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Recombinant escherichia coli for producing L-glutamine and construction method and application thereof

A technology for recombining Escherichia coli and glutamine, which is applied in the field of genetic engineering, can solve the problems of further improvement of the titer of fermentation method, increase of glutamine refining cost, complicated separation and purification steps, etc., and achieve single reaction system components and high yield The effect of high, short synthetic route

Active Publication Date: 2021-06-25
INST OF MICROBIOLOGY - CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the titer of the fermentation method still needs to be further improved. The components of the fermentation liquid are complex, and the separation and purification steps are relatively complicated. Because glutamine is easy to decompose at room temperature, it can only be carried out at low temperature, which further increases the cost of glutamine refining.

Method used

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  • Recombinant escherichia coli for producing L-glutamine and construction method and application thereof
  • Recombinant escherichia coli for producing L-glutamine and construction method and application thereof
  • Recombinant escherichia coli for producing L-glutamine and construction method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0179] Embodiment 1, construct the recombinant plasmid of overexpressing glutamine synthetase

[0180] 1. Gene CgglnA (nucleotide sequence shown in SEQ ID No.1), BpglnA (nucleotide sequence shown in SEQ ID No.2), Bb1glnA (nucleotide sequence shown in SEQ ID No.3), Bb2glnA (nucleotide sequence shown in SEQ ID No.4) was synthesized by Nanjing GenScript Biotechnology Co., Ltd. The gene fragments were respectively inserted between Xho I and EcoR I of the pYB1a vector after double digestion with Xho I and EcoR IHF, and the ligation product was transformed into Trans1-T1 competent cells (Beijing Quanshijin Biology, catalog number CD501), Spread LB solid plates containing the corresponding ampicillin resistance. Overnight at 37°C, single clones were picked to extract plasmids, and primer pairs P1 (5'-cggcgtcacactttgctatg-3') and P2 (5'-cgtttcacttctgagttcggc-3') were used for PCR verification, and the correct clones were sent for sequencing. The recombinant plasmids with correct seq...

Embodiment 2

[0185] Example 2, construction of L-glutamine-producing Escherichia coli mutants AQ02, AQ04, AQ06

[0186] Use CRISPR-Cas9 gene knockout technology to edit wild-type E. coli K12 to construct E. coli mutants: the Genbank number of knockout E. coli K12 strain BW25113 is NC_000913.3 (update date is 10-Oct-2019) The coding gene (glsA) of glutaminase A shown in the 511641-512573 positions obtained the Escherichia coli mutant AQ03, and the genotype was BW△GlsA; the Genbank number of the knockout Escherichia coli K12 bacterial strain BW25113 was No. 2687070 of NC_000913.3 The coding gene (glnB) of the PII-1 protein shown in -2687408, the GlnE shown in the 3196801-3199641 of Genbank No. NC_000913.3 (having both glutamine synthetase adenyltransferase activity (ATase) and The coding gene (glnE) of the bifunctional enzyme of glutamine synthetase deadenylation activity (ATd) obtains Escherichia coli mutant AQ02, and the genotype is BW△GlnEB; Knockout the SEQ ID No of Escherichia coli K12 ...

Embodiment 3

[0221] Embodiment 3, construct the recombinant escherichia coli producing L-glutamine

[0222] According to Table 2, the recombinant plasmids prepared in Example 1 were respectively transformed into Escherichia coli mutants AQ02, AQ04, AQ06 and Escherichia coli BW25113 constructed in Example 2 by the calcium chloride method. ml) resistant LB plate to screen positive clones (clones that can grow on the plate) to obtain recombinant Escherichia coli as shown in Table 2.

[0223] Table 2 shows the information of each recombinant Escherichia coli used in the above L-glutamine yield detection.

[0224] Table 2. Information of each recombinant Escherichia coli

[0225] recombinant plasmid host bacteria Recombinant Escherichia coli pYB1a-CgGlnA Escherichia coli K12 strain BW25113 pYB1a-CgGlnA / BW pYB1a-CgGlnA Escherichia coli mutant AQ02 pYB1a-CgGlnA / AQ02 pYB1a-CgGlnA Escherichia coli mutant AQ04 pYB1a-CgGlnA / AQ04 pYB1a-CgGlnA Escherichi...

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Abstract

The invention discloses recombinant escherichia coli for producing L-glutamine and a construction method and application thereof. The invention firstly discloses recombinant Escherichia coli, compared with Escherichia coli serving as a starting strain, the expression quantity and / or content and / or activity of genes of glutamine synthetase are increased, and / or the expression quantity and / or content and / or activity of genes of the following proteins are reduced; the protein is at least one of the following: glutaminase A; glutaminase A and glutaminase B; a difunctional enzyme which has the activity of glutamine synthetase adenine transferase and the activity of glutamine synthetase deadenylation at the same time; a myristoyl carrier protein dependent acyltransferase; and a PII-1 protein. The invention further provides a method of preparing L-glutamine. A set of method for efficiently synthesizing free L-glutamine by taking L-glutamic acid as a substrate is developed by overexpressing a gene of glutamine synthetase in escherichia coli and blocking a bypass pathway.

Description

technical field [0001] The invention relates to the field of genetic engineering, in particular to a recombinant Escherichia coli producing L-glutamine and its construction method and application. Background technique [0002] L-glutamine (L-glutamine, L-Gln), alias L-glutamine, scientific name 2-amino-4-carbamoyl butyric acid, is the amide of glutamic acid. Glutamine was first discovered in the human body in 1914. It is the most abundant non-essential amino acid in the human body, and the concentration in the blood can be as high as 0.4-0.9mM. Glutamine is mainly synthesized, stored and released by skeletal muscle, and a small part is synthesized in fat cells, liver and lung; it can be absorbed by intestinal cells, kidney, liver, pancreatic islet cells and immune cells. Glutamine is mainly obtained by de novo synthesis because the glutamine synthesis capacity greatly exceeds the glutamine content in proteins and exists in the cytoplasm. During physiological stress such as...

Claims

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Application Information

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IPC IPC(8): C12N1/21C12N15/70C12P13/14C12R1/19
CPCC12P13/14C12N9/93C12Y603/01002
Inventor 林白雪朱江明陶勇
Owner INST OF MICROBIOLOGY - CHINESE ACAD OF SCI
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