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Protein, DNA molecule, conversion host containing DNA and method for production of L-valine by utilization of conversion host

A DNA molecule, valine technology, applied in the field of L-valine production, can solve the problems of inability to meet market demand, low L-valine yield, etc.

Active Publication Date: 2014-03-19
MEIHUA BIOTECH LANGFANG CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the yield of L-valine produced by fermentation of existing bacterial strains is low, which cannot meet the market demand

Method used

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  • Protein, DNA molecule, conversion host containing DNA and method for production of L-valine by utilization of conversion host
  • Protein, DNA molecule, conversion host containing DNA and method for production of L-valine by utilization of conversion host
  • Protein, DNA molecule, conversion host containing DNA and method for production of L-valine by utilization of conversion host

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0042] Example 1 Construction of expression plasmid pXMJ19-leuDH

[0043] 1. Using the genomic DNA of Bacillus megaterium ATCC14945 as a template, PCR amplification was performed with a primer pair consisting of leuF and leuR to obtain PCR amplification products:

[0044] leuF: 5-'G AAGCTT ATGAAAATTTTTGACTAT-3' (the underline is the recognition site of HindIII digestion)

[0045] leuR: 5-'C GAATTC TTAGCGCGTATGACGTCTAC-3' (the underline is the recognition site of EcoRI digestion)

[0046] 2. Recover the PCR product of step 1 and connect it to the vector pEASY TM -T5 (purchased from Beijing Quanshijin Biotechnology Co., Ltd.), the recombinant plasmid pT5-leuDH was obtained, and the leuDH gene sequence was obtained by sequencing, as shown in SEQ ID No.1;

[0047] 3. Use two restriction endonucleases (HindIII and EcoRI) to double digest the plasmid pT5-leuDH and the vector pXMJ19, and obtain the leuDH fragment (about 1100bp) and pXMJ19 vector backbone (about 6600bp) after ge...

Embodiment 2

[0049] Example 2 Construction of Engineering Bacteria CGMCC1.299 / pXMJ19-leuDH

[0050] 1. Preparation of electric shock transformation competent cells of Corynebacterium;

[0051] 2. Electric shock transformation: Add 3-8 μL of pXMJ19-leuDH plasmid to competent cells, place on ice for 10 minutes; transfer to a 1 mm electric shock cup, and electric shock at 1.8 or 2.1 kV for 5-7 ms;

[0052] 3. Add 1 mL of LB medium, culture at 33°C and 150rpm for 1 hour; spread it on a brain-heart medium plate containing 25 μg / mL kanamycin after concentration, and culture at a constant temperature of 33°C for 36-48 hours;

[0053] 4. Screen the positive clones, and carry out PCR identification with a primer pair composed of leuF / leuR (the one with a specific band of about 1.1 kb is positive) to obtain recombinant bacteria.

[0054] The recombinant bacterium was named Corynebacterium pekinense MHZ-1010, and the strain was deposited in the General Microorganism Center (CGMCC) of China Committee...

Embodiment 3

[0055] Example 3 Fermentative production of L-valine by corynebacterium with preservation number CGMCC No.7041 provided by the present invention

[0056] 1. Medium

[0057] Seed activation medium: yeast extract 1%, peptone 1%, sodium chloride 0.5%, glucose 0.5%, agar 2%, pH7.2;

[0058] 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%, pH7.2;

[0059] 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 50ug / L, V B1 ·HCl100μg / L, pH7.2;

[0060] 2. Production of L-valine by shake flask fermentation

[0061] (1) Seed culture: Pick 1 loop of Corynebacterium slant seeds provided by the present invention and transfer them to a 500 mL Erlenmeyer flask containing 20 mL of seed culture medium, and shake and cultivate at 33° C. and 220 r / min for 16-22 hours;

[0062...

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Abstract

The invention relates to the biotechnology field, and especially relates to a protein, a DNA molecule, a conversion host containing the DNA and a method for production of L-valine by utilization of the conversion host. A coding gene leuDH (the nucleotide sequence is shown in SEQ ID No.2) of a leucine dehydrogenase with an auxiliary factor of NADH is converted and introduced into corynebacteria, and genetically engineered bacterium with changed redox equilibrium in cells are obtained. The bacterial strain is cultured and can be used for production of L-valine, and the yield of L-valine is raised remarkably.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a protein, a DNA molecule encoding the protein, a transformed host machine containing the DNA molecule, and a method for producing L-valine by the transformed host. 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 (L-Val) 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). Function has a particularly important position in the metabolism of human life. It can be widely used in pharmaceutical industry, food industry and fe...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/06C12N15/53C12N15/77C12N1/21C12P13/08C12R1/15C12R1/13C12R1/11
CPCC12N9/0016C12P13/08C12Y104/01009
Inventor 朱亚然胡炎华宫卫波
Owner MEIHUA BIOTECH LANGFANG CO LTD
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