Structural and Functional Dissection of the Dimerization-Dependent Secretion Mechanism of BsPdhD in Bacillus subtilis

Bacillus subtilis is widely recognized as a microbial chassis for industrial protein production due to its robust secretory capacity. In B. subtilis, most proteins are exported from the cytoplasm via classical secretion pathways, including the Sec and Tat systems, as well as ABC transporters. However, efficient secretion of heterologous proteins using signal peptides remains a major challenge, largely due to bottlenecks within these pathways. In this study, we demonstrate that the pyruvate dehydrogenase E3 subunit (PdhD) from B. subtilis (BsPdhD) exhibits significantly higher secretion efficiency compared to conventional signal peptides. Through systematic truncation analysis of BsPdhD's N-terminal FAD/NAD-binding domain and C-terminal dimerization domain, coupled with biophysical characterization of its oligomeric state, we uncover a unique dimerization-dependent secretion mechanism. Notably, disruption of BsPdhD dimerization via removal of its C-terminal dimerization domain completely abolished secretion, whereas the N-terminal domain was primarily responsible for protein expression. To our knowledge, this study provides the first mechanistic evidence that BsPdhD-mediated secretion strictly depends on C-terminal dimerization. These findings not only reveal a previously unrecognized mechanism of protein trafficking but also establish BsPdhD as a promising tool for engineering high-efficiency secretory systems for industrial protein production.