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Corynebacterium glutamicum and method for producing high-yield isoleucine with same

A glutamic acid rod and isoleucine technology, applied in the field of microorganisms, can solve the problems of limited knowledge, unreported and reported metabolic network dynamic regulation

Active Publication Date: 2016-08-24
TIANJIN UNIV OF SCI & TECH
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] The domestic production of L-isoleucine mainly relies on microbial fermentation, and the strains used are mainly Corynebacterium glutamicum. However, the current lack of understanding of the dynamic regulation of its metabolic network limits the progress of further research on the application of metabolic engineering.
The branched-chain amino acid (valine, isoleucine, and leucine) synthesis pathway to which the biosynthetic pathway of isoleucine belongs has always been a research hotspot for researchers, and most of the research focuses on the key enzymes in this metabolic network Amino acid changes in the active center, or overexpression of key enzymes, but so far, there is no report on the complete genome sequence of isoleucine-producing bacteria

Method used

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  • Corynebacterium glutamicum and method for producing high-yield isoleucine with same
  • Corynebacterium glutamicum and method for producing high-yield isoleucine with same
  • Corynebacterium glutamicum and method for producing high-yield isoleucine with same

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Experimental program
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Effect test

Embodiment 1

[0031] The chemical mutagenesis of embodiment 1 corynebacterium glutamicum

[0032] (1) Using the Corynebacterium glutamicum isolated in the soil as the starting strain, first activate it on the slope of the complete medium, and cultivate it at 32°C for 12 hours;

[0033] (2) Inoculate in a liquid seed medium from a slope, the seed medium is LB, and cultivate at 32° C. for 12 hours;

[0034] (3) Wash the cultured cells with physiological saline, and after repeating once, resuspend the bacteria with a phosphate buffer solution with a pH of 7.0 to make 10 7 -10 8 Cells / mL of bacterial suspension;

[0035] (4) Take 5-10 mL of bacterial suspension, add it to a sterile test tube, then add 1% (V / V) diethyl sulfate, seal the test tube with a cotton plug, and shake for 30-40 minutes;

[0036] (5) Dilute with an appropriate amount of sterile water, spread on the resistance screening medium containing sulfaguanidine and L-isoleucine structural analogues, and culture at 32°C until a s...

Embodiment 2

[0039] The genome sequencing of embodiment 2 Corynebacterium glutamicum YI

[0040] (1) Extraction of genome: use the bacterial genome DNA extraction kit of PROMEGA company Genomic DNA Purification Kit A1120, using a nucleic acid analyzer to ensure that the quality of genomic DNA meets the requirements of sequencing;

[0041] (2) The genome was sequenced using the third-generation sequencing platform PacBio RS II;

[0042] (3) Genome assembly was performed using the assembly software provided by PacBio, and routine comparative genome analysis was performed using related software.

Embodiment 3

[0043] Comparison of strain isoleucine synthesis ability before and after the gene mutation of embodiment 3

[0044] (1) Activated slant medium (g / L): glucose 10, beef extract 10, peptone 10, yeast powder 5, NaCl 5, agar strip 20, pH 6.7-7.2;

[0045] (2) Shake flask seed medium: glucose 30g, yeast powder 5g, bean cake powder hydrolyzate 20mL, corn steep liquor 20g, NaCl 5g, MgSO 4 ·7H 2 O 2g, KH 2 PO 4 12H 2 O 2g, FeSO 4 ·7H 2 O 0.01g, MnSO 4 ·H 2 O 0.01g, leucine 0.1g, valine 0.1g, phenol red solution (0.4g / L) 20mL, distilled water 1000mL, pH 6.7-7.2;

[0046] (3) Shake flask fermentation medium: glucose 60g, bean cake powder hydrolyzate 25mL, corn steep liquor 40g, MgSO 4 ·7H 2 O 2g, KH 2 PO 4 12H 2 O 2g, FeSO 4 ·7H 2 O 0.01g, MnSO 4 ·H 2 O 0.01g, V B1 0.01g, phenol red solution (0.4g / L) 20mL, distilled water 1000mL, pH 6.7-7.2;

[0047] (2) The bean cake powder hydrolyzate described in (3) was purchased from Shandong Linghua Monosodium Glutamate Co., Lt...

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Abstract

The invention relates to Corynebacterium glutamicum and a method for producing high-yield isoleucine with the same, and belongs to the field of microorganisms and genomics. The Corynebacterium glutamicum, specifically Corynebacterium glutamicum YI is preserved with a preservation number CGMCC NO. 12153. The Corynebacterium glutamicum is obtained by means of mutagenesis and is fermented in a 30L fermentation cylinder for 32-40 hours; the yield of isoleucine is up to 28g / L; accordingly, the method has the advantages of short time for fermentation and high yield and is superior to the prior art. The invention also discloses a gene mutation site of the strain relative to a standard strain, providing a new direction for correlational researches.

Description

Technical field: [0001] The invention relates to the fields of microorganisms and genomics, in particular to a Corynebacterium glutamicum and a method for high-yielding L-isoleucine. Background technique: [0002] Corynebacterium glutamicum is a Gram-positive bacterium of the order Actinomycetes, and its cells are arranged in a short rod shape. Corynebacterium glutamicum plays a pivotal role in the field of amino acid fermentation and has been safely used for nearly 60 years. Currently, most amino acids including L-lysine, L-valine, L-threonine, L-leucine, L-isoleucine, L-alanine, L-aspartic acid, etc. Both began to use Corynebacterium glutamicum fermentation for production. [0003] The domestic production of L-isoleucine mainly relies on microbial fermentation, and the strains used are mainly Corynebacterium glutamicum. However, the current lack of understanding of the dynamic regulation of its metabolic network limits the progress of further research on the application ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/20C12P13/06C12R1/15
CPCC12P13/06C12N1/205C12R2001/15
Inventor 谢希贤陈宁马跃超徐庆阳张成林李燕军范晓光马倩杜丽红陈启欣石拓
Owner TIANJIN UNIV OF SCI & TECH
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