复合诱变选育高效转化DHEA为7α,15α-diOH-DHEA的菌株及其转化工艺优化

Optimization of hydroxylating DHEA to 7α,15α-diOH-DHEA by compound mutation and fermentation optimization

摘要:

结合酮康唑抗性筛选法，采用亚硝基胍和低能氮离子注入复合诱变方法筛选得到一株高效生物转化去氢表雄酮 (DHEA) 为3β,7α,15α-三羟基雄甾-5-烯-17-酮 (7α,15α-diOH-DHEA) 的菌株亚麻刺盘孢Colletotrichum lini ST-1，该突变株在底物DHEA投料浓度为10 g/L时产物摩尔得率达到34.2%，较出发菌株提高了46.2%。在此基础上进行培养基组分的优化，采用Plackett-Burman实验设计考察转化培养基中各组分对产物摩尔得率的影响，有效筛选出葡萄糖、酵母粉和MgSO4·7H2O浓度对产物摩尔得率影响显著，继而采用最陡爬坡路径逼近最大响应区域，并利用中心组合响应面设计实验对3个显著性因素的最佳水平进行研究，得到最适转化培养基组分为 (g/L) ：葡萄糖26.34；酵母粉12.15；玉米浆3.00；FeSO4·7H2O 0.015；MgSO4·7H2O 0.14；KH2PO4 0.90。采用该优化培养基，菌株C. lini ST-1的产物摩尔得率达到49.3%，较优化前提高了44.2%。

Abstract:

Combined with method of ketoconazole resistance screening, a 7α,15α-diOH-DHEA high-producing mutant Colletotrichum lini ST-1 was obtained by compound mutation of NTG and low energy N+ ion beam implantation. With the substrate concentration of 10 g/L DHEA, the molar yield of 7α,15α-diOH-DHEA reached 34.2%, increased by 46.2% than that of the original strain. Then we optimized the medium. First, Plackett-Burman design was used to evaluate the effects of medium components on molar yield of the product. Results show that glucose, yeast extract and MgSO4·7H2O were the important parameters for the biotransformation process. Subsequently, the path of steepest ascent was used to approach the optimal levels. To obtain the optimal levels, central composite design and response surface analysis were carried out. The optimal medium was as follows (g/L): glucose 26.34, yeast extract 12.15, corn flour 3.00, FeSO4·7H2O 0.015, MgSO4·7H2O 0.14, KH2PO4 0.90. Under the optimal conditions, the molar yield of 7α,15α-diOH-DHEA reached 49.3%, which was 44.2% higher than that of using the medium before optimization.