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Recombinant dna, bacterial strain and method for fermentative production of l-valine

A valine, DNA sequence technology, applied in the field of microbial engineering, to achieve the effects of improving metabolic intensity, activity, yield and sugar-acid conversion rate

Inactive Publication Date: 2011-12-21
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0009] The method of expressing the enzyme gene of L-valine biosynthetic pathway in the wild-type strain and adopting high temperature and short time fermentation has not been established

Method used

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  • Recombinant dna, bacterial strain and method for fermentative production of l-valine
  • Recombinant dna, bacterial strain and method for fermentative production of l-valine
  • Recombinant dna, bacterial strain and method for fermentative production of l-valine

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0083] Example 1 Construction of expression plasmid pDXW-8-ilvEBN r C

[0084] a) Cloning of ilvBNC operon and ilvE gene: Brevibacterium flavum B. flavum NV128 genome as a template, SEQ ID NO: 1 (including SD sequence) and SEQ ID NO: 2 as primers, PCR to obtain ilvBNC operon, ilvBNC operon Connected with T vector to form pUCm-T-ilvBNC; B. flavum NV128 genome as template, SEQ ID NO: 5 (including SD sequence) and SEQ ID NO: 6 as primers, PCR to obtain ilvE gene, connected with ilvE gene T vector to form pUCm -T-ilvE.

[0085] b) ilvN site-directed mutation relieves the feedback inhibition of three branched-chain amino acids on acetolactate synthase: using pUCm-T-ilvBNC as a template, SEQ ID NO: 3 and SEQ ID NO: 4 as primers, PCR to obtain mutants, DpnI method Screen mutants. The mutant was verified by sequencing, and the mutant was named pUCm-T-ilvBN r c. Ensure that Gly-Ile-Ile at positions 20-22 of the regulatory subunit of acetolactate synthase is mutated to Asp-Asp-Phe....

Embodiment 2

[0089] Example 2 Construction of Escherichia coli JM109 / pDXW-8-ilvEBNC and JM109 / pDXW-8-ilvEBN r C

[0090] (1) Competent preparation of Escherichia coli JM109

[0091] a) LB medium JM109 was cultured to an OD value of 0.3-0.5.

[0092] b) 3,000g (or 6,000r / min) at 4°C for 5min. Discard the supernatant and harvest the bacteria.

[0093] c) Resuspend in TSB which is 1 / 10 of the original culture volume.

[0094] e) Place on ice for 10 minutes, aliquot on ice, 60 μL per tube, and store at -70°C.

[0095] TSB formula (requires high pressure), LB (pH6.1) contains: 10% PEG 3350 or 4000, 5% DMSO (dimethyl sulfoxide), 10mmol / L MgCl 2 , 10mmol / L MgSO 4 ·7H 2 O, 10% glycerin.

[0096] 5×KCM solution formula: 0.5mol / L KCl, 0.15mol / L CaCl 2 , 0.25mol / L MgCl 2 .

[0097] (2) Conversion:

[0098] a) Plasmids pDXW-8-ilvEBNC and pDXW-8-ilvEBN r C respectively 5 ~ 10 μ L, 5 × KCM solution 10 μ L, double distilled water 30 μ L, (total 50 μ L), mix well, put on ice.

[0099] b) Tak...

Embodiment 3

[0105] Example 3 Structural Engineering Bacteria B.flavum ATCC 14067 / pDXW-8-ilvEBN r C

[0106] (1) A single colony was inoculated in 30 mL LBG medium and cultured at 30°C for 16 hours.

[0107] (2) Inoculate in Epo medium to OD 600 =0.3, culture OD at 30℃ 600 = 0.9.

[0108] (3) Cool on ice for 10 minutes, and centrifuge at 4,000 g for 10 minutes at 4°C.

[0109] (4) Wash 4 times with 15 mL 10% glycerol, and resuspend with 0.2 mL 10% glycerol.

[0110] (5) Add appropriate amount of DNA to cool on ice, 0.1cm electric shock cup 1.8kV 10S.

[0111] (6) Add 1 mL of LBHIS medium and incubate at 30°C for 1 hour.

[0112] (7) Coat the LBHIS solid kana plate and incubate for 36 hours.

[0113] The aforementioned Epo medium formula: 10g / L trypton (peptone), 5g / L yeast extract (yeast extract), 10g / L NaCl, 4g / L isonicotinic acid hydrazide (isoniazid), 25g / L glycine (glycine), 0.1% Tween 80.

[0114] The aforementioned LBHIS medium formula: 5g / L trypton (peptone), 5g / L NaCl, 2.5...

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Abstract

The invention relates to a recombinant DNA (deoxyribonucleic acid), strain and method for producing L-valine by fermentation. The recombinant DNA comprises DNA sequence for coding acetohydroxy acid synthetase free of feedback inhibition of three branched-chain amino acids to acetohydroxy acid synthetase, DNA sequence for coding dihydroxy reduction isomerase, and DNA sequence for coding branched-chain amino acid aminotransferase. The strain is corynebacterium or brevibacterium transformed by introducing the recombinant DNA. The method is implemented by culturing the strain to produce the L-valine. In the invention, the expression L-valine is used for producing the bacterium L-valine biosynthetic gene, so the yield of the L-valine can be greatly increased; and the fermentation model, especially the high-temperature short-time fermentation model, is constructed according to the optimum temperature of the L-valine biosynthetic enzyme, so that the metabolism intensity of the strain is enhanced, the activity of the L-valine biosynthetic enzyme is enhanced, and the yield of the L-valine and the conversion rate of the sugar acid can be further increased.

Description

technical field [0001] The invention relates to a production method of L-valine, in particular to a DNA, bacterial strain and method for fermenting and producing L-valine, and belongs to the technical field of microbial engineering. Background technique [0002] Fermentative production of L-valine mostly uses microbial strains isolated from the natural environment or artificial mutants obtained from such microbial strains. Most of the known high-yielding artificial mutants are resistant to α-aminobutyric acid (α-AB), α-amino-β-hydroxyvaleric acid (AHV) and 2-thiazolealanine (2-TA), and belong to Corynebacterium, Brevibacterium and Escherichia, etc. [0003] As far as Corynebacterium or Brevibacterium are concerned, there are currently disclosed vector plasmids that are autonomously replicable in bacteria and have drug resistance marker genes (see US4514502), and have been disclosed for introducing genes into bacterial cells. method (for example JP2207791), and the possibil...

Claims

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Application Information

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IPC IPC(8): C12N15/61C12N15/54C12N15/60C12N1/21C12P13/08C12R1/13C12R1/15
Inventor 张伟国钱和侯小虎
Owner JIANGNAN UNIV
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