Investigation On The Oxidative Rearrangement Mechanism In Chartreusin Biosynthesis

The type ? polyketide synthases are widely present in actinomycetes.Natural products biosynthesized by them have diverse structures and biological activities,which play an indispensable role in the development of new drugs.With the gradual maturity of natural product separation methods and the continuous development of gene sequencing and bioinformatics analysis technology,increasing compounds with good biological activity which derived from type ? polyketide synthases have been found.Chartreusin,which was originally isolated from Streptomyces.chartreusis NA02069,is an aromatic polyketide with potent anti-tumor activities.It consists of a fucose,a digitalose,and a bislactone pentacyclic aglycone named chartarin.As early as 2005,its biosynthetic gene cluster(cha)had been disclosed by the research group of Christian Hertweck.However,the detailed biosynthetic steps remain enigmatic.According to the accumulated linear aromatic polyketide intermediate(resomycin C)from ?chaZ mutant strain,Hertweck group speculated that chartreusin was synthesized via the typical anthracycline pathway,followed by two novel oxidative rearrangement reaction steps.Here,we focus on the elucidation of the biosynthetic pathway and the critical oxidation rearrangement reaction through gene deletion,bioconversion,in vitro catalysis,protein crystallization and site-specific mutation.This dissertation consists of four chapters:In chapter one,we briefly indroduced the recent advances of structures and biosynthesis of type II polyketides from bacteria.In chapter two,we reported the isolation of chartreusin from marine S.chartreusis through bioassay screening.In chapter three,we mainly studied the biosynthetic pathway of chartreusin.Firstly,the chartreusin biosynthetic gene cluster in S.chartreusin was identified by sequence alignment,and the annotation of each gene on the cluster was conducted subsequently.Thereafter,the corresponding mutant strains were obtained by knocking out sixteen related genes,respectively.A total of twenty intermediates and by-products were obtained by isolating the fementation of mutant strains.Furthermore,the functions of the involved oxidases were verified by gene and chemical complementation and in vitro enzyme catalysis.From the above,the biosynthetic pathway of chartreusin was reasonably speculated.In chapter four,the author mainly investigated the mechanism of oxidative rearrangement reaction involving ChaP in the last step of chartreusin biosynthesis.First,the in vivo biotransformation process was conducted through heterogenous expression of chaP gene in S.albus J1074 strain.Next,author successfully carried out the above reaction using the recombinant ChaP as catalyst and FAD activated oxygen as oxidant.In addition,crystals of ChaP and its homologs provided the catalytic machinery of this unusual reaction.In summary,this dissertation reported the re-isolation of chartreusin from a marine Streptomyces strain.We revealed the biosynthetic pathway for chartreusin through detailed bioinformatics analysis,characterization of intermediates and/or shunt metabolites from gene deletion mutants.Then,the novel oxidative rearrangement mechanism in the final step of chartreusin biosynthetic pathway was further elucidated through gene heterogenous expression,in vitro biochemical reaction,protein crystal analysis,docking study and site-specific mutation.This work resolves the long-standing mystery in chartreusin biosynthesis and discloses a novel enzymatic reaction.