Encapsulation-enhanced genetic switches in lactobacilli

Lactiplantibacillus plantarum is known for its potential in healthcare, food production, and environmental biotechnology. However, its broader utility is constrained by a limited genetic toolbox, particularly lacking robust genetic switches for inducible gene expression. Addressing this gap, we developed a novel genetic switch for L. plantarum based on a strong bacteriophage-derived promoter and the food-grade inducer, cumate. However, the switch was susceptible to leaky expression in the late log phase of bacterial growth, which was correlated to a reduction in the culture pH. This leakiness was partially resolved by regulating culture conditions (temperature and nutrients) to limit growth below a certain bacterial density. More interestingly, leaky expression could be stably suppressed by encapsulating the bacteria in alginate as an engineered living material. This physically restricted growth and limited the pHdrop, thereby enhancing the switch performance. The possibilities to regulate protein secretion over several days, reversibly switch protein production, and establish dual functionalities by co-encapsulating strains with different switches were demonstrated. Thus, for the first time, we show a material-based strategy to enhance the performance of a genetic switch in bacteria. This strategy facilitates the development of L. plantarum for advanced applications in biotechnology, pharmaceutics, and living therapeutics.