Corynebacterium capable of realizing high yield of valine as well as construction method and application thereof

A technique of corynebacterium glutamicum and its construction method, which is applied in the field of corynebacteria with high valine production and its construction, and can solve the problems of poor fermentation performance, inability to meet large-scale industrial production, and low conversion rate of L-valine , to achieve the effect of increasing output, broad industrial application prospects, and improving production capacity

Pending Publication Date: 2020-04-10
新疆梅花氨基酸有限责任公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, the fermentation performance of L-valine strains is still poor, and the conversion ra...

Method used

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  • Corynebacterium capable of realizing high yield of valine as well as construction method and application thereof
  • Corynebacterium capable of realizing high yield of valine as well as construction method and application thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0030] Example 1: Recombinant plasmid pK18mobsacB-leuA A1G Construction of and introduction of point mutations in MHZ-1012-2

[0031] (1) pK18mobsacB-leuA A1G Plasmid construction

[0032] Using the MHZ-1012-2 genome as a template, using leuA A1G -f1 / leuA A1G -r1 primer pair for PCR amplification to obtain the upstream fragment leuA A1G -up; take the MHZ-1012-2 genome as a template, take leuA A1G -f2 / leuA A1G -r2 primer pair for PCR amplification to obtain the downstream fragment leuA A1G -dn. with leuA A1G -up, leuA A1G -dn two-fragment mixture as template, with leuA A1G -f1 / leuA A1G -r2 primer pair for PCR amplification to obtain mutated leuA A1G fragment. leuA A1G The fragment was double-digested with EcoRI and SmaI, and pK18mobsacB was double-digested with the same enzymes. The two digestion products were ligated with T4DNA Ligase, transformed into Trans1T1 competent cells, and the recombinant plasmid pK18mobsacB-leuA was obtained A1G .

[0033] Table 1 Pr...

Embodiment 2

[0037] Example 2: Fermentation of L-valine genetically engineered bacteria to produce L-valine.

[0038] 1. Medium

[0039] Seed medium: corn steep liquor 2.5wt%, glucose 1.0wt%, ammonium sulfate 0.4wt%, magnesium sulfate 0.04wt%, potassium dihydrogen phosphate 0.1wt%, urea 0.1wt%, CaCO 3 0.5wt%, the balance is water, pH 7.2.

[0040] Fermentation medium: corn steep liquor 0.5wt%, glucose 12.0wt%, ammonium sulfate 4.0wt%, magnesium sulfate 0.04wt%, potassium dihydrogen phosphate 0.1wt%, CaCO 3 4wt%, VH 50μg / L, VB1·HCl 100μg / L, balance water, pH 7.2.

[0041] 2. Production of L-valine by MHZ-1012-3 shake flask fermentation

[0042] (1) Seed culture: Pick 1 loop of MHZ-1012-2 and MHZ-1012-3 slanted seeds and transfer them to a 500mL Erlenmeyer flask containing 20mL of seed medium, and shake at 33°C and 220r / min for 16-22h;

[0043] (2) Fermentation culture: Inoculate 2 mL of seed solution into a 500 mL Erlenmeyer flask containing 20 mL of fermentation medium, and culture a...

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Abstract

The invention belongs to the technical field of bioengineering, and discloses corynebacterium capable of realizing high yield of valine as well as a construction method and an application thereof. Theinvention provides corynebacterium capable of realizing high yield of valine as well as the construction method and the application thereof. According to the corynebacterium capable of realizing highyield of valine, alanine (A) at the first site of a 2-isopropylmalate synthetase gene leuA is mutated into glycine (G). According to the corynebacterium capable of highly producing valine, accumulation of by-product leucine is reduced by weakening a leuA gene, and the production capacity of an L-valine strain is improved. The experiments show that the corynebacterium disclosed by the invention isan L-valine high-yielding strain, can effectively accumulate L-valine and increase the yield of L-valine, lays a foundation for industrial production of L-valine, and has a wide industrial application prospect.

Description

technical field [0001] The invention belongs to the technical field of bioengineering, and in particular relates to a high-valine-producing corynebacterium and a construction method and application thereof. Background technique [0002] L-valine (L-valine), the chemical name is L-α-aminoisovaleric acid, and the molecular formula is C 5 h 11 NO 2 , the relative molecular mass is 117.15. L-valine is white crystal or crystalline powder, odorless, bitter taste, solubility in water: 88.5g / L at 25°C, 96.2g / L at 50°C, insoluble in cold ethanol, ether, acetone. The isoelectric point of L-valine is 5.96, and the melting point is 315°C. [0003] L-valine is one of the eight essential amino acids for the human body, and one of the three branched-chain amino acids (including valine, leucine, and isoleucine). Because of its special structure and function, it is widely used in human life. Metabolism has a particularly important position. L-valine can be widely used in pharmaceutical...

Claims

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Application Information

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IPC IPC(8): C12N1/21C12N15/77C12P13/08C12R1/15C12R1/13
CPCC12N9/1025C12N15/77C12P13/08C12N1/20C12Y203/03013
Inventor 程江红张亚张晓云吴涛
Owner 新疆梅花氨基酸有限责任公司
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