Escherichia coli transformant for producing itaconate and uses thereof

Patent metadata

Field	Value
Publication number	US-2020325503-A1
Application number	US-201916516464-A
Country	US
Kind code	A1
Filing date	Jul 19, 2019
Priority date	Apr 11, 2019
Publication date	Oct 15, 2020
Grant date	—

Abstract

The present disclosure provides an Escherichia coli transformant and a method for producing itaconate using the Escherichia coli transformant.

First claim

What is claimed is:

1 . An Escherichia coli transformant, comprising at least one plasmid, wherein the at least one plasmid includes a D-xylose dehydrogenase (XDH) gene, a D-xylonolactonase (XL) gene, a 2-keto-3-deoxy-D-xylonate dehydratase (KdxD) gene, and a 2-ketoglutarate semialdehyde dehydrogenase (KGSADH) gene;
wherein an isocitrate dehydrogenase (icd) gene of the Escherichia coli transformant is deleted.

2 . The Escherichia coli transformant according to claim 1 , wherein the at least one plasmid further includes a D-xylonate dehydratase (XD) gene.

3 . The Escherichia coli transformant according to claim 2 , wherein the XDH gene, the XL gene, the KdxD gene, the KGSADH gene, and the XD gene are obtained from Burkholderia xenovorans strains.

4 . The Escherichia coli transformant according to claim 2 , wherein the XDH gene, the XL gene, the XD gene, the KdxD gene, and the KGSADH gene are sequentially constructed from their 5′ ends to 3′ ends in the at least one plasmid.

5 . The Escherichia coli transformant according to claim 1 , which is a transformant of Escherichia coli BW25113 or a transformant of Escherichia coli XL-1 Blue.

6 . The Escherichia coli transformant according to claim 1 , which converts xylose into α-ketoglutaric acid through a nonphosphorylative metabolic pathway.

7 . The Escherichia coli transformant according to claim 1 , wherein a xylose isomerase gene (xylA), a 2-dehydro-3-deoxy-D-pentonate aldolase gene (yjhH), and a 2-keto-3-deoxygluconate aldolase gene (yagE) of the Escherichia coli transformant are deleted.

8 . The Escherichia coli transformant according to claim 1 , further comprising a citrate synthase (gltA), an aconitase B (acn B), and a pyruvate carboxylase (pyc).

9 . The Escherichia coli transformant according to claim 1 , which produces itaconate.

10 . The Escherichia coli transformant according to claim 9 , wherein the Escherichia coli transformant produces itaconate without adding a yeast extract or glutamate.

11 . A method for producing itaconate using the Escherichia coli transformant according to claim 1 , comprising the following steps:
(a) transforming the at least one plasmid into cells of Escherichia coli to obtain the Escherichia coli transformant; (b) providing a substrate as a culture environment, wherein the substrate comprises a carbon source, a nitrogen source, minerals, and vitamins; and (c) cultivating the Escherichia coli transformant at a temperature ranging from 30° C. to 37° C. and a dissolved oxygen ranging from 20% to 30%, and inducing the Escherichia coli transformant to produce itaconate.

12 . The method according to claim 11 , wherein the carbon source in step (b) comprises a glycerol, and the nitrogen source is an ammonium salt.

13 . The method according to claim 12 , wherein the carbon source in step (b) further comprises a xylose, and an initial concentration of the xylose is ranging from 2 g/L to 10 g/L.

14 . The method according to claim 11 , wherein the Escherichia coli transformant produces 1.2 g/L to 2.2 g/L itaconate every 24 hours.

15 . The method according to claim 11 , wherein the Escherichia coli transformant produces 19 g/L to 21 g/L itaconate every 90 hours.

16 . The method according to claim 11 , wherein the Escherichia coli transformant produces itaconate without adding a yeast extract or glutamate.

Assignees

• Univ Nat Tsing Hua
• Chang Chun Plastics Co Ltd
• Chang Chun Petrochemical Co Ltd
• Dairen Chemical Corp

Inventors

• Shen Roa-Pu
• Lu Wei
• Wang Tung-Yu

Classifications

• C12P7/44 Primary

  Polycarboxylic acids · CPC title

• C12N1/20

  Bacteria; Culture media therefor · CPC title

• C12Y604/01001

  Pyruvate carboxylase (6.4.1.1) · CPC title

• C12Y402/01082

  Xylonate dehydratase (4.2.1.82) · CPC title

• C12Y402/01003

  Aconitate hydratase (4.2.1.3) · CPC title