Unraveling the relationship between population composition and metabolite induction in high-precision cellulosic bacterial–fungal co-cultivations

Co-cultures of filamentous microorganisms are a tool to unlock the biosynthetic machinery encoded in silent gene clusters. However, if bioactive compounds are to be produced biotechnologically, it is essential to understand the dynamics of microbial partnership. In this context, we have previously introduced a co-cultivation platform based on low-cost crystalline cellulose as a substrate. Here, the fungus Trichoderma reesei RUT-C30 degrades α-cellulose into soluble sugars to support the growth of the natural product-producing bacterium Streptomyces coelicolor A3(2), which is unable to utilize cellulose. This co-cultivation approach mimics a carbon-limited interaction scenario of natural habitats. We investigated how the population dynamics of both strains affect metabolite production by combining high-throughput online fluorescence monitoring with high-resolution liquid chromatography–tandem mass spectrometry (LC-MS/MS)-based metabolomics. We then determined the metabolic profile of the cultures and identified metabolites specifically induced during co-cultivation. The coupled analysis of population ratios and metabolomic data indicated that the highest number of induced metabolites compared to axenic cultures is observed in co-cultures with a balanced ratio of both strains. This analysis further led to deciphering a biotransformation of dimerumic acid, a hydroxamate siderophore produced by T. reesei , into the dehydroxylated derivatives eleutherazine B and talarazine A by S. coelicolor . Additionally, a small cluster of unique metabolites strongly induced in co-cultures was found to be structurally similar to steroidal lactones of the withanolide class, thus highlighting the potential for the discovery of natural products. Overall, the platform described combines natural product prospecting with a biotechnological production strategy that can be extended also to other co-cultures. IMPORTANCE This work provides a comprehensive analysis of population dynamics and metabolite production in a complex microbial co-culture. This approach integrates three key aspects. First, high-throughput online monitoring enables biomass tracking through fluorescence labeling in a system containing two filamentous microorganisms with distinct macro- and micromorphologies and a non-soluble fibrous substrate. Second, concomitant metabolomics analysis during co-cultivation settings helps to resolve metabolic interaction patterns. Interestingly, a metal-acquisition compound produced by the first microbe is chemically modified by the second, suggesting a competition strategy for an essential micronutrient. Third, from a biotechnological perspective, substrate solubilization resembles a fed-batch strategy that minimizes carbon catabolite repression during secondary metabolite production. Overall, this platform advances our understanding of microbial interactions and culture conditions that can be transferred to other culture platforms of relevance in bioprocess intensification.