Investigation Of Secondary Metabolites And Sesquiterpene Synthase From Two Microorganisms Isolated From Extreme Environment

Microbial natural products play a crucial role for drug discovery.Under the influence of various environmental stress factors,microorganisms may produce more natural products with novel structure and unique biological activity.Genome-based mining of these microorganisms has provided significant opportunities in the last decade to isolate new natural products.In this study,in-house phytopathogenic fungi and marine actinomy cete libray has been constructed and sequenced.The gene cluster prediction was carried out throuah the antiSMASH software.One phytopathogenic fungus,Bipolaris sp,11134 and one marine-derived actinobacteria,Verrucosispora sp.MS10037 were highlighted according to the unique gene clusters.Further activation of their biosynthetic gene clusters in a multi-directional manner was performed using One Strain-Many Compounds(OSMAC)approach.Six seco-sativene sesquiterpene compounds(1-6)were isolated from thefermentation broth of strain Bipolaris sp.11134,including three new compounds 1-3.Seco-sativene compounds belong to the sativene sesquiterpene skeleton with a unique[3/2/1]bridged ring structure.The structural complexity reveals that it might contain novel genes and biosynthetic mechanisms.Therefore,further studies we revealed that its key synthase enzyme.Using bioinformatics analysis,it was found that the genome of Bipolaris sp.11134 contains four potential sesquiterpene synthase gene sequences.and these genes were cloned and further expressed in three different heterologous expression systems such as Saccharomyces ceevisiae,Escherichia coli,and Aspergillus nidulans.The heterologous production in vivo was performed to identify the structure of the cyclized product,and in vitro enzymatic catalysis experiments was performed to characterize the function of sesquiterpene synthase,and further combine the gene cluster information and the identified chemical structure of the sesquiterpene compound to speculate its biosynthetic pathway.In addition,comparative genomics analysis showed that Verrucosispora sp.MS100137 contains almost the identical biosynthetic gene clusters as V.maris AB-18-032,which is capable of producing abyssomicin compounds with anti-MRSA activity.And this gene clusters has a proposed epoxidase,so the secondary metabolic potential of Verrucosispora sp.MS 10037 was further explored by OSMAC fermentation and LC-MS experiments.It was found that Verrucosispo,ra sp.MS 10037 produced a new abyssomicin-like compound,and 7 known compounds were obtained(7-13)by extraction and purification using a variety of modern spectroscopy techniques such as nuclear magnetic resonance and circular dichroism,identified as four abyssomicin compounds(7-10)and three proximicin compounds(11-13).of which abyssomicin Y(7).It is a new compound of abyssomicin having an epoxy group on the C8 and C9 double bonds,further gene function identification is underway.The results of this experiment indicate that the OSMAC strategy can effectively activate the expression of more silent genes to produce new compoundsBased on bioinformatics approach.we successfully isolated a variety of novel compounds from specific microbes.The new seco-sativene compounds was discovered with neuroprotective activity.The catalytic function of the corresponding sesquiterpene synthase was identified,which laid a solid foundation for further analysis of its cyclization mechanism and biosynthesis mechanism.In addition,by using the OSMAC strategy to successfully stimulate the production of new abyssomcin analogs with almost identical gene clusters reported,it was demonstrated that novel secondary metabolites can be efficiently exploited by changing the culture environment.