Abstract
Actinomycetes are powerhouses of natural product biosynthesis. Full realization of this biosynthetic potential requires approaches for recognizing novel metabolites and determining mediators of metabolite production. Herein, we develop an isotopic ratio outlier analysis (IROA) ultra-high performance liquid chromatography-mass spectrometry (UHPLC/MS) global metabolomics strategy for actinomycetes that facilitates recognition of novel metabolites and evaluation of production mediators. We demonstrate this approach by determining impacts of the iron chelator 2,2′-bipyridyl on the Nocardiopsis dassonvillei metabolome. Experimental and control cultures produced metabolites with isotopic carbon signatures that were distinct from corresponding “standard” culture metabolites, which were used as internal standards for LC/MS. This provided an isotopic MS peak pair for each metabolite, which revealed the number of carbon atoms and relative concentrations of metabolites and distinguished biosynthetic products from artifacts. Principal component analysis (PCA) and random forest (RF) differentiated bipyridyl-treated samples from controls. RF mean decrease accuracy (MDA) values supported perturbation of metabolites from multiple amino acid pathways and novel natural products. Evaluation of bipyridyl impacts on the nocazine/XR334 diketopiperazine (DKP) pathway revealed upregulation of amino acid precursors and downregulation of late stage intermediates and products. These results establish IROA as a tool in the actinomycete natural product chemistry arsenal and support broad metabolic consequences of bipyridyl.
Highlights
Bacteria from the order Actinomycetales, commonly known as actinomycetes, are diverse and widespread across marine, freshwater, and terrestrial habitats [1]
Metabolites 2019, 9, 181 low natural product yields [10], and repeated discovery of known metabolites [11]. These struggles have been tackled by approaches that include supplementation of cultures with small molecules hypothesized to mediate biosynthesis [12,13], co-cultivation of microorganisms to elicit metabolite production [12,14,15,16], heterologous expression of targeted biosynthetic pathways in hosts optimized for secondary metabolism [17,18], and creation of biosynthetic gene elimination mutants to facilitate recognition of metabolites encoded by these genes [18]
In published isotopic ratio outlier analysis (IROA) metabolomics protocols [32,33,34], organisms from experimental and control groups were grown in media containing uniformly labeled carbon sources with different 12 C:13 C ratios (i.e., 95:5 vs. 5:95) and pooled prior to chemical extraction and UHPLC/mass spectrometry (MS) or gas chromatography-mass spectrometry (GC/MS)
Summary
Bacteria from the order Actinomycetales, commonly known as actinomycetes, are diverse and widespread across marine, freshwater, and terrestrial habitats [1]. Actinomycetes are prolific producers of small molecules, accounting for over 75% of bioactive bacterial natural products identified to date [2] These structurally diverse metabolites serve as lead compounds for pharmaceutical and agrochemical development [3], inspire organic syntheses [4], mediate intra- and interspecies interactions [5], and provide chemical probes for elucidating mechanisms of biological processes [6]. While actinomycete metabolism has been intensely investigated for over 75 years [2], bioinformatics analyses support that actinomycete genomes encode a treasure trove of biosynthetic pathways with the potential of yielding as-yet unidentified secondary metabolites [7]. These struggles have been tackled by approaches that include supplementation of cultures with small molecules hypothesized to mediate biosynthesis [12,13], co-cultivation of microorganisms to elicit metabolite production [12,14,15,16], heterologous expression of targeted biosynthetic pathways in hosts optimized for secondary metabolism [17,18], and creation of biosynthetic gene elimination mutants to facilitate recognition of metabolites encoded by these genes [18]
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