Genetically modified microorganism for producing long-chain dicarboxylic acid and method of using thereof

Patent metadata

Field	Value
Publication number	US-9695404-B2
Application number	US-201514802282-A
Country	US
Kind code	B2
Filing date	Jul 17, 2015
Priority date	Jul 18, 2014
Publication date	Jul 4, 2017
Grant date	Jul 4, 2017

Abstract

Described herein are genetically-modified microorganisms for producing long-chain dicarboxylic acids and methods of using the microorganisms. The microorganisms contain a first nucleic acid encoding an Umbellularia californica lauroyl ACP-thioesterase (BTE) operably linked to a promoter or a second nucleic acid encoding a Cocos nucifera lauroyl ACP-thioesterase (FatB3) operably linked to a promoter.

First claim

What is claimed is:

1. A genetically modified microorganism, comprising a first nucleic acid encoding an Umbellularia californica lauroyl acyl carrier protein (ACP) thioesterase (BTE) operably linked to a promoter, a second nucleic acid encoding a Cocos nucifera lauroyl ACP-thioesterase (FatB3) operably linked to a promoter, and one or more exogenous nucleic acids each operably linked to a promoter, each exogenous nucleic acid encoding a protein selected from the group consisting of an acetyl-CoA carboxylase (ACC), an acetyl-CoA carboxylase carboxyl transferase subunit a (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 (FAQ1), a long-chain alcohol dehydrogenase (FADH), and an adenosine monophosphate-forming acetyl-coenzyme A synthetase (AceCS), wherein the microorganism is Escherichia coli and produces an increased amount of long-chain dicarboxylic acids as compared to the unmodified parent of the microorganism, wherein the genetically modified microorganism 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.

2. A genetically modified microorganism comprising a first nucleic acid encoding an Umbellularia californica lauroyl ACP thioesterase (BTE) operably linked to a promoter, and a second nucleic acid encoding a Cocos nucifera lauroyl ACP thioesterase (FatB3) operably linked to a promoter, and further contains a loss-of-function mutation in or expresses a lower level of one or more genes selected from the group consisting of a palmitoyl-ACP thioesterase gene, an acyl-coenzyme A oxidase gene, a citric synthetase (gltA) gene, or an acyl-coenzyme A synthetase (acs) gene, wherein the microorganism is Yarrowia lipolytica and produces an increased amount of long-chain dicarboxylic acids as compared to the unmodified parent of the microorganism, 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) further contains three exogenous nucleic acids each operably linked to a promoter and encoding a CPR, a FAO1, and a FADH, respectively.

3. The genetically modified microorganism of claim 2 , wherein the microorganism contains a loss-of-function mutation in or expresses a lower level of an ACP thioesterase gene.

4. The genetically modified microorganism of claim 3 , wherein the microorganism contains additional exogenous nucleic acids each operably linked to a promoter and encoding an AccD and a FAS subunit, respectively.

5. The genetically modified microorganism of claim 1 , wherein the one or more exogenous nucleic acids include three nucleic acids each encoding a CPR, a FAO1, and a FADH, respectively.

6. The genetically modified microorganism of claim 5 , wherein the one or more exogenous nucleic acids further include nucleic acids each encoding an AccA, an AccB, and an AccD.

7. The genetically modified microorganism of claim 5 , further comprising a loss-of-function mutation in or expressing a lower level of an acs gene or a gltA gene.

8. The genetically modified microorganism of claim 5 , wherein the BTE is BTEΔNC containing the sequence of SEQ ID NO:22 and the acyl-coenzyme A oxidase gene is fadD.

9. The genetically modified microorganism of claim 6 , wherein the BTE is BTEΔNC containing the sequence of SEQ ID NO:22 and the acyl-coenzyme A oxidase gene is fadD.

10. The genetically modified organism of claim 2 , wherein the acyl-coenzyme A oxidase gene is pox2 or pox5.

11. The genetically modified organism of claim 3 , wherein the acyl-coenzyme A oxidase gene is pox2 or pox5.

12. The genetically modified organism of claim 4 , wherein the acyl-coenzyme A oxidase gene is pox2 or pox5.

13. A method of producing a long-chain dicarboxylic 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 a long-chain dicarboxylic acid; whereby the microorganism produces the long-chain dicarboxylic acid.

14. The method of claim 13 , further comprising collecting the long-chain dicarboxylic acid.

15. The method of claim 14 , wherein the long-chain dicarboxylic acid is C10-C18 dicarboxylic acid.

16. The method of claim 15 , wherein the long-chain dicarboxylic acid is C12 dicarboxylic acid.

Assignees

• Ind Tech Res Inst

Inventors

• Hsieh Hsin-Ju
• Chien Liang-Jung
• Wang Jia-Hung
• Lin Yu-Ju

Classifications

• C12P7/6418

  by hydrolysis of fatty acid esters · CPC title

• C12P7/44

  Polycarboxylic acids · CPC title

• C12Y301/02014

  Oleoyl-[acyl-carrier-protein] hydrolase (3.1.2.14), i.e. ACP-thioesterase · CPC title

• C12N9/16 Primary

  acting on ester bonds (3.1) · CPC title

• C12Y301/02021

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