Research Of The Secondary Metabolites And The Interaction Mechanism Of Coculture Between Three Endophytic Fungi Associated With Gastrodia Elata And Armillaria Sp.

Microbial co-culture has become an important method to improve production titer and discover new compounds.It can enrich microbial chemical diversity and has important research significance and application prospect.This has also aroused great concern about the understanding of microbial co-culture in inducing the production of novel active chemicals.The research of metabolic interactions between microorganisms will contribute to deepen the understanding and application of microbial co-culture strategies.This di ssertation investigated the chemical composition and biological activity of the secondary metabolites of endophytic fungi associated with Gastrodia elata and mycorrhizal Armillaria sp.Based on the analysis of the metabolic characteristics of the two fungi under the interaction,the interaction mechanism of the two fungi was investigated.This dissertation was composed of four chapters.The first chapter reviews the frontier of co-culture strategy in the study of microbial secondary metabolites,introduces the trend of the mechanism of microbial co-culture,and provides a reference for further research.The second chapter and the third chapter select the Phoma sp.YUD17001-Armillaria sp.co-culture system synergisticaction effect on morphology and the Epicoccum sp.YUD17002-Armillaria sp.co-culture system with antagonistic effect on morphology.The chemical composition and biological activity of metabolites produced by co-cultures were systematically investigated.Then,based on the analysis of metabolic characteristics of single strain culture and co-culture,the interaction mechanism between co-culture strains was explored.The fourth chapter researches the mechanism of patulin toxicity reduction in Penicillium sp.TG-46 and Armillaria sp.co-culture system based on secondary metabolic characteristics.A total of 87 compounds were characterized,which included 28 new compounds.The plane structure and absolute configuration of these compounds were determined by modern spectral analysis techniques,single-crystal X-ray crystallography,Mosher method and ECD calculation.The chemical structure types of compounds are mainly terpenes,polyketones and phenols.Some of the isolated new compounds showed different cytotoxic activities,antimicrobial activities or acetylcholinase inhibits activities.The biosynthesis pathway of some new compounds was deduced.The qualitative analysis of the origin for new compounds were proceeded by HPLC and UHPLC-MS.Phomretone E（5）was detected in Phoma sp.YUD17001 single culture,the remaining new compounds were produced only in co-culture.The main progresses of this dissertation were as following.A series of new C₁₂polyketides,phomretones A-F（1-6）,were discovered from Phoma sp.YUD17001-Armillaria sp.coculture system,they featured high levels of oxidation and stereoisomerization functions.Phomretone G（7）featured novel cyclohexene parallel furan cyclic frameworks.Phomretone H（8）possessed a rare cycloheptanone scaffold.Armilaroma（14）exhibited strong cytotoxic activity against 5 human tumor cell lines（A-549,SMMC-7721,HL-60,MCF-7 and SW480）,the inhibitory activity on MCF-7and SW480 was stronger than that of cisplatin.Armilaroma also has moderate anti acetylcholinesterase activity and bacteriostatic activity.Phomretone G（7）showed moderate inhibitory activity against Escherichia coli,MIC 8μg/m L.Three novel polyoxygenated meroterpenoids,epiterenoids A-C（1-3）were isolated from a liquid co-culture of Armillaria sp.and the endophytic fungus Epicoccum sp.YUD17002associated with Gastrodia elata.Epiterenoids A-C（1-3）possessed a 5/6/4/5/5-fused pentacyclic novel skeleton.Armiloid A（4）represents the first example of an illudalane sesquiterpenoid derivative featuring a substituent group on C-1.Epicoterpene D（8）is the first example of an ent-protoilludane sesquiterpenoid scaffold bearing a five-membered lactone.Armiloid A（4）exhibited strong antiacetylcholinesterase activity(IC₅₀ value of 4.91μM).Armilliphatic A（10）exhibited moderate in vitro cytotoxic activities against five human cancer cell lines（HL-60,A-549,MCF-7,SMMC-7721,and SW480）with IC₅₀ values ranging from 15.80 to 23.03μM,it also showed moderate acetylcholinesterase inhibitory activity.Furthermore,armiloid A（4）,epicoterpene A（5）,epicoterpene C（7）,epicoterpene D（8）and armilliphatic A（10）also showed different levels of antibacterial activity.Researches of the interaction mechanism of the two culture systems show that the factors inducing the production of new compounds are not only determined by extracellular enzymatic or metabolic substrate molecular regulation of a single strain.Based on the experiments of bacteriostasis and feeding,the mechanism of Penicillium sp.TG-46 and Armillaria sp.co-cultured patulin detoxification was inferred from the inhibition of patulin biosynthesis precursor 3-（hydroxymethyl）phenol（3）by the release of extracellular enzymes during biological stress.The results of this dissertation not only provide a new reference for the discovery of novel active natural products,but also provide a new idea and direction for the investigate of symbiotic interaction mechanism between Gastrodia elata and Armillaria sp.