Manipulating the Epigenetic Machinery of Marine Fungi Through the Application of Epigenetic Modifiers to Induce the Biosynthesis of Novel Secondary Metabolites

Abstract

Fungi are among the most prolific producers of secondary metabolites, many of which have huge utility for society as various anti-infective and anticancer drugs. The genes encoding these secondary metabolites are arranged in continuous biosynthetic gene clusters (BGCs). Each fungus can have tens of different BGCs in its genome, possessing the ability to produce a multitude of secondary metabolites with potential for medical utility. However, most of the BGCs are silent under standard laboratory conditions, packaged in heterochromatin, inaccessible for transcription. To activate these clusters the heterochromatin must be converted to euchromatin, the transcription accessible form. The addition of small molecule epigenetic modifiers, inhibiting the activity of transcription repressive epigenetic enzymes, have been proven to effectively induce secondary metabolism in many fungi. As this approach does not require genetic manipulation, it is potentially applicable to a broad range of fungi, recalcitrant to genetic manipulation. This method could therefore have the potential for implementation. the high-throughput screening of microorganisms at Marbio.

In this thesis, the method of manipulating the epigenetic machinery of marine filamentous fungi by the application of epigenetic modifiers 5-azacytidine, SBHA, and nicotinamide were employed as a means for inducing activation of biosynthetic gene clusters. This is the first time epigenetic modifiers have been applied to any microbial cultures at Marbio. The fungi investigated were cultivated in solid and liquid media with added epigenetic inhibitors in different combinations to study the effects of the modifiers on the fungi. The fungal cultures were subsequently extracted and subjected to bioactivity testing. The bioactivity screening revealed that epigenetic modifiers did not induce the production of bioactive secondary metabolites in detectable amounts. However, SBHA was found to alter colony morphology and pigmentation in some cultures, indicating that these fungi are accessible for epigenetic manipulation by HDAC inhibitors. Increased concentrations or applications of more potent HDAC inhibitors such as SAHA, might be necessary to observe more evident changes in the fungal metabolomes.