Preliminary Exploration On The Secondary Matebolites From The Sea Anemone-derived Fungus Cochliobolus Lunatus Using Chemical Epigenetic Modification

Previous studies in our group on a sea anemone-derived fungus Cochlioboluslunatus (TA26-46) led to the isolation of a series of14-nembered resorcylic acidlactones. These metabolites with novel strucures also have diverse bioactivities, suchas antifouling activities against the larvae of Balanus amphtrite and antifungalactivities against different fungal strains. Present study maily focused on the influenceof epigenetic modifiers on C. lunatus (TA26-46). We tried to improve the diversity ofthe secondary metabolites and provide compound basis for post-study.Based on previous research, we tried to study the influence of the differentfermentation conditions of C. lunatus on the production of14-nembered resorcylicacid lactones. By changing medium compositions (rice medium, GPY medium, starchand synthesis mediums), adding precursor (sodium acetate，sodium malonate), andadding key metabolic enzyme inhibitors (the single cell oxidase inhibitor: metyrapon,and, the polyketide synthase inhibitor: iodoacetamide), we find that metabolism of C.lunatus is stable and suitable for subsequent study. Among them, in the solidoligotrophic culture conditions, the chromatographic peaks of metabolites wereseparate obviously, therefore the culture condition is good for chemical epigeneticmodification for post-study. The types of metabolites were single with C. lunatuscultured in the lipid fermentation conditions. While when C. lunatus was cultured byorthogonal experiment, the production of compound LL-Z1640-2reached at119.76mg/L, more than3.6times compared with before.The epigenetic modifiers were used to changing the secondary metabolites ofthis fungus. Histone deacetylase inhibitors, such as sodium butyrate andsuberoylanilide hydroxamic acid (SAHA) were selected for the research. Two new14-membered resorcylic acid lactones characterized with bromine substitution, 5-bromozeaenol (1) and3,5-dibromozeaenol (2), together with four known analogues(36), were isolated from the culture treated with sodium butyrate. The absoluteconfigurations of1and2were assigned by CD spectra and chemical conversion.Compounds1and2represent the first example of natural brominated resorcylic acidlactones. These brominated lactones were produced by C. lunatus (TA26-46) onlywhen treated with HDAC inhibitors, sodium butyrate or SAHA.While the resorcylic acid lactones are still the major metabolites of this fungus,the secondary metabolites of this fungus displayed a grate impact under the samemedium containing5–azacytidine, a DNA methyltransferase inhibitor.11diethyleneglycol phthalate esters were isolated: Diethylene glycol phthalate ester A (7),1,2-Benzenedicarboxylic acid,1,2-bis [2-(2-hydroxyethoxy) ethyl] ester (8),1,2-Benzenedicarboxylic acid, oxydi-2,1-ethanediyl dimethyl ester (9), Diethyleneglycol phthalate ester B (10),1,2-Benzenedicarboxylic acid,1,1’-(oxydi-2,1-ethanediyl)2,2’-bis [2-(2-hydroxyethoxy) ethyl] ester (11),Diethylene glycol phthalate ester C (12),1,2-Benzenedicarboxylic acid,1,2-bis[2-[[2-[[2-(2-hydroxyethoxy) ethoxy] carbonyl] oxy] ethoxy] ethyl] ester (13),Diethylene glycol phthalate ester D (14), Diethylene glycol phthalate ester E (15),Diethylene glycol phthalate ester F (16), and Diethylene glycol phthalate ester G (17).7,10,12,14,15,16, and17are new compounds.The isolated compounds were evaluated for their antibacterial activity,antifouling activity, zebrafish embryo teratogenicity and other biological activities.zeaenol (4), LL-Z1640-1(5) and LL-Z1640-2(6) exhibited potent antifouling activityagainst the larval settlement of the barnacle Balanus amphitrite with an EC50value of5.0μg/mL,5.3μg/mL and1.82μg/mL respectively. But5-Bromozeaenol (1) and3,5-Dibromozeaenol (2) were inactive (EC50>25μg/mL). Other compounds showednone activities with other active modoles.In conclusion, we study the C. lunatus (TA26-46) derived from a sea anemoneusing the method of epigenetic modification, discovering that it can producebrominated lactones induced by histone deacetylase inhibitors. It produced a series ofdiethylene glycol phthalate esters with the existence of resorcylic acid lactones when using5-Azacytidine.9new compounds including two brominated lactones whichrepresent the first natural example of brominated resorcylic acid lactones wereisolated from C. lunatus. The epigenetic modification screening methods provide anew approach for efficiently obtaing new compounds.