Multi-well fungal co-culture for de novo metabolite-induction in time-series studies based on untargeted metabolomics

Abstract

The induction of fungal metabolites by fungal co-cultures grown on solid media was explored using multi-well co-cultures in 2 cm diameter Petri dishes. Fungi were grown in 12-well plates to easily and rapidly obtain the large number of replicates necessary for employing metabolomic approaches. Fungal culture using such a format accelerated the production of metabolites by several weeks compared with using the large-format 9 cm Petri dishes. This strategy was applied to a co-culture of a Fusarium and an Aspergillus strain. The metabolite composition of the cultures was assessed using ultra-high pressure liquid chromatography coupled to electrospray ionisation and time-of-flight mass spectrometry, followed by automated data mining. The de novo production of metabolites was dramatically increased by nutriment reduction. A time-series study of the induction of the fungal metabolites of interest over nine days revealed that they exhibited various induction patterns. The concentrations of most of the de novo induced metabolites increased over time. However, interesting patterns were observed, such as with the presence of some compounds only at certain time points. This result indicates the complexity and dynamic nature of fungal metabolism. The large-scale production of the compounds of interest was verified by co-culture in 15 cm Petri dishes; most of the induced metabolites of interest (16/18) were found to be produced as effectively as on a small scale, although not in the same time frames. Large-scale production is a practical solution for the future production, identification and biological evaluation of these metabolites.

The secreted metabolome of Streptomyces chartreusis and implications for bacterial chemistry

“…It will be interesting to expand this LC-MS/MS-based approach by diversifying cultivation conditions as, e.g., in the OSMAC approach (14). Alternative cultivation conditions may result in the activation of additional BGCs that were silent under the three conditions tested here, an approach that has been employed successfully (18). The combination of metabolomics with global transcriptome or proteome analysis and targeted mutagenesis to match BGCs with their metabolite products will further expand our understanding of the chemical capabilities of microbes.…”

“…For most BGCs no expression has ever been observed, which has led to the assumption that they are silent or have lost the ability to produce secondary metabolites (12). To unlock the unused chemical potential of silent BGCs, various strategies are applied, such as genetic or cultivation-dependent activation, biosynthesis in heterologous hosts, or synthetic biology approaches (13)(14)(15)(16)(17)(18).…”

Gibepyrone Biosynthesis in the Rice Pathogen Fusarium fujikuroi Is Facilitated by a Small Polyketide Synthase Gene Cluster

“…Besides F. fujikuroi, gibepyrone A production was also reported for natural isolates of Fusarium keratoplasticum and Fusarium petroliphilum, two members of the Fusarium solani species complex (34). Gibepyrone D was found in cultures of endophytic isolates of Fusarium oxysporum and Fusarium proliferatum (35,36), whereas gibepyrone F was detected in co-cultures of Fusarium sp./Aspergillus clavatus (37). Moreover, gibepyrone F was isolated from extracts of the South China Sea sponge Jaspis stellifera (38) which possibly originates also from a fungal symbiont, as described for other marine sponge-derived compounds (39,40).…”

Predominately Uncultured Microbes as Sources of Bioactive Agents

“…These include, but are not limited to: discussion of eliciting secondary metabolism in actinomycetes (Seyedsayamdost et al, 2012; Abdelmohsen et al, 2015; Rutledge and Challis, 2015); methodologies for determining the compounds expressed (Adnani et al, 2014; Luzzatto-Knaan et al, 2015; Medema and Fischbach, 2015; Mohimani and Pevzner, 2016); analyses of the biosynthetic clusters identified in fungi to published natural product structures (Li et al, 2016b); small-scale plate-based techniques for fungal co-culture (Bertrand et al, 2014); on-demand production of secondary metabolites (Bode et al, 2015); mixed culture of endophytes (Chagas et al, 2013); metabolomics in induced cultures (Derewacz et al, 2015; van der Lee and Medema, 2016; Zhang et al, 2016a,b; Ziemert et al, 2016); use of synthetic biological techniques to further expand the chemical biodiversity discovered (Smanski et al, 2016); and a recent review of the array of approaches to study such cryptic cluster expression by Zarins-Tutt et al (2016).…”