Gene engineering bacterium for strengthening metabolic pathway of synthesizing tryptophan

A technology of genetically engineered bacteria and tryptophan synthase, applied in genetic engineering, plant genetic improvement, bacteria, etc.

Inactive Publication Date: 2007-10-10
INST OF HYGIENE & ENVIRONMENTAL MEDICINE PLA ACAD OF MILITARY MEDICAL
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

At present, there is no domestic patent application for the use of third-generation gene

Method used

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  • Gene engineering bacterium for strengthening metabolic pathway of synthesizing tryptophan
  • Gene engineering bacterium for strengthening metabolic pathway of synthesizing tryptophan
  • Gene engineering bacterium for strengthening metabolic pathway of synthesizing tryptophan

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0025] A recombinant plasmid containing a mutant of tryptophan synthase gene and phosphoglycerate dehydrogenase gene is prepared by the following method: using pET22b (+) single plasmid multi-gene tandem expression strategy to synthesize tryptophan The enzyme gene trpBA gene was concatenated with the phosphoglycerate dehydrogenase gene mutant M3serA gene to obtain a recombinant plasmid pET-T7 promoter-M3serA-T7promotor-trpBA-T7 terminator, which was named M3BAT-I. The construction process is shown in Figure 1.

[0026] The construction method of the serA gene mutant (M3serA gene) has been published in "Chinese Journal of Biochemistry and Molecular Biology" 2006, No. 10, pages 806-810.

[0027] The method for cloning the trpBA gene has been published on pages 12-14 of the first issue of "Biotechnology Letters" in 2006.

Embodiment 2

[0029] A genetically engineered bacterium with enhanced tryptophan synthesis and metabolism pathway is obtained by the following method: Infect the host bacterium Escherichisa coli 491 with λDE3 phage, and make the chromosome of the lysogenized bacterium named DE3-491 contain a Copy the T7 RNA polymerase gene controlled by lacUV5, and transfer a recombinant plasmid M3BAT-I containing tryptophan synthase gene and phosphoglycerate dehydrogenase gene mutant made in Example 1 into competent DE3-491 Afterwards, it became a genetically engineered bacterium named M3BAT-I+DE3-491 with enhanced tryptophan synthesis and metabolism pathway. Induced expression with IPTG, analyzed by SDS-PAGE, M3BAT-I can express 44kD, 37kD, 29kD proteins in DE3-491 (Figure 2), which correspond to tryptophan synthase β subunit and phosphoglycerate dehydrogenase M3 respectively Mutant, tryptophan synthase alpha subunit. Enzyme activity analysis showed that the activity of tryptophan synthetase was increase...

Embodiment 3

[0031] A kind of genetically engineered bacterium that knocks out the tryptophan synthetic metabolic pathway of tnaA gene and strengthens is obtained by the following method: (1) selecting tnaA gene upstream and downstream flanking sequence fragments and kanamycin resistance gene kan r , connected to construct the tandem gene cassette pET22b-tnaA5'-kan r -tnaA3', and the linearized fragment tnaA5'-kan r -tnaA3' replaces the tnaA gene on the chromosome of a genetically engineered bacterium (M3BAT-I+DE3-491) with enhanced tryptophan synthesis and metabolism pathway through the Red homologous recombination system, resulting in the loss of the normal function of the gene and obtaining tryptophan The acidase-deficient strain was named M3BAT-I+DE3-491ΔT.

[0032] PCR amplification (Figure 4) and sequence determination (Figure 5) confirmed that the tnaA gene had been successfully knocked out.

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Abstract

This invention discloses a kind of genetic engineering bacteria with enhanced tryptophan anabolism, and the recombinant plasmid contained in the genetic engineering bacteria. The recombinant plasmid is prepared by: connecting tryptophan synthetase gene trpBA and phosphoglycerate dehydrogenase gene mutant M3serA gene in series to obtain M3BAT-I, a kind of genetic engineering bacteria with enhanced tryptophan anabolism, expressing induced by IPTG, and analyzing with SDS-PAGE to express tryptophan synthetase beta subunit, phosphoglycerate dehydrogenase M3 mutant, and tryptophan synthetaselpha subunit. After culture of the genetic engineering bacteria with enhanced tryptophan anabolism (tnaA gene removed), the tryptophan content in the supernatant is increased from 2 mg/L to 99 mg/L.

Description

technical field [0001] The invention belongs to the field of biological fermentation engineering and relates to a genetically engineered bacterium with enhanced amino acid synthesis and metabolism pathway. Background technique [0002] L-tryptophan (tryptophan), also known as β-indole-α-alanine, is an aromatic amino acid. L-tryptophan plays an important role in the growth, development and metabolism of humans and animals. At present, L-tryptophan is widely used as a raw material in the processing and preparation of feed, health food, formula diet, amino acid tablets and amino acid injections, etc. [0003] The earliest production of L-tryptophan mainly relied on protein hydrolysis and chemical synthesis, and later fermentation and enzymatic production processes appeared. However, the above-mentioned methods have cumbersome processes, high costs, or the output cannot meet the needs of large-scale industrial production. The problem has caused the price of L-tryptophan to rem...

Claims

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Application Information

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IPC IPC(8): C12N15/63C12N15/52C12N15/53C12N1/21C12P13/22
Inventor 郭长江徐琪寿张绪梅刘云吴健全韦京豫杨继军
Owner INST OF HYGIENE & ENVIRONMENTAL MEDICINE PLA ACAD OF MILITARY MEDICAL
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