Method for building gene engineering strains of producing tryptophan

The technology of a genetically engineered strain and a construction method is applied in the construction field of tryptophan-producing genetically engineered strains, and can solve the problems of high cost, low tryptophan yield and the like

Inactive Publication Date: 2012-10-10
INST OF MICROBIOLOGY HEILONGJIANG ACADEMY OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0006] The present invention aims to solve the problems of low tryptophan yield and high cost in the current tryptophan production method, and provides a method for constructing tryptophan-producing genetically engineered strains

Method used

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  • Method for building gene engineering strains of producing tryptophan
  • Method for building gene engineering strains of producing tryptophan
  • Method for building gene engineering strains of producing tryptophan

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specific Embodiment approach 1

[0012] Specific embodiment one: the construction method of the tryptophan-producing genetically engineered strain of the present embodiment is carried out according to the following steps:

[0013] One, extract the genomic DNA of Escherichia coli JM109 with the bacterial genome extraction kit; Two, use Escherichia coli JM109 genomic DNA as template, carry out PCR amplification with P1, P2 as primer, use 1% agarose coagulation with PCR amplification product A Gel electrophoresis detection, and then use the gel recovery kit to purify to obtain the target gene aroG; 3. Connect the target gene aroG to the pMD18-T vector to construct the recombinant vector pMD18-T-aroG; 4. Use Escherichia coli JM109 genomic DNA as the Template, using P3 and P4 as primers for PCR amplification, PCR amplification product B is detected by 1% agarose gel electrophoresis, and then purified by using a gel recovery kit to obtain the target gene trpBA; 5. Combine the target gene trpBA with The pMD18-T vect...

specific Embodiment approach 2

[0021] Embodiment 2: This embodiment differs from Embodiment 1 in that the primer P1 used in the PCR amplification in step 2 is 5′-CGGAATTCCATCTCTCTCTAGA-3′, and the primer P2 is 5′-CGGGATCCACGTCATTCGTTT-3′. Others are the same as in the first embodiment.

[0022] Primers were designed according to the aroG gene sequence published by NCBI, and appropriate restriction sites EcoRI, BamH I and protective bases were added at both ends.

specific Embodiment approach 3

[0023] Embodiment 3: The difference between this embodiment and Embodiment 1 or 2 is that the primer P3 used for PCR amplification in step 4 is 5′-CGGAATTCTTTTCTTACCCCGGT-3′, and the primer P4 is 5′-CGGGATCCCGCTTGGCAACGTT-3′. Others are the same as in the first or second embodiment.

[0024] Primers were designed according to the trpBA gene sequence published by NCBI, and appropriate restriction sites EcoRI, BamH I and protective bases were added at both ends.

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Abstract

The invention discloses a method for building engineering strains of producing tryptophan and relates to the method for building gene engineering strains of producing tryptophan. The problem to be solved is that the method for producing tryptophan at present is low in yield of tryptophan and high in cost. The method comprises the steps of: carrying out PCR (polymerase chain reaction) augmentation by taking escherichia coli JM109 genome DNA as a template to obtain target genes aroG and trpBA; building pMD18-T-aroG, connecting with a pET-32a vector after enzyme digestion to obtain a connection product X; building pMD18-T-trpBA, connecting with a pET-30a vector after enzyme digestion, thus obtaining a connection product Y; converting the connection product X into a competent cell, and converting the connection product Y into a positive recombinant strain namely an engineering strain of producing tryptophan. The engineering strain built by the method can excessively express DAHP (EC 2.5.1.54,3-deoxy-D-arabino-heptulosonate-7-phosphate synthase) synthetase and tryptophan synthetase, reduce feedback inhibition of a reaction product on the tryptophan synthetase, and increase the yield of the tryptophan. The method is used for producing the tryptophan.

Description

technical field [0001] The invention relates to a method for constructing a tryptophan-producing genetic engineering strain. Background technique [0002] The chemical name of tryptophan is a-amino-p-indolepropionic acid, which has L-type and D-type isomers, and there is also racemic DL-tryptophan. L-Tryptophan is an essential amino acid for humans and animals, participates in the body's protein synthesis and metabolic network regulation, and widely exists in nature. Due to the extensive effects of L-tryptophan and its metabolites on the body, it has certain applications in many fields such as medicine, food industry, and feed industry. [0003] Traditional tryptophan production methods mainly include proteolytic extraction, chemical synthesis and enzymatic conversion. These three methods have the disadvantages of limited material sources, multi-step synthesis process, difficult optical resolution, long cycle time, and expensive substrates, etc., which are greatly restrict...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/70C12N1/21C12R1/19
Inventor 于冲夏海华曲晓军王金英孙建华李思明
Owner INST OF MICROBIOLOGY HEILONGJIANG ACADEMY OF SCI
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