Secondary Metabolites And Heterologous Expression Of Biosynthetic Gene Cluster Of Marine-derived Fungi Aspersillus Sp.

Marine microorganisms are an important potential pharmacological source of lead compound producers because of the novel structures and significant biological activities of their secondary metabolites.Among them,marine-derived Aspergillus spp.fungi,which offer a variety of molecules with novel structures and bioactivities,have attracted more attention.The bioinformatics data indicates that there are plenty of biosynthetic gene clusters(BGCs)in fungi.Considering Aspergillus and Penicillium alone,the number is approximately 25000,which means the secondary metalolites wealth of fungi is largely untapped.It is very important to practice traditional chemical strategies and innovative techniques based on genomic information for discovery of novel bioactive secondary metabolites.In this study,three Aspergillus sp.fungi Z3,Z4 and Z5,isolated from the gut of marine isopod Ligia exotica,were chosen for further study of their secondary metabolites.The OSMAC(One Strain Many Compounds)strategy was used to select the better media and fermentation conditions.As a result,a total of 34 compounds were purified including nine new ones and one compound first isolated from fungal metabolites.All the isolated aspochalasins and indole diketopiperazine dimers were tested for their cytotoxic,antibacterial and neuroprotection activities.Under the guidance of genome sequence information,one NRPS-like gene cluster was selected to express in heterologous host A.nidulans.Z4 was cultured in two different media,2216E and rice,according to the OSMAC strategy.Seventeen compounds including thirteen aspochalasins(1-13)and four diketopiperazines(14-17)were isolated.There are five new compounds(1-5),of which two(1 and 2)are novel skeleton aspochalasins.Compound 12 showed significant cytotoxic activity against human prostate cancer PC3 cell line with an IC50 of 11.14 ?M,and Compound 9 showed moderate cytotoxic activity with an IC50 of 31.72 ?M.Eight compounds were purified from the culture broth of fungus Z3.They are one indanone(18),four indole diketopiperazine dimers(19-22),one ergosterol(23),one diorcinol(24)and one diketopiperazine(25).Compound 18 is a new one.Compounds 20-22 are stereoisomers of compound 19.Their absolute configurations were elucidated by combination of X-ray crystallographic data,comparative and calculate ECD spectra and Marfey's methodOne NRPS-like gene cluster(cluster 24),whose backbone gene g8094 was under the control of constitutive promoter gpdAp,was successfully heterologous expressed into the host A.nidulan.s.Meanwhile,one inducible promoter alcAp was successfully insert before the gene g8094 to get the mutant Z5.The results of RT-PCR and relative quantitative q-PCR showed that the expression level of the backbone gene g 8094 was increased after the replacement of the strong promoters,but still not high.Some of the modifying genes didn't express or expressed at low level,and the metabolites encoded by this gene cluster was not discovered.