Synthetic niches enable co-culture bioprocessing but are prone to mutational escape

Abstract

Stabilizing microbial co-cultures is a central challenge for bioproduction. While division of labor between strains can enhance efficiency, it often results in population instability over time. Classical strategies, including cross-feeding, quorum sensing, and toxin-antitoxin modules, often rely on complex ecological interactions that are difficult to predict or maintain under bioprocess conditions. Here, we introduce synthetic niches as an alternative framework, using genetic toggle switches that couple growth to defined phenotypic states. We engineered two auxotrophic strains, TOGGLE_green and TOGGLE_yellow, in which growth is linked to either GFP- or YFP-expressing states and assessed their behavior in continuous bioreactor cultures using automated flow cytometry. Unexpectedly, the introduction of auxotrophic pressure reshaped circuit function i.e., instead of maintaining bistability, toggle strains behaved as unidirectional inducible systems that reverted upon inducer withdrawal. This feature enabled simplified control with a single input but also revealed a vulnerability to mutational escape under intensified cultivation. A simple repression-based ODE model recapitulated the reversible dynamics, but deviations under prolonged operation highlighted the rapid evolutionary erosion of control. Our findings demonstrate both the potential and the limitations of synthetic niches for co-culture engineering and emphasize the need to integrate evolutionary robustness into the design of next-generation bioprocess control strategies.