Recombinant strain as well as construction method and application thereof

A technology for recombinant strains and construction methods, applied in the field of microorganisms, can solve the problems of insufficient NADPH, accumulation of NADH, and inability to apply

Inactive Publication Date: 2016-06-15
MEIHUA BIOTECH LANGFANG CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

This process produces NADH, which cannot be used in the four-step reaction of valine terminal synthesis, resulting in a large accumulation of NADH on the one hand, and insufficient NADPH for synthesis on the other hand.

Method used

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  • Recombinant strain as well as construction method and application thereof
  • Recombinant strain as well as construction method and application thereof
  • Recombinant strain as well as construction method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0056] Example 1 Construction of expression plasmid pXMJ19-gapN

[0057] 1. With the genomic DNA of Streptococcus mutans (streptococcusmutans) ATCC25175 as template, carry out PCR amplification with the primer pair that gapNF and gapNR form, obtain PCR amplification product:

[0058] gapNF: 5-'T GGATCC ATGACAAAACAATATAAAATTA-3' (the underline is the recognition site for BamHI digestion), as shown in SEQ ID No.2;

[0059] gapNR: 5-'C GAATTC TTATTTGATATCAAATACGACGG-3' (the underline is the recognition site for EcoRI digestion), as shown in SEQ ID No.3;

[0060] 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-gapN was obtained, and the gapN gene sequence was obtained by sequencing, as shown in SEQIDNo.1, and its protein sequence was shown in SEQIDNo.4;

[0061] 3. Use two restriction endonucleases (BamHI and EcoRI) to double digest the plasmid pT5-gapN and...

Embodiment 2

[0063] Example 2 Construction of Engineering Bacteria CGMCC1.299 / pXMJ19-gapN

[0064] 1. Preparation of electric shock transformation competent cells of Corynebacterium;

[0065] 2. Electric shock transformation: Add 3-8 μL of pXMJ19-gapN 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;

[0066] 3. Add 1 mL of LB medium, incubate at 31°C and 150 rpm for 1 hour; spread it on a brain-heart medium plate containing 25 μg / mL kanamycin after concentration, and incubate at a constant temperature of 31°C for 36-48 hours;

[0067] 4. Screen the positive clones, and carry out PCR identification with a primer pair consisting of gapNF / gapNR (the one with a specific band of about 1.4 kb is positive) to obtain recombinant bacteria.

Embodiment 3

[0068] Example 3 Engineering bacteria CGMCC1.299 / pXMJ19-gapN (MHZ-1011) fermented to produce L-valine

[0069] 1. Medium

[0070] Seed activation medium: yeast extract 1%, peptone 1%, sodium chloride 0.5%, glucose 0.5%, agar 2%, pH7.2;

[0071] 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;

[0072] 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;

[0073] 2. Production of L-valine by shake flask fermentation

[0074] (1) Seed culture: Pick the engineering strain MHZ-1011 slant seeds 1 loop and connect them to a 500mL Erlenmeyer flask containing 20mL seed culture medium, and shake at 33°C and 220r / min for 16-22h;

[0075] (2) Fermentation culture: inoculate 2 mL of seed solution into a 500 mL Erlenmeyer flask c...

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Abstract

The invention relates to the field of microorganisms, in particular to recombinant strains, construction methods and applications thereof. The invention protects the engineered strain obtained by introducing the target gene into Corynebacterium or Brevibacterium by means of recombinant plasmid or insertion into chromosome. The result of the experiment of producing L-valine by shaking flask fermentation showed that the L-valine yield of the starting strain CGMCC 1.299 was 3.2g/L, while the L-valine yield of the engineering strain MHZ-1011 of the present invention was 6.5g/L. L, the yield was 103% higher than that of the starting strain, with a very significant difference (P<0.01). The experimental results of L-valine production by 50L tank fermentation show that: the L-valine yield of the starting strain CGMCC1.299 is 7.3g/L, while the L-valine yield of the engineering bacterium MHZ-1011 of the present invention is 18.4g /L, the yield was 152% higher than that of the starting strain, with a very significant difference (P<0.01).

Description

technical field [0001] The present invention relates to the field of microorganisms, in particular to recombinant bacterial strains and their construction methods and applications. 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. [0003] 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 feed industry, etc. [0004] In the pharmaceutical industry, it can b...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/21C12P13/08C12R1/15C12R1/13
CPCC12N9/0008C12P13/08C12Y102/01009
Inventor 朱亚然毛贤军刁刘洋
Owner MEIHUA BIOTECH LANGFANG CO LTD
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