Revelation Of The Balanol Biosynthetic Pathway In Tolypocladium Ophioglossoides

Fungi are vital resources of microbial medicine,their genome sequences have the potential to synthesize a large number of new secondary metabolites.Under normal laboratory conditions,most of these gene clusters remain silent.Methods to activate these silent gene clusters has become a highlight in the development of microbial natural products.Tolypocladium ophioglossoides is a filamentous fungi that has been used as a traditional Chinese medicine for the treatment of metrorrhagia and menstrual disorder.In 1977,ophiocordin was discovered in T.ophioglossoides,and its structure was proved to be identical with balanol,which was found in fungus Verticillium balanoides in 1993.Balanol has strong inhibitory activity against protein kinase C(PKC)as well as antifungal activity,and contains a unique hexahydroazepine ring structure.The total chemical synthesis of balanol was first reported in 1994,in later decades,improvements and optimizations were reported,yet the biosynthetic pathway of balanol is still unknown.We have isolated a strain of T.ophioglossoides from Xishuangbanna earlier.Through draft genome sequencing and prediction analysis by antiSMASH,we discovered that the genome of T.ophioglossoides contains 35 biosynthetic gene clusters,including 13 polyketide synthase(PKS)gene clusters,8 nonribosomal synthetase(NRPS)gene clusters,6 hybrid PKS-NRPS gene clusters,4 terpenoid synthetic gene clusters and 4 other type gene clusters.By spectrum analysis of the fermentation product of T.ophioglossoides,we find most of these clusters remain silent.By blast analysis of these predicted gene clusters,we find a hybrid PKS-NRPS gene cluster bln.Some genes inside bln show high homology to the genes inside monodictyphenone synthetic gene cluster from Aspergillus nidulans.The structure of the PKS module of balanol is similar to the structure of monodictyphenone,thus we speculate that bln is the biosynthetic gene cluster of balanol.There is a transcriptional factor blnR inside the bln cluster which belongs to the Zn2Cyc6 family,we acquired the coding sequece of blnR from cDNA,constructed the over-expression plasmid driven by the strong promoter pTEF.By agrobacterium mediated T-DNA transformation into T.ophioglossoides,positive transformants were identified by PCR.The transformant’s fermentation broth has several newly generated compounds compared to the wild type,according to HPLC analysis,showing that the bln cluster was activated.After separation,purification and identification by high-resolution MS and NMR,9 compounds were isolated,including balanol,3 new natural-source compounds and 1 new compound.By qRT-PCR analysis of the blnROE strain,we identified the boundary of the hybrid PKS-NRPS gene cluster bln.On the basis of the blnROE strain,several knockout strains of different bln genes were constructed and identified,each of their fermented products were analyzed by LC-MS in order to validate each genes’functions during the synthesis of balanol,a reasonable biosynthetic pathway of balanol was proposed.We elucidated the biosynthetic pathway of balanol for the first time,and our research shed a light on the biosynthetic mechanism of the unique hexahydroazepine ring structure inside balanol.We also found the unique mode of separated PKS module and NRPS modules inside the hybrid bln cluster,which provides foundations for the synthetic biological research.