Methods and materials for making simvastatin and related compounds

Patent metadata

Field	Value
Publication number	US-9970037-B2
Application number	US-201514599100-A
Country	US
Kind code	B2
Filing date	Jan 16, 2015
Priority date	May 24, 2006
Publication date	May 15, 2018
Grant date	May 15, 2018

Abstract

The invention disclosed herein relates to methods and materials for producing simvastatin and related compounds such as huvastatin.

First claim

The invention claimed is:

1. A composition of matter comprising a functional LovD acyltransferase that comprises an amino acid sequence having from 3 to 29 amino acid substitution mutations in SEQ ID NO:1.

2. The composition of claim 1 , further comprising:
monacolin J; and
an acyl thioester that donates an acyl moiety to the C8 hydroxyl group of monacolin J in the presence of LovD acyltransferase.

3. The composition of claim 2 , wherein the monacolin J, the acyl thioester, and the functional LovD acyltransferase are combined in a fermentation media in the presence of an isolated organism.

4. The composition of claim 3 , wherein the isolated organism is at least one of:
(a) Aspergillus terreus that expresses LovD polypeptide of claim 1 ;
(b) Aspergillus terreus that does not express LovF polypeptide of SEQ ID NO: 3;
(c) Escherichia coli that expresses LovD polypeptide of claim 1 ; or
(d) Escherichia coli that does not express bioH polypeptide of SEQ ID NO: 4.

5. The composition of claim 2 , wherein the acyl thioester is able to cross the cellular membranes of Escherichia coli or Aspergillus terreus cells growing within a fermentation media.

6. The composition of claim 5 , wherein the acyl thioester is α-dimethylbutyryl-S-methyl-mercaptopropionate, and wherein the α-dimethylbutyryl-S-methyl-mercaptopropionate is present at a concentration range of 1 mM-100 mM.

7. The composition of claim 2 , further comprising simvastatin, wherein:
a) simvastatin is made in vitro in the absence of an isolated organism, or
b) simvastatin is made in vivo in a fermentation media in the presence of an isolated organism, and the acyl thioester is derived from an exogenous source.

8. The composition of claim 1 , wherein the functional LovD acyltransferase comprises at least 3 amino acid substitution mutations.

9. The composition of claim 1 , wherein the functional LovD acyltransferase comprises at least 4 amino acid substitution mutations.

10. The composition of claim 2 , wherein the acyl thioester is a butyrlyl-thioester, a N-acetylcysteamine thioester or a methyl-thioglycolate thioester.

11. The composition of claim 2 , wherein the acyl thioester comprises medium chain length (C3-C6) acyl group moieties.

12. The composition of claim 2 , wherein the acyl thioester is selected from the group consisting of α-dimethylbutyryl-S-methyl-mercaptopropionate (DMB-S-MMP), dimethylbutyryl-S-ethyl mercaptopropionate (DMB-S-EMP) and dimethylbutyryl-S-methyl thioglycolate (DMB-S-MTG) and dimethylbutyryl-S-methyl mercaptobutyrate (DMB-S-MMB).

13. The composition of claim 1 , further comprising monacolin J in the absence of an isolated organism.

14. The composition of claim 1 , further comprising simvastatin in the absence of an isolated organism.

15. A composition of matter comprising:
a functional LovD acyltransferase that comprises an amino acid sequence having at least 93% identity to SEQ ID NO:1; and
simvastatin, in the absence of an isolated organism.

16. A composition of matter comprising:
a functional LovD acyltransferase that comprises an amino acid sequence having at least 93% identity to SEQ ID NO:1;
monacolin J;
an acyl thioester that donates an acyl moiety to the C8 hydroxyl group of monacolin J in the presence of LovD acyltransferase; and
simvastatin; in the absence of an isolated organism.

17. The composition of claim 16 , wherein the acyl thioester comprises medium chain length (C3-C6) acyl group moieties.

18. The composition of claim 1 , wherein the functional LovD acyltransferase comprises at least 5 amino acid substitution mutations.

19. The composition of claim 1 , wherein the functional LovD acyltransferase comprises at least 6 amino acid substitution mutations.

20. The composition of claim 1 , wherein the functional LovD acyltransferase comprises at least 7 amino acid substitution mutations.

21. The composition of claim 1 , wherein the functional LovD acyltransferase comprises at least 8 amino acid substitution mutations.

22. The composition of claim 1 , wherein the functional LovD acyltransferase comprises at least 9 amino acid substitution mutations.

23. The composition of claim 1 , wherein the functional LovD acyltransferase comprises at least 10 amino acid substitution mutations.

Assignees

• Univ California

Inventors

• Tang Yi
• Xie Xinkai

Classifications

• A61P9/10

  for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis · CPC title

• C12P17/06 Primary

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

• C12P7/42

  Hydroxy-carboxylic acids · CPC title

• C12N9/1025

  Acyltransferases (2.3) · CPC title

• C12P7/62

  Carboxylic acid esters · CPC title