Synthetic Biology Approaches To Discovery,Structural Optimization And Post-PKS Modifications Of Rifamycins

As a global problem,tuberculosis(TB)remains one of the top 10 causes of death worldwide as yet,and TB control is far from being done because of the global spread of AIDS and drug-resistant Mycobacterium tuberculosis.Semi-synthetic derivatives of rifamycin,such as rifampicin,rifapentine and rifambutin,are effective in treating tuberculosis and have long been first-line drugs for the clinical treatment of tuberculosis,leprosy and AIDS-related mycobacterial infection.However,M.tuberculosis has also gradually developed resistance to these compounds due to their extensive clinical use for the past few years,so it is in urgent need to discover or synthesize rifamycin derivatives against drug-resistant M.tuberculosis.In particular,the biosynthetic pathway of rifamycin have remained in hypothesis stage ever since discovered in 1957.In order to exploit the structure diversity of rifamycins and obtain rifamycin analogues for activity screening,this study mainly studied rifamycin biosynthesis from the following three aspects.Firstly,eleven rifamycin congeners(1-11)including six new ones(1-6)were isolated and identified from the YMG agar fermentation extract of A.mediterranei S699.Compounds 1 and 2 were rifamycin glycosides and named as rifamycinosides A and B,respectively.Their polyketide skeleton represented a novel cleavage pattern of rifamycin ansa chain.Compounds 6 and 8 showed potential inhibitory activity against Type III secretion system(T3SS)of Salmonella enterica Typhimurium UK-1 x8956,and could be applied for developing novel agent against gram-negative bacterial infection.In addition,compound 6 could induced G2/M phase arrest and caused DNA damage in HCT116 cells.Secondly,the extension units and ring system of rifamycin polyketide were modified to obtain rifamycin derivatives with long side chain or reduced ring.However,only premature polyketides were accumulated from these module swapping or module fusion mutants,which may due to the particularity of rifamycin releasing mechanism.While,series of module fusion and corresponding site-directed mutation results also domenstrated the importance of preserving KS-AT linker and the feasibility of module fusion through KR-ACP linker during polytide extension,thus further enhance our understanding of ansa PKS engineering.Finally,the complicated post-PKS modifications during rifamycin biosynthesis were further explored,including the shut pathway from proansamycin X to rifamycin W,the rifamycin W skeleton cleavage catalyzed by Rif-Orf5 and the naphthalene formation catalyzed by Rif-Orf19.These post-PKS modifications of rifamycin have been better understood through the above exploration,which could contribute to the synthetic biology or in vitro enzymatic modification of rifamycins.In summary,in order to expand the structure diversity of rifamycins and obtain rifamycin analogues exhibit better antibacterial activity against multidrug-resistant strains of M.tuberculosis,the secondary metabolites of rifamycin producing strain were rediscovered,the polyketide skeleton of rifamyicn were modified and the legacy of rifamyicn post-PKS modifications were further explored in this study.Our results promote a deepgoing understanding of the particularity during rifamycin polyketide backbone modification,the diverisity of rifamyicn polyketide backbone breakage and the mechanism of C-8 shut-pathway,C-12/C-29 double bond oxidative cleavage,naphthalene ring formation,double bond migration of rifamyicn post-PKS modifications,which then provide solid basis for subsequent generation of rifamyicn structural derivatives against drug-resistant M.tuberculosis via synthetic biology.