Adventures in Australian microbial biodiscovery

Abstract

New chemical scaffolds are needed to inspire the development of new chemotherapeutics to treat evolving pathogens. Despite hurdles facing natural product biodiscovery paradigms, recent advances in microbial genomics showed their potential to produce diverse metabolites more than estimated. This thesis is focused on exploration of the chemical diversity of microorganisms obtained from animal (sheep and fish) gastrointestinal tract content and in-house Australian Collection of Microorganisms (ACM) through applying two strategies (i) bioassay-guided biodiscovery, and (ii) variation of culture conditions. Microbial metabolites were purified using different chromatographic techniques such as SPE fractionation, size exclusion chromatography, semi-preparative, and preparative HPLC. In addition, different spectroscopic methods (UV-vis, HRMS, [a]D, circular dichroism (CD), 1D and 2D NMR), total synthesis, semi-synthesis, and chemical derivatization (C3 Marfey’s method) were utilized for structure elucidation of isolated metabolites. Chapter 1 highlights the importance of natural products especially the microbial natural products to enrich the chemical space with high hit-to-lead ratio. The chapter also gives an overview of the common challenges encountering microbial biodiscovery and selected opportunities to overcome them and access new chemical diversity. Chapter 2 outlines the construction of (i) microbial libraries from faecal samples of sheep infected with Haemonchus contortus nematode and gastrointestinal tract (GIT) content of Mugil mullet fish obtained from local commercial market and (ii) an extract library obtained from solid vs liquid-based cultivations. It also describes the chemical and biological profiling of the obtained crude extract library, and strains prioritization. This chapter also discusses bioassay-guided approach through the isolation and characterization of bioactive metabolites of three strains prioritized for the biological properties of their crude extracts. Chapter 3 describes an example of the chemical diversity of fish gastrointestinal tract microbiota. It illustrates the chemical investigation of Evlachovaea sp. CMB-F563 sourced from gastrointestinal tract of mullet fish leading to the isolation of thirteen metabolites belonging to different chemical classes. The structures were assigned on the basis of detailed spectroscopic analysis, total synthesis, semi-synthesis, analytical studies, and biosynthetic considerations. Chapter 4 describes the method development for the detection of the putative biosynthetic precursors of prolinimines A and B, N-amino-l-Pro hydrazinyl Schiff bases isolated from CMB-F563 in Chapter 3, that revealed N-amino-l-Pro methyl ester as the genuine natural product. This chapter discusses our concerns about the definition of natural products and artefacts. Additionally, it describes the synthesis, hydrolytic stability, and biological properties of a series of N-amino-l-Pro methyl ester Schiff bases. Chapter 5 demonstrates the effect of varying culture conditions (MATRIX) on the secondary metabolism of a fungus isolated from the gastrointestinal tract of mullet fish, Metarhizium sp. CMB-F624. Scale-up cultivation of CMB-F624 on rice solid medium afforded seven metabolites including three new macrocycles, metarhizolides A–C, together with two new 4'-O-methyl glucosides, metarhizides A and B. The structures were assigned on the basis of detailed spectroscopic analysis, and chemical derivatization (C3 Marfey’s analysis). The isolated metabolites were evaluated for cytotoxic properties against cancer cell lines and none showed cytotoxic activity. Chapter 6 illustrates the efficiency of varying culture conditions (MATRIX) in inducing the production of new antibacterial biantharyl atropisomers, lincolnenins, in Streptomyces lincolnensis ACM-4234. Analytical scale cultivations (under 34 different culture conditions) identified ISP2 broth shaken and oatmeal agar as the only conditions for production of lincolnenins with the latter as the optimum culture condition for scale-up cultivation. The planar structures of four biantharyl atropisomers, lincolnenins A and C–E, were elucidated by extensive analysis NMR spectroscopic data. The absolute configuration of lincolnenin A was fully assigned by ECD calculation. Lincolnenin A and C displayed good antibacterial activity against Gram-positive bacteria (IC50 1.3-4.1 mM) with no cytotoxic properties against cancer cell lines.