IP Library › Granted Patent US 12,509,667
Granted Patent B2
US 12,509,667 · App. 18/267,326 · Granted Dec 30, 2025

Cytochrome P450 enzyme mutant and application thereof

Inventors: Hao Hong (Morrisville, NC); Gage James (Morrisville, NC); Na Zhang (Tianjin, CN); Xuecheng Jiao (Tianjin, CN); Yulei Ma (Tianjin, CN); Shan Cao (Tianjin, CN); Yibing Cheng (Tianjin, CN); Kaihua Sun (Tianjin, CN)
Assignee: ASYMCHEM LIFE SCIENCE (TIANJIN) CO., LTD.
C12N9/0081C12N15/70C12P7/02C12P7/24
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Quick Facts
Patent No.
US 12,509,667
App. No.
18/267,326
Granted
Dec 30, 2025
Kind
B2
Abstract

Provided are a cytochrome P450 enzyme mutant and an application thereof. The enzyme activity and anti-Markov oxidation selectivity of P450 derived from a wild-type strain of Bacillus megaterium undergo protein modification by means of directed evolution, thus improving enzyme activity and selectivity, and developing a series of P450 enzyme mutants that may be used for industrial production.

Claims (143)

1 . A cytochrome P450 enzyme mutant, wherein the cytochrome P450 enzyme obtained from Bacillus megaterium;

wherein the cytochrome P450 enzyme mutant has the amino acid sequence of SEQ ID NO: 1 with a mutation of one or more amino acids:

V79A + F332A

V79A + F332A + A75V

V79A + F332A + A75F

V79A + F332A + L76A

V79A + F332A + A83F

V79A + F332A + A83V

V79A + F332A + F88A

V79A + F332A + F88V

V79A + F332A + T89A

V79A + F332A + T89V

V79A + F332A + A265V

V79A + F332A + A265F

V79A + F332A + T269V

V79A + F332A + T269A

V79A + F332A + T269F

V79A + F332A + A329V

V79A + F332A + A329F

V79A + F332A + A331F

V79A + F332A + A331V

V79A + F332A + L438A

V79A + F332A + L438F

V79A + F332A + C63Y

V79A + F332A + C63Y + S107C

V79A + F332A + C63Y + S107C + Q129L

V79A + F332A + C63Y + S107C + Q129L + E229K

V79A + F332A + C63Y + S107C + Q129L + E229K + Q388L

V79A + F332A + C63Y + S107C + Q129L + E229K + Q388L + T428I

V79A + F332A + C63Y + S107C + Q129L + E229K + Q388L + T428I +

N320I

V79A + F332A + C63Y + S107C + Q129L + E229K + Q388L + T428I +

S165G

V79A + F332A + C63Y + S107C + Q129L + E229K + Q388L + T428I +

F78L

V79A + F332A + C63Y + S107C + Q129L + E229K + Q388L + T428I +

F78L + I175S

V79A + F332A + C63Y + S107C + Q129L + E229K + Q388L + T428I +

F78L + I175S + A331P

V79A + F332A + C63Y + S107C + Q129L + E229K + Q388L + T428I +

F78L + I175S + A331G

V79A + F332A + C63Y + S107C + Q129L + E229K + Q388L + T428I +

F78L + I175S + A331D

V79A + F332A + C63Y + S107C + Q129L + E229K + Q388L + T428I +

F78L + I175S + A329K

V79A + F332A + C63Y + S107C + Q129L + E229K + Q388L + T428I +

F78L + I175S + A329L

V79A + F332A + C63Y + S107C + Q129L + E229K + Q388L + T428I +

F78L + I175S + A75R

V79A + F332A + C63Y + S107C + Q129L + E229K + Q388L + T428I +

F78L + I175S + A329Y + A331P

V79A + F332A + C63Y + S107C + Q129L + E229K + Q388L + T428I +

F78L + I175S + A329L + A331P

V79A + F332A + C63Y + S107C + Q129L + E229K + Q388L + T428I +

F78L + I175S + A329K + A331F

V79A + F332A + C63Y + S107C + Q129L + E229K + Q388L + T428I +

F78L + I175S + A329K + A331R

V79A + F332A + C63Y + S107C + Q129L + E229K + Q388L + T428I +

F78L + I175S + A329K + A331D

V79A + F332A + C63Y + S107C + Q129L + E229K + Q388L + T428I +

F78L + I175S + A329K + R48H

V79A + F332A + C63Y + S107C + Q129L + E229K + Q388L + T428I +

F78L + I175S + A329K + Y52G

V79A + F332A + C63Y + S107C + Q129L + E229K + Q388L + T428I +

F78L + I175S + A329K + Q74L

V79A + F332A + C63Y + S107C + Q129L + E229K + Q388L + T428I +

F78L + I175S + A329K + T269P

V79A + F332A + C63Y + S107C + Q129L + E229K + Q388L + T428I +

F78L + I175S + A329K + S333H

V79A + F332A + C63Y + S107C + Q129L + E229K + Q388L + T428I +

F78L + I175S + A329K + S333M

V79A + F332A + C63Y + S107C + Q129L + E229K + Q388L + T428I +

F78L + I175S + A329K + M355F

V79A + F332A + C63Y + S107C + Q129L + E229K + Q388L + T428I +

F78L + I175S + A329K + M355K

V79A + F332A + C63Y + S107C + Q129L + E229K + Q388L + T428I +

F78L + I175S + A329K + L182I

V79A + F332A + C63Y + S107C + Q129L + E229K + Q388L + T428I +

F78L + I175S + A329K + L182M

V79A + F332A + C63Y + S107C + Q129L + E229K + Q388L + T428I +

F78L + I175S + A329K + A331F + S333Q

V79A + F332A + C63Y + S107C + Q129L + E229K + Q388L + T428I +

F78L + I175S + A329K + A331F + S333V

V79A + F332A + C63Y + S107C + Q129L + E229K + Q388L + T428I +

F78L + I175S + A329K + A331F + W91M

V79A + F332A + C63Y + S107C + Q129L + E229K + Q388L + T428I +

F78L + I175S + A329K + A331F + S90C

V79A + F332A + C63Y + S107C + Q129L + E229K + Q388L + T428I +

F78L + I175S + A329K + A331F + L18V

V79A + F332A + C63Y + S107C + Q129L + E229K + Q388L + T428I +

F78L + I175S + A329K + A331F + S90V

V79A + F332A + C63Y + S107C + Q129L + E229K + Q388L + T428I +

F78L + I175S + A329K + A331F + S90G

V79A + F332A + C63Y + S107C + Q129L + E229K + Q388L + T428I +

F78L + I175S + A329K + S333H + L182A

V79A + F332A + C63Y + S107C + Q129L + E229K + Q388L + T428I +

F78L + I175S + A329K + S333H + L182G

V79A + F332A + C63Y + S107C + Q129L + E229K + Q388L + T428I +

F78L + I175S + A329K + S333H + L182M

V79A + F332A + C63Y + S107C + Q129L + E229K + Q388L + T428I +

F78L + I175S + A329K + S333H + L182F

V79A + F332A + C63Y + S107C + Q129L + E229K + Q388L + T428I +

F78L + I175S + A329K + S333H + A331P

V79A + F332A + C63Y + S107C + Q129L + E229K + Q388L + T428I +

F78L + I175S + A329K + S333H + A331S

V79A + F332A + C63Y + S107C + Q129L + E229K + Q388L + T428I +

F78L + I175S + A329K + S333M + M355W

V79A + F332A + C63Y + S107C + Q129L + E229K + Q388L + T428I +

F78L + I175S + A329K + S333M + M355G

V79A + F332A + C63Y + S107C + Q129L + E229K + Q388L + T428I +

F78L + I175S + A329K + S333M + A331D

V79A + F332A + C63Y + S107C + Q129L + E229K + Q388L + T428I +

F78L + I175S + A329K + S333H + L182F + S73G

V79A + F332A + C63Y + S107C + Q129L + E229K + Q388L + T428I +

F78L + I175S + A329K + S333H + L182F + W91F

V79A + F332A + C63Y + S107C + Q129L + E229K + Q388L + T428I +

F78L + I175S + A329K + S333H + L182F + P173Q

V79A + F332A + C63Y + S107C + Q129L + E229K + Q388L + T428I +

F78L + I175S + A329K + S333H + L182F + P173R

V79A + F332A + C63Y + S107C + Q129L + E229K + Q388L + T428I +

F78L + I175S + A329K + S333H + L182F + L72F

V79A + F332A + C63Y + S107C + Q129L + E229K + Q388L + T428I +

F78L + I175S + A329K + S333H + L182F + S73A.

2 . A DNA molecule encoding the mutant of claim 1 .

3 . A recombinant vector comprising the DNA molecule of claim 2 .

4 . The recombinant vector of claim 3 , wherein the recombinant vector is selected from the group consisting of: pET-21b(+), pET-22b(+), pET-3a(+), pET-3d(+), pET-11a(+), pET-12a(+), pET-14b(+), pET-15b(+), pET-16b(+), pET-17b(+), pET-19b(+), pET-20b(+), pET-21a(+), pET-23a(+), pET-23b(+), pET-24a(+), pET-25b(+), pET-26b(+), pET-27b(+), pET-28a(+), pET-29a(+), pET-30a(+), pET-31b(+), pET-32a(+), pET-35b(+), pET-38b(+), pET-39b(+), pET-40b(+), pET-41a(+), pET-41b(+), pET-42a(+), pET-43a(+), pET-43b(+), pET-44a(+), pET-49b(+), pQE2, pQE9, pQE30, pQE31, pQE32, pQE40, pQE70, pQE80, pRSET-A, pRSET-B, pRSET-C, pGEX-5X-1, pGEX-6p-1, pGEX-6p-2, pBV220, pBV221, pBV222, pTrc99A, pTwin1, pEZZ18, pKK232-18, pUC-18 and pUC-19.

5 . An isolated host cell, comprising the recombinant vector of claim 3 .

6 . The host cell of claim 5 , wherein the host cell is a prokaryotic cell or an eukaryotic cell, wherein the eukaryotic cell is a yeast cell.

7 . The host cell of claim 6 , wherein the host cell is a competent cell.

8 . The host cell of claim 6 , wherein the competent cell is E. coli BL21 cell or E. coli W3110.

9 . A method of producing a carbonyl compound or an alcohol compound, wherein the method comprises:

catalyzing an olefin compound

using the cytochrome P450 enzyme mutant of claim 1 for a direct anti-Markovnikov oxidation reaction to generate a carbonyl compound

and an alcohol compound

wherein R is selected from the group consisting of substituted of unsubstituted alkyl group, substituted or unsubstituted aralkyl group, and substituted or unsubstituted aryl group.

10 . The method of claim 9 , wherein R represents a group having 1-20 carbon atoms selected from the group consisting of substituted or unsubstituted alkyl group, substituted or unsubstituted aralkyl group, and substituted or unsubstituted aryl group.

11 . The method of claim 9 , wherein R represents a group having 1-10 carbon atoms selected from the group consisting of substituted or unsubstituted alkyl group, substituted or unsubstituted aralkyl group, and substituted or unsubstituted aryl group.

12 . The method of claim 11 , wherein the substituted refers to a substitution by a halogen atom, a nitrogen atom, a sulfur atom, a hydroxyl group, a nitro group, a cyano group, a methoxy group, an ethyoxyl group, a carboxyl group, a carboxymethyl group, a carboxyethyl group or a methylenedioxy group.

13 . The method of claim 9 , wherein the olefin compound is a styrene compound substituted or unsubstituted at any position on the benzene ring, and the reaction is an anti-Markovnikov oxidation reaction of to generate

to generate

wherein R1 represents that any position on the benzene ring is substituted with halogen, a nitro group, a methyl group or a methoxy group.

14 . The method of claim 13 , wherein the halogen is a chlorine atom.

15 . The method of claim 9 , wherein the olefinic compound is selected from any one of the groun consisting of

Assignments (1)
ASSIGNMENT OF ASSIGNOR'S INTEREST Recorded Jun 14, 2023
From: CHENG, YIBING; HONG, HAO; JAMES, GAGE; ZHANG, NA; JIAO, XUECHENG; MA, YULEI; CAO, SHAN; SUN, KAIHUA
To: ASYMCHEM LIFE SCIENCE (TIANJIN) CO., LTD.
Reel/Frame 063950/0966 →
Priority Claims (1)
CN 202110039458.8 · Jan 13, 2021 · national
Continuity (1)
Related Publication 20240010997A1 · Jan 11, 2024
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