摘要
9α-羟基雄甾-4-烯-3,17-二酮(9α-OH-AD)是一种重要的甾体药物中间体,分枝杆菌转化植物甾醇的过程中,9α-OH-AD的分解是导致其产率降低的一个关键因素,之前的研究已表明,3-甾酮-Δ1-脱氢酶(3-ketosteroid-Δ1-dehydrogenase,KstD)在9α-OH-AD的分解过程中起着重要的作用.耻垢分枝杆菌mc^2155(Mycobacterium smegmatismc^2155)菌株基因组中存在6个编码3-甾酮-Δ1-脱氢酶基因,通过对这6个脱氢酶基因分别进行外源表达和活性测定,发现只有KstD1、KstD2和KstD3具有脱氢活力;使用CRISPR-Cas12a辅助重组技术成功构建了失活kstD1、kstD2和kstD3的菌株,结果显示单独失活基因kstD1能够产生9α-OH-AD,但随着转化时间的延长,9α-OH-AD会降解,无法积累,同时失活kstD1、kstD2和kstD3的菌株以5 g/L植物甾醇为底物时,转化14 d内9α-OH-AD能够稳定存在,产物浓度达到2.86 g/L.可见,耻垢分枝杆菌mc^2155中kstD1基因对9α-OH-AD积累起关键作用,随着转化的进行和产物的积累,kstD2和kstD3的作用逐渐显现,同时失活3个3-甾酮-Δ1-脱氢酶有效提高了菌种生产9α-OH-AD的稳定性和产率,本研究结果可为构建9α-OH-AD生产菌种及产业化应用奠定基础.(图6表5参30)
The 9α-hydroxyandrost-4-ene-3,17-dione(9α-OH-AD)compound is an important medical steroid intermediate.Decomposing 9α-OH-AD causes low yields of phytosterol transformation by mycobacteria,in which 3-ketosteroid-Δ1-dehydrogenase(KstD)aids this decomposition.Hence,this study aimed to identify the KstDs in Mycobacterium smegmatis mc^2155 that were responsible for decomposing 9α-OH-AD.Six genes were found to encode the 3-ketosteroid-Δ1-dehydrogenase in the M.smegmatis mc^2155 genome.All of the genes were exogenous expressed,and the dehydrogenation activities were determined,in which only KstD1,KstD2,and KstD3 had the activities.Next,CRISPR-Cas12 a-assisted recombineering was used to successfully construct the strain to inactivate kstD1,kstD2,and kstD3,where the inactivation of the kstD1 gene alone produced 9α-OH-AD,but it degraded and did not accumulate when the transformation time was extended.When the strain with inactivated kstD1,kstD2 and kstD3 simultaneously was cultivated with phytosterols at 5 g/L,9α-OH-AD could stably accumulate at 2.86 g/L concentration within 14 days.Therefore,this study identified the 3-ketosteroid-Δ1-dehydrogenase in M.smegmatis mc^2155 that were critical for the accumulation of 9α-OH-AD.As the transformation progressed and the product accumulated,the activities of KstD2 and KstD3 were gradually carried out.The stability and yield of 9α-OH-AD are effectively improved by simultaneously inactivating the three 3-ketosteroid-Δ1-dehydrogenases,which laid the foundation for the construction of strains for the effective production of 9α-OH-AD.
作者
李梦
李雪梅
冯进辉
张瑞
吴洽庆
朱敦明
马延和
LI Meng;LI Xuemei;FENG Jinhui;ZHANG Rui;WU Qiaqing;ZHU Dunming;MA Yanhe(School of Life Sciences,University of Science and Technology of China,Hefei 230026,China;Tianjin Institute of Industrial Biotechnology,Chinese Academy of Sciences,Tianjin 300308,China;National Technology Innovation Center of Synthetic Biology,Tianjin 300308,China)
出处
《应用与环境生物学报》
CAS
CSCD
北大核心
2020年第4期739-746,共8页
Chinese Journal of Applied and Environmental Biology
基金
国家重点研发计划项目(2019YFA0905300)
天津市合成生物技术创新能力提升行动项目(TSBICIP-KJGG-001-01,TSBICIP-KJGG-001-04)
中国科学院科技服务网络计划项目(KFJ-STS-QYZX-O66)资助。
