Methods for incorporating formaldehyde into biomass

Abstract

A method for the incorporation of formaldehyde into biomass is described, comprising the following enzymatically catalyzed steps: (1) condensation of pyruvate and formaldehyde to 4-hydroxy-2-oxobutanoic acid (HOB); (2) amination of the resulting 4-hydroxy-2-oxobutanoic acid (HOB) to produce homoserine; (3) conversion of the resulting homoserine to threonine; (4) conversion of the resulting threonine to glycine and acetaldehyde or acetyl-CoA; (5) condensation of the resulting glycine with formaldehyde to produce serine; and (6) conversion of the resulting serine to produce pyruvate, which can then be used as a substrate in step (1).

Claims

1. The following enzymatically catalyzed steps (1) Condensation of pyruvate and formaldehyde to 4-hydroxy-2-oxobutanoic acid (HOB); (2) Production of homoserine by amination of the 4-hydroxy-2-oxobutanoic acid (HOB) thus produced; (3) The conversion of homoserine thus produced to threonine; (4) Conversion of the threonine thus produced to glycine and acetaldehyde or acetyl-CoA; (5) The formation of serine by condensation of the glycine thus produced with formaldehyde; and (6) Production of pyruvate by conversion of the serine thus produced, wherein the pyruvate can then be used as a substrate in step (1). Methods that include...

2. The conversion from homoserine to threonine in step (3) (i) the formation of o-phosphohomoserine by phosphorylation of homoserine thus produced; and (ii) The method according to claim 1, wherein the production of threonine occurs by dephosphorylation of the o-phosphohomoserine thus produced.

3. The method according to claim 1 or 2, wherein the conversion from serine to pyruvate in step (6) is achieved by deamination.

4. (a) In step (1), the condensation of pyruvate and formaldehyde to 4-hydroxy-2-oxobutanoic acid (HOB) is achieved using an aldolase classified as EC 4.1.

2. _; (b) In step (2), the production of homoserine by amination of the 4-hydroxy-2-oxobutanoic acid (HOB) thus produced is achieved by using an aminotransferase enzyme classified as EC 2.6.

1. or by an amino acid dehydrogenase (EC 1.4.1). (c) In step (3)(i) of claim 2, the production of o-phosphohomoserine by phosphorylation of the homoserine thus produced is achieved by using homoserine kinase (EC 2.7.1.39); (d) In step (3)(ii) of claim 2, the production of threonine by dephosphorylation of the thus produced o-phosphohomoserine is achieved by using threonine synthase (EC 4.2.3.1); (e) In step (4), the conversion of the thus produced threonine to glycine and acetaldehyde is achieved by using 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 in step (5), the conversion of the thus produced threonine to glycine and acetyl-CoA is achieved by a combination of threonine dehydrogenase (EC 1.1.1.103) and 2-amino-3-ketobutyrate CoA ligase (EC 2.3.1.29); (f) In step (5), the production of serine by condensation of the glycine thus produced with formaldehyde is achieved by using threonine aldolase (selected from the group consisting of EC4.1.2.5, EC4.1.2.6, EC4.1.2.48 and EC4.1.2.49); (g) In step (6), the production of pyruvate by conversion of the serine thus produced is achieved by using serine deaminase (EC 4.3.1.17) or threonine deaminase (EC 4.3.1.19); or any combination of (a) through (g), The method according to any one of claims 1 to 3.

5. The method according to any one of claims 1 to 4, wherein the formaldehyde used for condensation in step (1) and / or (5) is obtained by oxidation of methanol.

6. The formaldehyde in step (1) and / or (5) is (a) Enzymatically converting methanol to formaldehyde using methanol dehydrogenase (EC 1.1.1.244) or methanol dehydrogenase (cytochrome c) (EC 1.1.2.7); and / or (b) Enzymatically convert methanol to formaldehyde using alcohol oxidase (EC 1.1.3.13). The method according to any one of claims 1 to 5, as provided by [the present invention].

7. Next reaction: (1) Condensation of pyruvate and formaldehyde to 4-hydroxy-2-oxobutanoic acid (HOB) by aldolase classified as EC 4.1.2; (2) Production of homoserine by amination of 4-hydroxy-2-oxobutanoic acid (HOB) by an aminotransferase enzyme classified as EC2.6.1 or by an amino acid dehydrogenase (EC1.4.1). (3) Production of o-phosphohomoserine by phosphorylation of homoserine thus produced by homoserine kinase (EC2.7.1.39); (4) Production of threonine by dephosphorylation of the thus produced o-phosphohomoserine by threonine synthase (EC 4.2.3.1); (5) Conversion of the threonine thus produced to glycine by 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) or threonine dehydrogenase (EC 1.1.1.103) and 2-amino-3-ketobutyrate CoA ligase (EC 2.3.1.29); (6) Production of serine by condensation of the glycine thus produced with formaldehyde by threonine aldolase (selected from the group consisting of EC4.1.2.5, EC4.1.2.6, EC4.1.2.48 and EC4.1.2.49); and (7) Production of pyruvate by deamination of the serine thus produced with serine deaminase (EC 4.3.1.17) or threonine deaminase (EC 4.3.1.19), wherein the pyruvate can then be used as a substrate in step (1); A recombinant microorganism expressing an enzyme that catalyzes step (1), comprising at least one heterologous nucleic acid molecule encoding an aldolase that catalyzes step (6), i.e., an enzyme that catalyzes the production of serine by the condensation of glycine and formaldehyde, The aforementioned microorganism is Escherichia coli (E. coli). The recombinant microorganisms mentioned above.

8. The microorganism according to claim 7, further overexpressing at least one of the enzymes that catalyze step (3), step (4), or step (5).

9. A microorganism according to claim 7 or 8 that can convert methanol to formaldehyde.

10. The aforementioned microorganism, (a) Enzymatically converting methanol to formaldehyde with methanol dehydrogenase (EC 1.1.1.244) or methanol dehydrogenase (cytochrome c) (EC 1.1.2.7); and / or (b) Enzymatically convert methanol to formaldehyde using alcohol oxidase (EC 1.1.3.13). The microorganism according to claim 9.

11. A microorganism according to any one of claims 7 to 10, which lacks the enzyme activity to convert pyruvate to aspartic acid semialdehyde.

12. A microorganism according to any one of claims 7 to 11, which lacks the enzymatic activity of 3-phosphoglycerate dehydrogenase (EC 1.1.1.95).

13. A microorganism according to any one of claims 7 to 12, lacking the enzymatic activity of serine hydroxymethyltransferase (EC2.1.2.1) and / or the enzymatic activity of the glycine cleavage system (gcvTHP).

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