Discovery And Biosynthesis Of New PoTeM Products

With the emergence of drug-resistant bacteria,the discovery of new antibiotics with novel mode of action has become particularly urgent.Natural products isolated from microbes remain an important source of new antibiotics.The whole-genome sequencing of microbes showed that microbes contain a large number of cryptic biosynthetic gene clusters,and genome mining is a powerful tool to uncover these novel secondary metabolites.Polycyclic tetramate macrolactam(PoTeM)is a class of macrolactam compounds that have a tetramic acid unit and polycyclic systems.Most of PoTeMs show good biological activity.According to the different polycyclic systems,PoTeMs can be divided into three subgroups:5/5/6 tricyclic,5/6/5 tricyclic and 5/5 dicyclic,with HSAF(5/5/6),ikarugamycin(5/6/5)and alteramide A(5/5)as representatives.It has to be note that the first ring in tricyclic system can be formed in two ways,for example,C14/C18 in HSAF and C15/C19 in ikarugamycin.PoTeM compounds are forming a new family of antibiotics due to their novel chemical structures and unique mode of actions.This thesis reported the targeted discovery and combinatorial biosynthesis of new PoTeMs by genome mining based on the simplicity of their biosynthetic loci.Previously,we identified two new PoTeMs gene clusters,cbm and cft,in Streptomyces sp.S10 and Streptomyces sp.S23,respectively.However,large fermentation of strains S10 and S23 have not lead to the isolation of PoTeM compounds,indicating that both of the above two gene clusters were silent or low expressed.Therefore,we are planning to activation the two gene clustes through heterologous expression stratagy,including:4)Heterogenous expression of the cbm gene cluster of Streptomyces sp.S10 in Streptomyces sp.SR111;2)Heterogenous expression of the cft gene cluster of Streptomyces sp.S23 in Streptomyces sp.S001.Bioinformatics analysis showed that the cbm gene cluster of Streptomyces sp.S10 might encode 5/5 double ring PoTeM compounds.Previous studies showed that the"core genes" involved in formation of the skeleton of PoTeMs were in one operon,which laid a foundation for the activation of the cbm gene cluster.In this study,the following studies were conducted on the cbm gene cluster:1)Introduction of strong promoter kasOp*into the upstream of the cbm A-D operon to activate the cbm gene cluster,which led to the isolation of three new PoTeM compounds(combamides A-C)from the recombinant mutant;2)Deletion of the cbm D gene,which is responsible for the oxidative modification of combamides,led to the isolation of a new PoTeM compound(combamide D)from the recombinant mutant;3)The OX4 gene in the HSAF gene cluster has been proved to catalyze the formation of the six-membered ring of the 5/5/6 tricyclic system in HSAFs.Therefore,the OX4 gene and the cbm gene cluster were combined to form a hybrid gene cluster,wich led to the isolation of two new 5/5/6 tricyclic PoTeM compounds(combamides E and F)from the recombinant mutant.Bioinformatics analysis showed that Streptomyces sp.S23 contained cftS23 gene cluster that may encode 5/6/5 tricyclic PoTeM compounds.We carried out the following studies on the cftS23 gene cluster:1)the strong promoter kasOp*and ermEp*were introduced into the upstream of the cfts23 A-D operon and the upstream of the cfts23 E gene,respectively.The resulting recombinant plasmids were transformed into Streptomyces sp.S001 to obtain two recombinant mutants,which can produce new 5/6/5 trycyclic PoTeMs with oxidation modifications;2)Deletion of the cfts23 D gene led to the construction of the recombinant mutant,which are capable to produce 5/6 dicyclic PoTeM compounds with oxidation modifications;3)the cbmC gene in the cbm gene cluster was replaced by cfts23 C gene in situ to obtain novel 5/5 dicyclic ring system PoTeM compounds with the same ring formation mode as the first 5-member ring in ikarugamycins.In conclusion,six new PoTeMs combamides A-F were obtained by activation and combinatorial biosynthesis of the cbm gene cluster in Streptomyces sp.S10.For activation and combinatorial biosynthesis of cfts23 gene cluster in Streptomyces sp.S23,six heterologous expression mutants have been constructed,and the isolation of the target compounds are still undergoing.