional mutations selected from: A9V, K26E, M157V, L192I, R250K, G275S, Q297E/G, and A383V.

5. The non-naturally occurring polynucleotide encoding the variant LovD polypeptide of claim 4 , wherein the one or more additional mutations are selected from: A9V, K26E, and G275S.

6. The non-naturally occurring polynucleotide of claim 1 , wherein said polynucleotide comprises a nucleic acid sequence optimized for expression in E. coli.

7. An expression vector comprising the non-naturally occurring polynucleotide of claim 1 .

8. The expression vector of claim 7 , further comprising at least one control sequence.

9. The expression vector of claim 7 , wherein said vector comprises SEQ ID NO: 15.

10. A host cell comprising the non-naturally occurring polynucleotide of claim 1 .

11. A host cell comprising the expression vector of claim 7 .

12. The host cell of claim 10 , wherein the host cell is E. coli.

13. The host cell of claim 11 , wherein the host cell is E. coli.

Assignees

• Codexis Inc

Inventors

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

Classifications

• C12P17/06 Primary

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

• C07K14/00

  Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof · CPC title

• C12P7/62

  Carboxylic acid esters · CPC title

• C12Y203/01

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

• C12N9/1029

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