Genomic Analysis Of Phoma Sp.XZ068 And Biosynthetic Studies Of The Tropolone Meroterpenoids

Fungal secondary metabolites are an important source of drug leads,and have attracted much attention.Our previous investigations of Phoma sp.XZ068,a low-temperature altered species,brought to the discovery of a series of tropolonic sesquiterpenoids,which exhibited various biological activities.Eupenifeldin,a bistropolone meroterpenoid,was also isolated from the Phoma sp.XZ068,and showed potent inhibitory effects on the proliferation of glioma cells,suggesting that it could be used as a lead compound for further modification and evaluation.To explore the biosynthesis of the tropolone meroterpenoids,the genome of Phoma sp.XZ068 was sequenced and analyzed.Based on the genomic analysis,we provided insight into the genome characteristics of Phoma sp.XZ068.The results from in silico assays revealed that the fungus had a putative life strategy pathogenic to the plants,and was potential producers of diverse secondary metabolites when cultured on rice.In this study,we identified the eupenifeldin biosynthetic gene cluster eup,and predicated the functions for most of genes in the cluster based on genomic analysis,gene disruption,and product analysis.A terpene cyclase EupE of an unknown family was characterized to catalyze humulene formation,and a cytochrome P450 enzyme EupD was responsible for hydroxylation of humulene.A putative biosynthetic pathway for eupenifeldin was proposed on the basis of the putative intermediates,and the functional analysis of genes in eup cluster.The pathway-specific regulatory gene eupR was demonstrated to regulate the transcription of the eupenifeldin biosynthetic genes positively.By comparing the HPLC fingerprints of the gene eupA disruption mutant with the wild type strain,we found that the gene eupA disruption mutant produced a series of new compounds when eupenifeldin production was abolished.The structures of new compounds were elucidated,most of which are structurally related to the tetramic acid natural products containing decalin rings.In the current work,the biosynthetic gene cluster of the tetramic acid compounds in Phoma sp.XZ068 was primarily identified,and it was suggested that these compounds share the common substrates with eupenifeldin.Collectively,the work described in this dissertation not only provides information on the genome and strain characteristics,and the first insight into the biosynthesis gene cluster of eupenifeldin,but also paves the way to further decipher the tropolone meroterpenoids biosynthetic pathway and to generate new congeners for further evaluations.Identification of the biosynthetic gene cluster related to tetramic acid compounds also shed light on the further targeted separation of the secondary metabolites in Phoma sp.XZ068 via the modification of the metabolic network.