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Genetically engineered bacterium over-expressing heterogenous glutamine synthetase and construction method thereof

A technology of genetically engineered bacteria and glutamine, applied in the fields of microbial metabolism regulation and genetic engineering, can solve the problems of low enzyme activity, restricted L-glutamine synthesis and production, and reduced activity.

Active Publication Date: 2018-01-19
TIANJIN UNIVERSITY OF SCIENCE AND TECHNOLOGY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In addition, during the fermentation process of amino acids, organic acids, etc., the pH drops due to the accumulation of intracellular products, and the enzyme activity of conventional microbial glutamine synthetase is low at this time
At present, the activity of L-glutamine-producing strains will be sharply reduced due to the adenylation of glutamine synthetase, which severely limits the synthesis and production of L-glutamine.

Method used

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  • Genetically engineered bacterium over-expressing heterogenous glutamine synthetase and construction method thereof
  • Genetically engineered bacterium over-expressing heterogenous glutamine synthetase and construction method thereof
  • Genetically engineered bacterium over-expressing heterogenous glutamine synthetase and construction method thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0034] Example 1: Construction of recombinant bacteria GM34 / pXMJ19glnA, GM34 / pXMJ19glnA1 and GM34 / pXMJ19, and TP607 / pXMJ19glnA and TP607 / pXMJ19

[0035] 1. Construction of overexpression vector pXMJ19glnA

[0036] (1) glnA gene synthesis

[0037]According to the Lactobacillus acidophilus (Lactobacillus acidophilus) glutamine synthetase coding gene sequence (Gene ID: 3251394) published on GENBANK in NCBI, it is codon-optimized (code The sequences before and after suboptimization are respectively shown in SEQ ID NO: 1 and SEQ ID NO: 2), so that it can be efficiently transcribed in Corynebacterium glutamicum. The optimized sequence was sent to Jinweizhi Company for synthesis, and the recombinant plasmid pUC57glnA with glnA gene was obtained, in which the enzyme cutting sites were Hind III and BamH I, and it was stored in Escherichia coli.

[0038] (2) Digestion

[0039] The vector pUC57glnA carrying the gene glnA and the Escherichia coli-Corynebacterium glutamicum shuttle plas...

Embodiment 2

[0074] Embodiment 2: the mensuration of glutamine synthetase enzyme activity

[0075] 1. Determination of protein concentration in crude enzyme solution

[0076] (1) Bacterial culture and expression

[0077] Inoculate the strains Corynebacterium glutamicum GM34 and GM34 / pXMJ19glnA preserved at -80°C on a nutrient-rich petri dish (the strain GM34 / pXMJ19glnA is resistant to chloramphenicol) by streaking on the plate, and culture at a constant temperature of 32°C for 24 hours , pick a well-growing single colony and inoculate it on the corresponding slant medium, culture it at a constant temperature of 32°C for 24 hours, pick a ring and transfer it to the second-generation slant medium, cultivate it at a constant temperature of 32°C for 12 hours, scrape it off and connect it to the filling solution In a round-bottom conical flask with a volume of 30 mL, culture on a shaker at 32°C at 200 rpm. Grow in shake flasks to OD 600 When it was 12-15, adding IPTG with a final concentrati...

Embodiment 3

[0098] Embodiment 3: the fermenter fed-batch fermentation of L-glutamine

[0099] 1. Medium

[0100] (1) Activated slant medium (g / L): glucose 1.0, peptone 10, beef extract 10, NaCl 2.5, yeast extract powder 5, potassium dihydrogen phosphate 1.0, magnesium sulfate 0.4, agar strip 25, pH 7.0-7.2.

[0101] (2) Seed tank culture medium (g / L): oral glucose 25, corn steep liquor 25mL / L, KH 2 PO 4 2.2,V B1 0.01, yeast extract powder 3, magnesium sulfate 0.9, urea 0.3%.

[0102] (3) 5L tank fermentation medium (g / L): corn steep liquor 3mL / L, soybean meal hydrolyzate 20mL / L, KH 2 PO 4 1.5, initial glucose 100, V B1 0.013, MnSO 4 0.01, FeSO 4 0.01, ZnSO 4 0.01, MgSO 4 1.6, (NH 4 ) 2 SO 4 30, pH7.0-7.2.

[0103] 2. Determination of L-glutamine

[0104] (1) Derivation method: Take 1 mL of the treated fermentation broth, centrifuge at 12,000 r / min for 3 min, and take the supernatant to filter the membrane. Take an appropriate amount of the fermented liquid after p...

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Abstract

The invention provides a corynebacterium glutamicum genetically engineered bacterium over-expressing heterogenous glutamine synthetase and a construction method thereof. The construction method comprises the following steps: synthesizing and optimizing a glutamine synthetase glnA derived from lactobacillus acidophilus; by virtue of a restriction enzyme ligation method, connecting the gene glnA onto an escherichia coli-corynebacterium glutamicum shuttle type expression carrier pXMJ19, so that an expression carrier pXMJ19glnA is constructed; and introducing the expression carrier pXMJ19glnA intoa corynebacterium glutamicum GM34 or a corynebacterium glutamicum TP607, so that a genetically engineered bacterium GM34 / pXMJ19glnA or TP607 / pXMJ19glnA is constructed. The genetically engineered bacterium provided by the invention has the advantages that exogenous glutamine synthetase is over-expressed, so that yield of L-glutamine is obviously improved; meanwhile, the synthetase is over-expressed in an L-tryptophan producing strain, so that yield of L-tryptophan is obviously improved.

Description

technical field [0001] The invention relates to a genetic engineering bacterium of Corynebacterium glutamicum overexpressing heterologous glutamine synthetase and its construction method and application, belonging to the technical field of microbial metabolism regulation and genetic engineering. Background technique [0002] Some important enzymatic reactions in cells rely on L-glutamine to provide amino donor transamination reactions, and L-glutamine is deaminated to generate L-glutamic acid. The regeneration of L-glutamine is accomplished by the reaction of L-glutamic acid and ammonium salt catalyzed by glutamine synthetase. The synthesis of intracellular L-tryptophan, glucosamine, hyaluronic acid, etc. involves glutamine transamination reaction, and their massive accumulation requires the rapid synthesis of L-glutamine. [0003] However, glutamine synthetase will undergo adenylylation, resulting in a sharp decrease in its enzymatic activity. AMP covalently binds to the ...

Claims

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Application Information

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IPC IPC(8): C12N1/21C12N15/77C12P13/22C12P13/14C12R1/23
Inventor 谢希贤李燕军李娟陈宁徐庆阳张成林范晓光马倩
Owner TIANJIN UNIVERSITY OF SCIENCE AND TECHNOLOGY
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