Variant LovD polypeptides and their uses

Patent metadata

Field	Value
Publication number	US-9932616-B2
Application number	US-201615372788-A
Country	US
Kind code	B2
Filing date	Dec 8, 2016
Priority date	Sep 30, 2009
Publication date	Apr 3, 2018
Grant date	Apr 3, 2018

Abstract

The present disclosure provides acyltransferases useful for synthesizing therapeutically important statin compound.

First claim

What is claimed is:

1. An isolated recombinant variant LovD polypeptide having at least two-fold greater acyltransferase activity than the wild-type Aspergillus terreus acyltransferase of SEQ ID NO: 2, wherein said variant LovD polypeptide is at least 90% sequence identity to SEQ ID NO:2, wherein the amino acid sequence of said variant LovD polypeptide comprises the substitution S172N.

2. The isolated recombinant variant LovD polypeptide of claim 1 , wherein the amino acid sequence further comprises at least one additional substitution selected from 14N, A9V, K26E, R28S, 135L, N43R, D96R, S109C, A123P, M157V, S164G, L174L, A178L, N191G, L1921, Q241M, A247S, R250K, S256T, A261H, G275S, Q297G, L335M, L361M, V3701, A383V, N391S, and H404K, wherein the positions is numbered by correspondence with the amino acid sequence set forth in SEQ ID NO:2.

3. The isolated recombinant variant LovD polypeptide of claim 1 , wherein the amino acid sequence further comprises the substitutions 14N, A9V, K26E, R28S, 135L, N43R, D96R, S109C, A123P, M157V, S164G, L174F, A178L, N191G, L1921, Q241M, A247S, R250K, S256T, A261H, G275S, Q297G, L335M, L361M, V3701, A383V, N391S, and H404K, wherein the positions is numbered by correspondence with the amino acid sequence set forth in SEQ ID NO:2.

4. A method of making simvastatin comprising contacting monacolin J or lovastatin substrate with the isolated recombinant variant LovD polypeptide of claim 1 , in the presence of an α-dimethylbutyryl thioester co-substrate and under conditions in which simvastatin is produced.

5. The method of claim 4 , wherein the monacolin J is a sodium salt.

6. The method of claim 5 , wherein the monacolin J is a sodium salt and activated charcoal is added.

7. The method of claim 4 , wherein the monacolin J is an ammonium salt.

8. The method of claim 7 , wherein an agent for precipitating simvastatin is added.

9. The method of claim 8 , wherein said agent is ammonium hydroxide.

10. The method of claim 4 , wherein said lovastatin substrate, said variant LovD polypeptide and said thioester are added at substantially the same time into a vessel.

11. The method of claim 10 , wherein said lovastatin substrate and said variant LovD polypeptide are first added into a vessel and then said thioester is added into said vessel.

12. The method of claim 4 , wherein an agent for precipitating simvastatin is added.

13. The method of claim 12 , wherein said agent is ammonium hydroxide.

Assignees

• Codexis Inc

Inventors

• Gilson Lynne
• Collier Steven J
• Sukumaran Joly
• Yeo Wan Lin
• Alvizo Oscar
• Teo Ee Ling
• Wilson Robert J
• Xu Junye

Classifications

• 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

• C12N9/1029

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

• C07K14/00

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