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Corynebacterium glutamicum, and key tryptophan synthesis gene thereof

A technology of Corynebacterium glutamicum and tryptophan, which is applied in the field of microbial breeding and fermentation engineering, and can solve problems such as unclear mutation sites of key enzymes

Active Publication Date: 2017-03-22
TIANJIN UNIVERSITY OF SCIENCE AND TECHNOLOGY +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there was no related report on this strain, and the mutation site of the key enzyme was not clear

Method used

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  • Corynebacterium glutamicum, and key tryptophan synthesis gene thereof
  • Corynebacterium glutamicum, and key tryptophan synthesis gene thereof
  • Corynebacterium glutamicum, and key tryptophan synthesis gene thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0039] Example 1: Construction of C.glutamicumTP608

[0040] 1. Extraction of Genomic DNA from C. glutamicum TP607 (CGMCC No.12302)

[0041] A single colony was inoculated in an LB shake tube, and after overnight culture at 32°C for 12 hours, 2% of the inoculum was added to 30 mL of LB medium for overnight culture in a shake flask. The steps to extract the genome are as follows:

[0042] (1) Centrifuge the bacterial solution at 13000rpm for 2min, remove the supernatant, and collect it into a 1.5mL EP tube.

[0043] (2) Add 600 μL of TE buffer with pH=8.0 to suspend the bacteria, centrifuge at 13000 rpm for 2 min, and discard the supernatant.

[0044] (3) Add 600 μL TE to re-suspend the cells, add 6 μl RNAase (10 μg / mL), 30 μl lysozyme (50 μg / mL) and mix well, and incubate at 37°C for more than 30 minutes.

[0045] (4) Add 30 μL of 10% SDS and 10 μL of proteinase K, mix well, and incubate at 65° C. for more than 15 minutes.

[0046] (5) Add 100 μL 5mol / L NaCl solution and m...

Embodiment 2

[0104] Example 2: Shake flasks carry out tryptophan fermentation

[0105] 1. Strains

[0106] The strains used for shake flask fermentation are as follows:

[0107] C. glutamicum TP607; C. glutamicum TP607-trp1;

[0108] C. glutamicum TP607-trp1-trp2; C. glutamicum TP608;

[0109] 2. Activation of strains

[0110] Slant culture: Streak inoculation of -80°C preserved strains on the activated slant, culture at 32°C for 24 hours, pass passage once, and culture at 32°C for 12 hours.

[0111] Activation medium (g / L): peptone 10, beef extract 10, yeast powder 5, NaCl 2.5, corn steep liquor 15mL / L, agar 25, pH 7.0-7.2, sterilization conditions: 121°C, 20min.

[0112] 3. Seed culture

[0113] Seed culture: Use a 500mL triangular flask to cultivate seeds, the liquid volume is 30mL, the strain uses the second-generation slant, the inoculation amount is two rings of the second-generation slant, the inoculation method is to scrape the second-generation slant seeds with the inoculatio...

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Abstract

The invention belongs to the fields of microorganism breeding and fermentation engineering, and concretely relates to a Corynebacterium glutamicum, and a key tryptophan synthesis gene thereof. The above high-yielding strain is obtained through editing the genome of Corynebacterium glutamicum CGMCC No.12302 through a gene engineering means, and integrating the trpDCB mutation site in a tryptophan operon to the genome of the CGMCC No.12302. The Corynebacterium glutamicum provides a new mutation site for tryptophan synthesis related gene, and provides a new research direction for biosynthesis of the tryptophan; and a shaking bottle fermentation result shows that the tryptophan output of the obtained strain is about 3.4 times original output, so the tryptophan production performance of the strain is greatly improved.

Description

[0001] Technical field: [0002] The invention belongs to the field of microbial breeding and fermentation engineering, and in particular relates to the construction of a high-yield strain of Corynebacterium glutamicum for fermenting tryptophan and the mutation sites of genes trpD, trpC and trpB related to tryptophan synthesis. [0003] Background technique: [0004] Tryptophan is the only aromatic amino acid with an indole branch, and its chemical name is 2-amino-3-indolepropionic acid. Tryptophan is one of the 8 kinds of amino acids necessary for the human body. Its source depends on food supply. L-tryptophan has two main outlets, one is to synthesize protein, and the other is to enter catabolism. During its catabolism, L-tryptophan will produce various physiologically active substances such as 5-hydroxytryptophan, niacin, pineal gland hormone and xanthuric acid. These biologically active substances have important medical uses; in addition, L-tryptophan can be used as a food...

Claims

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Application Information

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IPC IPC(8): C12N1/21C12P13/22C12R1/15
CPCC12N9/1077C12N9/88C12N9/90C12P13/227C12Y204/02018C12Y401/01048C12Y402/0102C12Y503/01024
Inventor 李燕军陈宁韩洪军袁启发高立栋张顺棠李娟谢希贤张成林徐庆阳范晓光马倩
Owner TIANJIN UNIVERSITY OF SCIENCE AND TECHNOLOGY
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