De novo metabolite production through co-cultivation of different fungal species on solid media

In the field of natural products research, finding sources of novel bioactive compounds is of primary importance. In this respect, microorganisms have provided a large number of biologically active molecules, but due to continuous re-isolation of known secondary metabolites this source is losing attractiveness. To discover original microbial natural products, new strategies that switch on silent genes appear as an interesting alternative to yield more structurally diverse secondary metabolites. In this regard, the use of fungal co-cultures for the induction of new bioactive compounds has emerged as a promising field in drug discovery [1,2].

In this work, a method based on the use of 12-well-plate miniaturized Petri dishes (2 cm diameter) and compatible with UHPLC-HRMS metabolomics has been applied to screen for metabolite induction in co-cultures of four fungal species (Epicoccum sp., Eutypa sp., Fusarium sp., Aspergillus sp.) grown on solid media [3]. A miniaturized multi-well procedure was used as it allows growing and analysing with high-throughput a large number of samples (single- and co-cultures) necessary for statistical studies. PCA (Principal Component Analysis) was performed in order to explore the data through an unsupervised approach and reveal metabolome variation among single culture and co-cultures. The results of the screening showed that these fungal species produced new compounds when co-cultivated with another fungus. For example, de novo induced metabolites (detected in the co-culture but not in the single cultures) were revealed in the interaction of Fusarium sp. vs. Aspergillus sp. as well as in the interaction of Eutypa sp. vs. Epicoccum sp. and some fungal secondary metabolites, like O-methylmellein, were dereplicated. This miniaturized strategy provided a satisfactory reproducibility; moreover it is generic and can be applied to other types of microorganisms that can grow on solid media such as those that are part of the microbiome. This study demonstrates the consistent induction of new metabolites through fungal co-cultures.

Keywords: Fungal co-culture, de novo induction, screening, 12-well-plate.

References:

[1] Bertrand S, Bohni N, Schnee S, Schumpp O, Gindro K, Wolfender JL. Metabolite induction via microorganism co-culture: A potential way to enhance chemical diversity for drug discovery. Biotechnol Adv 2014; 32: 1180 – 1204

[2] Glauser G, Gindro K, Fringeli J, De Joffrey JP, Rudaz S, Wolfender JL. Differential analysis of mycoalexins in confrontation zones of grapevine fungal pathogens by ultrahigh pressure liquid chromatography/time-of-flight mass spectrometry and capillary nuclear magnetic resonance. J Agric Food Chem 2009; 57: 1127 – 1134

[3] Bertrand S, Azzollini A, Schumpp O, Bohni N, Schrenzel J, Monod M, Gindro K, Wolfender JL. Multi-well fungal co-culture for de novo metabolite-induction in time-series studies based on untargeted metabolomics. Mol BioSyst 2014; 10: 2289 – 2298