Genome Mining And Biosynthesis Of New Meroterpenoids From Two Fungi

Natural products with architecturally distinct scaffolds remain an important resource of new drugs.However,bioactive natural products that are easily accessible using traditional discovery strategy have already been isolated,rendering the exploration of natural products as drug leads inefficient.Advances in genome sequencing and bioinformatics analysis have revealed that filamentous fungi have greater potential to biosynthesize more secondary metabolites than currently reported ones.Thus,with the knowledge accumulation of "gene-structure",more and more natural product chemists pay more attention to utilize gene information for mining new natural products.Meroterpenoids are hybrid molecules partially derived from terpenoid pathways.As a major calss,the various chemical structures and a wide spectrum of biological activities of fungal meroterpenoids have inspired scientists to keep on exploring new meroterpenoids.During our genome mining efforts in our microbe collection,we found two homologous biosynthetic gene clusters in Arthrinium sp.NF2194 and Nectria sp.Z14-w,both of which encoded conserved putative meroterpenoid biosynthetic genes.As both gene clusters have not been reported yet,we postulated that the two fungi might be able to produce new meroterpenoids with structure difference catalyzed by diverse tailoring enzymes.Thus,our attention was focused on the characterization of presumed new meroterpenoids and their biosynthetic pathways as well as the understanding on the relatedness of the meroterpenoid profile/pattern with the gene cluster difference.The dissertation is composed of three chapters.Chapter one summerizes the advances of structures,bioactivites and biosynthesis of fungal meroterpenoids.Described in chapter two are the homologous meroterpenoid biosynthetic gene clusters(atn and ntn)in two fungal genome by comprehensive bioinformatics analysis.In order to isolate related compounds,we used RT-PCR to evaluate the gene expression of atn and ntn cluster when two fungal strains were cultivated in different media,and applied the optimized medium to culture them.As a result,six new cochiloquione type meroterpenoids named arthripenoids A-F(1-6)were isolated from Arthrinium sp.NF2194,and two new meroterpenoids named nectripenoids A-B(7-8)from Nectria sp.Z14-w.Arthripenoid C(3)was found to inhibit the proliferation of concanavalin(ConA)-induced T cells(IC50=8.8 μM)with a discernible selectivity since its inhibition of primary lymphocyte proliferation towards could be negligible.Moreover,3 showed potent inhibition against the inflammatory cytokine IFN-y(Interferon-y)with an IC50 value of 4.2 μM.In chapter three,the arthripenoid biosynthetic pathway is described.The PKS genes atnG and atnH were heterologously expressed in Aspergillus oryzae NSAR1 to afford the anticipated nascent intermediate(11).Nine genes in the atn cluster were knocked out individually with the mutants producing none of 1-6,but generating two early-stage intermediates(16a and 17c)and eight shunt metabolites(11a,14a-b,15a-c,16b,and 17d).The observation accommodated the proposal of the arthripenoid biosynthetic pathway.In order to characterize the functions of two key enzymes,AtnJ and AtnB,which were individually overexpressed and purified.The ensuing enzyme-promoted transformation tests showed that AtnJ catalyzes theOdecarboxylative hydroxylation of 11 into 12.The subsequent 02 labeled experiment reinforced the O2 origination of new hydroxyl(11-OH in 12).AtnB,a ketoreductase,was shown to be substrate promiscuous since it catalyzes the 4-keto reduction of all the four cochiloquiones.Comparative biosynthetic analysis enabled our proposal of the nectripenoid biosynthetic pathway.In summary,this work identified two homologous meroterpenoid biosynthetic gene clusters(atn and ntn)from two fungi Arthrinium sp.NF2194 and Nectria sp.Z14-w through genome mining,and characterized a collection of new cochlioqunione-type natural products,of which one is immunosuppressive.The arthripenoid biosynthetic pathway was established with certainty through a combination of bioinformatic analysis,gene inactivation,and heterologous expression.Our work provides an effective way to identify new bioactive molecules by genome mining.These data expand the structural and biosynthetic diversity of fungal meroterpenoids,which may be of value for the drug discovery endeavor.