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Coryneform bacteria capable of producing lysine at high yield as well as construction method and application thereof

A technology for high-yielding lysine and coryneform bacteria, applied in the field of bioengineering, can solve the problems of poor fermentation performance, inability to meet large-scale industrial production, and low conversion rate of L-lysine, achieve high activity, and enhance initial transcription Efficiency, increased level of effect

Pending Publication Date: 2020-04-03
新疆梅花氨基酸有限责任公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the fermentation performance of L-lysine strains is still poor, and the conversion rate of L-lysine is still low, which still cannot meet the needs of large-scale industrial production.

Method used

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  • Coryneform bacteria capable of producing lysine at high yield as well as construction method and application thereof
  • Coryneform bacteria capable of producing lysine at high yield as well as construction method and application thereof
  • Coryneform bacteria capable of producing lysine at high yield as well as construction method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0058] Example 1. Construction of recombinant vector containing type 1 mutant lysC promoter and its introduction in Corynebacterium glutamicum

[0059] Take ATCC 13032 genome as template, use PlysC mut1 -1 / PlysC mut1 -2 primer pair for PCR amplification to obtain the upstream fragment PlysC mut1 -up; Take ATCC 13032 genome as template, use PlysC mut1 -3 / PlysC mut1 -4 primer pair for PCR amplification to obtain fragment PlysC mut1 -dn. Take the above two-fragment mixture as a template, and use PlysC mut1 -1 / PlysC mut1 -4 primer pair for PCR amplification to obtain PlysC containing the target mutation mut1 Fragment. The fragment was double-cut with BamHI and PstI, and the vector pK18mobsacB was double-cut with the same enzyme. The two digested products were ligated with T4 DNA Ligase and transformed into Trans1 T1 competent cells to obtain the recombinant plasmid pK18mobsacB-lysC mut1 .

[0060] Competent cells of MHZ-0912-1 were prepared according to the Gubang classic method (C. g...

Embodiment 2

[0061] Example 2. Construction of recombinant vector containing type 2 mutant lysC promoter and introduction into Corynebacterium glutamicum

[0062] Take ATCC 13032 genome as template, use PlysC mut2 -1 / PlysC mut2 -2 primer pair for PCR amplification to obtain the upstream fragment PlysC mut2 -up; Take ATCC 13032 genome as template, use PlysC mut2 -3 / PlysC mut2 -4 primer pair for PCR amplification to obtain fragment PlysC mut2 -dn. Take the above two-fragment mixture as a template, and use PlysC mut2 -1 / PlysC mut2 -4 primer pair for PCR amplification to obtain PlysC containing the target mutation mut2 Fragment. The fragment was double-cut with BamHI and PstI, and the vector pK18mobsacB was double-cut with the same enzyme. The two digested products were ligated with T4 DNA Ligase and transformed into Trans1T1 competent cells to obtain the recombinant plasmid pK18mobsacB-lysC mut2 .

[0063] Competent cells of MHZ-0912-1 were prepared according to the Gubang classic metho...

Embodiment 3

[0064] Example 3. Construction of a recombinant vector containing a type 1 mutant pyc promoter and its introduction in Corynebacterium glutamicum

[0065] Take ATCC 13032 genome as template, use Ppyc mut1 -1 / PlysC mut1 -2 primer pair for PCR amplification to obtain the upstream fragment Ppyc mut1 -up; use ATCC 13032 genome as template and Ppyc mut1 -3 / Ppyc mut1 -4 primer pair for PCR amplification to obtain fragment Ppyc mut1 -dn. Using the above two fragment mixture as a template, using Ppyc mut1 -1 / Ppyc mut1 -4 primer pair for PCR amplification to obtain Ppyc containing the target mutation mut1 Fragment. The fragment was double-cut with BamHI and PstI, and the vector pK18mobsacB was double-cut with the same enzyme. The two digested products were ligated with T4 DNA Ligase and transformed into Trans1 T1 competent cells to obtain the recombinant plasmid pK18mobsacB-pyc mut1 .

[0066] Competent cells of MHZ-0912-1 were prepared according to the Gubang classic method (C. glutamicum...

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Abstract

The invention belongs to the technical field of bioengineering, and discloses coryneform bacteria capable of producing lysine at high yield as well as a construction method and an application thereof.The coryneform bacterium capable of producing lysine at high yield has L-lysine production capacity, and the activity of at least one of promoters of encoding genes of key enzymes of an intracellularL-lysine synthesis pathway and / or a TCA circulating back-supplement pathway of the coryneform bacterium is enhanced. According to the coryneform bacterium capable of producing lysine at high yield, the sequence of the promoter of the coding gene of the key enzyme in the lysine biosynthesis pathway or the TCA circulating back-supplement pathway is modified, compared with an endogenous promoter, the L-lysine promoter has higher activity, so that the initial transcription efficiency is enhanced, the lysine production level is further improved, and the production capacity of an L-lysine strain isimproved. The experiments show that the coryneform bacteria provided by the invention significantly improve the yield and conversion rate of L-lysine, lay a foundation for industrial production of L-lysine, and have a wide industrial application prospect.

Description

Technical field [0001] The invention belongs to the technical field of bioengineering, and specifically relates to a coryneform bacterium with high lysine production and a construction method and application thereof. Background technique [0002] The chemical name of Lysine is 2,6-diaminohexanoic acid. Lysine is a basic essential amino acid. Because the content of lysine in cereal food is very low, and it is easily destroyed and lacked during processing, it is called the first limiting amino acid. Generally speaking, lysine refers to L type. L-Lysine is needle-shaped crystals, darkens at 210℃, decomposes at 224.5℃, easily soluble in water, slightly soluble in alcohol, and insoluble in ether. [0003] Lysine is one of the essential amino acids for humans and mammals. The body cannot synthesize by itself and must be supplemented from food. Lysine is mainly found in animal foods and legumes. The content of lysine in cereals is very low. Lysine has positive nutritional significanc...

Claims

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Application Information

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IPC IPC(8): C12N1/21C12N15/77C12P13/08C12R1/15
CPCC12N15/77C12P13/08C07K14/34
Inventor 胡丹王成薛婷莉李岩
Owner 新疆梅花氨基酸有限责任公司
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