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Recombinant corynebacterium glutamicum in high L-lysine yield and construction method thereof

A technology of Corynebacterium glutamicum and its construction method, which is applied in the field of Corynebacterium glutamicum engineering bacteria and its construction, can solve problems such as doubts about the use of antibiotics, and achieve the effects of simple construction method, good application prospects, and reduced accumulation

Active Publication Date: 2019-02-22
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

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

Method used

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  • Recombinant corynebacterium glutamicum in high L-lysine yield and construction method thereof
  • Recombinant corynebacterium glutamicum in high L-lysine yield and construction method thereof
  • Recombinant corynebacterium glutamicum in high L-lysine yield and construction method thereof

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Embodiment 1

[0034] Construction of embodiment 1 recombinant fragment

[0035] The artificially synthesized nucleotide sequence is shown in SEQ ID NO.1 as the encoding gene gapC of glyceraldehyde phosphate dehydrogenase. According to Corynebacterium glutamicum ATCC13032 genome DNA sequence information, design primer gapC-1-F, gapC-1-R, gapC-3-F and gapC-3-R (see table 1), use above-mentioned primer to get from glutamic acid The homology arm gene sequences on both sides of the insertion position of gapC were amplified and inserted into the genome of Corynebacterium CICC 23604, and the obtained three amplified fragments were fused by fusion PCR technology to obtain the recombinant fragment GA1.

[0036] According to the genome DNA sequence information of Corynebacterium glutamicum ATCC13032, design primers thrB-1-F, thrB-1-R, thrB-2-F and thrB-2-R, use the above primers from the genome of Corynebacterium glutamicum CICC 23604 800 bp of homologous arm gene sequences on both sides of the thrB...

Embodiment 2

[0039] The construction of embodiment 2 recombinant plasmids

[0040]According to the vector pEC-XK99 sequence information, design primers ztgap-F, ztgap-R, ztthr-F and ztthr-R, use the above primers to carry out PCR, obtain the linearized vector pEC-XK99-G containing sgRNA respectively (nucleotide sequence As shown in SEQ ID NO.3) and pEC-XK99-T (nucleotide sequence shown in SEQ ID NO.4), they were ligated with the recombinant fragments GA1 and TH2 respectively to construct recombinant plasmids. After Eco32I, XmaJI double enzyme digestion verification and sequencing, it was confirmed that the recombinant plasmids pEC-XK99GA1 and pEC-XK99TH2 were successfully constructed.

Embodiment 3

[0041] The construction of embodiment 3 recombinant GA1 fragment Corynebacterium glutamicum

[0042] The pFSC plasmid containing the cas9 protein (nucleotide sequence shown in SEQ ID NO.5) was transformed into Corynebacterium glutamicum CICC 23604. The successfully transformed recombinant Corynebacterium glutamicum was screened with a kana resistance plate and named CGC9, and then the recombinant plasmid pEC-XK99GA1 was transformed into Corynebacterium glutamicum CGC9, and the expression of glyceraldehyde phosphate dehydrogenase derived from Acetobutylicum was obtained. The primers gapC-2-F and gapC-2-R were used to select the transformants for colony PCR, a 1005bp band appeared, and the recombinant Corynebacterium glutamicum was successfully constructed, named CGC9-EGA.

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Abstract

The invention discloses recombinant corynebacterium glutamicum in high L-lysine yield and a construction method thereof, and belongs to the technical field of genetic engineering. According to the recombinant corynebacterium glutamicum disclosed by the invention, a phosphoglyceraldehyde dehydrogenase encoding gene gapC derived from Clostridium acetobutylicum ATCC824 is heterogeneously expressed bytaking corynebacterium glutamicum CICC 23604 as an original strain and adopting CRISPR-Cas9 gene editing technology, and supply of intracellular NADPH (Nicotinamide Adenine Dinucleotide Phosphate) isincreased; a homoserine kinase encoding gene thrB is knocked out, accumulation of a byproduct-threonine during a fermentation process is reduced, corynebacterium glutamicum genetically engineered bacteria with the accumulation of L-lysine can be obtained, the yield is up to 42g / L, and foundation is laid for producing the L-lysine through further metabolism engineering reform of the corynebacterium glutamicum.

Description

technical field [0001] The invention relates to a high-production L-lysine Corynebacterium glutamicum engineering bacterium and a construction method thereof, belonging to the technical field of genetic engineering. Background technique [0002] L-Lysine is one of the eight essential amino acids, which are necessary for humans and animals and cannot be synthesized by themselves. L-lysine has the functions of balancing amino acid composition, regulating internal metabolic balance, improving the body's absorption and utilization of cereal protein, and promoting body growth and development, so it is widely used in feed, medicine and food industries. [0003] At present, the production of L-lysine mainly includes chemical synthesis, proteolysis and microbial fermentation. Among them, microbial fermentation has low production cost, high production intensity, and little environmental pollution, so it has become the current industrial production of L-lysine. the broadest method. ...

Claims

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Application Information

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IPC IPC(8): C12N1/21C12N15/53C12N15/77C12P13/08C12R1/15
CPCC12N9/0008C12N15/77C12P13/08C12Y102/01012
Inventor 刘龙陈泰驰李江华堵国成陈坚
Owner JIANGNAN UNIV
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