Bacterium for stably producing 2,3-butanediol at high yield and method for compound mutation by using low-temperature plasma and diethyl sulfate

[0020] This example illustrates the method of using plasma and diethyl sulfate to mutagenize microorganisms.

[0021] The experimental strain is Enterobacter cloacae (Enterobacter cloacae), screened from the soil. The plasma discharge device used in the experiment is the dielectric barrier discharge (DBD) device used in the first embodiment of patent 200610047213.5.

[0022] The seed medium used in this embodiment is: glucose 40g / L, (NH 4 ) 2 HPO 4 6g / L, KCl 1.8g / L, EDTA 0.51g / L, MgSO 4 0.88g / L, FeSO 4 ·7H 2 O 0.0225g / L, ZnSO 4 ·7H 2 O 0.0075g / L, MnSO 4 ·H 2 O 0.0023g / L, citric acid 0.21g / L, sodium citrate 0.294g / L, yeast powder 1g / L.

[0023] The fermentation medium is: glucose 120g / L, (NH 4 ) 2 HPO 4 18g / L, KCl 1.8g / L, EDTA 0.51g / L, MgSO 4 0.88g / L, ZnSO 4 ·7H 2 O 0.0075g / L, FeSO 4 ·7H 2 O 0.0225g / L, MnSO 4 ·H 2 O 0.0023g / L, citric acid 0.21g / L, sodium citrate 0.294g / L, yeast powder 1g / L.

[0024] The culture conditions are as follows: the amount of l...

[0030] This example illustrates the method for screening mutant strains with high production of 2,3-butanediol

[0031] Shake flask primary screening

[0032] Dilute the mutagenized bacterial solution onto a plate, place it in a constant temperature incubator at 37°C for 24 hours, pick colonies with good colonies on the plate and inoculate them into 50 mL of seed medium, and cultivate them at 37°C and 200 rpm for 36 hours. Detect the concentration of 2,3-butanediol, and select the strain whose product concentration is more than 20% higher than that of the original bacteria.

[0033] Shake flask re-screening

[0034] The strains obtained by primary screening in shake flasks were transferred to 200mL fermentation medium, and each sample was made in triplicate, placed in a constant temperature shaker at 37°C, and shaken at 200rpm for 36h. The concentration of 2,3-butanediol was detected by gas phase, and the strain whose product concentration was still more than 20% higher than...

[0038] This example illustrates the genetic stability of the mutant Enterobacter cloacae DLM.

[0039] The seed medium with glucose as the carbon source was continuously passaged, and the first, third and seventh passages were used for batch fed-batch experiments in fermenters to check the passage stability of the mutant strain DLM. The experimental results are shown in Table 1.

[0040] Table 1

[0041] Strain

[0042] It can be seen from the experimental results that the fermentation performance of the mutant strain is very stable after 7 consecutive generations of passage.