Molecular Cloning And Functional Analysis Of The Key Enzyme Genes Involved In The Biosynthesis Of Sesquiterpene Lactones(STLs)

Sesquiterpene lactones(STLs)constitute a group of secondary metabolites which accumulate primarily in the family of the Asteraceae.Due to the presence of STLs,these plant species show multiple pharmacological properties,including anti-tumor and anti-malarial activities.A large portion of STLs derive from germacrene A acid(GAA)and the formation of their lactone rings depends on the regio-(C6 or C8)and stereo-selective(a-or ?-)hydroxylations of GAA,producing STLs with four distinct stereo-configurations(12,6?-,12,6?-,12,8?-,and 12,8?-olide derivatives of GAA)in nature.Among the four stereo-isomers of STLs,only the biosynthesis of 12,6a-configured lactone has been most extensively studied,while the knowledge regarding the biogenesis of the other lactone rings remains scant so far.Through the integration of molecular,biochemical and phtochemical experiments,we report here the biochemical mechanism for the biosynthesis of the C12,8-configured STLs.From the Asteraceae plant species Inula hupehensis,we isolated a novel cytochrome P450 gene,designated Ih8H.When the Ih8H was expressed in yeast cells,the recombinant Ih8H catalyzed the hydroxylation of GAA in both 8a-and 8?-configurations,resulting in both 8?-and 8?-hydroxyl GAAs as products.Of these two products,only 8a-hydroxyl GAA is spontaneously lactonized to the 12,8?-configured STL(named as Inunolide)while the 8?-hydroxyl GAA remains unchanged without lactonization.The Ih8H displays 63-66%amino acid identity to the previously reported CYPs(CYP71BL1/2)catalyzing GAA 6a-or 8?-hydroxylation,indicating Ih8H shares the same evolutionary lineage with other stereo-selective CYPs,but it catalyzes hydroxylations uniquely in a non-stereo-selective manner(note:CYP enzymes usually catalyze the oxidizing reactions in a stereo-selective manner).The expression pattern of the Ih8H gene in different plant tissues matches the accumulation of the C12,8 STLs in vivo,suggesting that Ih8H plays a role in the biosynthesis of the C12,8 STLs.The correlation of the Ih8H to the C12,8 STLs biosynthesis was confirmed by applying a signal molecule methyl jasmonate(MeJA)to the I.hupehensis plantlets,in which the MeJA increased the Ih8H transcript levels and therefore elevated the biosynthesis of the C12,8 STLs.To test the universality of the Ih8H role,we isolated the Ih8H homologs from another two Asteraceae species-Xanthium strumarium and Inula lineariifolia.Two Ih8H homologs were cloned from X.strumarium and named as Xs8H-HB and Xs8H-HN,respectively,while one homologous gene Il8H was isolated from I.lineariifolia.Biochemical assays indicated that either Xs8H-HN or Il8H displays the same activity as Ih8H whereas Xs8H-HB is inactive with GAA.To explain why the Xs8H is inactive with GAA,the protein structure of the Xs8H was modeled and the amino acid residues interacting with the substrate GAA was predicted.Through several site-mutations,we concluded that the 116th amino acid residue in the Xs8H is essential for the GAA oxidizing activity,and this residue is naturally mutated in the wild Xs8H-HB,causing the loss of the Xs8H-HB activity.Taken together,we are the first to elucidate the mechanism underlying the biosynthesis of the C12,8 STLs,and the discovery of the Ih8H gene would pave the way to engineer the production of C12,8 STLs in microbes using synthetic biology approach in the future.In the other hand,here we also report the molecular cloning and functional analysis of a novel sesquiterpene synthase,designated IhsTPS1,from I.hupehensis.The recombinant IhsTPS1 converts the substrate farnesyl diphosphate into multiple sesquiterpenes with the product 10-epi-junenol being predominant.To the best of our knowledge,this is the first report concerning the cDNA cloning of a 10-epi-junenol synthase from plant kingdom.The identification of IhsTPS1 provides a valuable resource for further research into the biosynthesis of junenol based-eudesmanolides,and also provides a potential to increase the production of 10-epi-junenol in plants by overexpressing IhsTPS1 or facilitate its production in microbial hosts by synthetic biology.