Methods and microorganisms for making 2,3-butanediol and derivatives thereof from C1 carbons

Patent metadata

Field	Value
Publication number	US-11111496-B2
Application number	US-201816481799-A
Country	US
Kind code	B2
Filing date	Jan 30, 2018
Priority date	Jan 30, 2017
Publication date	Sep 7, 2021
Grant date	Sep 7, 2021

Abstract

Genetically modified microorganisms that have the ability to convert carbon substrates into chemical products such as 2,3-BDO are disclosed. For example, genetically modified methanotrophs that are capable of generating 2,3-BDO at high titers from a methane source are disclosed. Methods of making these genetically modified microorganisms and methods of using them are also disclosed.

First claim

What is claimed is:

1. A genetically modified microorganism capable of converting a Cl carbon to 2,3-BDO, wherein the microorganism is from the species Methylococcus capsulatus and comprises at least one heterologous gene encoding: i) an acetoin reductase; ii) an alpha-acetolactate decarboxylase; and/or iii) an acetolactate synthase.

2. The microorganism of claim 1 , comprising: (i) a heterologous acetoin reductase; (ii) a heterologous alpha-acetolactate decarboxylase; and/or (iii) a heterologous acetolactate synthase.

3. The microorganism of claim 2 , wherein the acetolactate synthase is non-constitutively expressed.

4. The microorganism of claim 2 , wherein: (i) the acetolactate synthase comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 1 or 19; (ii) the alpha-acetolactate decarboxylase comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 7; and/or (iii) the acetoin reductase comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 9.

5. The microorganism of claim 2 , wherein the acetoin reductase is NADPH-dependent.

6. The microorganism of claim 1 , wherein the heterologous gene(s) is/are integrated by an integration vector into the genome of said microorganism.

7. The microorganism of claim 1 , wherein the heterologous gene(s) is/are expressed on an episomal vector.

8. The microorganism of claim 1 comprising:
(a) a heterologous gene encoding acetolactate synthase that is 5′ in relation to any other heterologous gene;
(b) a heterologous gene encoding acetoin reductase that is 3′ in relation to any other heterologous gene; and/or
(c) a heterologous gene encoding alpha-acetolactate decarboxylase that is neither 5′ or 3′ in relation to any other heterologous gene.

9. The microorganism of claim 1 , wherein the heterologous gene(s) is/are under the control of a switch.

10. The microorganism of claim 1 , wherein: (i) the acetolactate synthase comprises an amino acid sequence at least 90% identical to any one of SEQ ID NOs: 1, 3, or 19; (ii) the alpha-acetolactate decarboxylase comprises an amino acid sequence at least 90% identical to SEQ ID NO:
5 or 7; and/or (iii) the acetoin reductase comprises an amino acid sequence at least 90% identical to any one of SEQ ID NO: 9, 11, or 13.

11. A method of making the microorganism of claim 1 , the method comprising transforming the microorganism with a heterologous nucleic acid comprising a gene encoding an acetoin reductase, an alpha-acetolactate decarboxylase, and/or an acetolactate synthase.

12. A method of making 2,3-BDO, the method comprising:
a) contacting the microorganism of claim 1 with a Cl carbon; and
b) growing the microorganism to produce 2,3-BDO.

13. The method of claim 12 , wherein the Cl carbon is methane.

14. The method of claim 12 , wherein the microorganism is grown at a temperature between 32° C. and 49° C.

15. The method of claim 12 , further comprising contacting the microorganism with media containing at least 1 μM lanthanum before growing the microorganism to produce 2,3-BDO.

16. The method of claim 15 , further comprising diluting the lanthanum in media before growing the microorganism to produce 2,3-BDO.

17. The method of claim 12 , wherein the microorganism comprises a heterologous gene encoding alpha-acetolactate decarboxylase and is grown to produce acetoin.

18. The method of claim 12 , further comprising contacting the 2,3-BDO with a catalyst to produce butadiene or methyl ethyl ketone.

Assignees

• Intrexon Corp
• Precigen Inc

Inventors

• Zhao Xinhua
• Held Mark Anton
• Huynh Tina
• Chao Lily Yuin
• Trinh Na
• Schmalisch Matthias Helmut
• Yeh Bryan
• Kealey James
• Dietzel Kevin Lee

Classifications

• C12N9/1022

  transferring aldehyde or ketonic groups (2.2) · CPC title

• C12P7/18 Primary

  polyhydric · CPC title

• C12N9/88

  Lyases (4.) · CPC title

• C12P7/16

  Butanols · CPC title

• C12N15/52 Primary

  Genes encoding for enzymes or proenzymes · CPC title