# US 10,870,835 B2 · Ketoreductase polypeptides for the preparation of phenylephrine

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- Number: US 10,870,835 B2
- Document ID: US10870835B2
- Status: Granted
- Kind: B2
- Application number: 16/778,368
- Filed: 2020-01-31
- Published: 2020-05-21
- Granted: 2020-12-22
- Earliest claimed: 2009-08-19
- Assignees: Codexis, Inc.
- Applicants: Codexis, Inc.
- Inventors: Oscar Alvizo; Steven J. Collier; Hans-Georg Joerg Hennemann; Seong Ho Oh; Wenjuan Zha
- Web page: https://patentgrep.com/patent/US10870835B2

## CPC

- C12N 9/0006: {acting on CH-OH groups as donors (1.1)}
- A61K 31/137: Arylalkylamines, e.g. amphetamine, epinephrine, salbutamol, ephedrine {or methadone}
- C12P 13/001: {Amines; Imines}
- C12Y 101/01184: Carbonyl reductase (NADPH) (1.1.1.184)
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- A61K: PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES (devices or methods specially adapted for bringing pharmaceutical products into particular physical or administering forms A61J3/00; chemical aspects of, or use of materials for deodorisation of air, for disinfection or sterilisation, or for bandages, dressings, absorbent pads or surgical articles A61L; soap compositions C11D)
- 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)}
- C12Y: ENZYMES

## Abstract

The disclosure relates to engineered ketoreductase polypeptides and processes of using the polypeptides for production of phenylephrine.

## Claims

### Claim 1

An engineered polypeptide capable of converting 1-(3-hydroxyphenyl)-2-(methylamino)ethanone to (R)-phenylephrine, wherein the amino acid sequence of the polypeptide has at least 95% identity to SEQ ID NO: 4 and comprises at least one residue difference selected from C190G, A202F, M206C, and Y249F.

### Claim 2 (depends on 1)

The engineered polypeptide of claim 1, wherein the at least one residue difference is M206C.

### Claim 3 (depends on 1)

The engineered polypeptide of claim 1, wherein the amino acid sequence comprises at least two further residue differences selected from V95M, S96L, L147I, C190G, A202F, and Y202F.

### Claim 4 (depends on 1)

The engineered polypeptide of claim 1, wherein the amino acid sequence comprises the residue differences: V95M, A202F, and M206C.

### Claim 5 (depends on 1)

The engineered polypeptide of claim 1, wherein the amino acid sequence comprises the residue differences: V95M, C190G, A202F, and M206C.

### Claim 6 (depends on 1)

The engineered polypeptide of claim 1, wherein the amino acid sequence comprises the residue differences: V95M, C190G, A202F, M206C, and Y249F.

### Claim 7 (depends on 1)

The engineered polypeptide of claim 1, wherein the amino acid sequence comprises the combination of residue differences of any one of SEQ ID NO: 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, or 34 as compared to SEQ ID NO: 2.

### Claim 8 (depends on 1)

The engineered polypeptide of claim 1, wherein the amino acid sequence further comprises at least one residue difference selected from T2S, I11L, A64V, T76I, V95M, S96L,V99L, V148L, T152A, L153M, S159T, D197A, and E200P.

### Claim 9 (depends on 1)

The engineered polypeptide of claim 1, wherein the polypeptide is capable of stereospecifically converting 1-(3-hydroxyphenyl)-2-(methylamino)ethanone to (R)-phenylephrine in a enantiomeric excess of at least 99%.

### Claim 10 (depends on 1)

The engineered polypeptide of claim 1, wherein the polypeptide comprises a sequence selected from the group consisting of SEQ ID NO: 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, and 34.

### Claim 11 (depends on 1)

An expression vector comprising a polynucleotide encoding the engineered polypeptide of claim 1.

### Claim 12 (depends on 11)

The expression vector of claim 11, operably linked to a control sequence suitable for directing expression in a host cell.

### Claim 13 (depends on 11)

A host cell comprising the expression vector of claim 11.

### Claim 14 (depends on 12)

A host cell comprising the expression vector of claim 12.

### Claim 15 (depends on 13)

A method for preparing an engineered polypeptide, comprising culturing a host cell of claim 13, and optionally isolating the polypeptide from the cell.

### Claim 16 (depends on 14)

A method for preparing an engineered polypeptide, comprising culturing a host cell of claim 14, and optionally isolating the polypeptide from the cell.