Screening Of Substrate-resistant Mutant Strain Producing Androst-4-ene-3,17-dione And Optimization Of Biotransformation Condition

Androst-4-ene-3,17-dione (AD), as an important raw material and keyintermediate in the synthesis of steroid hormones, was an indispensable drug inclinical. It can be obtained by chemical method and microbial conversion. Due to thecomplexity of synthesis process, high cost, environmental pollution, and rawmaterial’s drying up and rising prices in recent years, chemical synthesis method hasbeen replaced by microbial transformation gradually. AD can be obtained byselectively cleaving the side chain of phytosterol. Due to its mild reaction conditions,short cycle, high selection, and environmental friendly features, microbial conversionmethod has been increasingly used in the production of steroid drugs. But inindustrical production, many strain species’s biotransformation ability is low and havesereval kind of co-products. The substrate phytosterol has a low solubility in water.It’s high concentration also inhibit the growth of bacteria and the synthesis ofproducts.In this paper, the original strain M2which is capable of selectively cleaving theside-chain of phytosterol producing AD was induced by UV irradiation and dithylsulfate (DES), respectively. By selecting the mutant strains which could grow withhigher concentration substrate through gradient plating technology and detectingthose strains’ fermentation ability, one mutant strain MN4with better tolerance ofsubstrate and higher conversion ability was obtained. Then, the mutant strain MN4’smedium compositions and transformation conditions were optimized by the singlefactor experiment and response surface experiments to further improve the strainbiotransformation capabilities. The specific research contents were as follows:(1) The original strain M2was identified by morphology, physiological,biochemical characteristics and16S rDNA sequence analysis. Then the strain wasidentified as Mycobacterium neoaurum JXNU.(2) The AD producing strain M2was induced UV and DES compound mutationto screen the substrate-resistant strains. One strain MN4with better tolerance ofsubstrate and higher conversion ability was selected by gradient plating technologyand shaking flask fermentation. When the phytosterol concentration was20g/L andshaking flask fermentation was kept for144hours, the conversion rate of AD was40.9%, which was15.1%higher than that of the original strain. In addition, the mutant strain showed high genetic stability after five continuous passage experiment.(3) The biotransformation medium was optimized by the response surfaceexperiments. The best carbon source, nitrogen source, phosphate source and inorganicsalt types was determined. The optimum medium composition for AD production wasfound to be0.3%glucose,2.28%corn steep liquor,0.1%Na2HPO4,0.54%NaNO3and15.32%soya-bean oil through the response surface method. Under the optimiummedium composition, the AD conversion rate reached52.1%, which increased27.3%compared with the original level.(4) Besides, the strain’s fermentation conditions were optimized through singlefactor tests. The optimized biotransformation conditions were as follows: initial pHvalue7.0, seed cultivation time48h, inoculum volume14%, shaking speed230rpm,temperature31℃. Under the optimium conditions, the AD conversion rate reached55.2%, which was6%higher than the condition optimization.(5) The batchwise fermentation experiment was conducted in the5L bioreactorto investigated the pH change and the AD concentration during the fermentation time.It indicated that, when the biotransformation process was kept for90hours, the ADmaximum yield was9.56g/L and AD conversion rate reached79.8%. Generallyspeaking, the fermentation in the canisters had a higher AD conversion.