Recombinant corynebacterium glutamicum and application thereof in production of L-glutamine

A technology of Corynebacterium glutamicum and glutamine, applied in the field of bioengineering, can solve the problems of low yield and low yield of L-glutamine production strains, achieve enhanced ATP content, increase yield, and solve imbalance Effect

Active Publication Date: 2021-11-23
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] In order to solve the problems of low production and yield of L-glutamine production strains in the prior art, the present invention provides a strain of Corynebacterium glutamicum N01, which is stored in In the China Center for Type Culture Collection, the deposit number is CCTCC NO: M 2021931

Method used

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  • Recombinant corynebacterium glutamicum and application thereof in production of L-glutamine
  • Recombinant corynebacterium glutamicum and application thereof in production of L-glutamine
  • Recombinant corynebacterium glutamicum and application thereof in production of L-glutamine

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0060] Embodiment 1: the acquisition of Corynebacterium glutamicum (Corynebacterium glutamicum) N01 bacterial strain

[0061] Specific steps are as follows:

[0062] (1) Preparation of bacterial suspension: Pick Corynebacterium glutamicum ATCC 14067 from a glycerol tube at -80°C with an inoculation loop, draw lines in three sections on BHI solid medium, and culture in a 30°C incubator for 24 hours; Take the activated single colony from the plate, inoculate it into a 50 mL shake flask containing 10 mL of BHI liquid medium, and cultivate it to OD at 30°C and 220 rpm 600 About 10 (logarithmic growth phase); centrifuge at 8000rpm for 3min to collect cells, and wash twice with 50mL 0.1M phosphate buffer saline (PBS); finally add a certain amount of PBS solution to make the bacterial concentration about 10 8 CFU / mL, the bacterial suspension was prepared;

[0063] (2) Atmospheric room temperature plasma (ARTP) mutagenesis: use ARTP-IIIS instrument, use high-purity helium as the wor...

Embodiment 2

[0067] Embodiment 2: Construction of Corynebacterium glutamicum (Corynebacterium glutamicum) CGQ01

[0068] Specific steps are as follows:

[0069] (1) Construction of knockout plasmid pK18mobsacB-ΔproB

[0070] From the gene proB sequence (NCBI Genebank accession number BA000036.3) of the γ-glutamate kinase gene on the Corynebacterium glutamicum ATCC 13032 genome published on the NCBI website, determine the sequence position of the proB gene and determine that the missing fragment is 271-836, select proB Gene fragments of about 800 bp in size before and after the deletion fragment serve as upstream and downstream homology arms, and the sequences are shown in SEQ ID NO.6 and SEQ ID NO.7.

[0071] Using primers P1 / P2 and P3 / P4 to amplify the upstream and downstream homology arm fragments of the proB gene base deletion site from the genome of Corynebacterium glutamicum N01, the PCR program is: 95°C, 10min; 95°C, 30s; 58°C, 30s; 72°C, 1min; 72°C, 10min, cycle 30 times. After p...

Embodiment 3

[0080] Embodiment 3: Construction of Corynebacterium glutamicum (Corynebacterium glutamicum) CGQ02

[0081] Specific steps are as follows

[0082] (1) Construction of knockout plasmid pK18mobsacB-ΔNCgl1221

[0083] From the gene NCgl1221 sequence (NCBI Genebank accession number: NC_003450.3) of the glutamate mechanochannel protein on the Corynebacterium glutamicum ATCC 13032 genome published on the NCBI website, determine the sequence position of the NCgl1221 gene and determine that the missing fragment is 401-1203, select Gene fragments of about 900 bp in size before and after the NCgl1221 deletion fragment serve as upstream and downstream homology arms, and the sequences are shown in SEQ ID NO.8 and SEQ ID NO.9.

[0084] According to the method of embodiment 2 step (1), using primers P5 / P6 and P7 / P8 to amplify the upstream and downstream homology arm fragments of the NCgl1221 gene base deletion site from the genome of Corynebacterium glutamicum N01, two obtained After the ...

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Abstract

The invention discloses recombinant corynebacterium glutamicum and application thereof in production of L-glutamine, and belongs to the technical field of bioengineering. The activity of gamma-glutamate kinase, glutamic acid secretion mechanical channel protein and adenosine acylase of glutamine synthetase in cells of the recombinant corynebacterium glutamicum is lost, and plasmid pXMJ19 is used for co-expressing the glutamine synthetase from saccharomyces cerevisiae and polyphosphate kinase from escherichia coli. The invention provides the recombinant corynebacterium glutamicum CGQ03 / pXMJ19-glnASc-ppkEc capable of producing the L-glutamine at high yield; the recombinant corynebacterium glutamicum is inoculated into a 5L fermentation tank and is fermented for 66h, so that the yield of the L-glutamine in fermentation liquid reaches 73.5 + / -3.1g / L and the conversion rate of glucosamine reaches 0.368 + / -0.034.

Description

technical field [0001] The invention relates to a recombinant Corynebacterium glutamicum and its application in producing L-glutamine, belonging to the technical field of bioengineering. Background technique [0002] L-glutamine, also known as 2,5-diamino-5-oxopentanoic acid, is a semi-essential amino acid. It is not only one of the basic amino acids for protein synthesis, but also an amino donor for the synthesis of many important compounds such as purine and pyrimidine. Due to its special nutritional and immune functions, L-glutamine has been widely used in the fields of food, medicine and feed in recent years. There are mainly the following aspects: (1) Food and health products. It can improve human immunity, ensure the balance of nitrogen metabolism, maintain normal digestive function, and regulate the normal acid-base balance of the human body. (2) Clinical drugs. It is used for the repair treatment of diseases such as gastrointestinal ulcers and the recovery treatm...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/21C12N15/77C12P13/14C12R1/15
CPCC07K14/34C12Y207/02011C12Y207/07042C12Y603/01002C12Y207/04001C12N9/93C12N9/1241C12N9/1229C12N9/1217C12N15/77C12P13/14Y02E50/10
Inventor 饶志明吕青兰徐美娟杨套伟张显
Owner JIANGNAN UNIV
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