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Recombinant strain, method for preparing recombinant strain and method for producing L-valine from recombinant strain

A technology of recombinant strains and valine, applied in the field of microorganisms, can solve the problems of slow growth of strains, multiple by-products, and difficulty in obtaining high-yielding strains, and achieves the effect of reducing by-products

Inactive Publication Date: 2017-03-22
MEIHUA BIOTECH LANGFANG CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, traditional mutation breeding is difficult to obtain high-yield strains due to the slow growth of the strain and the production of more by-products due to random mutations.

Method used

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  • Recombinant strain, method for preparing recombinant strain and method for producing L-valine from recombinant strain
  • Recombinant strain, method for preparing recombinant strain and method for producing L-valine from recombinant strain

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0042] Example 1: Recombinant plasmid pK18mobsacB-ilvN M13 Construction of and introduction of point mutations in ATCC14067

[0043] (1) pK18mobsacB-ilvN M13 Plasmid construction

[0044] Using the ATCC14067 genome as a template, using ilvN M13 -f1 / ilvN M13 -r1 primer pair for PCR amplification to obtain the upstream fragment ilvN M13 -up.

[0045] Using the ATCC14067 genome as a template, using ilvN M13 -f2 / ilvN M13 -r2 primer pair for PCR amplification to obtain the downstream fragment ilvN M13 -dn.

[0046] to ilvN M13 -up, ilvN M13 -dn two-fragment mixture as template, ilvN M13 -f1 / ilvN M13 -r2 primer pair for PCR amplification to obtain mutated ilvN M13 fragment.

[0047] wxya M13 The fragment was double-digested with BamHI and HindIII, 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-ilvN was obtained M13 ...

Embodiment 2

[0050] Example 2: Construction of recombinant plasmid pK18mobsacB-ΔavtA and knockout of avtA in MHZ-1012-1

[0051] (1) Construction of pK18mobsacB-ΔavtA plasmid

[0052] Using the ATCC14067 genome as a template, the upstream fragment avtA-up was amplified by PCR with avtA-f1 / avtA-r1 primer pair.

[0053] The downstream fragment avtA-dn was amplified by PCR using the ATCC14067 genome as a template and avtA-f2 / avtA-r2 primer pair.

[0054] Using the mixture of avtA-up and avtA-dn fragments as a template, PCR amplification was carried out with the avtA-f1 / avtA-r2 primer pair to obtain the avtA knockout fragment.

[0055] The avtA knockout fragment was double-digested with BamHI and HindIII, and pK18mobsacB was double-digested with the same enzymes. The two digested products were connected with T4DNA Ligase, transformed into Trans1T1 competent cells, and the recombinant plasmid pK18mobsacB-ΔavtA( figure 2 ).

[0056] (2) Knockout of avtA in MHZ-1012-1

[0057] MHZ-1012-1 co...

Embodiment 3

[0058] Example 3: Fermentation of L-valine genetically engineered bacteria to produce L-valine

[0059] 1. Medium

[0060] Seed activation medium: yeast extract 1%, peptone 1%, sodium chloride 0.5%, glucose 0.5%, agar 2%, pH7.2.

[0061] Seed medium: corn steep liquor 2.5%, glucose 1.0%, ammonium sulfate 0.4%, magnesium sulfate 0.04%, potassium dihydrogen phosphate 0.1%, urea 0.1%, CaCO 3 0.5%, pH 7.2.

[0062] Fermentation medium: corn steep liquor 0.5%, glucose 12.0%, ammonium sulfate 4.0%, magnesium sulfate 0.04%, potassium dihydrogen phosphate 0.1%, CaCO 3 4%, V H 50μg / L, V B1 · HCl 100 μg / L, pH 7.2.

[0063] 2. Production of L-valine by MHZ1012-1 shake flask fermentation

[0064] (1) Seed culture: pick ATCC14067, MHZ1012-1, slant seed 1 loop and connect to a 500mL Erlenmeyer flask with 20mL seed medium, shake and culture at 33°C and 220r / min for 16-22h;

[0065] (2) Fermentation culture: Inoculate 2 mL of seed solution into a 500 mL Erlenmeyer flask containing 2...

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Abstract

The invention relates to the field of microbial technology, in particular to a recombinant strain, a method for preparing the recombinant strain and a method for producing L-valine from the recombinant strain. According to the recombinant strain, corynebacterium serves as a starting strain for transformation, and the transformation process includes the steps of conducting point mutation on the ilvN gene and knocking out the avtA gene. The recombinant strain is obtained by conducting point mutation on the ilvN in a valine metabolism path and knocking out the avtA gene for transaminase coding; through fermentation culture, the yield of L-valine can reach 12.2 g / L, and generation of the by-product alanine is greatly reduced.

Description

technical field [0001] The invention relates to the technical field of microbes, in particular to a recombinant bacterial strain, a preparation method thereof and a method for producing L-valine. 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. 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 is 5.96 and the melting point is 315°C. 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. It can be widely used in pharmaceutical industry, food industry and feed industry, etc. I...

Claims

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Application Information

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IPC IPC(8): C12N1/21C12P13/08C12R1/15
CPCC12N9/1096C12N9/1022C12P13/08C12Y202/01006
Inventor 康培程江红胡丹刁刘洋毛贤军
Owner MEIHUA BIOTECH LANGFANG CO LTD
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