Homoserine dehydrogenase gene inactivation method of Bacillus subtilis

A technology of homoserine dehydrogenase and serine dehydrogenase, which is applied in the field of genetic engineering, can solve the problems of competition for imported products, many steps, and small production scale, and achieve simple, fast and effective results

Inactive Publication Date: 2015-05-20
INST OF SUBTROPICAL AGRI CHINESE ACAD OF SCI
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The industrial production of lysine is mainly based on the fermentation method, but due to the low level of acid production of the production strains, small production scale, high production costs, and difficulty in competing with imported products, therefore, the selection of high-yielding lysine bacteria kind of urgent
[0003] At present, gene knockout technology is usually used in genetic engineering to obtain strains with higher lysine production than the original strain. The knockout bacteria are mainly obtained through homologous recombination, by transforming To competent cells, screened, but this method has more steps

Method used

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  • Homoserine dehydrogenase gene inactivation method of Bacillus subtilis
  • Homoserine dehydrogenase gene inactivation method of Bacillus subtilis
  • Homoserine dehydrogenase gene inactivation method of Bacillus subtilis

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0013] Embodiment 1 Bacillus subtilis PA105 homoserine dehydrogenase gene inactivation

[0014] Bacillus subtilis PA105 was provided by the experimental center of the applicant, and the deposit number is CCTCC: M2011328.

[0015] 1. Competent preparation medium for Bacillus subtilis:

[0016] GMI medium: 1 mL of 10×Spizizen salts, 0.1 mL of 10% yeast powder, 0.25 mL of 20% glucose, 0.2 mL of 1% hydrolyzed casein, 0.2 mL of 0.25% required amino acids, supplemented with sterilized distilled water to a total volume of 10 mL.

[0017] GMII medium: 1mL 10×Spizizensalts, 0.05mL 10% yeast powder, 0.25mL 20% glucose, 0.04mL 1% hydrolyzed casein, 0.2mL 0.25% required amino acids, 0.05mL 0.1mol / LCaCl2, 1mL 25mmol / LMgCl2, supplemented with sterilization Distill water to a total volume of 10 mL.

[0018] 10 x Spizizen salts stock solution: 15% K 2 HPO 4 ·3H 2 O, 6% KH 2 PO 4 , 2% (NH 4 ) 2 SO 4 , 0.2% MgSO 4 and 1% sodium citrate dissolved in 75.8% distilled water, 6.6×10 4 Au...

Embodiment 2

[0038] The preparation of embodiment 2 mutant strain fermented liquid

[0039] The preparation of shake flask fermentation seed liquid: get the mutant strain 2ml that embodiment 1 obtains (concentration of viable bacteria is 10 8 -10 10 CFU / ml), inoculated in 100ml LB liquid medium for shake flask fermentation culture, the fermentation temperature is 37°C, the pH value is 7.2, the rotation speed is 200r / min, and the fermentation time is 24h. Among them, the composition of LB liquid medium is: tryptone 10g, yeast extract 5g, sodium chloride 10g, distilled water 1000ml, pH value is 7.2.

[0040] Carry out fermentor pilot test after shake bottle fermentation finishes, get 100ml shake bottle fermentation seed liquid (the concentration of seed liquid is 10 8 CFU / ml) was inoculated into a 10L fermenter with a liquid volume of 5L, the fermentation temperature was 37°C, the pH value was 7.2, the stirring speed was 300r / min, the fermentation was 24h, and the aeration ratio was 1:1. ...

Embodiment 3

[0041] Lysine yield analysis in embodiment 3 mutant strain fermented liquid

[0042] Take 5ml of the fermented liquid obtained in Example 2, centrifuge at 12000r / min for 10min, take 1ml of supernatant, add 2.5ml of 7.5% (mass ratio) trichloroacetic acid per ml, mix well, centrifuge at 18000r / min at 4°C for 15min, draw 1ml of the supernatant was transferred to an amino acid analysis vial, and the contents of arginine and other amino acids were determined with a Hitachi L-8800 automatic amino acid analyzer. The results showed that the lysine content in the fermentation broth was 63g / L.

[0043] Adopt the method of Example 2 to prepare Bacillus subtilis PA105 (preservation number is CCTCC: M2011328) fermented liquid, and analyze the yield of lysine in the fermented liquid according to the method of Example 3, the result shows that the lysine in its fermented liquid The content is 3.03g / L.

[0044]

[0045]

[0046]

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Abstract

The invention relates to a homoserine dehydrogenase gene inactivation method of Bacillus subtilis, which comprises the following steps: establishing a PCR (polymerase chain reaction) product plasmid for homologous recombination, wherein the recombinant plasmid is composed of a pMD18-T vector, a kanamycin resistance gene and a homoserine dehydrogenase gene, and the homologous recombinant DNA (deoxyribonucleic acid) segment contains a homoserine dehydrogenase gene for substituting knock-out gene and a kanamycin resistance gene inserted in the middle of the homoserine dehydrogenase gene; and carrying out PCR amplification on the homoserine dehydrogenase gene and kanamycin resistance gene inserted in the middle of the homoserine dehydrogenase gene from the recombinant plasmid so as to obtain the single-chain PCR product, electrically transforming the single-chain PCR product into a Bacillus subtilis competent cell to obtain a transformant, and screening the positive transformant to obtain the homoserine dehydrogenase gene inactivated mutant strain. Compared with the existing technique for transforming the homoserine dehydrogenase recombinant plasmid into the Bacillus subtilis competent cell, the method is more simple and time-saving.

Description

technical field [0001] The invention relates to the field of genetic engineering, in particular to a method for inactivating the homoserine dehydrogenase gene of Bacillus subtilis. Background technique [0002] Lysine is one of the essential amino acids for humans and animals. When lysine is insufficient, the utilization of other amino acids will be limited. The development of lysine production is of great significance to improve the utilization efficiency of animal feed. The industrial production of lysine is mainly based on the fermentation method, but due to the low level of acid production of the production strains, small production scale, high production costs, and difficulty in competing with imported products, therefore, the selection of high-yielding lysine bacteria This has become a top priority. [0003] At present, gene knockout technology is usually used in genetic engineering to obtain strains with higher lysine production than the original strain. The knockout...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/74C12N1/21
Inventor 王升平李丽立张彬范觉鑫蒋国礼刘刚刘志强
Owner INST OF SUBTROPICAL AGRI CHINESE ACAD OF SCI
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