Analysis Of Volatile Components And Functional Identification Of Terpene Synthase Gene Family In Wolfiporia Cocos

Wolfiporia cocos,an important medicinal and edible fungus of Basidiomycota,has the functions of diuresis,excreting dampness,invigorating the spleen,and tranquilizing the mind.W.cocos contains abundant secondary metabolites,among which terpenes are a major class of important bioactive components that need to be further explored.Terpene synthase(TPS)is key enzyme in the biosynthetic pathway of terpene.Based on different types of products,TPSs are divided into monoterpene synthase,sesquiterpene synthase,and diterpene synthase.However,the genome-wide TPS gene family have not been identified in W.cocos.In this study,diverse methods were used to measure the volatile components of different parts of W.cocos and mycelium fermentation broth,and a variety of volatile terpenoids were detected.Based on genome data mining and manual correction,18 members of the W.cocos terpene synthase(WcTPS)gene family located in 15 terpenoid biosynthesis gene clusters identified.Totally of 9 WcTPS have been cloned in this study.To ascertain the potential biological roles of WcTPS genes in W.cocos,WcTPS genes was expressed in Escherichia coli and Saccharomyces cerevisiae,and the catalytic reaction of in vitro was carried out simultaneously.All catalytic products were analyzed by solidphase microextraction(SPME)and GC-MS.The results are shown as follows:1.The gene structure and protein conserved motif analysis of WcTPS family members showed that the number of introns was ranged from 1 to 5,mostly 4,which were less than the number of introns contained in plant TPS genes.Meanwhile,WcTPS located in the same branch had the same conserved motif type.Based on the phylogenetic relationship analysis,WcTPS gene family was divided into 4 clades,including of Clade Ⅰ(4 WcTPS genes),Clade Ⅱ(2 WcTPS genes),Clade Ⅲ(6 WcTPS genes),Clade Ⅳ(6 WcTPS genes).Multiple sequence alignment analysis of Basidiomycetes TPS revealed that the types of conserved domains in different clades were different.Clade Ⅰ is mainly DNXXD and DDXXD type,Clade Ⅱ and Clade Ⅲ are mainly DEXXD type.Clade Ⅳ has various types of conserved domains,and even contains DDXXXXXD and DDXXXXXXD types,however the NSE(DTE)domains were more conserved among the four clades.2.Based on the transcriptome data of W.cocos,WcTPS genes was expressed in different degrees in mycelium and sclerotium,among which WcTPS13 specific high expression in sclerotium.Gene expression was detected by RT-qPCR after methyl jasmonate(MeJA)induction in mycelium,and most genes were found to respond to MeJA induction.3.Using the endogenous substrates of the protein heterologous expression system,9 WcTPS were induced and expressed in the E.coli expression system and S.cerevisiae expression system.The catalytic products of WcTPS in heterologous expression system and in vitro enzymatic reaction system were collected by solid phase microextraction and detected by GC-MS.It was found that 7 WcTPSs had catalytic activities in different expression systems.Members belonging to Clade Ⅰ and Clade Ⅱ,such as WcTPS4 and WcTPS5,have the most diverse catalytic products,and can form more than 10 sesquiterpenoids in both E.coli and yeast.Clade Ⅲ branch members usually have important functions and have a single catalytic product,such as WcTPS 17 catalyzes the formation of Germacradienol,which was first identified in Basidiomycetes.Clade Ⅳmember WcTPS 13 is a bifunctional enzyme,which can catalyze the formation of monoterpene compound linalool,and can also catalyze the formation of sesquiterpene compounds nerolidol and farnesol.Molecular docking analysis found that WcTPS 13 had similar catalytic effects on the farnesyl diphosphate(FPP)and geranyl diphosphate(GPP).This study lays the foundation for analyzing the catalytic mechanism of Basidiomycete terpenoid synthase and constructing an efficient and fast heterologous production platform for terpenes.