Method for the incorporation of formaldehyde into biomass

Patent metadata

Field	Value
Publication number	US-11976310-B2
Application number	US-202117800484-A
Country	US
Kind code	B2
Filing date	Feb 16, 2021
Priority date	Feb 17, 2020
Publication date	May 7, 2024
Grant date	May 7, 2024

Abstract

The present disclosure relates to a method for the incorporation of formaldehyde into biomass comprising the following enzymatically catalyzed steps: (1) condensation of pyruvate with formaldehyde into 4-hydroxy-2-oxobutanoic acid (HOB); (2) amination of the thus produced 4-hydroxy-2-oxobutanoic acid (HOB) to produce homoserine; (3) conversion of thus produced homoserine to threonine; (4) conversion of the thus produced threonine into glycine and acetaldehyde or acetyl-CoA; (5) condensation of the thus produced glycine with formaldehyde to produce serine; and (6) conversion of the thus produced serine to produce pyruvate, wherein said pyruvate can then be used as a substrate in step (1). The disclosure also relates to enzymes for catalyzing the corresponding enzymatic reactions and recombinant microorganisms which express the enzymes for catalyzing the corresponding enzymatic reactions.

First claim

The invention claimed is:

1. A method for incorporation of formaldehyde into carbon compounds the can be assimilated into metabolism, said method comprising the following sequence of enzymatically catalyzed steps, wherein the method is carried out in vitro or in vivo:
(1) condensation of pyruvate with a first formaldehyde into 4-hydroxy-2-oxobutanoic acid (HOB);
(2) amination of the thus produced 4-hydroxy-2-oxobutanoic acid (HOB) to produce homoserine;
(3) conversion of the thus produced homoserine to threonine;
(4) conversion of the thus produced threonine into glycine and acetaldehyde or acetyl-CoA;
(5) condensation of the thus produced glycine with a second formaldehyde to produce serine; and
(6) conversion of the thus produced serine to produce pyruvate, wherein said pyruvate can then be used as a substrate in step (1);
wherein the method comprises at least one of
(a) in step (1) the condensation of pyruvate with formaldehyde into 4-hydroxy-2-oxobutanoic acid (HOB) is achieved using an aldolase classified in EC 4.1.2.;
(b) in step (2) the amination of the 4-hydroxy-2-oxobutanoic acid (HOB) to produce homoserine is achieved by using an aminotransferase enzyme classified in EC 2.6.1. or by an amino acid dehydrogenase (EC 1.4.1._);
(c) in step (3) the conversion of homoserine to threonine is achieved by (i) phosphorylation of the homoserine to produce o-phosphohomoserine achieved by using a homoserine kinase (EC 2.7.1.39); and (ii) dephosphorylation of the thus produced o-phosphohomoserine to produce threonine achieved by using a threonine synthase (EC 4.2.3.1);
(d) in step (4) the conversion of the threonine into glycine and acetaldehyde is achieved by using a threonine aldolase (selected from the group consisting of EC 4.1.2.5, EC 4.1.2.6, EC 4.1.2.48 and EC 4.1.2.49) and/or the conversion of the threonine into glycine and acetyl-CoA is achieved by a combination of a threonine dehydrogenase (EC 1.1.1.103) and a 2-amino-3-ketobutyrate CoA ligase (EC 2.3.1.29);
(e) in step (5) the condensation of the glycine with formaldehyde to produce serine is achieved by using a threonine aldolase selected from the group consisting of EC 4.1.2.5, EC 4.1.2.6, EC 4.1.2A8 and EC 4.1.2.49; and
(f) in step (6) the conversion of the thus produced serine to produce pyruvate is achieved by using a serine deaminase (EC 4.3.1.17) or a threonine deaminase (EC4.3.1.19); or any combination of (a) to (f).

2. The method of claim 1 , wherein the formaldehyde used for the condensation in step (1) and/or (5) is provided by the oxidation of methanol.

3. The method of claim 1 , wherein the formaldehyde in step (1) and/or (5) is provided by
(a) converting methanol enzymatically into formaldehyde using a methanol dehydrogenase (EC 1.1.1.244) or a methanol dehydrogenase (cytochrome c) (EC 1.1.2.7); and/or
(b) converting methanol enzymatically into formaldehyde using an alcohol oxidase (EC 1.1.3.13).

Assignees

• Scientist Of Fortune Sa
• Max Planck Gesellschaft

Inventors

• Bar-Even Arren
• He Hai
• Marliere Philippe

Classifications

• C12P13/04 Primary

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

• C12N15/52

  Genes encoding for enzymes or proenzymes · CPC title

• C12N15/70

  Vectors or expression systems specially adapted for E. coli · CPC title

• C12Y101/01103

  L-Threonine 3-dehydrogenase (1.1.1.103) · CPC title

• C12Y101/03013

  Alcohol oxidase (1.1.3.13) · CPC title