The Volatile Terpenome Investigation Of Ganoderma Lucidium

As a model medicinal fungus from basidiomycetes,Ganoderma lucidum has been widely used to treat diseases for more than 2000 in China.Modern pharmacological studies suggested that G.lucidum has significant anti-tumor,anti-aging,hypoglycemic,anti-hypertension,immunomodulatory and memory-improving effects.Up to date,polysaccharides and triterpenoids from G.lucidum have been widely studied.However,the volatile terpenoids from this species remain largely unknown.Based on genome data mining,23 sesquiterpene synthases(STSs)from G.lucidum and 26 STSs from G.sinense were found.In this study,all STSs from G.lucidum and 5 STSs from G.sinense were studied.Phylogenetic analysis suggested STSs in G.lucidum can be divided into five clades.Multiple sequence alignment analysis showed that all STSs in G.lucidum contained conserved NES/DET motif.However,another motif DDXXD was only conserved in clade Ⅰ,Ⅱ,Ⅲ,Ⅳ.Among 11 STSs of G.lucidum in clade V,two contained DDXXD motif,six contained DDXXXXD motif and three contained DDXXXXXD motif.In G.lucidum,the STSs were distributed in 9 of the 12 chromosomes and chromosomes 4,7 and 8 contain more STSs.Tandem duplication was regarded as the main way to expand STS family in this species.23 STS genes from G.lucidum were cloned and expressed in Saccharomyces cerevisiae.Volatile products were collected by solid phase microextraction(SPME)and detected by GC-MS.It was found that 16 STSs had catalytic activities and were promiscuous enzymes with multiple products,among which,GL22395 can produce two products and GL20791 are the most productive enzyme and can produce 24 different sesquiterpenes.The 16 STSs can produce 80 sesquiterpenes,including 25 main products.The structure of eight products was determined by comparing them with standard chemicals.Most of these sesquiterpenes were first found in G.lucidum,and nine sesquiterpenes have been reported to have significant pharmacological activities.Five STS genes from G.sinense were expressed heterogeneously in Escherichia.coli and their expressed proteins was detected by SDS-PAGE.Volatile products were collected by SPME and detected by GC-MS.It was found that three of five enzymes had catalytic activities and produced a total of 15 sesquiterpenes.G.lucidum STSs GL26009 and GL25909 were expressed in E.coli and the purified proteins were used to detect enzyme in vitro activity.The results showed that the purified enzymes under in vitro conditions can produce approximately the same products as those produced in the in vivo systems,except that the abundance of the products varied.GL26009 can produce the main product a-Cadinol in all the three systems.In addition,this enzymes can produce the other two main products δ-cadinene and Nerolidol in the in vitro system.GL25909 can produce the main product T-Muurolol in E.coli or yeast,while the main product changed to a-Cadinol in the in vitro system.These results suggested that the microenvironment of the reaction will have an effect on the product specificity of STSs.From the view of synthetic mechanism,STSs in G.lucidum involved C1-C6,C1-C10 and C1-C11 cyclization.The STSs in clade I and II produce monocyclic or bicyclic sesquiterpene compounds through the C1-C10 cyclization.All the STSs in CladeⅢ can produce β-Caryophyllen via a C1-C11 cyclization and the cyclization mechanism of some main products remain unknown.The STSs in clade IV was considered to be a y-Cadinene synthase and y-Cadinene was formed by FPP via a C1-C6 or C1-C10 cyclization.The STSs in clade V involved C1-C6 or C1-C10 cyclizations.The functional identification of STSs in G.lucidum can promote the study on correlation between their structure and function and then reveal the molecular mechanisms underlying their catalytic activity and product diversity,which will lay the foundation for the production of rare or novel sesquiterpenoids by modifying the catalytic activity with enzymatic engineering technology,thereby promoting the research and development of sesquiterpenoid-type drugs.