Mutation Breeding And Fermentation Research Of Androst-4-ene-3, 17-dione Producing Strain

Androst-4-ene-3, 17-dione (AD) was a key middle product of sterol hormones and could be obtained by selectively cleaving the side-chain of phytosterol. The microbial production of AD had the advantage of high purity, low cost and short cycle time, but it was restricted by the conversion ability of microbes and low solubility of phytosterol. In this experiment, a mutant with high productivity, Mycobacterium sp. BD-696 was obtained, with which AD output and transformation rate increased notably. Besides, aqueous-organic two-phase system was used as the fermentation system which boosted the concentration of phytosterol substrate greatly. The effects of different medium ingredients and fermentation conditions on transformation were also studied.The conclusions of the study were as follows:(1) The AD producing strain, the microorganism of Mycobacterium sp. BD-696 was treated by UV, DES and UV combined with DES. A mutant was obtained whose AD transformation productivity reached to 2.33g/l when the concentration of phytosterol was 6%o and the conversion rate was 61.4% which was 13.5% higher than that of its parent strain. Hereditary stability test demonstrated that the productivity of mutant strain from 1st to5th serial passage was stable.(2)The medium was optimized by singer factor experiments and orthogonal design of experiment. The obtained optimal medium ingredients were: sunflower oil 20%, molasses 6%, NH₄NO₃ 0.3%, (NH₄)₂HPO₄ 0.08%, FeSO₄·7H₂O 0.05 g/L, ZnSO₄·7H₂O 0.3 g/L, MgSO₄·7H₂O 0.2 g/L. Under the optimal medium determined, the conversion rate reached to 84.0% when the concentration of substrate phytosterol was 6%o, which was22.4%higher than the original medium.(3)The fermentation conditions with flask were optimized by unifactor experiment. The obtained optimal culture conditions were: incubation time 66h, incubation 12%, Liquid volume 50mL/250mL flask, rotating rate 220rpm, culture temperature 30～32°C, initial pH7.5.(4)The result of 5L bio-reactor identified the high output of shaking flask's fermentation. The conversion rate would decrease with the increasing concentration of phytosterol, but the total output would increase remarkably. When the concentration of phytosterol was 8%o, the conversion rate reached to 97.1% at 108h and the output was4.92g/L. When the concentration of phytosterol was 16%o, the conversion rate reached to 82.1% at 120h and the output was8.33g/L. It was economically acceptable to increase the concentration of phytosterol properly.