Guo Zhe, Hao Tianchao, Wang Ying, Pan Yuanyuan, Ren Fengxia, Liu Xingzhong, Che Yongsheng, Liu Gang
State Key Laboratory of Mycology, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100101, PR China.
University of Chinese Academy of Sciences, Beijing 100049, PR China.
Microbiology (Reading). 2017 Nov;163(11):1654-1663. doi: 10.1099/mic.0.000557.
Verticillins are the dimeric epipolythiodioxopiperazines (ETPs) produced by the fungus . Despite their profound biological effects, they are commonly produced in rice medium as complex mixtures that are difficult to separate, limiting further study and evaluation for this class of metabolites. Therefore, there is an urgent need to understand the regulation of verticillin biosynthesis. Recently, we cloned the biosynthetic gene cluster of verticillin (), and identified the only regulatory gene in this cluster. The deduced product of contains a basic Zn(II)Cys DNA-binding domain. Disruption of significantly reduced the production of 11'-deoxyverticillin A (C42) and decreased the transcriptional level of the verticillin biosynthetic genes. To further reveal its function, a recombinant gene encoding the DNA-binding domain of VerZ was expressed in and the His-tagged VerZbd was purified to homogeneity by Ni-NTA chromatography. Electrophoretic mobility shift assays (EMSAs) showed that VerZbd bound specifically to the promoter regions of the verticillin biosynthetic genes. Bioinformatic analysis of the VerZbd-binding regions revealed a conserved palindromic sequence of (T/C)(C/A)(G/T)GNCC(G/T)(A/G)(G/C). Base substitution of the conserved sequence completely abolished the binding activity of VerZbd to its targets. These results suggested that VerZ controls verticillin production through directly activating transcription of the biosynthetic genes in
轮枝菌素是由该真菌产生的二聚体环多硫代二氧哌嗪（ETP）。尽管它们具有深远的生物学效应，但它们通常在大米培养基中作为难以分离的复杂混合物产生，这限制了对这类代谢物的进一步研究和评估。因此，迫切需要了解轮枝菌素生物合成的调控机制。最近，我们克隆了轮枝菌素的生物合成基因簇，并鉴定出该簇中唯一的调控基因。该基因推导的产物含有一个碱性Zn（II）Cys DNA结合结构域。对该基因的破坏显著降低了11'-脱氧轮枝菌素A（C42）的产量，并降低了轮枝菌素生物合成基因的转录水平。为了进一步揭示其功能，在大肠杆菌中表达了一个编码VerZ DNA结合结构域的重组基因，并通过镍-氮三乙酸层析将带有His标签的VerZbd纯化至同质。电泳迁移率变动分析（EMSA）表明，VerZbd特异性结合轮枝菌素生物合成基因的启动子区域。对VerZbd结合区域的生物信息学分析揭示了一个保守的回文序列（T/C）（C/A）（G/T）GNCC（G/T）（A/G）（G/C）。该保守序列的碱基替换完全消除了VerZbd与其靶标的结合活性。这些结果表明，VerZ通过直接激活生物合成基因的转录来控制轮枝菌素的产生。
