Recombinant microorganism capable of growing using only carbon dioxide and formic acid and method for producing useful substances using the recombinant microorganism

Patent metadata

Field	Value
Publication number	US-2022017879-A1
Application number	US-202117366269-A
Country	US
Kind code	A1
Filing date	Jul 2, 2021
Priority date	Jul 14, 2020
Publication date	Jan 20, 2022
Grant date	—

Abstract

Disclosed is a recombinant microorganism capable of growing using only carbon dioxide and formic acid by introducing and improving a metabolic pathway for synthesizing pyruvic acid from carbon dioxide and formic acid to enhance pyruvic acid synthesis efficiency and performing additional genetic manipulation, and a method for producing useful substances using the same. Advantageously, the recombinant microorganism is capable of synthesizing pyruvic acid, a C3 organic compound, at a remarkably improved rate, and in particular, grows well even in a medium containing only carbon dioxide and formic acid as carbon sources without a glucose supply, and is thereby capable of synthesizing pyruvic acid and various high value-added compounds using the same as an intermediate product in an economically efficient manner.

First claim

What is claimed is:

1 . A recombinant microorganism, in which a gene encoding a glycine cleavage system transcriptional repressor, pyruvate formate lyase, or phosphoglycerate dehydrogenase is attenuated or deleted from a host microorganism having a formic acid assimilation pathway,
a gene encoding an enzyme involved in a glycine cleavage system reaction is enhancely expressed in the host microorganism having the formic acid assimilation pathway, and a gene encoding formate-tetrahydrofolate ligase, methenyl tetrahydrofolate cyclohydrolase, or methylene-tetrahydrofolate dehydrogenase is introduced into the host microorganism having the formic acid assimilation pathway.

2 . The recombinant microorganism according to claim 1 , wherein the host microorganism is selected from the group consisting of Escherichia, Mannheimia, Rhodobacter and Methylobacterium genera.

3 . The recombinant microorganism according to claim 1 , wherein the expression of the gene encoding the enzyme involved in the glycine cleavage system reaction is enhanced by substituting a native promoter with a strong promoter.

4 . The recombinant microorganism according to claim 3 , wherein the strong promoter is selected from the group consisting of a trc promoter, a tac promoter, a T7 promoter, a lac promoter and a trp promoter.

5 . The recombinant microorganism according to claim 1 , wherein the gene encoding formate-tetrahydrofolate ligase is represented by a nucleotide sequence of SEQ ID NO: 1, the gene encoding methenyl tetrahydrofolate cyclohydrolase is represented by a nucleotide sequence of SEQ ID NO: 2, and the gene encoding methylene-tetrahydrofolate dehydrogenase is represented by a nucleotide sequence of SEQ ID NO: 3.

6 . The recombinant microorganism according to claim 1 , wherein a gene encoding a phosphoenolpyruvate synthase regulatory protein or phosphoribosylglycinamide formyltransferase is further attenuated or deleted from the recombinant microorganism,
expression of a gene encoding phosphoenolpyruvate synthase (ppsA) or H + -translocating NAD(P) transhydrogenase (pntAB) is further enhanced in the recombinant microorganism, and a gene encoding formate dehydrogenase and/or a mutant thereof is further introduced into the recombinant microorganism.

7 . The recombinant microorganism according to claim 6 , wherein expression of the gene encoding the phosphoenolpyruvate synthase (ppsA) or H + -translocating NAD(P) transhydrogenase (pntAB) is enhanced by substituting a native promoter with a strong promoter.

8 . The recombinant microorganism according to claim 7 , wherein the strong promoter is selected from the group consisting of a trc promoter, a tac promoter, a T7 promoter, a lac promoter and a trp promoter.

9 . The recombinant microorganism according to claim 6 , wherein genes encoding one or more selected from the group consisting of the formate-tetrahydrofolate ligase, the methenyl tetrahydrofolate cyclohydrolase, the methylene-tetrahydrofolate dehydrogenase, the formate dehydrogenase, and the formate dehydrogenase mutant are introduced by being cloned into a vector including an origin of replication having 1 to 12 copies.

10 . The recombinant microorganism according to claim 9 , wherein the vector includes an origin of replication having 1 to 5 copies.

11 . The recombinant microorganism according to claim 9 , wherein the gene encoding formate dehydrogenase is represented by a nucleotide sequence of SEQ ID NO: 18, and the gene encoding the formate dehydrogenase mutant is represented by a nucleotide sequence of SEQ ID NO: 21.

12 . The recombinant microorganism according to claim 1 , wherein the recombinant microorganism is capable of producing a C3 compound using only formic acid and carbon dioxide as carbon sources.

13 . The recombinant microorganism according to claim 12 , wherein the C3 compound is pyruvic acid.

14 . A method for producing a C3 compound comprising:
(a) a step of culturing the recombinant microorganism according to claim 1 using formic acid and carbon dioxide as carbon sources to produce a C3 compound; and (b) a step of collecting the produced C3 compound.

15 . The method according to claim 14 , wherein 0.02 to 0.08 mM IPTG is added in the step of culturing the recombinant microorganism.

16 . The method according to claim 14 , wherein the recombinant microorganism is cultured at 31 to 33° C.

17 . The method according to claim 14 , wherein in the step of culturing, the formic acid is maintained at a concentration of 2 to 3 g/l, and the pH is maintained at 6.6 to 7.0.

18 . The method according to claim 14 , wherein the recombinant microorganism is initially cultured at a stirring speed of 450 to 550 rpm, and is then cultured while the stirring speed is increased to a final speed of 700 to 800 rpm.

19 . A method for producing a useful compound using pyruvic acid as an intermediate product comprising:
(a) a step of culturing the recombinant microorganism according to claim 1 using formic acid and carbon dioxide as carbon sources to produce a useful substance having a pyruvic acid as an intermediate product; and (b) a step of collecting the produced useful substance.

20 . The method according to claim 19 , wherein the useful substance is selected from the group consisting of butanol, isobutanol, hexanol, heptanol, octanol, nonanol, decanol, tert-butanol, 1-pentanol, 2-pentanol, 3-pentanol, 2-methyl-1-butanol, 3-methyl-1-butanol, 2-methyl-2-butanol, isobutanol, putrescine, L-ornithine, arginine, polycyclic aromatic hydrocarbons (PAHs), polylactate, polylactate-co-glycolate, polyisovalerate, polyhydroxybutyrate (PHB), 4-hydroxybutyrate, biodiesel, gasoline, olefin, 5-aminovaleric acid, gamma-iminobutyric acid, 3-hydroxypyropionic acid, 3-aminopropionic acid, acrylic acid, 1,3-diaminopropane, caprolactam, threonine, valine, isoleucine, fumaric acid, malic acid, succinic acid, ceramide, astaxanthin, silybin, lycopene, lutein, and retinol.

Assignees

• Korea Advanced Inst Sci & Tech

Inventors

• Lee Sang Yup
• Bang Junho
• Hwang Chang Hun
• Ahn Jung Ho
• Lee Jong An

Classifications

• Y02E50/10

  Biofuels, e.g. bio-diesel · CPC title

• C12Y305/04009

  Methenyltetrahydrofolate cyclohydrolase (3.5.4.9) · CPC title

• C12Y603/04003

  Formate--tetrahydrofolate ligase (6.3.4.3) · CPC title

• C12N9/78

  acting on carbon to nitrogen bonds other than peptide bonds (3.5) · CPC title

• C12N15/70 Primary

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