Llamas Ramírez, Reneé Martha; (2025) Establishing a systematic framework with syntrophy as principle to build a Streptomyces-Saccharomyces interaction in liquid culture using the natural capabilities of the microorganisms. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

In nature, microbial communities show characteristics that surpass those observed in axenic cultures that are the preferred option for bioprocessing applications. These include enhanced and/or extended metabolic capabilities, resilience to perturbations, and production of novel metabolites. However, replicating such characteristics under synthetic conditions is not an easy task. The knowledge about how these natural communities are established and the mechanisms responsible for the observed characteristics is still limited. Hence synthetic microbial communities are often challenging to assemble and maintain. This is mainly due to the dynamic nature of the processes involved―rendering them difficult to characterise―and the immense diversity of existing microorganisms, resulting in specific factors that are key for one community but not necessarily being relevant for another. The aim of this thesis was to establish a syntrophic microbial interaction between Streptomyces coelicolor and Saccharomyces cerevisiae, based on the exchange of iron and folate, using the natural capabilities of the microorganisms. Since syntrophy is a naturally occurring form of symbiotic relationship, it is proposed as a mechanism to stabilise microbial interactions, in the process of engineering synthetic communities. In this research, a multidisciplinary approach was used, applying principles of ecology, biochemistry, evolutionary ecology, and microbial physiology, to develop a framework that guided the decision-making of the experimental design, for which a Design of Experiments screening was the final step to determine the optimal condition for establishing the syntrophic relationship. As a first step, the literature available from axenic cultures was used for the selection of strains and initial culture conditions. However, given the limited knowledge about Streptomyces-Saccharomyces interactions, it was necessary to perform a series of experiments in axenic cultures to define conditions in which appropriate selective pressures could be established by controlling the availability of the metabolites of interest. The second step was to assess the competing capabilities of the interacting partners in mixed cultures without nutrient limitations. These experiments were carried out for a prolonged incubation time, with the aim to allow a natural competition for resources and gain insights into the population dynamics. The bacterium outcompeted the yeast in the end. In the final stage, a multi-response DoE model was generated, making it possible to identify factors with a significant effect on the selected response within the context of the microbial interaction. This can be used in the future to optimise the Streptomyces coelicolor-Saccharomyces cerevisiae collaboration or to optimise responses individually, such as the production of siderophores. Finally, bacterial-yeast growth ratios were selected as parameters for the analysis of the DoE results. This allowed to identify three conditions in which the growth of both microorganisms was positively impacted, with siderophores and folates cross-feeding playing a key role. Results strongly suggest that the metabolic collaboration is, in fact, a syntrophic interaction, facilitated by the iron limitations imposed.

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