Studies On The Metabolites Of Arthrobotrys Oligospora Mutants And Dactylella Alba

This thesis consisted of four parts:(1)P450 gene 282 was involved in the biosynthesis of Arthrosporols in nematode-trapping fungus Arthrobotrys oligospora and the targeted compounds in mutant(35)282 were purified and characterized;(2)Prenyltransferase gene 276 was heterologously expressed in Escherichia coli and the targeted compounds in the engineered strain were isolated and identified;(3)The effects of three precursor compounds on the biosynthesis of Arthrosporols in mutants?283,?282 and?281,and wide-type strain were evaluated;(4)Optimization of fermentation medium for Dactylella alba,an anamorphic nematode-trapping fungus of Orbilia alba was performed and the metabolites from this strain were isolated and determined.Main results and innovation:1.Four compounds including one targeted metabolite were isolated from mutant?282 strain.The targeted metabolite was characterized novel derivative of isopentenyl m-cresol and the known compounds were 3-methyl-1H-indole,ergosterol and 4-(hydroxymethyl)phenol.2.Three novel oxyanthracene compounds(276-1,276-2 and 276-3)were isolated and identified from an engeered Escherichia coli strain,which harbored a fungal prenyltransferase gene AOL_s00215g276 and was fed with toluquinol.Then,another six compounds were isolated from the engineering strain fed with xanthydrol,including three targeted compounds 9H-xanthen-9-one(28-1),them,3-(9H-xanthen-9-yl)-1H-indole(28-3),sulfamide,N-9H-xanthen-9-yl-(28-11)and a new one(28-5).Two other known compounds were benzoic acid and ergosterol.Compound 28-3 displayed strong tumor cytotoxic activity against human lung cancer cell line A549 with an IC₅₀ value of 1.47?M,and compound 28-5 exhibited strong tumor cytotoxic activity against human melanoma cells A375 and human osteosarcoma cells HOS,with IC₅₀ values of 4.13?M and 2.70?M,respectively.Moreover,both compounds were found to exhibt red fluorescence and to locate on the mitochondria in human 293T cell line.3.Metabolic profiles of mutant?283 fed with 6-MSA,m-cresol and toluquinol,respectively,at different concentrations of 0.125,0.25,0.5 and 1mM,displayed that no arthrobotrisins and arthrosporols were detected and no compounds exhibited between Rt 20–40 min in the HPLC spetra compared to those of wild-type strain.This result indicated that gene 283 was involved in not only the biosynthesis of the first precursor 6-MSA for arthrobotrisins and arthrosporols in Arthrobotrys oligospora,but also the regulation of the whole biosynthetic pathway for arthrobotrisins and arthrosporols.Toluquinol was found in all the mutant strains fed with three additive precursors,and its abundance was directly proportional to the concentration of three feeding precursors.m-cresol was also found in all the strains fed with m-cresol,and its abundance was also proportional to the concentration of the additive m-cresol.5.Metabolic profiles of the WT fed with 6-MSA,m-cresol and toluquinol,respectively,at different concentrations of 0.125,0.25,0.5 and 1mM,displayed that the strain fed with the additives were generally higher than the WT without additives.However,the abundances for the peaks between Rt 20–40 min in the HPLC profiles of WT with additive 1mM 6-MSA was lower than those of WT strain without additive,indicating that the high concentration of 6-MSA inhibited the biosynthesis of half amounts of compounds.Moreover,only under the concentrations of 1mM m-cresol and toluquinol,WT fed with additives displayed the accumulation of toluquinol.The results showed that WT strain exhibited better utilization for 6-MSA,m-cresol and toluquinol than mutant?283.It seems that the addition of the three precursors did not affect the biosynthesis of the types and amounts of arthrobotrisins and arthrosporols in the fungus.5.LC-MS analysis of metabolic profiles of Dactylella alba cultured in PDB medium from 7^th day to 12^th d revealed that the variety and abundance of compounds were relatively high at 11^th days.The metabolites of Dactylella alba fermented in potato medium with glucose,sucrose,maltose and galactose as the carbon sources and fermented in PDB medium with KNO₃,peptone and glutamine as nitrogen sources were analyzed by LC-MS.Glucose and sucrose were chosed to be added to potato medium for fungal fermentation.Three compounds,including aureonitol(OA-1),OA-2 and OA-3 were isolated from the PDB medium of Dactylella alba.Aureonitol was for the first time reported from this nematode-trapping fungus.