# US 11,999,976 B2 · Engineered ketoreductase polypeptides and uses thereof

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

- Number: US 11,999,976 B2
- Document ID: US11999976B2
- Status: Granted
- Kind: B2
- Application number: 17/290,916
- Filed: 2019-12-11
- Published: 2021-12-02
- Granted: 2024-06-04
- Earliest claimed: 2018-12-15
- Assignees: Enzymaster (Ningbo) Bio-Engineering Co., Ltd
- Applicants: Enzymaster (Ningbo) Bio-Engineering Co., Ltd
- Inventors: Haibin Chen; Chuanyang Shang; Baoqin Cai; Marco Bocola; Shumin Shi; Ruimei Hong; Lei Sun; Yong Koy Bong
- Web page: https://patentgrep.com/patent/US11999976B2

## CPC

- C12N 9/0006: {acting on CH-OH groups as donors (1.1)}
- C12N 11/00: Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof
- C12N 11/02: Enzymes or microbial cells immobilised on or in an organic carrier
- C12P 7/18: polyhydric
- C12P 7/22: aromatic
- C12P 7/62: Carboxylic acid esters
- C12P 41/002: {by oxidation/reduction reactions}
- C12Y 101/01184: Carbonyl reductase (NADPH) (1.1.1.184)
- Y02P 20/582: Recycling of unreacted starting or intermediate materials
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- 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
- Y02P: CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS

## Abstract

Provided are amino acid sequences of ketoreductase polypeptides that are useful for asymmetrically synthesizing chiral alcohol compounds, its preparation process as well as reaction process under industrial-relevant conditions. Also provided are polynucleotide sequences encoding engineered ketoreductase polypeptides, engineered host cells capable of expressing engineered ketoreductase polypeptides, and methods of producing chiral alcohol compounds using engineered ketoreductase polypeptides. Compared to other enzymes, the engineered ketoreductase polypeptides provided by the invention have better catalytic activity and thermal stability, allowing purification of the enzyme solution by heat treatment, which is advantageous for the production of enzymes and the industrial application of enzymatic reactions. The use of the engineered polypeptides of the present invention greatly simplifies the production process of chiral alcohol compounds, reduces the cost of production and the impact on the environment, and has good industrial application prospects.

## Claims

### Claim 1

An engineered ketoreductase polypeptide which is, under suitable reaction conditions, capable of catalyzing the conversion of 4-hydroxy-2-butanone to (R) -(-)-1,3-butanediol, wherein the amino acid sequence of said polypeptide comprises one or more of substitutions selected from the group consisting of substitutions corresponding to substitutions X46T, X185C, and X228A in the polypeptide of SEQ ID NO 2, wherein the numbering refers to SEQ ID NO:2, wherein said amino acid sequence is selected from the group consisting of SEQ ID NO: 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 66, 68, 70, 72, 74, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166, 168, 170, 172, 174, 176, 178, 180, 182, 184, 186, 188, 190, 192, 194, 196, 198, 200, 202, 204, 206, 208, 210, 212, 214, 216, 218, 220, 222, 224, 226, 228, 230, 232, 234, 236, 238, 240, 242, 244, 246, 248, 250, 252, 254, 256, 258, 260, 262, 264, 266, 268, 270, 272, 274, 276, 278, 280, 282, 284, 286, 288, 290, 292, 294, 296, 298, 300, 302, 304, 306, 308, 310, 312, 314, 316, 318, 320, 322, 324, 326, 328, 330, and 332.

### Claim 2 (depends on 1)

The engineered ketoreductase polypeptide of claim 1, wherein said suitable reaction conditions include the provision of about 5 g/L to 400 g/L of 4-hydroxy-2-butanone, about 0.01 g/L to 0.2 g/L of NADH, and about 10%-50% (v/v) of isopropanol, at a temperature of about 10-60° C.

### Claim 3

An engineered polypeptide, wherein said polypeptide is:
(a) a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 66, 68, 70, 72, 74, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166, 168, 170, 172, 174, 176, 178, 180, 182, 184, 186, 188, 190, 192, 194, 196, 198, 200, 202, 204, 206, 208, 210, 212, 214, 216, 218, 220, 222, 224, 226, 228, 230, 232, 234, 236, 238, 240, 242, 244, 246, 248, 250, 252, 254, 256, 258, 260, 262, 264, 266, 268, 270, 272, 274, 276, 278, 280, 282, 284, 286, 288, 290, 292, 294, 296, 298, 300, 302, 304, 306, 308, 310, 312, 314, 316, 318, 320, 322, 324, 326, 328, 330, and 332; or
(b) a polypeptide having ketoreductase activity, which comprises an amino acid sequence having (i) at least 95% sequence identity to one of the amino acid sequences recited in (a), and (ii) one or more substitutions selected from the group consisting of substitutions corresponding to substitutions X46T, X185C and X228A in the polypeptide of SEQ ID NO: 2.

### Claim 4 (depends on 3)

The engineered ketoreductase polypeptide of claim 3, wherein said polypeptide is capable of catalyzing the conversion of 4-hydroxy-2-butanone to (R) -(-)-1,3-butanediol under suitable reaction conditions with greater catalytic activity and/or stability than the polypeptide of SEQ ID NO: 2, and producing (R)-(-)-1,3-butanediol in an enantiomeric excess of at least 90%.

### Claim 5 (depends on 1)

A polypeptide immobilized on a solid material by a chemical bond or a physical adsorption method, wherein the polypeptide is the engineered ketoreductase polypeptide according to claim 1.

### Claim 6 (depends on 1)

A ketoreductase catalyst obtained by culturing a host cell comprising an expression vector comprising a polynucleotide encoding the engineered ketoreductase polypeptide of claim 1 , wherein said ketoreductase catalyst comprises (a) cells or culture fluid obtained from said host cell culture and that contain said engineered ketoreductase polypeptide, (b) an extract that contains said engineered ketoreductase polypeptide and is obtained from said host cell culture, (c) an isolated product that contains said engineered ketoreductase polypeptide and is obtained by isolating or purifying said engineered ketoreductase from said extract, or (d) an immobilization product that contains said engineered ketoreductase polypeptide and is obtained by immobilizing said host cell, an extract of said host cell, or isolated product of the host cell extract.

### Claim 7 (depends on 1)

A process for converting a carbonyl substrate of formula (II)
to a chiral alcohol compound of formula (I)
using the engineered ketoreductase polypeptide of claim 1, wherein said chiral alcohol compound of formula (I) has the indicated stereochemical configuration shown at the chiral center marked with an * and is in an enantiomeric excess over its other isomer, further wherein:
R1 is an optionally substituted or unsubstituted aryl or heteroaryl, an optionally substituted or unsubstituted C1-C8 hydrocarbyl, a cycloalkyl or a heterocyclic group; and
R2 is (a) an optionally substituted or unsubstituted C1-C6 hydrocarbyl, (b) a halogen selected from —F, —Cl, —Br, and —I, (c) an alkenyl, an alkynyl, an aryl, or a heteroaryl, or (d) —NO2, —NO, —SO2R′, —SOR′, —SR′, —NR′R′, —OR′, —CO2R′, —COR′, —C(O)NR′, —SO2NH2, —SONH2, —CN, or —CF3, wherein each R′ is independently selected from hydrogen, a C1-C4 hydrocarbyl, a halogen, a C1-C8 hydrocarbyl, a C2-C12 alkenyl, a C2-C12 alkynyl, a cycloalkyl, an aryl, and a heterocyclic substituent;
further wherein the process comprises the step of contacting under suitable reaction conditions said carbonyl substrate of formula (II) with the engineered ketoreductase polypeptide of claim 1.

### Claim 8 (depends on 7)

The process of claim 7, wherein the chiral alcohol compound of formula (I) is
further wherein,
(i) R2 is as defined in claim 7; and
(ii) R3 and R4 are each (a) hydrogen, (b) an optionally substituted or non-substituted C1-C4 hydrocarbyl, (c) a halogen selected from —F, —Cl, —Br, and —I, (d) an aryl or a heteroaryl, or (e) —NO2, —NO, —SO2R′, —SOR′, —SR′, —NR′R′, —OR′, —CO2R′, —COR′, —C(O)NR′, —SO2NH2, —SONH2, —CN, or —CF3, wherein each R′ is independently selected from hydrogen, a C1-C4 hydrocarbyl, a cycloalkyl, an aryl, and a heterocyclic substituent; and
(iii) the carbonyl substrate of formula (II) is:

### Claim 9 (depends on 8)

The process of claim 8, wherein R3 is (a) in the para position of the phenyl ring, (b) in the meta position of the phenyl ring, or (c) in the ortho position of the phenyl ring.

### Claim 10 (depends on 7)

The process of claim 7, wherein the compound of formula (I) is:

### Claim 11 (depends on 1)

A process of preparing an (R)-(-)-1,3-butanediol of formula A2:
wherein the process comprises the step of converting, under suitable reaction conditions, a 4-hydroxy-2-butanone compound of formula A1 to (R)-(-)-1,3-butanediol,
by reacting said 4-hydroxy-2-butanone compound of formula A1 with the engineered ketoreductase polypeptide of claim 1.

### Claim 12 (depends on 7)

The process of claim 7, wherein the chiral alcohol compound is present in an enantiomeric excess of at least 90%.

### Claim 13 (depends on 7)

The process of claim 7, wherein said engineered ketoreductase polypeptide converts said carbonyl substrate to said chiral alcohol in the presence of a co-factor NADH is regenerated during the conversion by (a) a process of converting isopropanol to acetone by a ketoreductase; (b) a process of converting glucose to gluconic acid by glucose dehydrogenase; or (c) by converting formic acid to carbon dioxide by formate dehydrogenase.

### Claim 14 (depends on 7)

The process of claim 7, wherein the process requires a reaction solvent selected from water, methanol, ethanol, propanol, isopropanol, isopropyl acetate, dimethyl sulfoxide (DMSO) and dimethylformamide (DMF).

### Claim 15 (depends on 7)

The process of claim 7, wherein the reaction conditions include a temperature of 10° C.to 60° C.

### Claim 16 (depends on 7)

The process of claim 7, wherein the reaction conditions include a pH 4.0 to pH 11.0.

### Claim 17 (depends on 7)

The process of claim 7, wherein the carbonyl substrate is present at a loading of 5 g/L to 400 g/L.

### Claim 18 (depends on 1)

A process for the resolution of racemic 1,3-butanediol, said process comprising the step of contacting under suitable reaction conditions a racemic 1,3-butanediol with an the engineered polypeptide of claim 1 .

### Claim 19 (depends on 18)

The process of claim 18, wherein the reaction conditions include a loading of about 5 g/L to 400 g/L of racemic 1,3-butanediol, about 0.01 g/L to 0.2 g/L of NAD+, and about 10%-50% v/v of acetone, at a temperature of about 10-60° C.