# US 2021/0277432 A1 · Variant lovd polypeptides and their uses

> Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt

- Number: US 2021/0277432 A1
- Document ID: US20210277432A1
- Status: Published application
- Kind: A1
- Application number: 17/330,172
- Filed: 2021-05-25
- Published: 2021-09-09
- Earliest claimed: 2009-09-30
- Assignees: Codexis, Inc.
- Applicants: Codexis, Inc.
- Inventors: Lynne Gilson; Steven J. Collier; Joly Sukumaran; Wan Lin Yeo; Oscar Alvizo; Ee Ling Teo; Robert J. Wilson; Junye Xu
- Web page: https://patentgrep.com/patent/US20210277432A1

## CPC

- C12P 17/06: containing a six-membered hetero ring, e.g. fluorescein
- C07K 14/00: Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C12N 9/1025: {Acyltransferases (2.3)}
- C12N 9/1029: {transferring groups other than amino-acyl groups (2.3.1)}
- C12N 15/52: Genes encoding for enzymes or proenzymes
- C12P 7/62: Carboxylic acid esters
- C12Y 203/01: transferring groups other than amino-acyl groups (2.3.1)
- Y02P 20/52: using catalysts, e.g. selective catalysts
- C12P: FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE {(brewing of beer C12C; producing vinegar C12J; producing specific peptides or proteins C07K; producing enzymes C12N9/00; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification C12N15/00; measuring or testing processes involving enzymes or microorganisms C12Q; measuring or testing processes involving nucleic acid amplification reactions C12Q1/6844; fermentation processes to form a food composition, A21 or A23; compounds in general, see the relevant compound class, e.g. C01, C07)}
- 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)
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- C12Y: ENZYMES
- Y02P: CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS

## Abstract

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

## Claims

### Claim 1

An engineered variant LovD polypeptide having at least 90% sequence identity to the wild-type Aspergillus terreus acyltransferase of SEQ ID NO:2

### Claim 2 (depends on 1)

The variant LovD polypeptide of claim 1, wherein the amino acid sequence of said polypeptide comprises a residue difference of A123P, as compared to the amino acid sequence of SEQ ID NO:2.

### Claim 3 (depends on 1)

The variant LovD polypeptide of claim 1, having at least two-fold greater catalytic activity than the wild-type A. terreus acyltransferase of SEQ ID NO:2.

### Claim 4 (depends on 1)

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

### Claim 5 (depends on 1)

The variant LovD polypeptide of claim 1, which comprises the amino acid sequence of SEQ ID NO:2 that includes one or more of the following characteristics:
(i) one or more mutations that correlate with an increase in catalytic activity;
(ii) one or more mutations that correlate with increased thermal stability;
(iii) one or more mutations that correlate with reduced aggregation;
(iv) one or more mutations that correlate with increased enzyme stability; and
(v) one or more mutations selected from the group consisting of 14N, 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.

### Claim 6 (depends on 1)

The variant LovD polypeptide of claim 1, which comprises the amino acid sequence of SEQ ID NO:2 that includes one or more of the following features:
(i) at least one mutation selected from the group consisting of M157V, S164G, S172N, L174F, A178L, N191G, L192I, A247S, R250K, S256T, A261H, G275S, Q297G, L361M, V370I and N391S;
(ii) at least one mutation selected from the group consisting of Q241M, A261H, Q295R and Q412R;
(iii) at least one mutation selected from the group consisting of N43R, D96R and H404K;
(iv) at least one mutation selected from the group consisting of C40R, C60R and D245E; and
(v) at least one mutation 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.

### Claim 7 (depends on 1)

The variant LovD polypeptide of claim 1 comprising an amino acid sequence selected from Table 1.

### Claim 8 (depends on 1)

A polynucleotide encoding a variant LovD polypeptide according to claim 1.

### Claim 9 (depends on 8)

The polynucleotide of claim 8 which is an expression vector suitable for expressing the variant LovD polypeptide in a host cell.

### Claim 10 (depends on 9)

The polynucleotide of claim 9 in which the encoding sequence is codon optimized for expression in the host cell.

### Claim 11 (depends on 1)

A host cell comprising a polynucleotide encoding a variant LovD polypeptide according to claim 1.

### Claim 12 (depends on 11)

A method of making a variant LovD polypeptide comprising culturing a host cell according to claim 11 under conditions in which the variant LovD polypeptide is expressed and optionally recovering the variant LovD polypeptide.

### Claim 13 (depends on 1)

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

### Claim 14 (depends on 13)

The method of claim 13, wherein the monacolin J is a sodium salt.

### Claim 15 (depends on 14)

The method of claim 14, wherein the monacolin J is a sodium salt and activated charcoal is added.

### Claim 16 (depends on 15)

The method of claim 15, wherein the monacolin J is an ammonium salt.

### Claim 17 (depends on 13)

The method of claim 13 wherein an agent for precipitating simvastatin is added.

### Claim 18 (depends on 17)

The method of claim 17, wherein said agent is ammonium hydroxide.