Functional Investigation On Terpenoid Synthases Of An Endophytic Fungal Trichoderma Atroviride

The genus Trichoderma is an important class of filamentous fungi,which has attracted much attention because of its ability to antagonize a variety of plant pathogenic fungi.Trichoderma has been widely studied and used as biological pesticides,biological fertilizers and soil conditioners.Trichoderma can produce many kinds of secondary metabolites including terpenoids and non-ribosomal peptides.Many studies have focused on separation and identification of terpenoids as well as their biologcial activities in Trichoderma,but there are only few studies about terpenoid biosynthesis.A number of bisabolane sesquiterpenoids and harziane diterpenoids have been already isolated and identified from the frementation of T.atroviride B7 in previous works of our group.Harziane diterpenoids are characteristic secondary metabolites of Trichoderma fungi,which has a unique 4/7/5/6 four-ring skeleton,showed antibacterial and cytotoxic activities.In addition,bisabolane sesquiterpenoids have multiple biological acitivities and high application values.Investigation on the biosynthesis of harziane diterpenoids and bisabolane sesquiterpenoids can enrich the diversity of terpene synthases and lay the foundation for their synthetic biology research and application.In this study,four predicted terpene synthase genes TRI1,TRI2,TRI5 and TRI7 were selected as candidate genes through bioinformatics analysis of the genome and transcriptome of T.atroviride B7.TRI2,TRI5 and TRI7 were individually constructed into the expression system of Escherichia coli.GC-MS anaylsis indicated that ?-bisabolene,6 monoterpenes likely containing a 6-member ring and another 4 unidentified sesquiterpenes could be detected in the fermentation products of E.coli containing TRI5.Thus,TRI5 may encode a multifunctional sesqui-/mono-terpene synthase,catalyzing the formation of bisabolane sesquiterpenes as well as other sesquiterpenes and monoterpenes.However,no specific terpene product was detected using GC-MS anaylsis in the fermentation products of E.coli containing TRI2 and TRI7.The PEG-mediated protoplast transformation method was used to knock out the TRI5 genes in T.atroviride B7,and it was found that the growth rate showed no obvious difference between wild-type and mutant strains.LC-MS analysis found that bisabolanol A and bisabolanol D,the major bisabolane sesquiterpenoids of T.atroviride B7,can not be detected in the fermentation products of TRI5 knockout strains,indicating that genes TRI5 is likely involved in the biosynthesis of bisabolane sesquiterpenoids.In addition,TRI1 was heterologously expressed in Aspergillus oryzae M-2-3,but no specific product was found using GC-MS analysis.In summary,this study has identified a multifunctional sesqui-/mono-terpene synthase which is likely involved in bisabolane biosynthesis from T.atroviride B7 through heterologous expression in E.coli in combination with the PEG-mediated protoplast transformation.The results will provide foundation for further study on terpene biosynthesis in T.atroviride.