# US 10,246,689 B2 · Variant LovD polypeptide

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- Number: US 10,246,689 B2
- Document ID: US10246689B2
- Status: Granted
- Kind: B2
- Application number: 15/296,844
- Filed: 2016-10-18
- Published: 2017-02-02
- Granted: 2019-04-02
- Earliest claimed: 2009-10-08
- Assignees: The Regents of the University of California
- Applicants: The Regents of the University of California
- Inventors: Yi Tang; Xue Gao; Xinkai Xie
- Web page: https://patentgrep.com/patent/US10246689B2

## CPC

- C12N 9/1029: {transferring groups other than amino-acyl groups (2.3.1)}
- C07K 2319/00: Fusion polypeptide
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- C07K: PEPTIDES (peptides in foodstuffs A23; obtaining protein compositions for foodstuffs, working-up proteins for foodstuffs A23J; preparations for medicinal purposes A61K; peptides containing beta-lactam rings C07D; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, C07D; ergot alkaloids of the cyclic peptide type C07D519/02; macromolecular compounds having statistically distributed amino acid units in their molecules, i.e. when the preparation does not provide for a specific; but for a random sequence of the amino acid units, homopolyamides and block copolyamides derived from amino acids C08G69/00; macromolecular products derived from proteins C08H1/00; preparation of glue or gelatine C09H; single cell proteins, enzymes C12N; genetic engineering processes for obtaining peptides C12N15/00; compositions for measuring or testing processes involving enzymes C12Q; investigation or analysis of biological material G01N33/00)

## Abstract

The invention disclosed herein relates to methods and materials for producing simvastatin and related compounds such as huvastatin. In particular, the disclosure teaches that variants of the LovD acyltransferase polypeptide can be engineered to exhibit properties that facilitate their use in the production of simvastatin and/or huvastatin. The materials and processes disclosed herein are designed so that fermentation facilities lovastatin being converted to simvastatin and related compounds with minimal modifications.

## Claims

### Claim 1

A variant of a LovD polypeptide, wherein:
the variant LovD polypeptide comprises a functional LovD acyltransferase with an amino acid sequence having at least 90% identity to SEQ ID NO:1; and
the variant LovD polypeptide comprises an amino acid substitution at amino acid residue position D12 and at least one further amino acid substitution selected from the group consisting of K26E, G275S, and L361M.

### Claim 2 (depends on 1)

The variant LovD polypeptide of claim 1, wherein the variant LovD polypeptide comprises a K26E amino acid substitution.

### Claim 3 (depends on 1)

The variant LovD polypeptide of claim 1, wherein the variant LovD polypeptide comprises a G275S amino acid substitution.

### Claim 4 (depends on 1)

The variant LovD polypeptide of claim 1, wherein the variant LovD polypeptide comprises a L361M amino acid substitution.

### Claim 5 (depends on 1)

The variant LovD polypeptide of claim 1, wherein the variant LovD polypeptide further comprises at least one amino acid substitution at an amino acid residue position selected from the group consisting of: A10; C40; C60; A86; H161; A190; K227; and V334.

### Claim 6 (depends on 1)

The variant LovD polypeptide of claim 1, wherein the variant exhibits:
a decreased aggregation;
an improved catalytic activity;
an improved kcat/Km value;
an improved soluble expression level; or
an improved whole cell activity at 25° C.;
as compared to the LovD polypeptide shown in SEQ ID NO:1.

### Claim 7 (depends on 5)

The variant LovD polypeptide of claim 5, wherein the variant LovD polypeptide further comprises amino acid substitutions at:
amino acid residue positions D12G, and G275S;
amino acid residue positions D12G, A190, and G275S;
amino acid residue positions D12G, K26E, H161, A190, and G275S;
amino acid residue positions A10, D12, K26E, A190, and G275S;
amino acid residue positions D12G, K26E, H161, A190, and G275S;
amino acid residue positions D12G, K26E, H161, A190, G275S, V334, and L361 M; or
amino acid residue positions D12G, K26E, H161, A190, G275S, and V334 of SEQ ID NO:1.

### Claim 8 (depends on 5)

The variant LovD polypeptide of claim 5, wherein the variant LovD polypeptide further comprises amino acid substitutions at:
D12G, C40A, C60N, A86V, A190T, and G275S;
D12G, K26E, C40A, C60N, A86V, H161Y, A190T, and G275S; or
D12G, K26E, C40A, C60N, A86V, H161Y, A190T, G275S, and V334F of SEQ ID NO:1.

### Claim 9

A variant of a LovD polypeptide, wherein:
the variant LovD polypeptide comprises a functional LovD acyltransferase with an amino acid sequence having at least 90% identity to SEQ ID NO:1; and
the variant LovD polypeptide comprises an amino acid substitution at amino acid residue position D12.