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Corynebacterium glutamicum mutant strain and application thereof in L-leucine production

A technology of Corynebacterium glutamicum and mutant strains, applied in the biological field, can solve problems such as unstable genetic information of strains, environmental pollution, and extensive cultivation techniques, and achieve simple and easy control of the production process, reduce the production of para-acids, and improve product purity. high effect

Active Publication Date: 2018-11-20
INNOBIO CORP LTD +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] At present, the production process of L-leucine in China mostly adopts the hydrolysis method, and there are problems such as serious environmental pollution.
Domestic production of L-leucine by fermentation started late and lags behind. Compared with Japan and other countries, there is still a certain gap. The reasons are that the genetic information of the strains is unstable, the cultivation process is extensive, and the heterogeneous acids affect the product yield and production rate. Purity etc.

Method used

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Examples

Experimental program
Comparison scheme
Effect test

preparation example Construction

[0020] (2) Preparation of bacterial suspension

[0021] Take 10ml of the culture solution prepared in step (1) in a sterile centrifuge tube, centrifuge at 5000rpm for 10min, discard the supernatant, resuspend the cells in normal saline, pour them into an Erlenmeyer flask, shake for 10min, and adjust the cell concentration to 5×10 8 pieces / ml.

[0022] (3) UV mutagenesis

[0023] Take the bacterial suspension prepared in step (2) and put it under 30W ultraviolet lamp for 30-120s mutagenesis, take samples every 10s, dilute and spread, culture on the plate in the dark and calculate the lethality. Elimination of wild-type and concentrated deficient strains by starvation culture and penicillin method, and detection of deficient strains (Glu - +Met - +Ile - ), and test fermentation acid production capacity according to step (5).

[0024] (4) Diethyl sulfate mutagenesis

[0025] Prepare the bacterial suspension from the deficient strain obtained in step (3) according to step (...

Embodiment 1

[0038] The CM slant lawn of Corynebacterium glutamicum mutant strain IBBH-15 was inoculated with tetracycline in a 3L shake flask, and cultured at 80r / min and 30°C until OD 600 =15, to obtain the seed solution of the shaker bottle, for subsequent use. According to the 10% (V / V) inoculum amount, the shake flask seed solution was inserted into the fermentation medium of a 30L fermenter, the initial temperature was 30°C, and the initial ventilation rate was 0.8L / min. The dissolved oxygen is 10-20% in the first 24 hours of the process, and the dissolved oxygen is 5-15% after 24 hours; the pH of the fermentation process is controlled by automatic feeding of ammonia water, the initial pH value is 6.7, and the OD of the bacteria 600 After growing to 20, the pH increased to 6.9, and the cell OD 600 After growing to 25, the pH increased to 7.0, and the cell OD 600 After increasing to 36, the pH increased to 7.2; during the fermentation process, the residual sugar concentration was co...

Embodiment 2

[0040] Study on the ability of Corynebacterium glutamicum mutant strain IBBH-15 to produce L-leucine under fermentation conditions.

[0041] This embodiment sets 2 test groups:

[0042] Group 1: the culture conditions described in Example 1 were tested; test results: the L-leucine output was the highest at 40.6g / L when fermented for 45h, the sugar-acid conversion rate was 26.6% (W / W), and the total para-acid The content is about 2.8g / L.

[0043] The second group is the control test group, and the test is carried out according to the following conditions: insert the slant strain IBBH-15 into a 3L shaker flask, and culture it at 80r / min and 30°C for 16h to obtain a seed solution for later use. According to the 10% (V / V) inoculum amount, the seed solution was put into a 50L fermenter with fermentation medium, the initial temperature was 30°C, and the initial ventilation rate was 1.0L / min. By controlling the stirring speed and air volume, the fermentation process The dissolved o...

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Abstract

The invention discloses a corynebacterium glutamicum mutant strain and application thereof in L-leucine production. The corynebacterium glutamicum mutant strain has a collection number of CGMCC No. 15720. By utilizing the mutant strain disclosed by the invention and a fermentation method, high-efficiency production of the L-leucine can be realized, and the yield of by-product acids can be effectively reduced in the fermentation production process. Furthermore, since the amount of heteroacids in fermentation liquor is small, a seed crystal, a crystal washing agent or a crystallization additivedoes not need to be added according to the conventional scheme in the field in the purification process, and a recrystallization step is not needed. The fermentation liquor is simply separated and extracted, and the product purity is high. The whole production technology is simple and controllable in process, low in cost and suitable for large-scale industrial application.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a mutant strain of Corynebacterium glutamicum used for L-leucine production and its application in microbial fermentation engineering. Background technique [0002] L-leucine is one of the eight essential amino acids that humans and animals cannot synthesize themselves and need to rely on external sources. L-leucine has a variety of physiological functions and is an indispensable raw material for compound amino acid intravenous injection in clinical practice. Maintaining the nutritional needs of critically ill patients takes an active role. In addition, L-leucine, L-valine, and L-isoleucine belong to branched-chain amino acids, and are also widely used in food, health products, and feed. [0003] At present, the production process of L-leucine in China mostly adopts the hydrolysis method, which has problems such as serious environmental pollution. Domestic production of L-leucine b...

Claims

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Application Information

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IPC IPC(8): C12N1/20C12N13/00C12N15/01C12P13/06C12R1/15
CPCC12N13/00C12N15/01C12P13/06C12N1/205C12R2001/15
Inventor 范超洪皓刘军陈剑彬吴文忠
Owner INNOBIO CORP LTD
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