- Natural products from plants, bacteria, and fungi play an important role in society. Over the last 35 years, natural products and their derivatives have inspired over 60% of all FDA approved small-molecule drugs, and are among the top prescribed therapeutics.1-2 Fleming’s discovery of the fungal antibiotic penicillin started the “Golden Age of Antibiotics” that lead to the discovery of a multitude of natural product based anti-infectives from the 1940s to the 1970s, but that rate has drastically fallen in the previous decades. This is worsened due to society facing an ever-increasing threat from new diseases like Zika fever or from emerging drug-resistant strains of human pathogens such as tetracycline-resistant Neisseria gonorrhoeae.3 There is a growing need to discover and develop novel compounds with antibiotic, antiviral, and cytotoxic properties to prevent life-threatening drug-resistant infections and cancers. Recent sequencing of over 1,000 fungal genomes indicate that fungi possess a large number of biosynthetic gene clusters (BGCs), granting them the potential to produce numerous bioactive natural products.4 However, fungi do not express their full metabolic potential under standard laboratory settings, so different approaches are necessary to comprehensively explore their metabolic space.5
In this thesis, the metabolic profile of two different fungi were explored. In the saprophyte Clonostachys sp. (internal designation MUSL 1/3), bioassay-guided fractionation and compound isolation led to the characterization of the known bioactive natural products verticillin D, pullularin E, and chloro-pullularin E.6-7 Bioassay-guided fractionation further suggests that there are potentially two novel bioactive molecules which have not been isolated and characterized. In the endophyte Phoma sp. 7204, multiple methods have been applied to activate silent biosynthetic gene clusters. The OSMAC (one strain-many compounds) approach of growing the same fungus in various environmental conditions has been an excellent method of altering fungal metabolic profiles.8 In addition, epigenetic modification using DNA methyltransferase (DNMT) and histone deacetylase (HDAC) inhibitors has been shown to upregulate silent NP pathways in some fungi.9 Previously, we published novel antibiotics from the endophytic fungus Phoma sp. 7204.10 Here, both nutritional and epigenetic approaches have been applied to expand its chemical profile. LCMS-based multivariate statistical analysis of the organic extracts revealed that the altered conditions significantly changed the metabolic profile of Phoma. The production of the antibiotic (+)-flavipucine was restricted to cultures grown in small shaking flasks of a maltose/glucose media type. In addition, when treated with the HDAC inhibitor sodium butyrate, (+)-flavipucine and three previously unseen compounds were produced. Epigenetic modification and the OSMAC approaches have shown to be effective in eliciting the production of bioactive natural products in Phoma sp.