Encapsulation-enhanced switchable protein release from engineered probiotic lactobacilli

Living microbial therapeutics promise precise, programmable interventions at disease sites, yet most demonstrations of on demand drug release still rely on Escherichia coli, whose rich genetic toolkit is unmatched among probiotics. In particular, genetic parts to regulate in situ protein production are severely lacking in non-model probiotic bacteria like lactobacilli. Here, we equip the probiotic Lactiplantibacillus plantarum with high-performance genetic switches and show how material encapsulation can further enhance their behavior. By integrating cumate or vanillate-responsive operators and repressors with the strongest constitutive promoter in L. plantarum (P_tec), we generated two switches that support micromolar range induction. In rapidly growing culture conditions, acidification-associated leakiness of the switch was observed, which could compromise their applicability for precise on-demand delivery of drugs. Furthermore, such leakiness also limits the duration for which these engineered probiotics can be reliably used. By restricting growth through mild temperature or nutrient limitation, acidification and leakiness were suppressed. Strikingly, immobilizing the engineered cells in core-shell alginate beads (Protein Eluting Alginate with Recombinant Lactobacilli, PEARLs) almost eliminated leakiness, enabling day-scale, reversible control of intracellular reporters and secreted enzymes. This leakiness suppression persisted when two strains carrying orthogonal switches were co-encapsulated and even after miniaturization to submillimeter beads. These results expand the genetic toolbox of probiotic L. plantarum, demonstrate the synergy between genetic circuit design and material encapsulation, and advance lactobacilli toward stimuli-responsive therapeutic platforms.