Research On Identification And Secondary Metabolites Of Several Antarctic Fungal Strains

In Polar Regions,the unique geography,climate and environmental characteristics,especially the great changes of light radiation,seasonal light time and extremely low temperature,contribute to the special biological diversity,and the special biological diversity often results in great diversities of special chemical molecules and their biological activities.The molecular systematics reports of active polar fungal strains showed interesting diversity of the polar fungi,which covers some genus of Penicillium,aspergillus,Coniophora,Acremonium,Piricaulalia,Cladosporium,Mortierella,Rhizoscyphus,Antarctomyces,and so on.The bioactive secondary metabolites of different groups of polar fungi,including steroids,alkaloids,terpenoids,cyclic peptides,benzoquinones,and polyketides,show potent antibacterial,anti-tumor,antiviral,immunoregulatory,antioxidant and other biological activities.The discovery of novel structures and bio-active secondary metabolites provides valuable medicinal resources for the research of microorganisms from extreme environments,especially some important leading compounds for innovative drug study.Three fungal strains were isolated from the Antarctic sea mud,and identified as Aspergillus sp.NJF3,Cladosporium sp.NJF4,and Cladosporium sp.NJF6 by morphology,18 s r DNA-ITS sequence analysis.Early screening of protein phosphatase activity were carried out with P-NPP as substrate,and the inhibitory effect on protein phosphatase was tested with low concentration of OA.It shows that there is a remarkable protein phosphatase activity in these three fungi strains,while no inhibitory effect was observed with 100 n M OA.The genes of coding PP2 A protein phosphatase are primarily screened from Aspergillus sp.NJF3.The analysis of fatty acid constituents shows that there are high levels of total unsaturated fatty acids,oleic acid and linoleic acid in NJF4 and NJF6,and quite low levels of total saturated fatty acids,palmitic acid and stearic acid.It may indicate a good application potential of fatty acid constituents in these two strains.The metabolite extracts were obtained from fermentation broth of NJF3 and NJF4.By combination use of TLC,column chromatography on positive/reversed-phase silica gel and Sephadex LH-20 gel,and semi-prearative HPLC methods,respectively eight compounds were purified from the extracts of NJF3 and NJF4.By modern spectroscopy methods(LC-TOF MS,1H-and 13C-NMR,COSY,HSQC,HMBC,ROESY,UV,etc.),and their physical and chemical properties,fourteen structures were identified,including pyrimidine nucleoside,cyclo-heptapeptide,leporin C,leporin A,?-cyclopiazonic acid,1-methyl-?-carbolin,3-(3-benzofuryl)-2-alanine,5,22-diene-ergosta-3?,7?,8?-triol,N-hydroperoxy-2-(1H-indol-3-yl)ethenamine,cyclo-(phenylalanine-aspartic acid),cyclo(tryptophan-aspartic acid),and cyclo(phenylalanine-serine),etc.To explore the influence on secondary metabolites diversity and target compounds quantity by protein posttranslational modification regulation technology,different concentration of protease inhibitors,such as 100/200 n M OA,50/100 ?M SAHA,2.5/5 ?M EPZ and 50/100 ?M CBHA were added into different culture medium groups,and fermented products were analized by HPLC-TOF MS.Results revealed that the amounts and type of metabolites is significantly increased in 100 n M SAHA group,especially the S-containing compounds,and little effect on the synthesis of four target compounds incuding cyclo-heptapeptide,leporin C,leporin A,and ?-cyclopiazonic acid.FOR purpose of producing more new compound,there is little difference between 50 n M and 100 n M OA groups,so we need further research to find the optimal concentration.While the amounts of four target compounds were significantly increased in the 100 n M OA group.The results may provide basis for further investigation of interesting secondary metabolites from these strains.Protein posttranslational modification regulation is a kind of effective epigenetic method,which has been proved that cryptic biosynthetic gene cluster of secondary metabolites can be activated,and the amount and type of secondary metabolites can be also enriched.But the interaction mechanisms of protein posttranslational modification including methylation,acetylation,phosphorylation and ubiquitination have not been clear in polar fungi,without forming a relatively complete gene regulatory network.The first focus of our future study is to get a lot of group of protease genes by sequencing of total fungal genome.And then with the reference of some special secondary metabolites,the role of protein posttranslational modification in secondary metabolite biosynthesis will be discussed on molecular level.We hope to lay a foundation for widely discovering novel and bio-active compounds,to provide more leading compounds for new drug development,and finally increase the competitiveness of our country to creat original new drugs.