Variant LovD polypeptides and their uses

Patent metadata

Field	Value
Publication number	US-11046982-B2
Application number	US-202016784011-A
Country	US
Kind code	B2
Filing date	Feb 6, 2020
Priority date	Sep 30, 2009
Publication date	Jun 29, 2021
Grant date	Jun 29, 2021

Abstract

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

First claim

What is claimed is:

1. An engineered variant LovD polypeptide having at least two-fold greater catalytic activity than the wild-type Aspergillus terreus acyltransferase of the amino acid sequence of SEQ ID NO: 2, wherein said engineered variant LovD polypeptide has at least 90% sequence identity to the wild-type A. terreus acyltransferase of the amino acid sequence of SEQ ID NO: 2, and wherein said engineered variant LovD polypeptide comprises the substitution G275S.

2. The engineered variant LovD polypeptide of claim 1 , wherein said engineered variant LovD polypeptide has at least 10-fold greater catalytic activity than the wild-type A. terreus acyltransferase of the amino acid sequence of SEQ ID NO: 2.

3. The engineered variant LovD polypeptide of claim 1 , wherein said engineered variant LovD polypeptide further comprises one or more substitutions selected from the group consisting of I4N, A9V, K26E, R28K, R28S, I35L, C40A, C40V, C40F, S41R, N43Y, C60F, C60Y, C60N, C60H, S109C, S142N, A184T, A184V, N191S, A261T, A261E, A261V, L292R, Q297E, L335M, A377V, A383V, N391D, and H404R.

4. The engineered variant LovD polypeptide of claim 1 , further comprising one or more of the following features:
(i) at least one substitution selected from the group consisting of A123P, M157V, S164G, S172N, L174F, A178L, N191G, L192I, A247S, R250K, S256T, A261H, Q297G, L361M, V370I and N391S;
(ii) at least one substitution selected from the group consisting of Q124M, A261H, Q295R AND 0412R;
(iii) at least one substitution selected from the group consisting of N43R, D96R and H404K;
(iv) at least one substitution selected from the group consisting of C40R, C60R AND D245E; and
(v) at least one substitution selected from the group consisting of I4N, A9V, K26E, R28K, R28S, I35L, C40A, C40V, C40F, S41R, N43Y, C60F, C60Y, C60N, C60H, S109C, S142N, A184T, A184V, N191S, A261T, A261E, A261V, L292R, Q297E, L335M, A377V, A383V, N391D and H404R.

5. A method of making an engineered variant LovD polypeptide comprising culturing a host cell under conditions in which the engineered variant LovD polypeptide of claim 1 is expressed and optionally recovering the said engineered variant LovD polypeptide.

6. A method of making simvastatin comprising contacting a monacolin J substrate with the engineered variant LovD polypeptide of claim 1 in the presence of an a-dimethylbutyryl thioester co-substrate and under conditions in which the monacolin J is converted to simvastatin.

7. The method of claim 6 , wherein said monacolin J is a sodium salt.

8. The method of claim 7 , wherein said monacolin J is a sodium salt and activated charcoal is added.

9. The method of claim 8 , wherein said monacolin J is an ammonium salt.

10. The method of claim 6 , wherein an agent for precipitating simvastatin is added.

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

• C07K14/00

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

• Y02P20/52

  using catalysts, e.g. selective catalysts · 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

• C12N9/1025

  Acyltransferases (2.3) · CPC title