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Glutamine synthetase and its dedicated expression engineered bacteria and uses

A technology of glutamine and synthase, applied in the direction of enzymes, bacteria, enzymes, etc., can solve the problems of loss, reduction of GS enzyme activity, inactivation and other problems

Inactive Publication Date: 2008-06-04
TSINGHUA UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Adenylylation will reduce or lose the activity of GS enzyme
In addition, the adenylylation modification and inactivation of GS enzyme and its reverse process are also regulated by the concentration of ammonium salt, and the GS enzyme in cells grown to the stationary phase under the culture condition of limited ammonium salt is not modified by adenosine; Under the culture condition of excess ammonium salt, the degree of adenylylation is enhanced, and the activity of GS enzyme is decreased or even inactivated

Method used

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  • Glutamine synthetase and its dedicated expression engineered bacteria and uses
  • Glutamine synthetase and its dedicated expression engineered bacteria and uses
  • Glutamine synthetase and its dedicated expression engineered bacteria and uses

Examples

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Effect test

Embodiment 1

[0026] Embodiment 1, the acquisition of glutamine synthetase site-directed mutation gene

[0027] The glutamine synthetase gene (NCBIGenBank No.: Y13221) of Corynebacterium glutamicum (C. glutamicum) was subjected to site-directed mutation by overlapping PCR method, and the adenylation site tyrosine from the 1213-1215th position of the 5' end Acid codons are mutated into phenylalanine codons, and recognition sites for restriction endonucleases Nde I and Hind III are respectively introduced at both ends of the sequence. The specific method includes the following steps:

[0028] 1. The first round of amplification

[0029] Using the genomic DNA of Corynebacterium glutamicum (C. glutamicum) as a template, primer 1: 5'-ATAAGGGAGGAGTG CATATG GCGTTTGAAA-3' (underlined base is restriction endonuclease Nde I recognition site, SEQ ID No. in the sequence listing: 1) and primer 3:

[0030] 5'-GGTAG Under the guidance of a primer pair composed of GAAGAGGTCCTTGTCCACTGGAG-3' (the base ...

Embodiment 2

[0036] Embodiment 2, the construction of glutamine synthetase special-purpose expression engineering bacterium

[0037] 1. Construction of glutamine synthetase gene recombinant Escherichia coli expression vector

[0038] After the glutamine synthetase mutant gene amplified in Example 1 was digested with restriction endonucleases Nde I and Hind III, it was combined with the plasmid pET containing ampicillin (Amp) resistance marker through double digestion with the same enzymes. -3a (Novagen) for ligation, the ligation product was transformed into E.coli BL21 (DE3) competent cells, and the transformant with Amp resistance was screened to obtain a recombinant expression vector containing glutamine synthetase mutant gene, which was named pET-3a / GSIM, its construction flow chart see figure 1 .

[0039] 2. Transform the recombinant expression vector pET-3a / GSIM into Escherichia coli

[0040] Use the recombinant expression vector pET-3a / GSIM constructed in step 1 with CaCl 2 E.c...

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Abstract

The invention discloses a glutamine synthetase and its special-purpose expression project bacterium and its allocation, which is to provide a glutamine synthetase and its special-purpose expression project bacterium and its allocation. The said glutamine synthetase is prepared by abrupt change of the 405th amino acid residue of amino end to phenylalanine corynebacterium glutamicum. The engineering bacteria prepare the recombination bacteria by importing the recombination expression carrier containing the said glutamine synthetase gene into host bacteria. The glutamine synthetase produced by the engineering bacteria can specially act on NH4+ and aminoglutaric acid, the durability to the high concentration ammonium ion has been distinctively improved because of the dissolution of adenosine acylation modification, the major bottleneck of glutamine with enzymatical production has been solved, and the concentration of the obtained glutamine can reach 47.6g / L. The invention has higher practicability and flackery and has wide application foreground in glutamine commercial manufacture.

Description

technical field [0001] The invention relates to glutamine synthetase and its special expression engineering bacteria and application thereof, in particular to a glutamine synthetase and its special expression engineering bacteria and the application of the engineering bacteria in glutamine production. Background technique [0002] Glutamine is an important food additive and pharmaceutical raw material with high nutritional value and medicinal value. The market demand for glutamine is growing day by day. At present, the market price of glutamine is about RMB 100,000-150,000 per ton. The domestic demand for glutamine is about 5,000 tons per year, and the market value exceeds RMB 500 million. Pharmaceuticals or health care products will create greater economic benefits and have a broad market prospect. The main glutamine producers in the world are Japan, South Korea and China. The fermentation concentration in Japan is as high as 60g / L, and South Korea has reached 50g / L. At p...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/00C12N15/52C12N1/21C12P13/02C12R1/19
Inventor 曹竹安黄星刘铭
Owner TSINGHUA UNIV
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