Unlocking Green Biomanufacturing Potential: Superior Heterologous Gene Expression with a T7 Integration Overexpression System in <i>Bacillus subtilis</i>

Industrial biotechnology employs cells for producing valuable products and serving as biocatalysts sustainably, addressing resource, energy, and environmental issues. <i>Bacillus subtilis</i> is a preferred host for creating microbial chassis cells and producing industrial enzymes and functional nutritional products. In this study, a dual-module T7 integration expression system in <i>B. subtilis</i> was established. The first module, driven by the T7 RNA polymerase, was integrated into the genome via the CRISPR/Cas9 system. Another module responsible for expression control was systematically integrated into 28 discrete chromosomal loci and the impact of different genomic positions on gene expression was explored, resulting in a high-intensity integrated expression system. Furthermore, by modifying the LacI repressor factor for biological regulation, we achieved a strong expression intensity without the inducer addition. This system was successfully used to express phospholipase D and hyaluronic acid lyase, resulting in extracellular enzyme activities of 339.12 U/mL and 2.60 × 10⁴ U/mL, respectively. Additionally, by exclusively targeting the HA gene cluster for expression, a production yield of 6.86 g/L was achieved on a 5 L fermentation scale. The system eliminates the use of antibiotics and inducers, offering a controllable, efficient, and promising gene expression regulation tool in <i>B. subtilis</i>, enhancing its potential for biomanufacturing applications.