Exploring the natural products of Australian sheep coprophilous fungi

Abstract

Microbial natural products are a fundamental source of new chemical entities and are well represented as a substantial component of today’s pharmaceutical and agrochemical compendium. Despite the past problems of microbial biodiversity with the excessive rediscovery of known metabolites, recent advances in the genome sequencing have revealed the hidden potential of microorganisms to produce more diverse secondary metabolites than ever imagined. This thesis is focused on the application of approaches for the exploration of silent secondary metabolism of microorganisms obtained from sheep faeces. This includes the high-throughput cultivation and extraction of microbes under various cultivation media and conditions (called MATRIX), followed by advanced mass spectrometry (UHPLC-QTOF-MS/MS) and Global Natural Product Social (GNPS) molecular networking based dereplication strategies. Microbial co-cultivation based activation of silent secondary metabolites was also investigated to explore the microbial chemical diversity. Targeted microbial metabolites were then purified using different chromatographic techniques such as solid phase fractionation, preparative and semi-preparative HPLC. In addition, different spectroscopic methods (UV-vis, HRMS, [a]D, 1D and 2D NMR), total synthesis, chemical derivatization (C3 Marfey’s analysis) were adopted for structure elucidation and characterization of isolated microbial metabolites. Chapter 1 highlights the opportunities of microbial natural products and the strategies to explore the hidden capacities of microbial secondary metabolism. This chapter reviews some commonly used strategies reported in the recent literature, including (i) chemical dereplication, (ii) exploring microbes from unusual sources, (iii) variation in the culture and media conditions, (iv) microbial cocultivation and (v) bioassay-guided isolation of metabolites. Chapter 2 summarizes the assemble of a microbial library from sheep faecal samples supplied from sheep farms in NSW, Australia; and assembly of a microbial crude extract library obtained from the solid vs liquid cultivation of isolated microbes. It also describes prioritization of isolates by the chemical and biological profiling of microbial crude extracts including the co-cultivation and bioassay-guided isolation methods. This chapter briefly discusses one example of bioassay-guided approach through the isolation and characterization of bioactive metabolites. Chapter 3 describes the MATRIX cultivation and identification of suitable culture conditions for the production of metabolites by fungus, Talaromyces sp. CMB-NF091. Scaled-up cultivation on ISP2 agar medium afforded the isolation and identification of a novel cyclic hexapeptide, talaropyrolide A, which exhibits an unusual binary conformation. The structure was assigned on the basis of detailed spectroscopic analysis (1D and 2D NMR, HRESIMS) and chemical derivatization (C3 Marfey’s iii analysis). The binary conformation was confirmed by analysing the NMR spectroscopic data in different solvents and comparing with the synthetic model compounds. The isolated peptide was evaluated for antimicrobial properties against MDR strains and showed bacteriostatic activity against methicillin resistant Staphylococcus aureus (MRSA) and vancomycin resistant Enterococcus faecalis (VRE). The compound was also tested for cytotoxicity properties against cancer cell lines and showed no cytotoxic activity. Chapter 4 discusses the isolation and characterization of eight new chlorinated depsidone analogues, spiromastixones P-W, along with four known depsidones, from a sheep faeces derived fungus, Spiromastix sp. CMB-NB162. The anti-microbial and cytotoxic potential of the isolated depsidones are also described. Chapter 5 illustrates the effects of varying culture conditions (MATRIX) on the secondary metabolism of fungus Spiromastix sp. CMB-NB162 and GNPS molecular networking based identification and purification of new cyclic tetrapeptides, spiromastides A-E. Chemical derivatization (C3 Marfey’s analysis) of the cyclic tetrapeptides was used to confirm the absolute configuration of the amino acid residues. Chapter 6 investigates the application of microbial co-cultivation based activation of silent secondary metabolisms. Co-cultivation of fungus Purpureocillium sp. CMB-NF122 with co-isolated Streptomyces sp. CMB-NB198 resulted in the activation of antibacterial alternariol. Another cocultivation of a co-isolated fungus Penicillium sp. CMB-NF125 with the same Streptomyces sp. CMB-NB198 resulted up-regulation in the production of a fungal mycotoxin trypacidin by the fungus, and a new congener of the hazimycin class, pre-hazimycin (2-2¢-N-N-diformyl-dityrosine-diamide) by the Streptomyces CMB-NB198. The detailed study was performed to understand the basic mechanism of activation/upregulation of the metabolites involved in the co-cultivation. Chapter 7 describes the total synthesis and structure rearrangement of anthranilic acid derived hydrazone natural products, first reported as penipacids A-E, isolated from a marine derived fungus Penicillium paneum. This chapter draws attention to the chemically reactive nature of hydrazides and their potential to form hydrazone artefacts during extraction and handling.