N-Methylation Mechanism Of HSAF And Combinatorial Biosynthesis Of Combamides

Structure modification based on the biosynthetic mechanism of natural products is an important approach to discover new bioactive compounds.Polycyclic tetramate macrolactam(PoTeM)is a class of nitrogen-containing natural products identified from bacteria and sponges.PoTeMs are characterized by a tetramic acid moiety and a polycyclic system.With significant physiological activity such as antiprotozoal,antibacterial,antifungal,antioxidative and cytotoxic,PoTeMs show promising potential for application in the field of biological control and medicine.Based on the biosynthesis of PoTeMs,this thesis carried out the following three aspects of research:1.Biosynthetic mechanism of N-28 methylation of HSAF.Heat-stable antifungal factor(HSAF)is a broad-spectrum antifungal PoTeM isolated from Lysobacter enzymogenes C3.Previous studies have shown that N-28 methylation can enhance the antifungal activity of HSAF and its analogs.However,the catalytic mechanism of N-28 methylation of HSAF has not been reported.By gene deletion and overexpression,we identified the methyltransferase gene,which is responsible for the N-28 methylation modification of HSAF and its analogs,in Lysobacter enzymogenes C3.Meanwhile,a new methylated derivative 3-deOH alteramide E(2)was isolated.In addition,in vitro biochemical experiments were performed to determine the catalytic function,enzymatic properties and substrate promiscuity of the methyltransferase.2.Combinatorial biosynthesis of combamides.Based on the simplicity and conservation of the PoTeM gene clusters,we performed the structure modifications of 5-5-bicyclic combamides through a combinatorial biosynthesis strategy.1)Combamide K(3),a new product with N-28 methylation,was obtained by combining the methyltransferase gene ceqC with the cbm gene cluster;2)Combamide L(4),a new compound with enhanced antifungal activity against filamentous fungi,was obtained by hydroxylation at the C-3 position of combamide J with C3 hydroxylase SahE;3)Clifednamide K(5),a new compound which was obtained through exchanging the OX genes involved in the formation of the polycyclic system,although it is not the expected one.3.Targeted discovery of novel PKS/NRPS hybrid compounds in Bacillus gobiensis.Bioinformatics analysis revealed that Bacillus gobiensis contained a hybrid PKS/NRPS gene cluster(bgo),which was similar to PoTeM gene cluster.Discovery of products encoded by this gene cluster may be useful for modification of the PKS/NRPS skeleton of PoTeMs.Therefore,we reconstructed the bgo gene cluster and heterologous expressed in Streptomyces sp.S001 and E.coli BAPI,respectively,but no target compound was detected up to now.