Genetically Modified Microorganism For Producing medium-chain lauric acid and/or dodecanedioic acid And Method of Using Thereof

Patent metadata

Field	Value
Publication number	US-2017191092-A1
Application number	US-201615394047-A
Country	US
Kind code	A1
Filing date	Dec 29, 2016
Priority date	Jul 18, 2014
Publication date	Jul 6, 2017
Grant date	—

Abstract

Described herein are genetically-modified microorganisms for producing medium chain lauric acid and/or dodecanedioic acid and methods of using the microorganisms. The microorganisms contain a nucleic acid encoding an Umbellularia californica lauroyl ACP-thioesterase (BTE) operably linked to a promoter.

First claim

What is claimed is:

1 . A genetically modified microorganism, comprising a nucleic acid encoding an Umbellularia californica lauroyl ACP-thioesterase (BTE) operably linked to a promoter, wherein the microorganism produces an increased amount of medium-chain lauric acid and/or dodecanedioic acid as compared to the unmodified parent of the microorganism.

2 . The genetically modified microorganism of claim 1 , wherein the Umbellularia californica lauroyl ACP-thioesterase (BTE) is a modified Umbellularia californica lauroyl ACP-thioesterase (BTE).

3 . The genetically modified microorganism of claim 2 , wherein the modified Umbellularia californica lauroyl ACP-thioesterase (BTE) comprises residues 17-283 of a wild-type Umbellularia californica lauroyl ACP-thioesterase (BTE).

4 . The genetically modified microorganism of claim 3 , wherein the modified Umbellularia californica lauroyl ACP-thioesterase (BTE) further comprises a C-terminal His-tag fusion.

5 . The genetically modified microorganism of claim 2 , wherein the modified Umbellularia californica lauroyl ACP-thioesterase (BTE) comprises residues 17-295 of a wild-type Umbellularia californica lauroyl ACP-thioesterase (BTE).

6 . The genetically modified microorganism of claim 5 , wherein the modified Umbellularia californica lauroyl ACP-thioesterase (BTE) further comprises a C-terminal His-tag fusion.

7 . The genetically modified microorganism of claim 2 , wherein the modified Umbellularia californica lauroyl ACP-thioesterase (BTE) consists of residues 17-283 of a wild-type Umbellularia californica lauroyl ACP-thioesterase (BTE) and a C-terminal His-tag fusion.

8 . The genetically modified microorganism of claim 7 , wherein residue 124 of the wild-type Umbellularia californica lauroyl ACP-thioesterase (BTE), cysteine, is further replaced with threonine.

9 . The genetically modified microorganism of claim 2 , wherein the modified Umbellularia californica lauroyl ACP-thioesterase (BTE) consists of residues 17-295 of a wild-type Umbellularia californica lauroyl ACP-thioesterase (BTE) and a C-terminal His-tag fusion.

10 . The genetically modified microorganism of claim 9 , wherein residue 124 of the wild-type Umbellularia californica lauroyl ACP-thioesterase (BTE), cysteine, is further replaced with threonine.

11 . The genetically modified microorganism of claim 1 , further comprising one or more additional nucleic acids each operably linked to a promoter, each additional nucleic acid encoding a protein selected from the group consisting of an acetyl-CoA carboxylase (ACC), an acetyl-CoA carboxylase carboxyl transferase subunit α (AccA), an acetyl-CoA carboxylase biotin carboxyl carrier protein (AccB), an acetyl-CoA biotin carboxylase (AccC), an acetyl-CoA carboxylase transferase subunit β (AccD), a fatty acid synthase (FAS) subunit, a cytochrome P450 reductase (CPR), a long-chain alcohol oxidase (FAO1), a long-chain alcohol dehydrogenase (FADH), and an adenosine monophosphate-forming acetyl-coenzyme A synthetase (AceCS).

12 . The genetically modified microorganism of claim 1 , further comprising additional nucleic acids each encoding a CPR, a FAO1, and a FADH, respectively.

13 . The genetically modified microorganism of claim 1 , further comprising a loss-of-function mutation in or expressing a lower level of one or more genes selected from the group consisting of a palmitoyl-acyl carrier protein (ACP) thioesterase gene, an acyl-coenzyme A oxidase gene, a citric synthetase (gltA) gene, or an acyl-coenzyme A synthetase (acs) gene.

14 . The genetically modified microorganism of claim 1 , further comprising a loss-of-function mutation in or expressing a lower level of an acyl-coenzyme A oxidase gene.

15 . The genetically modified microorganism of claim 14 , wherein the acyl-coenzyme A oxidase gene is pox2, pox5, or fadD.

16 . The genetically modified microorganism of claim 1 , wherein the microorganism (1) contains a loss-of-function mutation in or expresses a lower level of an acyl-coenzyme A oxidase gene and (2) contains additional nucleic acids each encoding a CPR, a FAO1, and a FADH, respectively.

17 . The genetically modified microorganism of claim 2 , wherein the microorganism (1) contains a loss-of-function mutation in or expresses a lower level of an acyl-coenzyme A oxidase gene and (2) contains additional nucleic acids each encoding a CPR, a FAO1, and a FADH, respectively.

18 . The genetically modified microorganism of claim 17 , wherein the acyl-coenzyme A oxidase gene is fadD.

19 . The genetically modified microorganism of claim 1 , wherein the microorganism is Yarrowia lipolytica or Escherichia coli.

20 . A method of producing a medium-chain lauric acid and/or dodecanedioic acid, the method comprising:
providing the genetically-modified microorganism of claim 1 ; and culturing the microorganism in a culture medium containing glucose or glycerol at pH 6 to 8 under conditions that allow production of the medium-chain lauric acid and/or dodecanedioic acid; whereby the microorganism produces the medium-chain lauric acid and/or dodecanedioic acid.

Assignees

• Ind Tech Res Inst

Inventors

• Hsieh Hsin-Ju
• Lin Yu-Ju
• Wang Hwei-Jiung
• Hsiao Chwan-Deng
• Lee Cheng-Chung
• Chen Ya-Wen

Classifications

• C12Y301/02021

  Dodecanoyl-[acyl-carrier-protein] hydrolase (3.1.2.21) · CPC title

• C12P7/6418

  by hydrolysis of fatty acid esters · CPC title

• C12N9/16

  acting on ester bonds (3.1) · CPC title

• C12P7/6409 Primary

  Fatty acids · CPC title

• C12P7/44

  Polycarboxylic acids · CPC title