Multi‐Level Engineering of Bacillus Subtilis for High‐Level Extracellular Production of Functional Expansins

Expansins, whose cell wall-loosening function contributes to plant cell growth, biomass deconstruction, and substrate accessibility, have attracted increasing attention in agricultural biotechnology and bioprocessing. However, their predominant sourcing from plants, with limited low abundance and inconsistency, presents a challenge for industrial production. In this study, we developed a multi-level engineering strategy in Bacillus subtilis to enhance the extracellular production of two expansins: B. subtilis expansin-like group X1 (BsEXLX1) and Solanum lycopersicum α-expansin (LeEXP2). By systematically screening temporal promoters, optimizing ribosome-binding site (RBS) and signal peptides (SPs), and expressing secretion machinery components, the expression levels of BsEXLX1 and LeEXP2 reached 232.3 mg/L and 15.6 mg/L, respectively, in shake flasks. Further fed-batch culture increased the extracellular BsEXLX1 and LeEXP2 to 1.3 g/L and 43.7 mg/L in a 5-L bioreactor, representing the highest level reported in B. subtilis to date. Furthermore, both microbial-generated expansins exhibited a strong synergistic effect on cellulose degradation, enhancing total sugar release by 42.6% (BsEXLX1) and 5.7% (LeEXP2) when combined with commercial cellulase. Collectively, this study establishes a scalable B. subtilis-based secretion platform for the high-level production of functional expansins and provides a transferable framework for efficient extracellular protein production in B. subtilis.