Corynebacterium glutamicum and application

A technology of Corynebacterium glutamicum and glutamic acid rods, applied in the field of microorganisms

Inactive Publication Date: 2016-12-07
TIANJIN UNIV OF SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, L-valine high-yielding strains are mainly obtained through traditional mutagenesis breeding, or combined with part of genetic engineering on this basis, but there is no report that clearly describes the complete L-valine production strain. Gene Information and Efficient Synthesis Mechanism of L-Valine

Method used

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  • Corynebacterium glutamicum and application
  • Corynebacterium glutamicum and application
  • Corynebacterium glutamicum and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0049] Chemical mutagenesis of Corynebacterium glutamicum

[0050] (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;

[0051] (2) Inoculate in a liquid seed medium from a slope, the seed medium is LB, and cultivate at 32° C. for 12 hours;

[0052] (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;

[0053] (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;

[0054] (5) Dilute with an appropriate amount of sterile water, spread on the resistance screening medium containing sulfaguanidine and L-valine structural analogues, cultivate at 32°C, and wait for a single colony to...

Embodiment 2

[0058] Genome sequencing of Corynebacterium glutamicum XV

[0059] (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;

[0060] (2) The genome was sequenced using the third-generation sequencing platform PacBio RS II;

[0061] (3) Genome assembly was performed using the assembly software provided by PacBio, and routine comparative genome analysis was performed using related software. On the basis of the genome sequence, by comparing the genome sequences of the valine-producing strain Corynebacterium glutamicum XV and the standard strain of Corynebacterium glutamicum ATCC 13032, it was found that there are more bases in the gene sequence related to valine synthesis Base mutations, base mutations lead to the following changes in the amino acid sequences of each gene-encoded product (using "amino ...

Embodiment 3

[0073] Comparison of valine synthesis ability of strains before and after gene mutation

[0074] (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, and the rest is distilled water;

[0075] (2) Shake flask seed medium (g / L): glucose 30, yeast powder 5, bean cake powder hydrolyzate 20, corn steep liquor 20, NaCl5, MgSO 4 ·7H 2 O2, KH 2 PO 4 12H 2 O2, FeSO 4 ·7H 2 O 0.01, MnSO 4 ·H 2 O 0.01, leucine 0.1, isoleucine 0.1, phenol red solution (0.4g / L) 20mL, pH 6.7-7.2, the rest is distilled water;

[0076] (3) Shake flask fermentation medium (g / L): glucose 60, bean cake powder hydrolyzate 25, corn steep liquor 40, MgSO 4 ·7H 2 O2, KH 2 PO 4 12H 2 O2, FeSO 4 ·7H 2 O 0.01, MnSO 4 ·H 2 O 0.01, V B1 0.01, phenol red solution (0.4g / L) 20mL, pH 6.7-7.2, the rest is distilled water;

[0077] (4) Incline culture: place in a 32°C incubator, cultivate the first-generation activated slope for 24 hou...

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Abstract

The invention provides corynebacterium glutamicum and application. The corynebacterium glutamicum is compared with a genomic sequence of a corynebacterium glutamicum standard strain ATCC 13032, and multiple base mutations exist in an amino acid sequence of an enzyme related to valine synthesis. The preservation number of the corynebacterium glutamicum is CGMCC No.12152, and the corynebacterium glutamicum has an important application value in the aspect of preparing valine.

Description

technical field [0001] The invention relates to the technical field of microorganisms, in particular to a coryneform bacterium glutamicum and its application. Background technique [0002] Corynebacterium glutamicum is a kind of aerobic Gram-positive bacteria that mainly lives in soil and is not pathogenic. The main application field is microbial fermentation, and its products include amino acids, organic acids, vitamins, etc., which are widely used all over the world. At present, most amino acids, including L-glutamic acid, L-lysine, L-valine, L-isoleucine, L-leucine, etc., can be produced by fermentation of Corynebacterium glutamicum. [0003] Domestic L-valine production is mainly based on the fermentation method of Corynebacterium glutamicum, and the average acid production can reach 60g / L or higher. At present, L-valine high-yielding strains are mainly obtained through traditional mutagenesis breeding, or combined with part of genetic engineering on this basis, but th...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/20C12P13/08C12R1/15
CPCC12N9/0006C12N9/1025C12N9/1096C12N9/88C12P13/08C12Y101/01085C12Y101/01086C12Y203/03013C12Y206/01042C12Y401/03C12Y402/01009C12Y402/01033C12Y403/01019
Inventor 谢希贤陈宁马跃超徐庆阳陈启欣杜丽红石拓
Owner TIANJIN UNIV OF SCI & TECH
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