Special bacillus licheniformis engineering bacteria for producing meso-2,3-butanediol and application thereof
A technology of Bacillus licheniformis and meso-2, which is applied in the direction of microorganism-based methods, bacteria, microorganisms, etc., to achieve the effects of high production intensity, low probability of contamination by miscellaneous bacteria, and high yield
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Embodiment 1
[0032] Example 1: Construction of engineering bacteria producing meso-2,3-butanediol - Bacillus licheniformis (Bacillus licheniformis) 10-1-A / △gdh
[0033]The starting strain was Bacillus licheniformis 10-1-A, which was deposited by the applicant on November 14, 2011 in the General Microorganism Center of China Committee for the Collection of Microorganisms, and the preservation number is: CGMCC NO.5461.
[0034] The genomic DNA of B. licheniformis 10-1-A was prepared by a conventional method. This process referred to the method for small-scale preparation of the bacterial genome in the "Guide to Molecular Biology" published by Science Press, and extracted Bacillus licheniformis (Bacillus licheniformis ) Genomic DNA of 10-1-A; Use synthetic primers Kgdh1-f and Kgdh1-r to obtain glycerol dehydrogenase gene gdh (GenBank: The front section of KF250430). Use the synthetic primers Kgdh2-f and Kgdh2-r to PCR amplify the latter segment of the glycerol dehydrogenase gene from the Bac...
Embodiment 2
[0044] Embodiment 2: prepare the cell liquid culture of Bacillus licheniformis CCTCC NO:M 2014559 bacterial strain
[0045] 1. Plate culture: Streak the B.licheniformis 10-1-A / △gdh obtained in Example 1 onto an LB plate containing 1.5% agar by mass volume ratio, and culture at 50°C for 12 hours;
[0046] 2. Seed culture: under sterile conditions, use a sterile toothpick to pick a single colony on the plate in step (1), then inoculate it into LB medium, and culture it on a shaker at 50°C for 12 hours;
[0047] 3. Shake flask culture: under aseptic conditions, get the bacterium liquid cultivated in step (2) with an inoculum size of 1 to 3% by volume, inoculate 30 to 150 milliliters of 60 grams per liter of glucose seed medium, Shaking culture at 50±1° C. for 11 to 13 hours; the cell liquid culture of Bacillus licheniformis CCTCC NO:M 2014559 strain is obtained.
[0048] Among them, the formula of the above-mentioned seed medium is: glucose 50-70 g / L, yeast powder 8 g / L, anhydro...
Embodiment 3
[0050] Embodiment 3: Utilize bacillus licheniformis CCTCC NO:M 2014559 bacterial strain to produce meso-2,3-butanediol by fermentation medium in 5L fermenter
[0051] The cell liquid culture of the bacillus licheniformis CCTCC NO:M 2014559 bacterial strain obtained by embodiment 2 is inoculated in 5 liters of fermentors that 4 liters of fermentation mediums that have been sterilized are housed with the inoculum size of volume percentage 10%, in Under the condition of 50°C, the stirring speed of the fermenter is 500 rpm, the ventilation rate is 0.5vvm-1.0vvm, and the fermentation is 60 hours.
[0052] During the fermentation, samples were taken every 3 hours, and the samples were centrifuged at 8,000×g for 10 minutes to detect the concentration of meso-2,3-butanediol and glucose in the supernatant, and dry glucose powder was added according to the concentration of glucose to maintain the concentration of glucose at 20-50 g / L; the supernatant was analyzed by gas chromatography t...
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