Method for the incorporation of formaldehyde into biomass
View Patent ↗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.
1 . A recombinant microorganism expressing enzymes for catalyzing the following sequence of reactions for in vivo incorporation of formaldehyde into carbon compounds that can be assimilated into metabolism:
(1) condensation of pyruvate with a first formaldehyde into 4-hydroxy-2-oxobutanoic acid (HOB) by an aldolase classified in EC 4.1.2._;
(2) amination of the thus produced 4-hydroxy-2-oxobutanoic acid (HOB) to produce homoserine by an aminotransferase enzyme classified in EC 2.6.1._ or by an amino acid dehydrogenase classified in EC 1.4.1._;
(3) phosphorylation of thus produced homoserine to produce o-phosphohomoserine by a homoserine kinase classified in EC 2.7.1.39;
(4) dephosphorylation of the thus produced o-phosphohomoserine to produce threonine by a threonine synthase classified in EC 4.2.3.1;
(5) conversion of the thus produced threonine into glycine by a threonine aldolase (selected from the group consisting of a threonine aldolase classified in EC 4.1.2.5, a threonine aldolase classified in EC 4.1.2.6, a threonine aldolase classified in EC 4.1.2.48, and a threonine aldolase classified in EC 4.1.2.49, or by a combination of a threonine dehydrogenase classified in EC 1.1.1.103 and a 2-amino-3-ketybutyrate CoA ligase classified in EC 2.3.1.29;
(6) condensation of the thus produced glycine with a second formaldehyde to produce serine by a threonine aldolase selected from the group consisting of a threonine aldolase classified in EC 4.1.2.5, a threonine aldolase classified in EC 4.1.2.6, a threonine aldolase classified in EC 4.1.2.48, and a threonine aldolase classified in EC 4.1.2.49; and
(7) deamination of the thus produced serine to produce pyruvate by a serine deaminase classified in EC 4.3.1.17 or a threonine deaminase classified in EC 4.3.1.19, wherein said pyruvate can then be used as a substrate in step (1),
wherein said microorganism contains at least one heterologous nucleic acid molecule encoding the aldolase catalyzing step (1) and overexpresses the enzyme catalyzing step (6) for the condensation of glycine with formaldehyde to form serine.
2 . The microorganism of claim 1 which furthermore overexpresses at least one of the enzymes catalyzing step (3), step (4) or step (5).
3 . The microorganism of claim 1 which is capable of converting methanol into formaldehyde.
4 . The microorganism of claim 1 , wherein said microorganism
(a) converts methanol enzymatically into formaldehyde by a methanol dehydrogenase classified in EC 1.1.1.244 or a methanol dehydrogenase (cytochrome c) classified in EC 1.1.2.7; and/or
(b) converts methanol enzymatically into formaldehyde by an alcohol oxidase that is a methanol oxidase classified in EC 1.1.3.13.
5 . The microorganism of claim 1 , wherein said microorganism is deficient in an enzyme activity converting pyruvate into aspartate semialdehyde.
6 . The microorganism of claim 1 , wherein said microorganism is deficient in the enzyme activity of 3-phosphoglycerate dehydrogenase classified in EC 1.1.1.95.
7 . The microorganism of claim 1 , wherein said microorganism is deficient in the enzyme activity of serine hydroxymethyltransferase classified in EC 2.1.2.1 and/or in the glycine cleavage system (gcvTHP).
8 . The microorganism of claim 1 which is E. coli.
9 . An extract of the microorganism of claim 1 , wherein the extract is a cell-free extract that provides enzymes for catalyzing the sequence of reactions for in vitro incorporation of formaldehyde into carbon compounds that can be assimilated into metabolism.