Recombinant bacterium for producing L-lysine, constructing method of recombinant bacterium and method for producing L-lysine

A technology of recombinant bacteria and lysine, applied in the field of microbial fermentation, to achieve the effects of increasing production, facilitating popularization and application, and increasing fermentation production

Pending Publication Date: 2019-05-14
BEIJING ZHONGKE EPPEN BIOTECH CO LTD +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, to date, there is no prior art for metabolic engineering of lysine producing strains from the perspective of affecting aspartic acid supply

Method used

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  • Recombinant bacterium for producing L-lysine, constructing method of recombinant bacterium and method for producing L-lysine
  • Recombinant bacterium for producing L-lysine, constructing method of recombinant bacterium and method for producing L-lysine
  • Recombinant bacterium for producing L-lysine, constructing method of recombinant bacterium and method for producing L-lysine

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Experimental program
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Embodiment 1

[0052] The construction of embodiment 1 lysine chassis engineering bacteria

[0053] In this example, Corynebacterium glutamicum wild-type ATCC13032 was used as the starting strain, and the metabolic flow of the branched threonine synthesis pathway was weakened by site-directed mutation of the hom (homoserine dehydrogenase, GenBank: CAF19887.1) gene; site-directed mutation of pyc (pyruvate carboxylase, GenBank: CAF19394.1) gene increases the supply of oxaloacetate, a precursor for lysine synthesis; knockout of the pck (phosphoenolpyruvate carboxykinase, GenBank: CAF20888.1) gene increases pyc* and dapB (dihydrodipicolinate reductase, GenBank: CAF20314.1) gene copy number; plasmid overexpression lysC (aspartokinase, GenBank: CAF18822.1), ddh (diaminopimelic Enzyme, GenBank: CAF21279.1), lysA (diaminoheptanoic acid decarboxylase, GenBank: CAF19884.1) genes, further enhance the synthesis pathway of lysine, and construct lysine-producing chassis engineering bacteria.

[0054] (1)...

Embodiment 2

[0094] Promoter replacement of asparaginase coding gene NCgl2026 in embodiment 2 lysine chassis engineering bacteria

[0095] According to the upstream and downstream sequences of the NCgl2026 gene promoter of Corynebacterium glutamicum ATCC13032 in Genbank and tuf Primers were designed for the promoter sequence (SEQ ID NO.40).

[0096] Using the genomic DNA of Corynebacterium glutamicum ATCC13032 as a template, using P31 and P32 as primers, PCR amplified the upstream homology arm of the NCgl2026 gene promoter; using P33 and P34 as primers to amplify the promoter P tuf ; Use P35 and P36 as primers to amplify the homology arm downstream of the NCgl2026 gene promoter. Using the above-mentioned purified PCR product as a template, using P31 and P36 as primers, and using overlap extension PCR technology (SOE) to amplify, a PCR product of 1800 bp was obtained, which contained a replacement promoter P tuf And fragments of the upstream and downstream homology arms of the replaced pr...

Embodiment 3

[0103] Increased copy of asparaginase coding gene NCgl2026 in embodiment 3 lysine chassis engineering bacteria

[0104] Primers were designed according to the NCgl2026 gene of Corynebacterium glutamicum ATCC13032 in Genbank and its upstream and downstream sequences.

[0105] Using Corynebacterium glutamicum ATCC13032 genomic DNA as a template, using P37 and P38 as primers, PCR amplified the upstream sequence of the target insertion site as the upstream homology arm of the increased copy of the NCgl2026 gene; using P39 and P40 as primers to amplify the NCgl2026 gene; Use P41 and P42 as primers to amplify the downstream sequence of the target insertion site as the downstream homology arm of the increased copy of NCgl2026 gene. Using the purified PCR product as a template, using P37 and P42 as primers, the overlap extension PCR technique (SOE) was used to amplify to obtain a 2778bp PCR product, which was a fragment containing the upstream and downstream homology arms of the target ...

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Abstract

The invention relates to a recombinant bacterium for producing L-lysine, a constructing method of the recombinant bacterium and a method for producing the L-lysine. Compared with a starting bacterium,the recombinant bacterium has the effect of improving the expression and / or activity of asparaginase, and the starting bacterium is a strain capable of accumulating lysine. It is observed through fermentation culture that the recombinant bacterium can have the effect of multiply improving the yield and the yield of the L-lysine is obviously improved.

Description

technical field [0001] The present invention generally relates to the field of microbial fermentation, and specifically relates to a recombinant bacterium producing L-lysine, a construction method thereof and a production method of L-lysine. Background technique [0002] L-Lysine is one of the nine essential amino acids for the human body. It has various physiological functions such as regulating the metabolic balance of the body and promoting growth and development. It is widely used in the fields of food, feed and medicine. In the feed industry, lysine is the first limiting amino acid for the growth of pigs and poultry. Adding L-lysine in the feed can increase the utilization rate of amino acid and protein in the feed, improve the nutritional value of the feed, and promote the growth of livestock and poultry. In the food industry, L-lysine is mainly used in nutrition enhancers and deodorants. In the field of medicine, L-lysine is one of the main components of compound am...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12P13/08C12N1/21C12R1/15
CPCC12P13/08C12R2001/15C12N15/77C12N9/82C12N15/52C12N15/64C12N15/69C12N1/165C12N1/185C12N1/205C12R2001/13C12R2001/72C12R2001/78C12R2001/84C12R2001/865
Inventor 温廷益张琛商秀玲柴欣张芸刘树文王国强李忠财
Owner BEIJING ZHONGKE EPPEN BIOTECH CO LTD
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