Investigation Of Naphthopyrone Derivatives From Endophytic Aspergillus Sp.D And Their Biological Synthesis

The ocean is a complex and special environment with low temperature,high pressure,and high salinity,marine microorganisms have gradually formed different genetic genes and metabolic mechanisms compared to terrestrial organisms,which generates an unique metabolic and defensive system.It is a common phenomenon that many marine microorganisms co-exist with marine algae and invertebrates,symbiotic microorganisms have potential to generate compounds with novel structures and diverse activities.Marine symbiotic Aspergillus sp.is widely studied among marine fungi in recent years due to a wide range of chemical diversity,which is an important source for the screening of drug leads.Secondary metabolites from Aspergillus sp.are produced by a series of enzymes,which constitute the metabolic pathway.Further study on its biosynthetic pathway would facilitate to explore the metabolic mechanism and improve the yield.The paper will systematically study marine symbiotic Aspergillus sp.D isolated from the coastal plant Edgeworthia chrysantha Lindl.,including the biosynthetic gene clusters,isolation,bioactivity of their secondary metabolites,and biosynthesis of their polyketones.Aspergillus sp.D was cultured in potato dextrose broth and inoculated on rice solid medium,and then extracted with ethyl acetate to obtain the crude extract I.Eight secondary metabolites（1–8）by pre-HPLC and semiprep-HPLC were obtained from the crude extracts I.By comparing with spectroscopic data（1D NMR and ESI-MS）as well as literature data,eight compounds were identified as naphthopyrone derivatives,including rubrofusarin B（1）,alternariol 9-O-methyl ether（2）,fonsecinone D（3）,asperpyrone A（4）,asperpyrone D（5）,fonsecinone B（6）,fonsecinone A（7）,and aurasperone A（8）.Biological assays indicated that all compounds exhibited weak activities against Candida albicans AY 204006,Escherichia coli AB 94012,and Staphyloccocus aureus AB 2010021 with MIC values of more than 100μg·mL^-1,but compound 8 showed moderate inhibitory effect on Helicobacter Pylori,with a MIC value of 50μg·mL^-1.The whole gene sequencing of Aspergillus sp.D had been completed,which displayed the strain had a strong ability to produce abundant secondary metabolites.Analysis results from antiSMASH showed there are 11 key gene clusters of PKS biosynthesis,corresponding gene clusters was revealed the strain D could generate betaenone,equisetin and alternapyrone derivatives.According to biological information analysis,gene scaffold8.t287 could be involved in producing naphthopyrone derivatives.Therefore,gene scaffold8.t287 was knocked out by screening resistance marker,recombined vector,and transformation.Finally,the strain?HY44 was successfully obtained and verified by PCR sequencing technique,and crude extracts II was obtained by the fermentation of rice solid medium at the same condition,and it was isolated by pre-HPLC as the same as the condition of crude extracts I.the result of each fragment was detected by UPLC and ¹H NMR.Finally,it showed both types and contents of metabolites were changed comparing to strain D,suggesting that the gene scaffold8.t287 was involved in biosynthesis of polyketides.