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Bacterial strain capable of realizing high yield of L-alanine

A high-yielding strain, alanine technology, applied in the field of bioengineering, can solve the problems of high mortality, low efficiency of positive mutation rate, difficult to control dose size, etc., and achieves significant economic value, increased yield, and good stability.

Inactive Publication Date: 2016-10-26
QILU UNIV OF TECH +1
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  • Abstract
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  • Claims
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Problems solved by technology

Gao Lisuo et al. reported the method of screening L-alanine high-yield strains by combining ultraviolet and laser lamp mutagenesis (Journal of Anhui Institute of Engineering Science and Technology, 2008, 23 (1) 19-24), but due to ultraviolet light on various The mutagenic effect of microorganisms is different, and it is difficult to grasp the dose of ultraviolet light, and there is a photorepair effect after mutation, so the effect of ultraviolet mutagenesis is not good, the efficiency of positive mutation rate is low, and the mortality rate is high

Method used

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  • Bacterial strain capable of realizing high yield of L-alanine

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Experimental program
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Effect test

Embodiment 1

[0032] Embodiment 1 Utilizes normal temperature and pressure plasma (ARTP) mutagenesis starting strain

[0033] Use MG1655 as the starting strain to culture to the late logarithmic period, add sterile water to dilute to the cell density OD 600mm If the value is greater than 1, dry it under sterile air, and then place it in the ARTP mutagenesis system for plasma irradiation for 0.5-6min; wash the sample with normal saline, and spread it on a plate LB solid medium after conventional gradient dilution, 37 Cultivate at ℃ for 15h.

Embodiment 2

[0034] Example 2 High-throughput preliminary screening of mutant strains

[0035] Pick the single-colony mutant strain obtained in the above steps, and inoculate them in a 96-well plate containing 0.5ml LB solid medium and a 96-well plate containing 0.5ml LB liquid fermentation medium respectively, and place the former at 35-37°C for static culture 10 to 18 hours, the latter placed at 35 to 37 °C, 200 to 300 r / min shaking culture for 12 to 20 hours; after cultivation, the content of L-alanine in the fermentation broth of each well was detected, and the output of the selected strain MG1655 was 40% higher than that of the starting strain MG1655. % of the mutant strains are primary screening high-yield strains.

Embodiment 3

[0036] Example 3 High-throughput re-screening of mutant strains

[0037] Inoculate the solid culture on the LB solid medium of the high-yield mutant strain obtained in step (2) into a 96-well plate equipped with 0.5ml LB liquid fermentation medium, and culture it with shaking at 35~37°C and 200~300r / min for 12~ 20h; transfer 0.05ml of inoculum into a 96-well plate containing 0.5ml of LB liquid fermentation medium, culture at 35-37°C, 200-300r / min shaking for 12-20h; after cultivation, detect the fermentation broth in each well The content of L-alanine is selected, and the mutant strain whose L-alanine content is 40% higher than the output of the starting strain MG1655 is selected as a high-yielding strain for re-screening.

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Abstract

The invention provides a bacterial strain capable of realizing high yield of L-alanine, belonging to the field of bioengineering technology. The bacterial strain capable of realizing high yield of L-alanine is named as Escherichia coli (EcQLS1) with an accession number of CGMCC No. 1764. The Escherichia coli (EcQLS1) strain is obtained through a combination of normal-temperature normal-pressure plasma mutation and high-flux screening, can greatly improve the yield of L-alanine and has good stability, wherein the yield of L-alanine has no obvious change after serial passage of the strain for five generations. Results of shake-flask experiments show that the yield of L-alanine reaches 120 g / L. The bacterial strain provided by the invention totally meets industrial mutation and screening demands on an L-alanine strain, is applicable to industrial fermentation production and has significant economic value.

Description

technical field [0001] The invention belongs to the technical field of bioengineering and provides an L-alanine high-yield bacterial strain. Background technique [0002] L-alanine, also known as α-alanine, is a left-handed isomer of α-amino acid, which is a white crystal with a special sweet taste. It is widely used in the fields of biology, medicine, chemical industry, food, polymer materials, etc., for the preparation of pharmaceutical compounds, synthetic sweeteners, and determination of liver function. It is an important chemical product with a wide range of applications and great development potential. More and more more popular and valued by people. [0003] Currently, L-alanine can be produced by chemical synthesis, enzymatic and fermentation methods. It is a common method in the chemical industry to produce various amino acids through different chemical synthesis routes, but the synthetic method has poor product quality, low recovery rate, high cost and environmen...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/20C12N13/00C12R1/19
CPCC12N13/00C12P13/06C12N1/205C12R2001/19
Inventor 邱磊李娇娇战伟伟赵佩玉李敬龙
Owner QILU UNIV OF TECH
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