Programmable Synthetic Condensates to Enhance Circuit Robustness for Improved Bioproduction in Bacillus subtilis

Genetic circuit reliability is often limited by growth-mediated dilution. However, developing an efficient strategy for maintaining reliable circuit function against dilution effects remains a challenge. Here, we introduce programmable condensates to stabilize circuits in Bacillus subtilis. First, we engineered synthetic condensates and employed RIAD/RIDD peptide pairs to selectively sequester the transcription factor (TF). Spatial recruitment of these TF-filled condensates to target promoters facilitated a significant enhancement in gene expression. By tailoring the physicochemical properties and spatial distribution of the condensates, we substantially reinforced the buffering capacity of the circuits at higher temperatures. As a proof of concept, we harnessed this strategy to significantly improve the bioproduction of alkaline protease and 2′-fucosyllactose, achieving a 109.3% increase in enzyme activity and a 108.9% increase in product titer, respectively. Overall, this work offers a valuable toolkit for B. subtilis applications and a programmable framework for robust circuit redesign in other hosts.