Rapid dissection of the biosynthesis of antiMRSA antibiotics produced in co-culture by extremophilic fungi through the development of Fungal Artificial Chromosomes

Project Summary The goal of this SBIR Phase I project is to develop a novelrobust method that combines fungal artificial chromosomesFACswith next generation sequencingNGStechnologies and an advanced fungal heterologous host to allow the direct capture and heterologous expression of a complete set of large intact biosynthetic gene clustersBGCsof fungal natural productsNPsfrom un sequenced fungiIn Phase I we will focus on the capture and heterologous expression of the BGCs of two un sequenced extremophilic fungi that produce a novelselective family of antibioticsthe berkelylactoneswhen grown in co cultureThe berkeleylactones selectively target methicillin erythromycin resistant Staphylococcus aureusMRSAAs an initial milestonethe capture of these gene clusters and expression in a single heterologous host will facilitate production of these important new antibioticsFungal natural products are important sources of antimicrobial agentsanticancer compoundsand other categories of drugs in clinical useHoweverhigh through put sequencing has shown that only aboutof fungal NP BGCs are expressed under laboratory conditionsThereforerevolutionary technologies and tools are urgently needed to more effectively dissect the biosynthesis of fungal NPs in order to more efficiently access novel fungal metabolites as potential pharmaceutical agentsRecentlywe developed a novel FAC method that allows the direct capture and heterologous expression of an entire set of large intact NP BGCs from a sequenced fungus efficientlyWe have also shown that FAC or shuttle bacterial artificial chromosomeBACtechnology combined with FAC BAC pooling and NGS index sequencing can achievekb single molecule sequencing and assemblingwhich will further improve fungal NP discovery from un sequenced fungifoldHencewe propose this SBIR Phase I project to examine the feasibility of employing this new strategy to rapidly access the large intact NP BGCs of un sequenced extremophilic fungi from an extremely unique environmentSince this project is designed to overcome a barrier to implementing FACNGS in un sequenced fungi for efficient NP discovery for anti MRSA antibioticsanti cancer compounds and moreif successfulit will have a profound impact on filamentous fungi for NPdrug discoveryMore broadlyour novel breakthrough can initiate a critical paradigm shift in largescale NP drug discoveryshaping the landscape of our nation s healthcare system as it tackles the major public health issuesincluding infectious diseasescancersand many other diseases and pathologies Project Narrative This project will develop novel technologies to enable the rapid dissection of the large intact biosynthetic gene cluster of the berkeleylactonesantiMRSA antibiotics produced by the coculture of two un sequenced extremophilic fungi from an extremely unique environmentthe Berkeley PitThis innovate methodology has many applications in genomicssynthetic biologylarge scale natural product drug discoveryand public healthincluding the rapid discovery and dissemination of new antimicrobial agentsanticancer agents and other drugs