The Biosynthetic Mechanism For Antibacterial Indolizidine Alkaloids

Deciphering the unprecedented biosynthetic mechanism of natural products is important for understanding how nature could use simple starting units to generate complex natural products,discover enzymes that can catalyze special chemical reactions with novel catalytic mechanism,and use the combinatorial biology to produce more valuable natural products.The fungal polyketide alkaloid curvulamine(1)was isolated from the Curvularia sp.IFB-Z10 and represents a new family of polyketide alkaloids composed of two undescribed building blocks 7 with pyrrole moiety and 8 with indolizidine moiety.Based on the feeding experiment of isotope labelled precursors,we proposed that polyketide synthase(PKS)and8-amino-7-oxononanoate synthase(AONS)might be involved in the biosynthesis of 1.However,the detailed biosynthetic pathway for 1 remains unknown.In this dissertation,we elucidate the biosynthetic mechanism of curvulamine and find an unprecedented way to forge the indolizidine framwork.We also discover the deletion of COX15 exerts a significant impact on the structural diversity of microbial secondary metabolites.The dissertation contains four parts.In chapter one,we reviewed the research progress of new strategies to discover microbial secondary metabolites and the biosynthetic mechanisms of indolizidine alkaloids.In chapter two,we used combination of bioinformatics analysis,gene deletion,heterologous expression in A.oryzae NSAR1 and biochemical characterization of tailoring enzymes to elucidate the biosynthetic mechanism of curvulamine.The biosynthetic gene cluster for curvulamine includes cur A(PKS),cur B(AONS),cur C(short chain alcohol dehydrogenase/reductase),cur D(FAD-dependent oxidoreductase),cur E(MFS general substrate transporter),cur F(transcription factor)and cur G(cytochrome c assembly protein COX15).Cur A and cur B were collaboratively responsible for the formation of intermediate 5,and then 5 was epoxidized into 6 by cur D.Compound 6 was catalysed into 7 by the reduction of cur C,which spontaneously transformed into 8.The building blocks 7 and 8 were connected by the formation of C-N bond and the C-C bond to construct curvulamine via successive Michael addition,dehydration,expoxidation and nucleophilic attacktion.Notely,the formation of 8 represents an unprecedented biosynthetic pathway for the construction of the indolizidine framework.COX15 plays a critical role in the biosynthesis of Heme A,however,we find that the deletion of COX15 exerts a significant impact on the structural diversity of microbial secondary metabolites in chapter three,which has never been reported before.Based on HPLC analysis,we found the titer of 1 was significantly improved and more absorption peaks were presented in Δcur G than that of wild-type Z10.We scaled up the fermentation of Δcur G,and characterized 15 new alkaloids.More importantly,compounds 17,18 and 20 have impressive antibacterial activity.The m RNA expression levels of cur A-F in Δcur G were higher than that of wild-type by q RT-PCR analysis.We also found the HPLC profiles between OE::cur F strain and Δcur G mutant were similar.These results indicated that cur G might play a regulatory role in the expression of the biosynthetic gene cluster for 1.The highlight of this work is the discovery of an unrecognized biosynthetic mechanism for the indolizidine framework construction.In addition,we also find the unusual role of COX15 in the regulation of secondary metabolisms production in microorganisms,which has never been reported before.The work also sets stage for engineering biosynthetic pathway of fungal polyketide alkaloids for drug discovery.