Biosynthesis Of The Polycyclic Systems Of HSAF And Pseudoamides And Improvement Of The Biocontrol Effect Of Lysobacter Enzymogenes C3

The Gram-negative Lysobacter in the Xanthomonadaceae family has significant antagonistic activity against a variety of plant pathogenic fungi,bacteria,and nematodes,and has emerged as new biocontrol agents for crop protection in agriculture.Moreover,Lysobacter has become a new source of bioactive natural products due to their largely unexplored cryptic biosynthetic gene clusters.Lysobacter enzymogenes C3 showed significant activity against plant pathogenic fungi and nematodes.Elucidation of the biosynthetic mechanism of the antifungal compound HSAF will facilitate the synthesis of new derivatives with improved antifungal activity.The development of efficient genetic manipulation tools for strain C3 will promote the heterologous expression of other biocontrol agents in strain C3.and facilitate the genome mining of novel bioactive natural products from strain C3.These genetic modifications will improve the biocontrol efficiency and expand the biocontrol range of strain C3.Based on the above background,this thesis will elucidate the biosynthetic mechanism of the polycyclic system formation of HSAF and pseudoamides,respectively,and improve the biocontrol activity of C3 through genetic manipulations.1.Biosynthetic mechanism of the polycyclic system formation of HSAF:Through the isotope labeling experiments and the identification of the labeled product,the regioselectivity and stereoselectivity of the OX4-catalyzed reductive cyclization that forms the inner sixmembered ring of HSAF,were revealed.The results support a mechanism consistent with a 1,6Michael addition reaction.Then,we provide the first experimental evidence in vivo to support that the inner five-membered ring formation of HSAF is coupled with C20-hydroxylation,which was catalyzed by OX1 or OX2.In addition,by co-expression of OX1,OX2,OX3,and Cbm A in E.coli BAP1,we found that the recombinant OX proteins produced by E.coli are active.2.Biosynthetic mechanism of the 5/5 bicyclic system formation of pseudoamides:Through combinatorial biosynthesis,in vitro biochemical assays and isotope labeling experiments,we demonstrated that redox partners are required for the activation of Pell and Pel3 to generate the 5-5 bicyclic system.Pel1 catalyzes a reductive[2+3]cycloaddition to form the outer 5-membered ring,which involves the incorporation of the hydride of FMNH2 and one proton from water.Pel3 catalyzes inner 5-membered ring formation coupled with C12 hydroxylation,and is proposed to use the nucleophilic FMN-C4a-hydroxyperoxide to attack and ultimately incorporate oxygen into the substrate.3.Improvement of the biocontrol effect of L.enzymogenes C3 and discovery of bioactive products:First,we developed the efficient genetic manipulation tools for C3,such as active promoters,site-specific recombination system and CRISPR/Cpfl system for gene editing.Then.the phenazines gene cluster of L.antibioticus ATCC 29479 was refactored using active promoters and successfully heterologous expressed with site-specific recombination system in strain C3.As a result,the engineered strains C3-cophz and C3-phz showed significantly inhibition agaist the plant pathogenic bacteria.Additionally,we constructed the mutant strain C3AHWL,in which three biosynthetic gene clusters were deleted,to facilitate the identification of novel natural products from strain C3.The known compound pyrrole-2-carboxylate with antibacterial and antifungal activity was isolated from strain C3ΔHWL through bioactivity guided assay.In conclusion,the biosynthetic mechanism of the formation of the 5/5/6 tricyclic system in HSAF and the 5/5 bicyclic system in pseudoamides have been elucidated through in vivo and in vitro experiments,which may be generally applicable to the corresponding ring formation in other PoTeMs.This work will not only help to simplify the components of HSAFs and improve its antifungal activity,but will also facilitate the engineering approach to generate PoTeMs bearing novel polycyclic systems.In addition,the biocontrol effect of C3 was significantly improved by introduction of the phenazines cluster,which lays a foundation for further development and application of Lysobacter as biocontrol agents.