Recombinant microorganisms having a methanol elongation cycle (MEC)

Patent metadata

Field	Value
Publication number	US-9518278-B2
Application number	US-201414211415-A
Country	US
Kind code	B2
Filing date	Mar 14, 2014
Priority date	Mar 14, 2013
Publication date	Dec 13, 2016
Grant date	Dec 13, 2016

Abstract

Provided are microorganisms that catalyze the synthesis of chemicals and biochemicals from a methanol, methane and/or formaldehyde. Also provided are methods of generating such organisms and methods of synthesizing chemicals and biochemicals using such organisms.

First claim

What is claimed is:

1. A recombinant prokaryote or yeast microorganism expressing enzymes that catalyze the conversion described in (i)-(xi), wherein at least one enzyme or the regulation of at least one enzyme that performs a conversion described in (i)-(xi) is heterologous to the microorganism:
(i) the production of acetyl-phosphate and erythrose-4-phosphate (E4P) from fructose-6-phosphate and/or the production of acetyl-phosphate and glyceraldehyde 3-phosphate (G3P) from xylulose 5-phosphate;
(ii) the reversible conversion of fructose-6-phosphate and E4P to sedoheptulose 7-phosphate (S7P) and (G3P);
(iii) the reversible conversion of S7P and G3P to ribose-5-phosphate and xylulose-5-phosphate;
(iv) the reversible conversion of ribose-5-phosphate to ribulose-5-phosphate;
(v) the reversible conversion of ribulose-5-phosphate to xylulose-5-phosphate;
(vi) the reversible conversion of xylulose-5-phosphate and E4P to fructose-6-phosphate and glyceraldehyde-3-phosphate;
(vii) the conversion of formaldehyde and ribulose-5-phosphate to D-arabino-3-Hexulose 6-phosphate;
(viii) the reversible conversion of D-arabino-3-Hexulose 6-phosphate to fructose-6-phosphate;
(ix) the conversion of formaldehyde and xylulose-5-phosphate to glyceraldehyde-3-phosphate and dihydroxyacetone;
(x) the conversion of glyceraldehyde-3-phosphate and dihydroxyacetone to fructose-6-phosphate; and
(xi) the conversion of methanol and a oxidized electron acceptor to formaldehyde and a reduced electron acceptor;
wherein the microorganism produces acetyl-phosphate, or compounds derived from acetyl-phosphate using a carbon source selected from the group consisting of methanol, methane, and formaldehyde and any combination thereof.

2. The recombinant prokaryote or yeast microorganism of claim 1 , wherein the microorganism is derived from an E. coli microorganism.

3. The recombinant prokaryote or yeast microorganism of claim 2 , wherein the E. coli is engineered to express a phosphoketolase.

4. The recombinant prokaryote or yeast microorganism of claim 3 , wherein the phosphoketolase is Fpk, Xpk or a bifunctional F/Xpk enzyme.

5. The recombinant prokaryote or yeast microorganism of claim 1 , wherein the microorganism is engineered to heterologously expresses one or more of the following enzymes:
(a) a phosphoketolase (F/Xpk);
(b) a transaldolase (Tal);
(c) a transketolase (Tkt);
(d) a ribose-5-phosphate isomerase (Rpi);
(e) a ribulose-5-phosphate epimerase (Rpe);
(f) a methanol dehydrogenase (Mdh);
(g) a hexulose-6-phosphate synthase (Hps);
(h) a hexulose-6-phosphate isomerase (Phi);
(i) a dihydroxyacetone synthase (Das); and
(j) a fructose-6-phosphate aldolase (Fsa).

6. The recombinant prokaryote or yeast microorganism of claim 5 , wherein the microorganism is further engineered to express a phosphotransacetylase.

7. The recombinant prokaryote or yeast microorganism of claim 5 , wherein the microorganism is engineered to express a phosphoketolase derived from Bifidobacterium adolescentis.

8. The recombinant prokaryote or yeast microorganism of claim 7 , wherein the phosphoketolase comprises a sequence that is at least 49% identical to SEQ ID NO:2 and has phosphoketolase activity.

9. The recombinant prokaryote or yeast microorganism of claim 5 , wherein the microorganism is engineered to express or over express a hexulose-6-phosphate synthase.

10. The recombinant prokaryote or yeast microorganism of claim 5 , wherein the microorganism is engineered to express or over express a hexulose-6-phosphate isomerase.

11. The recombinant prokaryote or yeast microorganism of claim 5 , wherein the microorganism is engineered to express or over express a dihydroxyacetone synthase.

12. The recombinant prokaryote or yeast microorganism of claim 5 , wherein the microorganism is engineered to express or over express a fructose-6-phosphate aldolase.

13. The recombinant prokaryote or yeast microorganism of claim 1 , wherein the microorganism expresses enzymes selected from the group consisting of:
(a) a phosphoketolase having at least 49% identity to SEQ ID NO:2;
(b) a transaldolase having at least 30% identity to SEQ ID NO:10;
(c) a transketolase having at least 41% identity to SEQ ID NO:12;
(d) a ribose-5-phosphate isomerase having at least 37% identity to SEQ ID NO:8;
(e) a ribulose-5-phosphate epimerase having at least 51% identity to SEQ ID NO:6;
(f) a methanol dehydrogenase having at least 95% identity to SEQ ID NO:4;
(g) a hexulose-6-phosphate synthase having at least 95% identity to SEQ ID NO:14;
(h) a hexulose-6-phosphate isomerase having at least 95% identity to SEQ ID NO:16;
(i) a dihydroxyacetone synthase having at least 95% identity to SEQ ID NO:18;
(j) a fructose-6-phosphate aldolase having at least 95% identity to SEQ ID NO:20; and
(k) any combination of (a)-(j).

Assignees

• Univ California

Inventors

• Liao James C
• Bogorad Igor

Classifications

• C12P9/00 Primary

  Preparation of organic compounds containing a metal or atom other than H, N, C, O, S or halogen {(phosphoglycerides, C12P7/6481)} · CPC title

• C12N15/52 Primary

  Genes encoding for enzymes or proenzymes · CPC title

• C12N15/70

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

• C12Y401/02022

  Fructose-6-phosphate phosphoketolase (4.1.2.22) · CPC title

• C12N15/63

  Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression · CPC title