Escherichia coli for synthesizing l-valine, construction method and use thereof

Patent metadata

Field	Value
Publication number	US-12473528-B2
Application number	US-202418420028-A
Country	US
Kind code	B2
Filing date	Jan 23, 2024
Priority date	Mar 21, 2022
Publication date	Nov 18, 2025
Grant date	Nov 18, 2025

Abstract

The invention provides an Escherichia coli for synthesizing L-valine, a construction method and use thereof. The Escherichia coli of the invention is designated as Escherichia coli W3110 and was deposited in China Center for Type Culture Collection (Address: Bayi Road, Wuchang District, Wuhan City, Hubei Province) under the Accession No. CCTCC M 2022293 on Mar. 18, 2022. The recombinant Escherichia coli takes Escherichia coli as a starting strain, and a transcription regulation factor is overexpressed to obtain a recombinant Escherichia coli . The recombinant Escherichia coli for synthesizing L-valine of the invention is fermented in a 5 L fermentor with trace dissolved oxygen to test strains, the yield of L-valine reaches 112 g/L, and the OD of the bacterium is 104.

First claim

What is claimed is:

1 . An Escherichia coli for synthesizing L-valine, wherein the Escherichia coli is designated as Escherichia coli W3110 and was deposited in China Center for Type Culture Collection (Address: Bayi Road, Wuchang District, Wuhan City, Hubei Province) under the Accession No. CCTCC M 2022293 on Mar. 18, 2022.

2 . A recombinant Escherichia coli for synthesizing L-valine, wherein the recombinant Escherichia coli is an Escherichia coli for synthesizing L-valine designated as Escherichia coli W3110 as deposited in China Center for Type Culture Collection (Address: Bavi Road, Wuchang District, Wuhan City, Hubei Province) under the Accession No. CCTCC M 2022293 on Mar. 18, 2022, and comprising further modifications as follows:
dicarboxylic acid reductoisomerase gene ilvC, dihydroxy acid dehydratase gene ilvD and branched chain amino acid aminotransferase gene ilvE are overexpressed; branched-chain amino acid transporter gene brnQ is knocked out, and branched-chain amino acid output protein gene brnFE is integrated into the site of the knocked-out branched-chain amino acid transporter gene brnQ; phosphogluconate dehydratase gene edd and KHG/KDPG aldolase gene eda are integrated; and a transcription regulation factor is overexpressed to obtain the recombinant Escherichia coli, wherein the transcription regulation factor is a positive transcription regulation factor and/or a negative transcription regulation factor; the positive transcription regulation factor is selected form the group consisting of a DNA binding transcription double regulator pdhR, a DNA binding transcription double regulator crp and a DNA binding transcription double regulator lrp; and the negative transcription regulation factor is RNA polymerase sigma factor rpoS, and wherein the transcription regulation factor is overexpressed from a ptrc99A or ptrc28A vector.

3 . A method for fermentation synthesis of L-valine, comprising:
providing the recombinant Escherichia coli according to claim 2 ; and performing a fermentation synthesis by fermenting a fermentation medium with the recombinant Escherichia coli to synthesize L-valine.

4 . The method according to claim 3 , wherein the fermentation synthesis is carried out in conditions of: pyruvate as a precursor, a dissolved oxygen content of 10-20%, a fermentation time of 24-72 h, a temperature of 35-40° C., and a rotational speed of 210-230 rpm.

5 . A method for constructing a recombinant Escherichia coli for synthesizing L-valine, comprising:
providing an Escherichia coli for synthesizing L-valine designated as Escherichia coli W3110 as deposited in China Center for Type Culture Collection (Address: Bavi Road, Wuchang District, Wuhan City, Hubei Province) under the Accession No. CCTCC M 2022293 on Mar. 18, 2022, and in the Escherichia coli for synthesizing L-valine further introducing the following: overexpressing a transcription regulation factor, wherein the transcription regulation factor is a positive transcription regulation factor and/or a negative transcription regulation factor; the positive transcription regulation factor is selected from the group consisting of a DNA binding transcription double regulator pdhR, a DNA binding transcription double regulator crp and a DNA binding transcription double regulator Irp; and the negative transcription regulation factor is RNA polymerase sigma factor rpoS; and/or overexpressing dicarboxylic acid reductoisomerase gene ilvC, dihydroxy acid dehydratase gene ilvD and branched chain amino acid aminotransferase gene ilvE; and/or knocking out branched-chain amino acid transporter gene brnQ, integrating branched-chain amino acid output protein gene brnFE into the site of the knocked-out branched-chain amino acid transporter gene brnQ, and making double copies of the brnFE gene; and/or integrating phosphogluconate dehydratase gene edd and KHG/KDPG aldolase gene eda to obtain the recombinant Escherichia coli.

Assignees

• Univ Jiangnan

Inventors

• Rao Zhiming
• Hao Yanan
• Xu Meijuan
• Pan Xuewei
• You Jiajia
• Yang Taowei
• Zhang Xian
• Shao Mingling

Classifications

• C12Y402/01012

  Phosphogluconate dehydratase (4.2.1.12) · CPC title

• C12Y402/01009

  Dihydroxy-acid dehydratase (4.2.1.9), i.e. acetohydroxyacid dehydratase · CPC title

• C12Y206/01042

  Branched-chain-amino-acid transaminase (2.6.1.42) · CPC title

• C12P13/08

  Lysine; Diaminopimelic acid; Threonine; Valine · CPC title

• C12N15/70

  Vectors or expression systems specially adapted for E. coli · CPC title