The Functional Diversity Of Terpene Synthases In Ganoderma Lucidum Based On The Whole Genomic Analysis

Ganoderma lucidum is a famous medicinal mushroom in China, belonging to basidiomycete, which was firstly described in the Chinese medical book "Shen Nong’s Herbal Classic". Modern studies have reported that G.lucidum possesses multiple pharmacological activities, such as antitumor, anti-hypertensive, anti-inflammatory and immunomodulatory activities. G.lucidum has been well-known as a "cellular factory for biologically useful compounds" and more than 400 bioactive compounds have been isolated from this fungus.In this study, we found that three sequenced fungi from Ganoderma genus encode plenty of terpene synthases (TPSs) by mining their genomes,23 from G.lucidum,26 from G. sinense and 26 from G. sp. Evolution analysis suggested all TPSs Ganoderma genus can be clustered into three branches in the phylogenetic tree. The TPS genes in the same branch have similar gene structure and intron phase. Further studying the position of genes in the chromosomes indicated that TPS gene duplication lead to the expansion of TPS family in G.lucidum. We also studied the expression of all TPS genes during G.lucidum development, and 12 TPS genes showed the highest expression in mycelia,4 in primordial and 7 in fruiting body, suggesting these TPSs were tightly controlled during development. We collected the volatile metabolites produced by liquid cultures of G.lucidum by SPME technology and characterized them by GC/MS. From day 4, G.lucidum mycelia began to produce sesquiterpenoids. Until day 18, a total of 13 different sesquiterpenes were found. Most of them were first found in G.lucidum. We obtained the full-length cDNAs of 21 TPSs, the remaining two TPSs can’t be amplified from total RNA by RT-PCR technology. So we synthesized them by chemical method according their predicted gene models. All TPS genes were herterologously expressed in E.coli. SDS-PAGE indicated 22 TPSs can be translated into soluble proteins. Fifteen TPSs can produce 1-5 sesquiterpenes, respectively. A total of 17 different sesquiterpenes were synthesized by all TPSs expressed in E.coli. We found that 14 TPSs contain two conserved motifs DDXX(D,E) and (N,D)DXX(S,T)XXXE, the remaining 9 TPSs have noly one motif (N,D)DXX(S,T)XXXE. The former belongs to branch I and III in the tree and the latter belongs to branch II. The TPSs in branch I and III can produce sesquiterpenes with two six-membered rings, which were synthesized by 1,10 and 1,6 closure, while TPSs in branch II can produce more diverse compounds, some of which were synthesized by 1,7 closure.Our study suggested that combination of the whole genome sequencing search and heterologous expression is an effective way to elucidate the biosynthetic pathway of bioactive compounds in traditional Chinese medicines. In the meanwhile, exploring the functional diversity of G. lucidum TPSs and the underlined molecular mechanism will lay the foundation for improving enzymes’activity or designing novel enzymes to produce "unnatural" natural products, thus greatly contributing to the development of new drugs.