Redox Self-Sufficient Biocatalytic Amination of Alcohols

Patent metadata

Field	Value
Publication number	US-2017145451-A1
Application number	US-201515323515-A
Country	US
Kind code	A1
Filing date	Jul 2, 2015
Priority date	Jul 3, 2014
Publication date	May 25, 2017
Grant date	—

Abstract

The present invention relates to a novel biocatalytic method for the production of primary and secondary amines, comprising coupled enzymatic oxidation and reduction processes simultaneously regenerating the required cofactor; making use of two enzymes—i.e. an alcohol dehydrogenase and an amine dehydrogenase—operating simultaneously in a one pot process (i.e. biocatalytic cascade). Furthermore, the overall process is redox neutral, since the hydride generated from the first oxidative step is internally recycled in the second reductive step. The invention also relates to recombinant expression systems and microorganisms procuring the required enzyme activities; and bioreactors for performing such methods.

First claim

1 . A method of preparing an amine, wherein said amine is of the general formula I
(R 1 )(R 2 )HC—NH 2   (I)
wherein R 1 and R 2 are identical or different and selected from H, alkyl, cycloalkyl, alkenyl, aryl, aralkyl, alkoxyalkyl, or aryloxyalkyl provided that R 1 and R 2 are not simultaneously H, and wherein aryl moiety optionally may be substituted, which method comprises: a) the enzymatic oxidation of the corresponding alcohol of said amine in the presence of the cofactor NAD(P) + and of an alcohol dehydrogenase (ADH) (E.C.1.1.1.x) to form the corresponding carbonyl compound of said alcohol, while the cofactor NAD(P) + is reduced to form NAD(P)H; and simultaneously the enzymatic reduction of said carbonyl compound in the presence of i)) ammonia or an ammonia source, and ii) an amine dehydrogenase (AmDH) to form the desired amine, while the cofactor NAD(P) + is regenerated from NAD(P)H; and optionally b) isolating said amine; wherein said AmDH is a mutant of a phenylalanine dehydrogenase (Ph-AmDH) (E.C. 1.4.1.20) or a mutant of a leucine dehydrogenase (L-AmDH) (E.C. 1.4.1.9); selected from a(1) a mutant of a phenyl alanine dehydrogenase comprises an amino acid sequence of SEQ ID NO: 1, wherein said mutant comprises the double mutation K78S, N277L, and wherein said mutant has a sequence identity of at least 80% to SEQ ID NO: 1, and (2) a mutant of a leucine dehydrogenase comprises an amino acid sequence of SEQ ID NO: 3, wherein said mutant comprises the four-fold mutation K68S, E114V, N262L, V291C, and wherein said mutant has a sequence identity of at least 80% to SEQ ID NO: 3.

2 . The method of claim 1 wherein said AmDH
a) is a mutant of a phenyl alanine dehydrogenase and comprises an amino acid sequence of SEQ ID NO: 2, or of amino acid residues 21 to 400 of SEQ ID NO:2 or
b) is a mutant of a leucine alanine dehydrogenase and comprises an amino acid sequence of SEQ ID NO: 4.

3 . The method of claim 1 wherein said ADH is selected from:
a) AA-ADH comprising an amino acid sequence of SEQ ID NO:5 or an amino acid sequence having a sequence identity of at least 80% sequence identity, to SEQ ID NO: 5;
b) LBv-ADH comprising an amino acid sequence of SEQ ID NO:6 or an amino acid sequence having a sequence identity of at least 80% sequence identity, to SEQ ID NO: 6;
c) HL-ADH comprising an amino acid sequence of SEQ ID NO: 8 or an amino acid sequence having a sequence identity of at least 80% sequence identity, to SEQ ID NO: 8;
d) ADH-A comprising an amino acid sequence of SEQ ID NO: 9 or an amino acid sequence having a sequence identity of at least 80% sequence identity, to SEQ ID NO: 9; or
e) ADH-hT comprising an amino acid sequence of SEQ ID NO: 10 or an amino acid sequence having a sequence identity of at least 80% sequence identity, to SEQ ID NO: 10.

4 . The method of claim 1 , which is performed in an aqueous or aqueous-organic mixture reaction medium.

5 . The method of claim 1 , wherein the method is performed in the presence of the set of required enzymes in isolated form, one or more recombinant microorganisms expressing at least one enzyme of the required set of enzymes or cell homogenates or cell lysates of one or more microorganisms expressing the required set of enzymes or at least one of the enzymes of the required set of enzymes.

6 . The method of claim 1 , wherein the cofactor is employed in sub-stoichiometric amounts.

7 . The method of claim 1 , wherein the reaction is performed in the presence of an excess of ammonia or of said ammonia source.

8 . The method of claim 1 , wherein the reaction is performed at a pH in the range of lower than 9.6.

9 . The method of claim 1 , wherein the reaction is performed at a pH in the range of about 8.0 to 8.7.

10 . The method of claim 1 , wherein the pH is controlled by means of a buffer which does not show metal chelating properties.

11 . The method of claim 1 , wherein the employed enzymes are NAD(H) dependent.

12 . A recombinant expression vector carrying under the control of at least one regulatory element the coding sequence of at least one ADH and at least one AmDH as defined in claim 1 .

13 . A recombinant microorganism carrying at least one expression vector according to claim 12 and functionally expressing said encoded enzymes.

14 . (canceled)

15 . (canceled)

16 . A recombinant microorganism carrying at least one expression vector containing under the control of at least one regulatory element the coding sequence of at least one ADH as defined in claim 1 and functionally expressing said encoded enzyme.

17 . A recombinant microorganism carrying under the control of at least one regulatory element the coding sequence of at least one AmDH as defined in claim 1 and functionally expressing said encoded enzyme.

Assignees

• Basf Se

Inventors

• Baldenius Kai-Uwe
• Breuer Michael
• Ditrich Klaus
• Navickas Vaidotas
• Mutti Francesco
• Knaus Tanja
• Turner Nicholas

Classifications

• C12P13/001 Primary

  Amines; Imines · CPC title

• C12Y104/01009

  Leucine dehydrogenase (1.4.1.9) · CPC title

• C12Y101/01001

  Alcohol dehydrogenase (1.1.1.1) · CPC title

• C12N9/0006

  acting on CH-OH groups as donors (1.1) · CPC title

• C12N9/0016

  with NAD or NADP as acceptor (1.4.1) · CPC title