The Study On The Side-chain Cleavage Of Phytosterols To Androstenedione And Androstadienedione By Mycobacterium Neoaurum

Steroid drugs are widely used to treat various diseases in the world with a range near to that of antibiotics.Androst-4-ene-3,17-dione(AD)and Androst-1,4-ene-3,17-dione(ADD)are the representatives of 17-keto steroids and were used as precursors for the production of steroidal drugs.AD and ADD can be produced through chemical process.However,chemical synthesis is being replaced by biochemical procedures for sustainability and being eco-friendly.Different kinds of micrograms have been developed to prepare AD and ADD through side-chain cleavage of phytosterols.Presently researchers are focusing on obtaining efficient strains and improving the biotransformation efficiency.Two strains Mycobacterium neoaurum R10 and MS261 accumulating AD and ADD respectively were obtained through genetic manipulation and were kept in our lab.The objective of this paper is to optimize the biotransformation process to improve the biotransformation efficiency for further industrialization.As for ADD production strains MS261,the highest biomass was obtained when the inoculum age was 24 h and inoculum volume is 16%.The most efficient biotransformation was obtained when 2 g/L substrate was loaded in the beginning and 10 g/L glucose and 5 g/L yeast extract.Instead of ADD,testosterone and dehydrotestosterone were accumulated when high concentration of glucose was used.The viability of MS261 used as resting cell for side-chain cleavage was investigated.Compared with growing cells,resting cells can utilize phytosterols more efficiently while giving a lower yield for intense product degradation.For AD accumulating strain Mycobacterium neoaurum R10,the effect of substrate loading and medium composition on the biotransformation process were investigated.The yield reached 31.7%when 3 g/L substrate was added in the broth in the second day after inoculation.AD cannot be accumulated when citrate were absent in the medium.AD yield was improved by 29.8%since 50mM NaHCO3 was added in the medium.10 g/L glucose and 5 g/L yeast extract were used as carbon sources and nitrogen sources and the yield of AD reached 35%when 5 g/L substrate was added in the medium.Meantime,we discovered that glucose supplementation after depletion can inhibit product degradation thus improving the yield.Later,different supplementation modes were applied and one time supplementation of glucose to 5 g/L after depletion gave the highest yield,39.1%higher than that of the control.Resting cells were also used for side-chain cleavage.Substrate and product degradation were found to be degraded simultaneously.Application of EDTA inhibited product degradation while side-chain cleavage was also inhibited.