Targeted Discovery Of New PoTeMs And Discovery Of New Natural Products From Lysobacter Sp. DSM 3655

Polycyclic tetramate macrolactams(PoTeMs)is a class of natural products with a polycyclic system,and is forming a new family of antibiotics with complex structures and diverse biological activities.Bioinformatics analysis revealed that there are a large number of cryptic PoTeM gene clusters in the database,indicating that the targeted discovery and combinatorial biosynthesis of new PoTeMs will expand the chemical diversity of PoTeMs.Lysobacter is a genus of Gram-negative bacteria in the Xanthomonadaceae family,and are prolific producers of new bioactive natural products that are largely underexplored.Since most of the secondary metabolic pathways in Lysobacter are "silent" or low expressed,genome mining of Lysobacter will lead to the identification of new bioactive natural products.Based on the above background,this thesis carried out the following two projects:1.Targeted discovery of new PoTeMs.Previously,we identified four PoTeM gene clusters(pel,sah,ceq and gsu)in the database that are proposed to encode new PoTeMs.In this study,we used the "pathway refactoring-heterologous expression" strategy to activate the above gene clusters.1)A new PoTeM compound with cytotoxic activity,pseudoamide B(2),was obtained from the metabolites of the recombinant strain SR111-pel123.2)Four recombinant plasmids containing different sah gene clusters were constructed and heterologous expressed in Streptomyces sp.SR111.Six PoTeM compounds were obtained from the metabolites of the recombinant strains,and sahamide A(3)showed weak antifungal activity.In addition,the biosynthetic pathway of sahamides was proposed.Remarkably,sahamides A-C(3-5)represent the first one-ring PoTeMs with oxidative modifications,and SahE is the first CYP decorating the ornithine unit of PoTeMs.3)Six recombinant plasmids containing different ceq gene clusters were constructed and heterologous expressed in Streptomyces sp.SR111.The targeted compounds are still being isolated.Additionally,combination of the methyltransferase gene ceqC with the combamide gene cluster lead to the identification of "unnatural" new PoTeM combamide K(16).4)Two known PoTeMs with antifungal activity,compounds a and c(1718),were isolated from the recombinant strain J1074-gsul2.2.Discovery of new natural products from Lysobacter sp.DSM 3655.Three gene clusters BGC1/4/9 of Lysobacter sp.DSM 3655 were selected and heterologous expressed in Streptomyces,Among them,only BGC9 was successfully activated,which lead to the isolation of two new polyketide-nonribosomal peptides,lysohexaenetides A and B(19-20),and the discovery of a new iterative bacterial type I PKS.In addition,the biosynthetic pathway of lysohexaenetides was speculated.In conclusion,seven new PoTeMs and two new polyketide-nonribosomal peptides were identified through genome mining and combinatory biosynthesis.Our results again indicated that the "pathway reconstruction-heterologous expression" strategy is effective for discovery of new PoTeMs,and the "promoter engineering and heterologous expression" strategy can be used for activating the "silent" gene clusters of Lysobacter,which provides an example for further exploration of the secondary metabolites of Lysobacter.