雄烯二酮耐底物突变株的筛选及生物转化培养基优化

采用紫外、硫酸二乙酯复合诱变的方法对能选择性降解植物甾醇侧链的新金分支杆菌MN2进行诱变处理，利用梯度平板技术筛选高底物耐受性突变株，通过摇瓶发酵实验检测其转化性能，以期筛选出具有较高底物耐受性且转化生成雄烯二酮（androst-4-ene-3,17-dione，AD）能力提高的突变株。最终筛选到1 株能在含高浓度底物平板上生长的AD突变株MN4。在底物甾醇投料量为20 g/L，摇瓶发酵144 h时AD生成率为40.9%，较出发菌株提高了15.1%。经斜面接种连续传代5 次，MN4突变株遗传性状稳定。对突变株MN4的发酵培养基进行优化，通过正交试验确定的最佳培养基配比为：葡萄糖1 g/L，玉米浆25 g/L，磷酸二氢钠0.6 g/L，硝酸钠6.2 g/L，豆油160 g/L。根据该配方进行转化，得到AD的生成率为50.6%。

Screening of Substrate-resistant Mutant Strains Producing Androst-4-ene-3,17-dione and Optimization of Biotransformation Medium

The original strain Mycobacterium MN2 which is capable of selectively cleaving the side chain of phytosterol to
generate androst-4-ene-3,17-dione (AD) and androsta-1,4-diene-3,17-dione (ADD) was induced by UV irradiation and dithyl
sulfate (DES). The mutant strains were selected and grown with higher concentration substrate by gradient plate method
and their fermentation performance was evaluated. Then the biotransformation medium of the selected mutant strains was
optimized by orthogonal array design. One mutant strain MN4 was obtained. When the concentration of substrate phytosterol
was 20 g/L and shaking flask fermentation was proceeded for 144 hours, the conversion rate of androst-4-ene-3,17-dione
(AD) from phytosterol by MN4 was 40.9%, which was 15.1% higher than that of the original strain. In addition, the mutant
strain showed high genetic stability after five continuous passages. The optimum medium composition for AD production
was found to be 1 g/L glucose, 25 g/L corn steep liquor, 0.6 g/L Na2HPO4, 6.2 g/L NaNO3, and 160 g/L soybean oil. Under
the optimal conditions, the conversion rate of androst-4-ene-3,17-dione was 50.6%. A method for screening substrateresistant
strain with high yield of androst-4-ene-3,17-dione has been established, and the mutant strain MN4 with high
biotransformation ability and excellent genetic stability had been obtained after UV and DES mutation.