Activation Of Natural Products Biosynthetic Pathways Via Protein-Modification Level Regulation And Combinatorial Biosynthesis Of C-3 Aromatic Pyrrolidine Indole Alkaloids

Two topics are discussed in this dissertation,including 1)Slient gene clusters are activated though overexpressing Sfp type phosphopantetheinyl transferases,which contribute to mining new natural product.2)We identify key enzymes when we analyze the synthesis mechanism of Naseseazine A,B isolated from Streptomyces sp.CMB-MQ030.The key enzyme is used to construct the whole cell catalytic system which catalyze cyclic dipeptide to produce pyrrolidine indole alkaloids.Natural products are critical for drug discovery and development,however their discovery is challenged by the wide inactivation or silence of biosynthetic pathways in microbes.Currently strategies targeting on this problem are mainly concentrated on chromosome dissembling,transcription and translation-stage regulations as well as elicitor stimulation.In this study,we identified that augmentation of the conserved protein modification step-phosphopantetheinylation in microbes is able to effectively regulate the biosynthesis of natural products.By overexpression of two broad-selective phosphopantetheinyl transferase(PPtase)genes in vivo,we achieved activation of metabolite production in 23 of 33(70%)Actinomycetes strains tested.This further allowed us to discover seven compounds from the two activated strains,and there were two new products.Our study provides a protein-modification level approach for efficient activation of cryptic/silenced biosynthetic pathways which will be useful for the discovery and study of secondary metabolite production.C-3 aromatic pyrrolidine indole alkaloids,a kind of important natural product,generally have excellent physiological activity.But,it is that lacking efficient methods to expand molecular skeleton limit the research of the compounds in pharmacy and biology.In this study,we analyzed the synthesis mechanism of NAZ-A and NAZ-B isolated from S sp.CMB-MQ030.We indentified that CDPS and p450(NAZ-p450)were the key enzymes in the pathway,then NAZ-p450 and ferrodoxin/ferrodoxin reductase from spinach were used to construct the whole cell catalytic system,which could catalyze 20 synthetic cyclic dipeptide to produce 27 new C-3 aromatic pyrrolidine indole compounds.The method,which combined chemistry and enzyme together,not only break the structural limitation of natural cyclic dipeptide,but also overcome the bottleneck synthesizing C2-C3 chrial tertiary carbon and quaternary carbon center through chemical methods.And it allows to synthesize the complex pyrrolidine indole alkaloids rapidly and effiently,which provides a solid foundation for the pharmaceutical and biological research of the alkaloids.