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A kind of recombinant Escherichia coli with high production of l-tryptophan and its construction method

A technology for recombining Escherichia coli and Escherichia coli, which is applied in the field of genetic engineering, can solve problems such as doubts about the use of antibiotics, and achieve the effect of simple construction method, good application prospects, and easy use

Active Publication Date: 2020-09-04
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the use of expression plasmids to mediate gene overexpression will inevitably introduce antibiotic resistance genes into E. coli cells and add certain antibiotics during the growth process, causing people's doubts about the use of antibiotics

Method used

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  • A kind of recombinant Escherichia coli with high production of l-tryptophan and its construction method
  • A kind of recombinant Escherichia coli with high production of l-tryptophan and its construction method
  • A kind of recombinant Escherichia coli with high production of l-tryptophan and its construction method

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Experimental program
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Effect test

Embodiment 1

[0048] Construction of embodiment 1 recombinant fragment

[0049] According to the Escherichia coli genome sequence information, design primers pT-aroK-1R and pT-aroK-2F, pT-aroK-1F, pT-aroK-2R (see Table 1), use the above four primers from the Escherichia coli CICC 10303 genome Amplify 600 bp of the homologous arm gene sequence of the aroK gene with the T7 promoter, and fuse the obtained two amplified fragments by fusion PCR technology to obtain the recombinant fragment T7AROK;

[0050] According to the genome sequence information of Escherichia coli, primers pT-pheA-1F, pT-pheA-1R, pT-pheA-2F and pT-pheA-2R were designed to amplify the start codon of pheA gene from the genome of Escherichia coli CICC 10303 The homologous arm gene sequences on both sides of the gene were 600 bp, and the obtained two amplified fragments were fused by fusion PCR technology to obtain the recombinant fragment TACPHE.

[0051] According to the Escherichia coli genome sequence information, design ...

Embodiment 2

[0054] The construction of embodiment 2 recombinant plasmids

[0055] According to the vector pTarget sequence information, design primers pT-aroK-F, pT-aroK-R, and perform PCR to obtain the linearized vector pTT7A containing sgRNA (sequence information is shown in SEQ ID NO.5); design primer pT-pheA-F , pT-pheA-R, carry out PCR to obtain the linearized carrier pTPH containing sgRNA (sequence information is shown in SEQ ID NO.6); design primers pT-mtr-F, pT-mtr-R, carry out PCR to obtain the linearized carrier containing The pTMT of the sgRNA (sequence information is shown in SEQ ID NO.7), pTT7A, pTPH, and pTMT were respectively connected with the recombinant fragments T7AROK, TACPHE, and MTRD to construct a recombinant plasmid, and the BamHI and BsgI double enzyme digestion verification and sequencing confirmed the successful construction of the recombinant plasmid . Plasmid maps of pTT7A, pTPH, pTMT such as figure 1 shown.

Embodiment 3

[0056] Example 3 Construction of Recombinant T7AROK Fragment Escherichia coli

[0057] The pCas9 plasmid containing the cas9 protein was transformed into Escherichia coli CICC 10303. The Kana resistance plate was used to screen the successfully transformed recombinant Escherichia coli CICC 10303-cas9, and then the recombinant plasmid pTargetT7AROK was transformed into Escherichia coli CICC 10303-cas9, and the screening confirmed the successful integration of the T7 promoter, and 0.05mM IPTG was added to induce it at 30°C for 12 hours, and then removed For the recombinant plasmid pTargetT7AROK, primers pT-aroK-2F and pT-aroK-2R were used to select transformants for colony PCR, and a band of about 600 bp appeared. After the sequence was correct, the recombinant Escherichia coli CICC 10303-aroKT with aroK gene promoter T7 was obtained.

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Abstract

The invention discloses recombinant escherichia coli for high yield of L-tryptophan and a construction method thereof, and belongs to the technical field of genetic engineering. According to the method, escherichia coli CICC 10303 is used as an original strain, the CRISPR-Cas9 gene editing technology is adopted, a shikimate kinase encoding gene aroK promoter is replaced by a strong promoter T7; aprephenate dehydrogenase encoding gene pheA promoter is replaced by a weak promoter tac; a tryptophan transporter encoding gene mtr is knocked out, and tryptophan is stopped from transporting back toa cell in the fermenting process. Finally, the escherichia coli genetically engineered bacterium accumulated with L-tryptophan is obtained, the yield can be 36g / L, and foundation is laid for further production of L-tryptophan by metabolic engineering modified escherichia coli.

Description

technical field [0001] The invention relates to a high-production L-tryptophan recombinant Escherichia coli and a construction method thereof, belonging to the technical field of genetic engineering. Background technique [0002] As a very important aromatic amino acid, L-tryptophan is one of the eight essential amino acids for the human body. In organisms, L-tryptophan can synthesize important biologically active substances such as 5-hydroxytryptamine, niacin, pigments, alkaloids, coenzymes, indole acetic acid, etc., which play an important role in the growth and development of humans and animals, and are widely used In food, medicine, feed, etc. The production of L-tryptophan mainly relied on chemical synthesis and protein hydrolysis at the beginning, but these methods had shortcomings such as limited material sources, long cycle time, and complicated processes, so they were gradually eliminated. Due to the characteristics of low cost, wide range of raw material sources,...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N1/21C12N15/113C12N15/70C12N15/90C12P13/22C12R1/19
CPCC07K14/245C12N9/001C12N9/1205C12N15/113C12N15/902C12N2310/10C12N2310/20C12P13/227C12Y103/01012C12Y207/01071
Inventor 刘龙陈泰驰李江华堵国成陈坚
Owner JIANGNAN UNIV
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