Finely Tuned CRISPRi Module for Upgrading the Performance of Constitutive Promoters in the <i>Bacillus subtilis</i> Protein Expression System

<i>Bacillus subtilis</i> is a critical host for protein production, with many industrial strains relying on strong constitutive promoters. However, this kind of promoter typically imposes a heavy burden on the host from the early stage of fermentation, leading to reduced growth rate and biomass. To overcome the drawbacks of these promoters, we developed a xylose-inducible CRISPRi module to dynamically control the activity of these promoters. The strength of this module was finely tuned via promoter engineering and the xylose concentration. The addition of xylose inhibited the target promoter and favored cell growth at an early stage, while the consumption of xylose recovered the strength of the promoter and facilitated protein expression, resulting in better balance between cell growth and protein production. The yield of a target protein was increased by 38% using this module. Our work provides a simple and effective method to upgrade industrial strains driven by strong constitutive promoters.