Unlocking Green\nBiomanufacturing Potential: Superior\nHeterologous Gene Expression with a T7 Integration Overexpression\nSystem in Bacillus subtilis

Industrial\nbiotechnology employs cells for producing valuable products\nand serving as biocatalysts sustainably, addressing resource, energy,\nand environmental issues. Bacillus subtilis is a preferred host for creating microbial chassis cells and producing\nindustrial enzymes and functional nutritional products. In this study,\na dual-module T7 integration expression system in B.\nsubtilis was established. The first module, driven\nby the T7 RNA polymerase, was integrated into the genome via the CRISPR/Cas9\nsystem. Another module responsible for expression control was systematically\nintegrated into 28 discrete chromosomal loci and the impact of different\ngenomic positions on gene expression was explored, resulting in a\nhigh-intensity integrated expression system. Furthermore, by modifying\nthe LacI repressor factor for biological regulation, we achieved a\nstrong expression intensity without the inducer addition. This system\nwas successfully used to express phospholipase D and hyaluronic acid\nlyase, resulting in extracellular enzyme activities of 339.12 U/mL\nand 2.60 × 10<sup>4</sup> U/mL, respectively. Additionally, by\nexclusively targeting the HA gene cluster for expression, a production\nyield of 6.86 g/L was achieved on a 5 L fermentation scale. The system\neliminates the use of antibiotics and inducers, offering a controllable,\nefficient, and promising gene expression regulation tool in B. subtilis, enhancing its potential for biomanufacturing\napplications.