Glutamic acid corynebacterium and construction method and application thereof

A Corynebacterium glutamicum and lysine technology, applied in the field of microorganisms, can solve the problems of low acid production rate, inability to meet large-scale industrial production, and poor fermentation performance of L-lysine, so as to increase production and enhance activity , the effect of increasing production

Inactive Publication Date: 2017-05-10
MEIHUA BIOTECH LANGFANG CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

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

Method used

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  • Glutamic acid corynebacterium and construction method and application thereof
  • Glutamic acid corynebacterium and construction method and application thereof
  • Glutamic acid corynebacterium and construction method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] Example 1: Construction of recombinant plasmid pK18mobsacB-lysE and addition of a copy of lysE in the starting strain MHZ-0912-1

[0037]Using the ATCC 13032 genome as a template, PCR amplification was performed with the lysE-1f / lysE-1r primer pair to obtain the upstream fragment lysE-up+lysE; using the ATCC 13032 genome as a template, PCR was performed with the lysE-2f / lysE-2r primer pair Amplify to obtain fragment lysE, use ATCC 13032 genome as a template, and perform PCR amplification with lysE-3f / lysE-3r primer pair to obtain fragment lysE-dn. The lysE-up+lysE, lysE, lysE-dn three-fragment mixture was used as a template, and the lysE-1f / lysE-3r primer pair was used for PCR amplification to obtain a lysE fragment with two copies. The fragment was double-digested with BamHI and PstI, and the vector pK18mobsacB was double-digested with the same enzymes. The two digested products were ligated with T4DNA Ligase, transformed into Trans1T1 competent cells, and the recombi...

Embodiment 2

[0039] Example 2: Recombinant plasmid pK18mobsacB-pyc P458S Construction of MHZ-0912-1 and the introduction of point mutations to enhance the activity of pyruvate carboxylase

[0040] Using the ATCC 13032 genome as a template, PCR amplification was performed with the pyc-1f / pyc-1r primer pair to obtain the upstream fragment pyc-up; using the ATCC 13032 genome as a template, PCR amplification was performed with the pyc-2f / pyc-2r primer pair , to get the downstream fragment pyc-dn. Using the mixture of pyc-up and pyc-dn fragments as a template, the pyc-1f / pyc-2r primer pair was used for PCR amplification to obtain the mutated pyc P458S fragment. pyc P458S The fragment was double-digested with SphI and NheI, and pK18mobsacB was double-digested with the same enzymes. The two digested products were ligated with T4DNA Ligase, transformed into Trans1T1 competent cells, and the recombinant plasmid pK18mobsacB-pyc was obtained P458S .

[0041] Prepare MHZ-0912-1 competent cells a...

Embodiment 3

[0042] Example 3: Recombinant plasmid pK18mobsacB-pyc P458S Construction of MHZ-0913-1 and the introduction of point mutations to enhance the activity of pyruvate carboxylase

[0043] Construct recombinant plasmid pK18mobsacB-pyc according to the method described in Example 2 P458S : Using the ATCC13032 genome as a template, PCR amplification was performed with the pyc-1f / pyc-1r primer pair to obtain the upstream fragment pyc-up; using the ATCC 13032 genome as a template, PCR amplification was performed with the pyc-2f / pyc-2r primer pair , to get the downstream fragment pyc-dn. Using the mixture of pyc-up and pyc-dn fragments as a template, the pyc-1f / pyc-2r primer pair was used for PCR amplification to obtain the mutated pyc P458S fragment. pyc P458S The fragment was double-digested with SphI and NheI, and pK18mobsacB was double-digested with the same enzymes. The two digestion products were ligated with T4 DNALigase, transformed into Trans1T1 competent cells, and the re...

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Abstract

The invention belongs to the field of microorganisms, and particularly relates to a glutamic acid corynebacterium and a construction method and application of the glutamic acid corynebacterium. The glutamic acid corynebacterium has the capability of producing L-lysine, and the activities of pyruvic carboxylase exportin and pyruvic carboxylase in a cell are both strengthened. The glutamic acid corynebacterium strengthens the activity of the pyruvic carboxylase exportin, so that the accumulation of the L-lysine is promoted, the yield of the L-lysine is increased, the activity of pyruvic carboxylase is strengthened at the same time, more precursors are provided for the synthesis of the L-lysine, the accumulation capability of the L-lysine is further improved, the amount of the L-lysine in a culture medium is increased, and the capability of production of the L-lysine through the fermentation of the strain is further improved. The experiment shows that the glutamic acid corynebacterium is a strain with high yield of L-lysine, the L-lysine is effectively accumulated, the yield of the L-lysine is improved, and a foundation is laid for the industrialized production of the L-lysine.

Description

technical field [0001] The invention belongs to the field of microorganisms, and in particular relates to a Corynebacterium glutamicum and its construction method and application, in particular to an L-lysine-producing Corynebacterium glutamicum and its construction method and application Background technique [0002] L-lysine (L-Lysine), the scientific name is L-2,6-diaminocaproic acid, the molecular formula is C 6 h 14 N 2 o 2 .HCL with a molecular weight of 182.65. L-lysine is one of the important components of protein. It is one of the eight kinds of amino acids that the human body cannot synthesize by itself but is very necessary. It can improve the utilization rate of protein, thereby greatly strengthening the nutrition of food, and promoting growth and development. Increase appetite, reduce disease, and enhance physical fitness. [0003] L-Lysine is the second largest amino acid production variety in the world and is widely used in animal feed, medicine and food ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/21C12N15/77C12P13/08C12R1/15
CPCC07K14/34C12N9/93C12P13/08C12Y604/01001
Inventor 孙雪胡丹刁刘洋毛贤军
Owner MEIHUA BIOTECH LANGFANG CO LTD
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