Methods in Microbial Biodiscovery

Abstract

This review presents an account of the microbial biodiscovery methodology developed and applied in our laboratory at The University of Queensland, Institute for Molecular Bioscience, with examples drawn from our experiences studying natural products produced by Australian marine-derived (and terrestrial) fungi and bacteria.

Chrysosporazines Revisited: Regioisomeric Phenylpropanoid Piperazine P-Glycoprotein Inhibitors from Australian Marine Fish-Derived Fungi

“…As a first step in furthering our investigations into chrysosporazines we set out to determine if Global Natural Products Social (GNPS) molecular networking [ 21 , 22 ] could be used to detect chrysosporazines in unfractionated cultivation extracts. A GNPS molecular network analysis of PDA solid phase cultivations of the known chrysosporazine producing fungi CMB-F214 and CMB-F294 ( Figure 4 blue highlights), was mapped against authentic standards of chrysosporazines ( Figure 4 red highlights), azachrysosporazines ( Figure 4 purple highlights), hancockiamide C ( Figure 4 pink highlight), as well as ×100 other marine fish GIT-derived co-isolated fungi.…”

“…UPLC-QTOF-(+)MS/MS data acquired for all samples at collision energy of 10, 20 and 40 eV were converted from Agilent MassHunter (Agilent Technologies Inc., Mulgrave, VIC, Australia) data files (.d) to mzXML file format using MSConvert software, and transferred to the GNPS server (gnps.ucsd.edu). Molecular networking was performed using the GNPS data analysis workflow [ 21 , 22 ], using the spectral clustering algorithm with a cosine score of 0.6 and a minimum of five matched peaks. The resulting spectral network was imported into Cytoscape software (version 3.7.1) [ 23 ] and visualized using a ball-stick layout where nodes represent parent masses and the cosine score was reflected by edge thickness.…”

“…The fungus CMB-F661 was cultured in a 24-well microbioreactor format using ×11 different culture media, across both static and shaken broths (1.5 mL) and solid agar (1 g slant), and extracted in situ with EtOAc (2 mL) to provide ×33 culture extracts [ 22 ]. The culture extracts were individually decanted, filtered, dried under N 2 and the resulting residue subjected to UPLC-DAD and UPLC-QTOF analysis with an internal calibrant, and GNPS analysis, to detect and quantify levels of metabolite production ( Figures S4–S6 and Table S1 ).…”

Glenthmycins A–M: Macrocyclic Spirotetronate Polyketide Antibacterials from the Australian Pasture Plant-Derived Streptomyces sp. CMB-PB041

“…CMB-PB041 was cultured in a microbioreactor format under 12 different media in solid phase, as well as static and shaken broths (media MATRIX) . After 6 days, individual cultivations were extracted with EtOAc (2 mL) and concentrated to dryness under N 2 at 40 °C.…”

“…Prior to embarking on a large-scale cultivation and chemical investigation of CMB-PB041, we first applied a media profiling approach where CMB-PB041 was cultivated in a miniaturized 24-well plate microbioreactor format (MATRIX) 36 using 12 different media compositions, under solid phase, as well as static and shaken broth conditions. Each culture was individually extracted in situ with EtOAc, filtered, and subjected to UPLC-DAD analysis with an internal calibrant, to provide a qualitative and quantitative comparison of metabolite production (Figure S3).…”