基于辅酶调控产9α-OH-AD工程菌株构建及其发酵工艺优化  

Construction of 9α-OH-AD Engineering Strain Based on Coenzyme Regulation and Optimization of Fermentation Process

作　　者：高园园 孙康 石畅 王雪梅 申雁冰[1] 王敏[1] GAO Yuanyuan;SUN Kang;SHI Chang;WANG Xuemei;SHEN Yanbing;WANG Min(Key Laboratory of Industrial Fermentation Microbiology,Ministry of Education,Tianjin Key Laboratory of Industrial Microbiology,College of Biotechnology,Tianjin University of Science&Technology,Tianjin 300457,China)

机构地区：[1]工业发酵微生物教育部重点实验室,天津市工业微生物重点实验室,天津科技大学生物工程学院,天津300457

出　　处：《天津科技大学学报》2024年第5期14-21,共8页Journal of Tianjin University of Science & Technology

基　　金：国家自然科学基金项目(21978221);天津市研究生科研创新项目(2022SKY099)。

摘　　要：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。In the process of using Mycobacterium fortuitum(MFT)to degrade phytosterols to produce 9α-hydroxyandrostenedione(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.

关 键 词：9α-羟基-雄烯二酮 植物甾醇 丙酰辅酶A 琥珀酰辅酶A 偶发分枝杆菌 

分 类 号：Q815[生物学—生物工程]