An operator-based expression toolkit for Bacillus subtilis enables fine-tuning of gene expression and biosynthetic pathway regulation

Genetic elements are key components of metabolic engineering and synthetic biological applications, allowing the development of organisms as biosensors and for manufacturing valuable chemicals and protein products. In contrast to the gram-negative model bacterium Escherichia coli, the gram-positive model bacterium Bacillus subtilis lacks such elements with precise and flexible characteristics, which is a great barrier to employing B. subtilis for laboratory studies and industrial applications. Here, we report the development of a malO-based genetic toolbox that is derived from the operator box in the malA promoter, enabling gene regulation via compatible "ON" and "OFF" switches. This engineered toolbox combines promoter-based mutagenesis and host specific metabolic engineering of transactivation components upon maltose induction to achieve stringent, robust, and homogeneous gene regulation in B. subtilis. We further demonstrate the synthetic biological applications of the toolbox by utilizing these genetic elements as a gene switch, a promoter enhancer, and an ON-OFF dual-control device in biosynthetic pathway optimization. Collectively, this regulatory system provides a comprehensive genetic toolbox for controlling the expression of genes in biosynthetic pathways and regulatory networks to optimize the production of valuable chemicals and proteins in B. subtilis.