Asymmetric metabolic adaptations undermine stability in microbial syntrophy.

Syntrophic interaction, driven by metabolite exchange, is widespread within microbial communities. However, co-inoculation of most auxotrophic microorganisms often fails to establish a stable metabolite exchange relationship. Here, we engineered two auxotrophic Escherichia coli strains, each dependent on the other for essential amino acid production, to investigate the dynamics of syntrophic relationships. Through invasion-from-rare experiments, we observed the rapid formation of syntrophic consortia stabilized by frequency-dependent selection, converging to a 2:1 ratio of lysine-to-arginine auxotrophs. However, laboratory evolution over 25 days revealed that syntrophic interactions were evolutionarily unstable, with cocultures collapsing as ΔL cells dominated the population. Reduced fitness in cocultures was driven by the emergence of a "selfish" ΔL phenotype, characterized by decreased arginine production and exploitation of lysine produced by ΔA cells. Dynamic metabolic assays revealed that metabolite production and utilization patterns strongly influenced the fitness of each strain. ΔL cells displayed metabolic plasticity, adjusting lysine utilization in response to lysine availability, which enabled them to outcompete ΔA cells. In contrast, ΔA cells lacked similar plasticity, resulting in their negative selection. These findings demonstrate that asymmetric metabolic responses and the emergence of selfish phenotypes destabilize syntrophic relationships. Our work underscores the importance of balanced metabolic exchanges for developing sustainable synthetic microbial consortia and offers insights into the evolutionary dynamics of microbial cooperation.