Oligomerization of fbaA Is Essential for Nonclassical Protein Export in <i>Bacillus subtilis</i>

<i>Bacillus subtilis</i> is a widely used microbial host for both fundamental research and the industrial production of enzymes and biopharmaceuticals. However, its four known signal peptide-dependent secretion pathways impose inherent limitations on the efficient extracellular expression of heterologous proteins. Here, we identify fructose 1,6-bisphosphate aldolase (FbaA) as a nonclassically secreted protein that achieves high-level expression and efficient extracellular export in <i>B. subtilis</i>. Structural and biochemical data indicate tetrameric FbaA as the predominant secreted form. Site-directed mutagenesis shows that the hydrophobic residue methionine 66 (M66) is critical for tetramer formation; substituting M66 disrupts oligomerization, abolishes secretion, and eliminates FbaA's ability to mediate the export of heterologous proteins. Together, these results establish that FbaA oligomerization is essential for its nonclassical secretion and that FbaA functions as a modular export element capable of facilitating the secretion of fused heterologous proteins. This work provides mechanistic insight into oligomerization-dependent protein export and offers a promising strategy for engineering efficient secretion systems in <i>B. subtilis</i>.