Recombinant microorganism having heterologous genes introduced thereto and method for producing useful material from formic acid and carbon dioxide using same microorganism

Patent metadata

Field	Value
Publication number	US-11214816-B2
Application number	US-201716472876-A
Country	US
Kind code	B2
Filing date	Nov 20, 2017
Priority date	Dec 27, 2016
Publication date	Jan 4, 2022
Grant date	Jan 4, 2022

Abstract

The present invention relates to a recombinant microorganism having heterologous genes introduced thereto and a method for producing a useful material from formic acid and carbon dioxide using the microorganism. The present invention provides a novel microorganism having a cyclic metabolic pathway introduced thereto through which C3 or higher carbon organic compounds can be synthesized from formic acid and carbon dioxide, whereby carbon dioxide rich in nature and formic acid that is of low toxicity and suitable for anabolic reaction in view of reaction kinetics and which can be easily and rapidly synthesized from carbon dioxide can be used to effectively synthesize the C3 organic compound pyruvic acid from which various high-value added compound can be synthesized.

First claim

The invention claimed is:

1. A recombinant microorganism having improved assimilation from formic acid and carbon dioxide, obtained by introducing a gene encoding an enzyme involved in a formic acid assimilation pathway,
wherein the microorganism is Escherichia coli capable of synthesizing a C3 or larger carbon compound under the formic acid assimilation pathway combined with a cyclic pathway for fixing carbon dioxide,
wherein the gene encoding an enzyme involved in a formic acid assimilation pathway comprises:
the gene encoding formate-tetrahydrofolate ligase comprising the nucleic acid molecule of SEQ ID NO: 7;
the gene encoding methenyl tetrahydrofolate cyclohydrolase comprising the nucleic acid molecule of SEQ ID NO: 8; and
the gene encoding methylene-tetrahydrofolate dehydrogenase comprising the nucleic acid of SEQ ID NO: 9 or SEQ ID NO: 19; and
wherein the gene encoding an enzyme involved in the cyclic pathway for fixing carbon dioxide comprises one or more genes selected from:
the gene encoding a serine-glyoxylate transaminase comprising the nucleic acid of SEQ ID NO:
6, SEQ ID NO: 13, SEQ ID NO: 16, or SEQ ID NO: 21;
the gene encoding a malyl-CoA lyase comprising the nucleic acid of SEQ ID NO: 5, SEQ ID NO:
14, or SEQ ID NO: 15; and
the gene encoding a malate-CoA ligase comprising the nucleic acid of SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 11, or SEQ ID NO: 12.

2. The recombinant microorganism according to claim 1 , wherein the gene encoding an enzyme involved in a cyclic pathway for fixing carbon dioxide further comprises:
the gene encoding a succinyl-CoA:(S)-malate CoA-transferase comprising the nucleic acid of SEQ ID NO: 17 or SEQ ID NO: 18;
the gene encoding a phosphoenolpyruvate carboxykinase comprising the nucleic acid of SEQ ID NO: 20; and/or
the gene encoding hydroxypyruvate reductase comprising the nucleic acid of SEQ ID NO: 10.

3. The recombinant microorganism according to claim 1 , wherein a gene encoding pyruvate formate lyase or a recombinant vector containing the gene is further introduced into or overexpressed in the recombinant microorganism.

4. The recombinant microorganism according to claim 3 , wherein the gene encoding pyruvate formate lyase is the nucleic acid molecule of SEQ ID NO: 22.

5. The recombinant microorganism according to claim 3 , wherein the gene is overexpressed by any one strong promoter selected from the group consisting of a trc promoter, a tac promoter, a T7 promoter, a lac promoter and a trp promoter.

6. The recombinant microorganism according to claim 1 , wherein a glycine cleavage complex is enhanced, amplified or further introduced into the recombinant microorganism.

7. The recombinant microorganism according to claim 6 , wherein the glycine cleavage complex is the nucleic acid molecule of SEQ ID NO: 63.

8. The recombinant microorganism according to claim 6 , wherein the expression of the genes constituting the glycine cleavage complex is enhanced by introducing a plasmid containing the genes constituting the glycine cleavage complex, or substituting an intrinsic promoter of the gene with any one strong promoter 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 a gcvR gene is further deleted from the recombinant microorganism.

10. The recombinant microorganism according to claim 9 , wherein the gcvR gene is the nucleic acid molecule of SEQ ID NO: 66.

11. The recombinant microorganism according to claim 1 , wherein serine deaminase is enhanced, amplified or further introduced into the recombinant microorganism.

12. The recombinant microorganism according to claim 11 , wherein the serine deaminase is the nucleic acid molecule of SEQ ID NO: 69.

13. The recombinant microorganism according to claim 1 , wherein the recombinant microorganism is capable of biosynthesizing pyruvate, glycine and serine assimilated from formic acid and carbon dioxide.

14. A method for producing a useful compound having a C3 compound as an intermediate product comprising:
(a) culturing the recombinant microorganism according to claim 1 with formic acid and carbon dioxide as a carbon source to produce a useful substance having a C3 compound as an intermediate product; and
(b) recovering the resulting useful substance.

15. The method according to claim 14 , wherein the C3 compound is pyruvate and 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, putrescine, L-ornithine, arginine, polycyclic aromatic hydrocarbons (PAHs), polylactate, polylactate-co-glycolate, poly(2-hydroxyisovalerate-co-lactate), polyhydroxybutyrate (PHB), 4-hydroxybutyrate, biodiesel, gasoline, olefin, 5-aminovaleric acid, gamma-butyric acid, 3-hydroxypropionic acid, 3-aminopropionic acid, acrylic acid, 1,3-aminopropane, caprolactam, threonine, valine, isoleucine, fumaric acid, malic acid, succinic acid, ceramide, astaxanthin, silybin, lycopene, lutein, and retinol.

16. A method for producing a C3 compound comprising:
(a) culturing the recombinant microorganism according to claim 1 with formic acid and carbon dioxide as a carbon source to produce a C3 compound; and
(b) recovering the produced C3 compound.

Assignees

• Korea Advanced Inst Sci & Tech

Inventors

• Lee Sang Yup
• Bang Junho

Classifications

• C12P13/001

  Amines; Imines · CPC title

• C12P7/04 Primary

  acyclic · CPC title

• C12Y603/04003

  Formate--tetrahydrofolate ligase (6.3.4.3) · CPC title

• C12Y305/04009

  Methenyltetrahydrofolate cyclohydrolase (3.5.4.9) · CPC title

• C12Y208/03015

  Succinyl-CoA:(R)-benzylsuccinate CoA-transferase (2.8.3.15) · CPC title