Function-orientated Structure Optimizations Of Bacterial Maytansinoids Via Synthetic Biology

The maytansinoids are members of the ansamacrolide group of natural products.Both the plant-derived maytansinoids and their bacterial counterparts showed strong antibiotic and antitumor activities.In 2013,the FDA licensed ado-trastuzumab emtansine(Kadcyla;Genentech,Inc.),an antibody maytansinoid conjugate,for use as a single agent for the treatment of patients with human epidermal growth factor receptor 2(HER2)-positive metastatic breast cancer,which triggered wide attention.Howver,the important intermediate of antibody maytansinoid conjugates,alanylated maytansinol,is still produced by tranditianal chemical synthesis which is a high-cost and low-yield method.Conrespondingly,the scientific research,production and clinical application of antibody maytansinoid conjugates have been seriously hindered.Therefore,in this research,we mananged to find new lead compounds of antibody maytansinoid conjugates and tried to optimize the product of alanylated maytansinol via synthetic biology methods.This dissertation was comprised of five chapters.In the first chapter,we presented the overview of ansamycins,including their chemical structures,biological activities and biosynthesis.Specifically,the second part of this chapter covered investigations of maytansinoids biosynthesis at the biochemical and genetic level,use of maytansinoids as "warheads" in antibody drug conjugates,and ecological issues related to the occurrence of such typical microbial metabolites in higher plants.In the second chapter,detail information of experimental materials and methods was provided.In Chapter 3,seven biosynthetic gene clusters(BGCs)of bacterial maytansinoids were analyzed and categorized into three groups.Ancestral reconstruction suggested a putative evolutionary scenario resulted from gene reshuffling.In particular we reported the identification and annotation of the ansacarbamitocin(ASCs)BGC(asc)from Amycolatopsis alba DSM 44262.In Chapter 4,the BGC for the bacterial maytansinoids asc was comprehensively studied.Specific gene disruption and heterologous expression led to the isolation of seven new bacterial maytansinoids.Subsequently,post-PKS modifications of ASCs were elucidated based on bioinformatic analysis and intermediates accumulated in selected gene disruption mutants.The 3'-O-methyltransferase gene orf9 and 3-0-carbamyltransferase gene asc21b involved in bacterial maytansinoid biosynthesis were identified for the first time.The new bacterial maytansinoids 7 and 15,showing strong antitumor activities against four human cancer cell lines,are potential candidates of the lead compounds of antibody maytansinoid conjugates.Additionally,the heterologous expression of asc11 led to a higher yield of maytansinol.In Chapter 5,we studied the alanylase AstC of ansatrienins.Three new ansatrienin analogues,representing three different polyketide chains,were isolated from the mutant strain Streptomyces sp.XZQH13O?astC?astF2.Accordingly,pathways for the biosynthesis of these new ansatrienins were proposed.The heterologous expression of astC led to the isolation of alanylated N-desmethyl-4,5-desepoxy-maytansinol,a new analogue with strong antitumor activity,indicating that AstC is a potential tool for alanylated maytansinoids.Additionally,dozens of truncations of TE-domain of AstC were constructed,and crystals of TE-domain were obtained.In summary,we studied the biosynthesis and function-orientated structure optimizations of bacterial maytansinoids via integrated application of bioinformatics,chemical biology and synthetic biology.Our results provide not only new ideas about the orgin of maytansinoids,but also novel insights into the structure activity relationships of these family of natural products,enabling the engineering of more derivatives with improved pharmacological properties.Moreover,this study revealed the diversity of ansamycin polyketide chains,substrate compatibility of the polyketide synthases and amide synthases,and the biosynthetic pathway of alanylated maytansinoid,facilitating the discovery of novel compounds and biosynthetic pathways of natural products.