Combinatorial Biosynthesis Of Combamides And Targeted Discovery Of Montamides

Natural products are an important source of new antibiotics.Polycyclic tetramate macrolactams(PoTeM)containing a tetramate moiety and a polycyclic system are synthesized by the polyketide-nonribosomal peptide pathway,and has a variety of biological activities,such as antibacterial,antifungal,antiprotozoal,antiulcer and antitumor activities.The structural diversity of PoTeMs is expanded by the different polycyclic systems,including 5/6/5 tricyclic,5/5/6 tricyclic and 5/5 bicyclic units,and post-modification reactions such as hydroxylation,carbonylation and epoxidation catalyzed by cytochrome P450 enzymes.PoTeMs is gradually forming a new family of antibiotics because of their complex structures,various biological activities and novel mode of action.Based on the biosynthesis of PoTeMs,this study carried out the following two parts:1.Combinatorial biosynthesis of combamides.(1)First,five cytochrome P450 genes related to PoTeMs biosynthesis were selected for structural modification of combamides.Then,these gene with different oxidative modification types were individually combined with the combamides biosynthetic genes with or without the OX4 gene to give ten recombinant plasmids.The constructs were introduced into Streptomyces sp.SR111 and Streptomyces sp.S001 to produce twenty recombinant strains.As a result,eight combamides(1-8)were identified from the metabolites of four recombinant strains,and five of them were new ones.In addition,by analyzing the structure of the isolated compounds,we found that the PoTeMs related P450s had substrate specificity toward the polycyclic systems.Biological activity evaluation showed that combamide J(6)has weak antifungal activity,combamides G(2),combamides J(6)and combamides H(8)have strong cytotoxic activity,combamides J(6)and combamides H(8)have strong inhibitory activity on T3SS secretion system of Salmonella typhimurium.(2)Five recombinant strains containing hybrid combamides biosynthetic gene clusters,which are expected to encode combamides with a novel polycyclic system,were constructed.As a result,two PoTeMs were identified from the metabolites of two recombinant strains.2.Targeted discovery of montamides in Streptomyces sp.S001.Previously,our group introduced the cftS023 gene cluster,which encodes the 5/6/5 tricyclic clifednamides,into Streptomyces sp.S001 to give the recombinant strain S001-PoTeMS023.In this study,a new 5/6/5 tricyclic PoTeMs montamide A was identified from S001-PoTeMS023,and the mtm gene cluster located in S001 was presumed to be responsible for the biosynthesis of montamide A by bioinformatics analysis.In order to obtain other new montamides,we try to activate the mtm gene cluster through promoter replacement and local expression.As a result,the yield of target product was increased.Attempt is being made to change host to further increase production.In conclusion,through combinatorial biosynthesis and targeted discovery strategy guided by gene sequence,the oxidative modification of combamides and activation of mtm gene cluster were achieved,and five new combamides were obtained.In one hand,our results indicated that combinatorial biosynthesis is an effective way to create " unnatural " new PoTeMs;on the other hand,it shows that promoter replacement is effective to obtain new PoTeMs,which lays a foundation for further structural modifications and yield improvement of PoTeMs.