Synthetic Niches Enable Coculture Bioprocessing but Are Prone To Mutational Escape

Stabilizing microbial cocultures 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. We report the first implementation, to our knowledge, of a synthetic-niche-based coculture operated in a continuous bioreactor, 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 under continuous bioreactor conditions using automated and reactive flow cytometry. Unexpectedly, the introduction of auxotrophic pressure reshaped circuit function, i.e., rather than maintaining bistability as typically reported in batch or microfluidic systems, toggle strains behaved as unidirectional inducible systems that reverted upon inducer withdrawal. This emergent behavior enabled simplified single-input control at the bioreactor scale, but also revealed a critical limitation for long-term operation, namely rapid mutational escape of the growth-impaired strain occurring within fewer than 10 generations in coculture, a markedly shorter time scale than the ∼50 generations typically reported in the literature. A simple repression-based ODE model recapitulated the reversible dynamics, capturing the effective inducible behavior observed under continuous cultivation, whereas deviations under prolonged operation highlighted the rapid evolutionary erosion of synthetic control. Our findings demonstrate both the potential and the limitations of synthetic niches as a scalable coculture control strategy.