Fungal biodiscovery: Strategies to explore fungal secondary metabolite potential

Abstract

Fungi have been an inspiring source of diverse secondary metabolites, with a number of metabolites developed as pharmaceuticals and agrochemicals. Despite the past success, fungal biodiscovery has been challenged with the excessive rediscovery of known metabolites. This thesis is focused on investigating four selected strategies to explore fungal secondary metabolite potential to discover novel and bioactive metabolites. This includes, bioactivity-guided isolation, varying cultivation conditions, application of advance analytical techniques and microbial co-cultivation. A library of ×185 microbes, including fungi (×132) and Streptomyces (×53), was constructed from a total of ×28 samples collected from terrestrial and marine samples. High-throughput miniaturized 24-well microbioreactor culture approach was used to generate extracts from solid (agar) and liquid (broth shaken) cultures to prepare a crude extract library, which was subjected to chemical and bioactivity profiling using HPLC-DAD-MS and UHPLC-QTOF. Two innovative bioassays to discover bioactive metabolites in the extracts were employed; (i) The larval development assay (LDA), used to detect anti-parasitic metabolites against drug (monepantel) resistant sheep intestinal parasite Haemonchus contortus and (ii) GABA (β-aminobutyric acid) receptor inhibitory activity, to detect molecules that may have application as anti-anxiety drugs. Selected strains were subjected to a program of analytical miniaturized cultivation, supported by UHPLC-DAD-MS and UHPLC-QTOF profiling to explore the wider secondary metabolite potential of fungi. The metabolite production was visualised using the Global Natural Product Social (GNPS) molecular networking analysis. Based on the chemical and bioactivity profiling, a total of ×6 fungi were selected for further investigation, and the isolation and characterization of metabolites from these strains are discussed in this PhD thesis.Chapter 1 highlights the importance of understanding, and exploring silent fungal secondary metabolism. This chapter also reviews different strategies reported in the literature for the discovery of novel and bioactive metabolites from fungi, with main focus on (i) bioactivity guided isolation, (ii) varying cultivation condition, (iii) application of advance analytical techniques and (iv) microbial co-cultivation.Chapter 2 focuses on investigating bioactivity-guided isolation as a prioritizing tool for microbial extracts to isolate biologically active metabolites. Anthelmintic metabolites identified from Penicillium sp. CMB-TS015 and Purpureocillium sp. CMB-F551 are discussed in this chapter.Chapter 3 discusses continued chemical and bioactivity investigation on Penicillium sp. CMB-TS015. Isolation, characterization and bioactivity profiling of three new fungal metabolites (+)-spiroquinazoline B, (+)-fumiquinazoline J and the tetramic acid chaunolidine D, are described in this chapter. Chapter 4 illustrates the application of innovative 24-well microbioreactor plate miniaturised cultivation approach (MATRIX) and GNPS molecular networking analyses for discovering new cyclic peptides from tunicate–derived fungus Talaromyces sp. CMB-TU011. Isolation and characterization of novel cyclic peptide talarolides A–C are discussed.Chapter 5 describes the isolation and characterization of novel linear peptides talaropeptides A–D from Talaromyces sp. CMB-TU011. The chemical profiling revealed that CMB-TU011 suppressed the production of cyclic peptides, talarolides, in YES static broth medium in favour of novel linear peptides talaropeptides A–D. The talaropeptide mega non-ribosomal peptide synthetase (NRPS) is described, as second only in size to that for the fungus-derived immunosuppressant cyclosporine (an 11-residue extensively N-methylated cyclic peptide).Chapter 6 discusses chemical and biological investigations of new 16-residues peptaibols, emeramides A–G, together with antiamoebin I, isolated from Emericellopsis spp. CMB-F057 and, CMB-F206, fungi isolated from sea mullet intestinal tissues. Here we demonstrated that UHPLC-QTOF-MS and MS/MS analysis coupled with C3 Marfey’s analysis is a powerful tool in complete structure elucidation of peptides, with limited materials and overlapping NMR resonances. Structure-activity relationship studies revealed that the number of D-Isovaline (D-Iva) presence in the structure is important for their cytotoxic and antibacterial activities.Chapter 7 investigates the application of co-cultivation of fungi and Streptomyces for isolating silent fungal metabolites. Co-cultivation of Fusarium sp. CMB-NF041 with co-existing antifungal amycin B producing Streptomyces sp. CMB-NB090 resulted in activating two silent antibacterial fungal metabolites, 8-O-methylbostrycoidin and bostrycoidin, which were only detected with single ion extraction (SIE) in the monoculture. Detailed analysis revealed that antifungal amycin B was not responsible for the activation of defensive fungal metabolites. Isolation, characterization and bioactivity profiling of three new amides, and known metabolites, 8-O-methylbostrycoidin, bostrycoidin and amycin B are discussed.