Study Of Biosynthesis Of Polysaccharides And Glycolipids From Fungi

Co-evolution of symbionts with hosts may increase their productivity of natural products with novel structures and/or significant bioactivities.The knowledge regarding the nature’s way of manufacturing compounds may help us figure out the strategy for their chemical syntheses,manipulation of microbial productions of target products,and structure diversifications of complex natural compounds,all being important in the discovery of new drugs.In this thesis,the biosynthesis of three types of symbiont-derived natural products has been investigated owing to their unique chemical structures,potent bioactivities,and supply issues that come into being.The first chapter is a mini-review concerning the biosynthetic research of polysaccharides and cerebrosides.The second chapter presents the biosynthesis of fusaruside,a sphingolipid derived from a Quercus variabilis endophytic fungus Fusarium sp.IFB-121.Fusaruside is unique in its possession of a 9-methyl-4,8,10-sphingatrienine chain and efficacy in treating liver injuries through selective-immunosuppression.We cloned and expressed a Δ10(E)-sphingolipid desaturase and employed gene-knockout,enzyme catalysis in vitro,and LC-MS analysis to prove it catalyze the generation of 10,11-double bond of fusaruside in F.graminearum.Low temperature and high salt culture experiments uncovers a mechanism for fungal cells’ aptations to "hostile" environments through sphingolipid desaturation.The 3rd chapter describes the biosynthesis of phoman,a novel cell wall glycan from a marine fungus Phoma herbarum YS4108.Phoman has a backbone of a-(1,4)-glucan with alternating branches composed respectively of a-(1,6)-monoglucose and a-(1,6)-monoglucuronic acid.The whole genoming approach assigned the genes governing its biosynthetic pathway,facilitating the optimisation for the scalable production of phoman.We cloned and expressed the UDP-glucose pyrophosphorylase(UGPase)and glycogen synthase(GS),and proved their involvement in phoman synthesis,employing immunochemical methods and in vitro synthesis.The glucuronosyltransferase(UGT)genes responsible for the attachment of glucuronic acid were identified using gene-knockout strategy and HPIC-PAD analysis.Finally,the biosynthetic pathway was proposed.Firstly,glucose 1-phosphate is activated by UGPase to uridine diphosphate glucose,part of which is oxidized to UDP-glucuronic acid by dehydrogenase.Glycosyltransferases are required to construct the repeating unit of phoman,including GS and UGT responsible for the main and side chains,respectively.The 4th chapter addresses the clarification of the biosynthetic pathway of dalesconols,immunosuppressive polyketides characterized from the culture of Daldinia eschscholzii IFB-TL01 residing in Tenodora aridifolia gut.Whole genome sequencing facilitates the identification of a laccase encoding gene lacTL downstreaming the polyketide synthase gene cluster.Knockout of lacTL deprived the fungus of its productivity of dalesconols implying the involvement of laccase in generating naphthol radicals.The free radical scavenging experimentations further substantiate the participation of radicals.The experimentations corroborate collectively that dalesconols are generated through the promiscuous couplings of naphthol radicals with different oxygenation patterns.