Two-Step Engineering of Food-Grade Aspergillus oryzae via Endogenous Signal Peptides and Vesicle Trafficking Proteins to Enhance Carrier-Free Protein Secretion.

Heterologous protein secretion in filamentous fungi is often constrained by limitations in signal peptide recognition and intracellular trafficking. Aspergillus oryzae, a food-grade industrial fungus, has a robust native secretory system. However, its capacity for recombinant protein secretion remains suboptimal. Here, we developed a two-step, carrier-free engineering strategy to enhance protein secretion in A. oryzae. We identified endogenous signal peptides among highly secreted proteins using a green fluorescent protein (GFP) reporter. The oryzin signal peptide SPAoalp1 increased GFP secretion 5.50-fold compared with a no-signal-peptide control. We co-overexpressed Aosly1, a Sec1/Munc18 family protein that regulates soluble N-ethylmaleimide-sensitive factor attachment protein receptor-mediated vesicle trafficking, which, in combination with SPAoalp1, increased secretion approximately two-fold compared with SPAlp1 control and ten-fold with no-SP control. Applying the engineered platform for genetic improvement of heterologous bovine κ-casein increased secretion from 0.11 to 0.24 mg/L. Physiological optimization further increased secretion. The developed system provided initial evidence for secretion of a ~12 kDa band consistent with Aopafb transcription, with MIC90 values of 4.56-8.24% (v/v) against two Candida albicans strains and 4.68% (v/v) against Aspergillus niger. The system offers a modular framework for engineering fungal secretion and expands the utility of A. oryzae for recombinant protein production.