利用偶发分枝杆菌(Mycobacterium fortuitum)MFT 降解植物甾醇生产 9α-羟基-雄烯二酮(9α-OH-AD)过程中，存在丙酰辅酶 A 过量积累及辅因子不平衡等问题，影响菌株的转化效率。通过表达 scpC 降低胞内丙酰辅酶 A 含量，并通过串联表达 ndh 解决植物甾醇侧链降解过程中胞内辅因子 I 不平衡的问题，在此基础上表达 AarC 将琥珀酰辅酶 A 和乙酸盐转化为琥珀酸盐和乙酰辅酶 A，增强三羟酸(TCA)循环。构建的重组工程菌株 MFT-scpC-AarC-ndh经初步优化发酵后，9α-OH-AD产率达到 89。56%，比原始菌株转化率提高了 37。86%。本研究为 9α-OH-AD的工业化生产提供了理论基础和技术支持，也为其他植物甾醇转化工程菌株的构建提供新策略。
Construction of 9α-OH-AD Engineering Strain Based on Coenzyme Regulation and Optimization of Fermentation Process
In the process of using Mycobacterium fortuitum(MFT)to degrade phytosterols to produce 9α-hydroxy-androstenedione(9α-OH-AD),issues such as excessive accumulation of propionyl-CoA and an imbalance of cofactors exist,affecting the efficiency of strain transformation.By expressing scpC to reduce intracellular propionyl-CoA content and by tandemly expressing ndh to address the intracellular cofactor I imbalance during the degradation of phytosterol side chains,and then AarC was expressed on this basis to convert succinyl-CoA and acetate into succinate and acetyl-CoA to enhancing the tricarboxylic acid(TCA)cycle.After preliminary optimization of the fermentation process,the constructed recombinant engineering strain MFT-scpC-AarC-ndh achieved a 9α-OH-AD conversion rate of 89.56%,which was 37.86%higher than that of the original strain.This study has provided a theoretical foundation and technical support for the industrial production of 9α-OH-AD,and also introduced new strategies for the construction of other phytosterol transformation engineering strains.
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