Multi-parameter optimization of recombinant protein secretion in Bacillus subtilis: a review

Abstract Bacillus subtilis is widely recognized as one of the most robust and versatile hosts for recombinant protein production. This review provides a comprehensive overview of the protein secretion pathway in B. subtilis , detailing its mechanistic basis and key limiting steps. We discuss the structural features of signal peptides (SPs) that govern efficient translocation and highlight recent advances in de novo promoter and signal peptide engineering. A particular focus is placed on the integration of artificial intelligence (AI) to predict and design optimal genetic elements. Drawing on findings from combinatorial and multi-parameter studies, we propose that future engineering strategies should move beyond maximizing individual parameters. Instead, achieving a dynamic balance across transcriptional, translational and secretion layers is essential to sustain high productivity while minimizing cellular secretion stress. This review offers a roadmap for systems-level engineering of B. subtilis as a next-generation protein production platform.