Targeted Discovery Of Novel PoTeMs

Polycyclic tetramate macrolactam(PoTeM)is a class of macrolactam compounds with a tetramate moiety and a polycyclic ring system.PoTeMs have a variable set of stereochemical rich carbocyclic rings,including 5/5,5/5/6,5/6/5 or 5/4/6 cyclization pattern.The structural diversity of PoTeMs can be further expanded by post modifica-tions,such as hydroxylation,epoxidation and N-methylation.PoTeMs has attracted considerable attention among chemists,biochemists and pharmacologists because of their distinctive chemical structures,impressive physiological activities and unique bi-osynthetic mechanisms.Based on the conserved biosynthetic genes of PoTeMs,we carried out the targeted discovery and biosynthesis of new PoTeMs,and the main achievements include the following three parts:1.Targeted discovery of new clifednamides in Kitasatospora sp.S023.Bioinfor-matics analysis showed that a putative clifednamides biosynthetic gene cluster cfts023 was in the genome of Kitasatospora sp.S023.Previously,our group have constructed four recombinant strains of Streptomyces sp.S001,which contained varies refactored cfts023 gene cluster.In this thesis,we performed the isolation and characterization of the target compounds in the above strains.As a result,10 PoTeMs,clifednamides A-B.clifednamides D-J and montamide A,were obtained,and 8 of which were new ones In addition,clifednamides D,G,H are the first discovered macrocyclic ring-opened PoTeMs.Clifednamides A-B and clifednamide H have cytotoxicity.Clifednamide B and clifednamides I-J inhibit the secretion of toxic protein of Salmonella T3SS.2.Targeted discovery of new PoTeMs in Pseudomonas alternatus.Previously,we identified a novel PoTeM biosynthetic gene cluster pel in the database,and constructed two recombinant strains of Streptomyces sp.SR111,which contained the refactored pel gene cluster.In this study,two novel PoTeM compounds,pseudoamides A and B,were obtained and characterized from the metabolites of the above strains.Both compounds have cytotoxicity,and pseudoamide B has weakly inhibits antifungal and the secretion of toxic protein of Salmonella T3SS3.Study on the catalytic function of OX4 in HSAF biosynthesis.Firstly,we iso-lated two 5/5 bicyclic PoTeMs,3-deOH alteramide B and new compound 3-deOH al-teramide C,with different stereo configurations from the metabolites of the mutants OH11?OX2 and C3?OX1.Then.OX4 protein was reacted with 3-deOH alteramide B or 3-deOH alteramide C in the presence of NAD(P)H,respectively.We found that OX4 can transform 3-deOH alteramide C to 5/5/6 tricyclic 3-deOH HSAFs,but only reduces the diene systems in 3-deOH alteramide B,instead of forming the 6-membered ring.The result clearly demonstrates the function of OX4 as the enzyme responsible for for-mation of the 6-membered ring in HSAF biosynthesis.In conclusion,we have obtained 14 PoTeMs through targeted strategies guided by gene sequences,including 12 new ones.The bioactivity evaluation of the isolated Po-TeMs was carried out,including antibacterial,antifungal,cytotoxic,and type three se-cretion system inhibition activities.This thesis laid a good foundation for further ex-ploration of novel PoTeMs,interpretation of biosynthetic mechanisms and structure modifications through synthetic biology approaches.