Synthetic methylotrophy to liquid fuels and chemicals

Patent metadata

Field	Value
Publication number	US-10059920-B2
Application number	US-201515112364-A
Country	US
Kind code	B2
Filing date	Jan 9, 2015
Priority date	Jan 16, 2014
Publication date	Aug 28, 2018
Grant date	Aug 28, 2018

Abstract

A non-naturally occurring microbe capable of growing in a medium comprising methanol is provided. The methanol contributes to a significant percentage (e.g., at least 40%) of the carbon source for the non-naturally occurring microbe, which expresses heterologous methanol dehydrogenase (MDH) and heterologous ribulose monophosphate (RuMP) pathway enzymes. Methods for producing liquid fuels and chemicals by the non-naturally occurring microbe and methods for preparing the non-naturally occurring microbe are also provided.

First claim

What is claimed:

1. A method for producing a metabolite, comprising
(a) growing a non-naturally occurring microbe in a medium comprising methanol, wherein the non-naturally occurring microbe expresses heterologous Bacillus stearothermophilus methanol dehydrogenase (MDH) and one or more heterologous ribulose monophosphate (RuMP) pathway enzymes, wherein the one or more heterologous RuMP pathway enzymes consist of Bacillus methanolicus 3-hexulose-6-phosphate synthase (HPS) and Bacillus methanolicus 3-hexulose-6-phosphate isomerase (PHI), and wherein the non-naturally occurring microbe is Escherichia coli,
(b) producing a metabolite by the non-naturally occurring microbe, and
(c) incorporating carbon from methanol into the metabolite, wherein at least 40% of the carbon in the metabolite is derived from the methanol.

2. The method of claim 1 , wherein the expression of the one or more heterologous RuMP pathway enzymes is under the control of a formaldehyde responsive promoter.

3. The method of claim 1 , wherein the non-naturally occurring microbe contains a deletion of a frmRAB operon.

4. The method of claim 1 , wherein the non-naturally occurring microbe further expresses one or more heterologous pentose-phosphate pathway (PPP) enzymes.

5. The method of claim 4 , wherein the expression of the one or more heterologous PPP enzymes is under the control of a formaldehyde responsive promoter.

6. The method of claim 4 , wherein the one or more heterologous PPP enzymes consist of heterologous phosphofructokinase (PFK), heterologous fructose bisphosphate aldolase (FBA), heterologous transketolase (TKT), heterologous fructose/sedoheptulose biphosphatase (GLPX), heterologous transaldolase (TAL), heterologous ribose-5-phospate isomerase (RPI) and heterologous ribulose phosphate epimerase (RPE).

7. The method of claim 1 , wherein the non-naturally occurring microbe further expresses one or more heterologous cyclic formaldehyde dissimilation enzymes.

8. The method of claim 7 , wherein the expression of the one or more heterologous cyclic formaldehyde dissimilation enzymes is under the control of a formaldehyde responsive promoter.

9. The method of claim 7 , wherein the one or more heterologous cyclic formaldehyde dissimilation enzymes consist of heterologous glucose-6-phosphate isomerase (PGI), glucose-6-phosphate-1-dehydrogenase (ZWF), 6-phosphogluconolactonase (PGL), and 6-phosphogluconate dehydrogenase (GND).

10. The method of claim 7 , wherein the non-naturally occurring microbe contains a deletion of a phosphogluconate dehydratase gene (edd).

11. The method of claim 1 , wherein the non-naturally occurring microbe further expresses one or more heterologous CO 2 fixation pathway enzymes.

12. The method of claim 11 , wherein the expression of the one or more heterologous CO 2 fixation pathway enzymes is under the control of a formaldehyde responsive promoter.

13. The method of claim 11 , wherein the one or more heterologous CO 2 fixation pathway enzymes consist of heterologous carbonic anhydrase (CA), heterologous formate dehydrogenase (FDH), and heterologous formaldehyde dehydrogenase (FLD).

14. The method of claim 1 , wherein the methanol is the sole carbon source of the non-naturally occurring microbe.

Assignees

• Univ Delaware

Inventors

• Papoutsakis Eleftherios T
• Nicolaou Sergios
• Fast Alan
• Falara Vasiliki
• Bennett Robert Kyle
• Whitaker William Brian
• Sandoval Nicholas Richard
• Gonzalez Jacqueline
• Antoniewicz Maciek

Classifications

• C12P7/16

  Butanols · CPC title

• C12N9/0006

  acting on CH-OH groups as donors (1.1) · CPC title

• C12P7/625

  Polyesters of hydroxy carboxylic acids · CPC title

• C12P7/44

  Polycarboxylic acids · CPC title

• C12N15/52

  Genes encoding for enzymes or proenzymes · CPC title