Bioproduction of enantiopure (r)- and (s)-2-phenylglycinol from styrene and renewable feedstocks via artificial enzyme cascade

Patent metadata

Field	Value
Publication number	US-2024052394-A1
Application number	US-202118265381-A
Country	US
Kind code	A1
Filing date	Dec 6, 2021
Priority date	Dec 7, 2020
Publication date	Feb 15, 2024
Grant date	—

Abstract

Disclosed herein are methods of bioproduction of enantiomerically pure or enantiomerically enriched 2-phenylglycinol or a derivative thereof by multiple enzyme-catalyzed chemical transformations in a one-pot reaction system.

First claim

1 . A method for producing an enantiomerically pure or enantiomerically enriched (R)- or (S)-2-phenylglycinol or a derivative thereof using one or more recombinant microbial cells genetically engineered to overexpress multiple enzymes, which method comprises subjecting styrene or a derivative thereof to multiple enzyme-catalyzed chemical transformations in a one-pot reaction system.

2 . The method of claim 1 , wherein the method produces enantiomerically pure or enantiomerically enriched 2-phenylglycinol or a derivative thereof, comprising the steps of:
(a) generating phenylethane diol or a derivative thereof from styrene or a derivative thereof by conducting an epoxidation reaction catalyzed by an epoxidase to form styrene oxide or a derivative thereof and conducting a hydrolysis reaction catalyzed by an epoxide hydrolase on the epoxide, or by a dihydroxylation reaction catalyzed by a dioxygenase: (b) generating 2-hydroxyacetophenone or a derivative thereof from the phenylethane diol or a derivative thereof by an oxidation reaction catalyzed by an alcohol dehydrogenase or a secondary alcohol oxidase; and (c) generating enantiomerically pure or enantiomerically enriched 2-phenylglycinol or a derivative thereof from 2-hydroxyacetophenone or a derivative thereof by a transamination reaction catalyzed by a transaminase or a reductive amination reaction catalyzed by an amine dehydrogenase.

3 . The method according to claim 2 , wherein one or more of the following apply:
(a) the epoxidase is a styrene monooxygenase; (b) the epoxide hydrolase, when used, is from Solanum tuberosum or its mutants or similar enzymes with more than 50% identity: (c) the alcohol dehydrogenase, when used, is from Candida parapsilosis or, more preferably, Bacillus subtilis or their mutants or similar enzymes with more than 50% identity: and/or (d) the transaminase is from Bacillus megaterium, Chromobacterium violaceum, Vibrio fluvialis, Martelella mediterranea, Neosartora fischeri, Arthrobacter sp., or Aspergillus terreus or their mutants or similar enzymes with more than 50% identity.

4 . (canceled)

5 . The method according to claim 1 , wherein one of the one or more recombinant microbial cells genetically engineered to overexpress multiple enzymes (i.e. the one or more recombinant microbial cells genetically engineered to overexpress multiple enzymes) is selected from one or more of the group consisting of:
(a) E. coli T7-pCDF-SMO-StEH, pET-BDHA-AlaDH, pRSF-BmTA; (b) E. coli T7-pCDF-SMO-StEH, pET-BmTA, pRSF-BDHA-AlaDH; (c) E. coli T7-pCDF-BDHA-AlaDH, pET-SMO-StEH, pRSF-BmTA; (d) E. coli T7-pCDF-BDHA-AlaDH, pET-BmTA, pRSF-SMO-StEH; (e) E. coli T7-pCDF-BmTA, pET-SMO-StEH, pRSF-BDHA-AlaDH; and (f) E. coli T7-pCDF-BmTA, pET-BDHA-AlaDH, pRSF-SMO-StEH.

6 . (canceled)

7 . The method according to claim 1 , wherein one of the one or more recombinant microbial cells genetically engineered to overexpress multiple enzymes (i.e. the one or more recombinant microbial cells genetically engineered to overexpress multiple enzymes) is selected from one or more of the group consisting of:
(a) E. coli T7-pCDF-SMO-StEH, pET-BDHA-AlaDH, pRSF-NFTA; (b) E. coli T7-pCDF-SMO-StEH, pET-NFTA, pRSF-BDHA-AlaDH; (c) E. coli T7-pCDF-BDHA-AlaDH, pET-SMO-StEH, pRSF-NFTA; (d) E. coli T7-pCDF-BDHA-AlaDH, pET-NFTA, pRSF-SMO-StEH; (e) E. coli T7-pCDF-NFTA, pET-SMO-StEH, pRSF-BDHA-AlaDH; and (f) E. coli T7-pCDF-NFTA, pET-BDHA-AlaDH, pRSF-SMO-StEH.

8 . The method according to claim 1 , wherein one of the one or more recombinant microbial cells genetically engineered to overexpress multiple enzymes (i.e. the one or more recombinant microbial cells genetically engineered to overexpress multiple enzymes) is E. coli T7-pCDF-SMO-StEH, pET-NFTA, pRSF-BDHA-AlaDH.

9 . The method according to claim 1 , wherein the method further comprises providing styrene or a derivative thereof by generating trans-cinnamic acid or a derivative thereof from L-phenylalanine or a derivative thereof by a deamination reaction catalyzed by an ammonia lyase and generating styrene or a derivative thereof from the trans-cinnamic acid or a derivative thereof in a decarboxylation reaction catalyzed by a decarboxylase.

10 . (canceled)

11 . The method according to claim 1 , wherein one of the one or more recombinant microbial cells genetically engineered to overexpress multiple enzymes (i.e. the one or more recombinant microbial cells genetically engineered to overexpress multiple enzymes) is E. coli T7-pACYC-PAL-PAD, pCDF-SMO-SIEH, pET-BDHA-AlaDH, pRSF-BmTA.

12 . The method according to claim 1 , wherein one of the one or more recombinant microbial cells genetically engineered to overexpress multiple enzymes (i.e. the one or more recombinant microbial cells genetically engineered to overexpress multiple enzymes) is E. coli T7-pACYC-PAL-PAD, pCDF-SMO-StEH, pET-BDHA-AlaDH, pRSF-NfTA.

13 . The method according to claim 9 , wherein the method further comprises cells that overexpress the natural L-phenylalanine biosynthetic pathway, which cells convert glucose or glycerol to L-phenylalanine.

14 - 15 . (canceled)

16 . The method according to claim 13 , wherein the microbial cells producing L-phenylalanine from glucose or glycerol that overexpress at least one enzyme is E. coli NST74-pACYC-PAL-PAD, pCDF-SMO-StEH, pET-BDHA-AlaDH, pRSF-BmTA or E. coli NST74-pACYC-PAL-PAD, pCDF-SMO-StEH, pET-BDHA-AlaDH, pRSF-NfTA.

17 . The method according to claim 13 , wherein the microbial cells producing L-phenylalanine from glucose or glycerol that overexpress at least one enzyme is a combination of E. coli NST74-Phe with E. coli T7-pACYC-PAL-PAD, pCDF-SMO-StEH, pET-BDHA-AlaDH, pRSF-BmTA or is a combination of E. coli NST74-Phe with E. coli T7-pACYC-PAL-PAD, pCDF-SMO-StEH, pET-BDHA-AlaDH, pRSF-NfTA.

18 - 20 . (canceled)

21 . An isolated nucleic acid molecule encoding at least one heterologous catalytic enzyme selected from the group comprising:
(a) an epoxidase for conducting an epoxidation reaction to form styrene oxide or a derivative thereof from styrene or a derivative thereof and an epoxide hydrolase for conducting a hydrolysis reaction on the epoxide to provide phenylethanediol or a derivative thereof, or an oxygenase for generating phenylethanediol or a derivative thereof from styrene or a derivative thereof by a dihydroxylation reaction: (b) an alcohol dehydrogenase or a secondary alcohol oxidase for generating 2-hydroxyacetophenone or a derivative thereof from phenylethanediol by an oxidation reaction: (c) a transaminase or an amine dehydrogenase for generating enantiomerically pure or enantiomerically enriched 2-phenylglycinol or a derivative thereof by a transamination reaction or a reductive amination from 2-hydroxyacetophenone or a derivative thereof; of (d) an ammonia lyase for generating trans-cinnamic acid or a derivative thereof from L-phenylalanine or a derivative thereof by a deamination reaction; and (e) a decarboxylase for generating styrene or a derivative thereof from trans-cinnamic acid or a derivative thereof in a decarboxylation reaction.

22 - 23 . (canceled)

24 . An expression construct comprising at least one nucleic acid molecule as described in claim 21 .

25 . One or more recombinant prokaryotic or eukaryotic cells selected from the group comprising bacterial cells, yeast cells, mammalian cells and insect cells, wherein said cells comprise at least one expression construct as described in claim 24 .

Assignees

• Nat Univ Singapore

Inventors

• Li Zhi
• Sundara Sekar Balaji
• Lukito Benedict Ryan

Classifications

• C12P41/006 Primary

  by reactions involving C-N bonds, e.g. nitriles, amides, hydantoins, carbamates, lactames, transamination reactions, or keto group formation from racemic mixtures · CPC title

• C12P13/001

  Amines; Imines · CPC title

• C12N9/0071

  acting on paired donors with incorporation of molecular oxygen (1.14) · CPC title

• C12Y114/14011

  Styrene monooxygenase (1.14.14.11) · CPC title

• C12N9/14

  Hydrolases (3) · CPC title