Functional Elucidation Of Sesquiterpene Synthase In Hericium Erinaceus

Hericium erinaceus,as a large edible and medicinal fungus,has been used for more than3,000 years,and it has many physiological functions,such as benefiting the five internal organs,anti-aging,anti-cancer,treating neurasthenia and diabetes.So far,more than 200 compounds have been isolated and identified,including polysaccharides,terpenes,alkaloids,sterols,phenols,etc.,but no sesquiterpenes have been reported.With the publication of the genome sequence of Hericium erinaceus,the biosynthesis of secondary metabolites has also received extensive attention,and through the mining of the Hericium erinaceus genome database,it was found that there are 7 genes encoding sesquiterpene synthase(He STS1-7)in Hericium erinaceus.We investigated the function of these 7 sesquiterpene synthases using heterologous expression of Aspergillus oryzae and in vitro enzyme activation reaction.The specific research content and main results are as follows:(1)Through NCBI,anti SMASH and other bioinformatics analysis methods,it was found that there are 7 sesquiterpene synthesis genes in the genome of Hericium erinaceus,which can be compared with the known sesquiterpene synthase sequence,and they can be divided into 3different types.The multiplex sequence comparison showed that all 7 sesquiterpene synthases contained D(D/E)XX(D/E)conserved sequences,while for the other N(D/E)XX(S/T)XXX(D/E)conserved sequence,except for He STS2 sesquiterpene synthase,which did not contain this conserved motif,the remaining 6 sesquiterpene synthases had this conserved sequence.(2)In order to verify the function of these 7 genes,we constructed recombinant plasmids that could be used for transfection of Aspergillus oryzae,and transferred them into Aspergillus oryzae,respectively,AO-He STS1-7 Aspergillus strains were obtained,and TLC and GC-MS detection of metabolites were performed through rice fermentation,and the results showed that4 of the 7 sesquiterpene synthases functioned,of which He STS1 produced 5 sesquiterpenes;He STS5 produces 4 sesquiterpenes and He STS6 produces 3 sesquiterpenes;He STS7 produces5 sesquiterpenes.The structure of these compounds is inferred by calculating RI values.In addition,we isolated and extracted the metabolites of He STS1 and performed nuclear magnetic resonance(NMR)analysis of the purified compounds to determine that one of the main products of He STS1 is δ-cadinene.(3)In the prokaryotic expression system Escherichia coli,we performed protein expression on these 7 sesquiterpene synthases,of which 5 sesquiterpene synthases were successfully expressed,namely He STS1,He STS4,He STS5,He STS6 and He STS7,and the protein purification of He STS1 sesquiterpene synthase,the isolated and purified protein in vitro enzyme reaction experiments,through GC-MS analysis showed that He STS1 in vitro enzymatic reactions are consistent with the compounds produced by Aspergillus oryzae expressed in vivo,but in fewer quantities than Aspergillus oryzae as a host product.To date,no sesquiterpenoids have been reported in Hericium erinaceus,and our results suggest that the sesquiterpene gene of Hericium erinaceus has the ability to produce complex and diverse sesquiterpene compounds.At the same time,it was proved that the Aspergillus oryzae system is an excellent host for expressing the genes of the fungi of the basidiomycetes.