Production of chiral 1,2-amino alcohols and alpha-amino acids from alkenes by cascade biocatalysis

Patent metadata

Field	Value
Publication number	US-2017067084-A1
Application number	US-201615257223-A
Country	US
Kind code	A1
Filing date	Sep 6, 2016
Priority date	Sep 3, 2015
Publication date	Mar 9, 2017
Grant date	—

Abstract

Disclosed herein are methods of forming chiral 1,2-aminoalcohols and α-aminoacids from alkene starting materials by way of an enzymatic cascade reaction sequence that may be accomplished in a single reaction vessel without the need to isolate any intermediates. Also disclosed herein are recombinant nucleic acids, vectors and host cells for use in the methods of the invention.

First claim

1 . A method for producing an enantiomerically pure or enantiomerically enriched 1,2-amino alcohol or α-amino acid, which method comprises subjecting an alkene starting material to multiple enzyme-catalyzed chemical transformations in a one-pot reaction system, wherein the method comprises generating a vicinal diol from the alkene and an α-hydroxyaldehyde from the vicinal diol.

2 . The method of claim 1 , wherein the method produces an enantiomerically pure or enantiomerically enriched 1,2-amino alcohol, comprising the steps of:
(a) generating a vicinal diol from an alkene by a dihydroxylation reaction catalyzed by a dioxygenase, or by conducting an epoxidation reaction catalyzed by an epoxidase to form an epoxide and conducting a hydrolysis reaction catalyzed by an epoxide hydrolase on the epoxide; (b) generating an α-hydroxyaldehyde or a or an α-hydroxyketone from the vicinal diol by an oxidation reaction catalyzed by an alcohol oxidase or alcohol dehydrogenase; and (c) generating a 1,2-aminoalcohol from the α-hydroxyaldehyde or α-hydroxyketone by a transamination reaction catalyzed by a transaminase or a reductive amination reaction catalyzed by an amine dehydrogenase.

3 . The method of claim 2 , wherein the method further comprises providing the alkene by generating a vinyl carboxylic acid from an α-amino acid by a deamination reaction catalyzed by an ammonia lyase and generating the alkene from the vinyl carboxylic acid in a decarboxylation reaction catalyzed by a decarboxylase.

4 . The method of claim 2 , wherein the alcohol oxidase is alditol oxidase or its mutants, or the alcohol dehydrogenase is selected from the group consisting of AlkJ from Pseudomonas putida , AlkJ homologue from Sphingomonas sp. HXN-200, dihydrodiol dehydrogenase, and mutants thereof.

5 . The method of claim 2 , wherein the transaminase is a ω-transaminase, or the amine dehydrogenase is a phenylalanine dehydrogenase, a leucine dehydrogenase or their mutants.

6 . The method of claim 2 , wherein the alkene has the formula (I):

where:
R 1 to R 3 independently represent H, an alkyl group, an alkenyl group, an alkynyl group, a cycloalkyl group, a cycloalkenyl group, an aryl group, an aryl alkyl group, a heterocyclic group, and a heterocyclic alkyl group, which groups are substituted or unsubstituted by one or more substituents, provided that at least one of R 1 to R 3 is not H.

7 . The method of claim 2 , wherein the method comprises the use of one or more reactive components selected from the group consisting of cells, immobilized cells, cell extract, isolated enzymes and immobilized enzymes.

8 . The method of claim 1 , wherein the method produces an enantiomerically pure or enantiomerically enriched α-amino acid, comprising the steps of:
(a) generating a vicinal diol from an alkene by a dihydroxylation reaction catalyzed by a dioxygenase, or by conducting an epoxidation reaction catalyzed by an epoxidase to form an epoxide and conducting a hydrolysis reaction catalyzed by an epoxide hydrolase on the epoxide;
(b) generating an α-hydroxyaldehyde from the vicinal diol by an oxidation reaction catalyzed by an alcohol oxidase or alcohol dehydrogenase;
(c) generating an α-hydroxy acid from the α-hydroxyaldehyde in an oxidation reaction catalyzed by an aldehyde dehydrogenase or an aldehyde oxidase;
(d) generating an α-ketoacid from the α-hydroxy acid in an oxidation reaction catalyzed by a hydroxy acid dehydrogenase or a hydroxy acid oxidase; and
(e) generating an α-amino acid from the α-ketoacid by a transamination reaction catalyzed by a transaminase or a reductive amination reaction catalyzed by an amino acid dehydrogenase.

9 . The method of claim 8 , wherein the method further comprises providing the alkene by generating a vinyl carboxylic acid from an α-amino acid by a deamination reaction catalyzed by an ammonia lyase and generating the alkene from the vinyl carboxylic acid in a decarboxylation reaction catalyzed by a decarboxylase.

10 . The method of claim 8 , wherein the alcohol oxidase is alditol oxidase or its mutants, or the alcohol dehydrogenase is selected from the group consisting of AlkJ from Pseudomonas putida , AlkJ homologue from Sphingomonas sp. HXN-200, dihydrodiol dehydrogenase, and mutants thereof.

11 . The method of claim 8 , wherein the aldehyde dehydrogenase is AlkH from Pseudomonas putida or its mutants and/or phenyl aldehyde dehydrogenase from Escherichia coli or its mutants.

12 . The method of claim 8 , wherein the hydroxy acid dehydrogenase is mandelate dehydrogenase or its mutants, and the hydroxy acid oxidase is mandelate oxidase or its mutants and/or hydroxymandelate oxidase from S. coelicolor and its mutants.

13 . The method of claim 8 , wherein the transaminase is an α-transaminase.

14 . The method of claim 8 , wherein the alkene has the formula (II):

where:
R 4 and R 5 independently represent H, a straight chain or branched alkyl group, a straight chain or branched alkenyl group, a straight chain or branched alkynyl group, a cycloalkyl group, a cycloalkenyl group, an aryl group, an aryl alkyl group, and a heterocyclic group, which groups are substituted or unsubstituted by one or more substituents, provided that at least one of R 4 and R 5 is not H.

15 . The method of claim 8 , wherein the method comprises the use of one or more reactive components selected from the group consisting of cells, immobilized cells, cell extract, isolated enzymes and immobilized enzymes.

16 . An isolated nucleic acid molecule encoding at least one heterologous catalytic enzyme selected from the group comprising:
(a) a dioxygenase for generating a vicinal diol from an alkene by a dihydroxylation reaction, or an epoxidase for conducting an epoxidation reaction to form an epoxide and an epoxide hydrolase for conducting a hydrolysis reaction on the epoxide; (b) an alcohol oxidase or alcohol dehydrogenase for generating an α-hydroxyaldehyde or an α-hydroxyketone from the vicinal diol by an oxidation reaction; and (c) a transaminase or an amine dehydrogenase for generating a 1,2-aminoalcohol from the α-hydroxyaldehyde or the α-hydroxyketone by a transamination reaction or a reductive amination; or (d) a dioxygenase for generating a vicinal diol from an alkene by a dihydroxylation reaction, or an epoxidase for conducting an epoxidation reaction to form an epoxide and an epoxide hydrolase for conducting a hydrolysis reaction on the epoxide; (e) an alcohol oxidase or alcohol dehydrogenase for generating an α-hydroxyaldehyde from the vicinal diol by an oxidation reaction; (f) an aldehyde dehydrogenase or an aldehyde oxidase for generating an α-hydroxy acid from the α-hydroxyaldehyde in an oxidation reaction; (g) a hydroxy acid dehydrogenase or a hydroxy acid oxidase for generating an α-ketoacid from the α-hydroxy acid in an oxidation reaction; and (h) a transaminase or an amino acid dehydrogenase for generating an α-amino acid from the α-ketoacid by a transamination reaction or reductive amination reaction.

17 . The isolated nucleic acid of claim 16 , encoding a plurality of said catalytic enzymes.

18 . The isolated nucleic acid molecule of claim 17 , wherein said plurality of catalytic enzymes is arranged as at least one module selected from the group comprising:
i) a module comprising heterolo

Assignees

• Nat Univ Singapore

Inventors

• Li Zhi
• Wu Shuke

Classifications

• C12P13/04 Primary

  Alpha- or beta- amino acids {(other amino acids C12P13/005)} · CPC title

• C12P13/001

  Amines; Imines · CPC title

• C12N15/52

  Genes encoding for enzymes or proenzymes · CPC title

• C12Y101/03013

  Alcohol oxidase (1.1.3.13) · CPC title

• C12N9/1096

  transferring nitrogenous groups (2.6) · CPC title