VerZ, a Zn(II)2Cys6 DNA-binding protein, regulates the biosynthesis of verticillin in Clonostachys rogersoniana

Verticillins are the dimeric epipolythiodioxopiperazines (ETPs) produced by the fungus Clonostachys rogersoniana . 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 ( ver ), and identified the only regulatory gene verZ in this cluster. The deduced product of verZ contains a basic Zn(II) 2 Cys 6 DNA-binding domain. Disruption of verZ 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 E. coli and the His 6 -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)GN 3 CC(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 C. rogersoniana.