Monitoring heterologous protein expression and trafficking in Aspergillus niger

Filamentous fungi such as Aspergillus niger are widely used hosts for heterologous protein production owing to their strong secretion capacity and ability towards post-translational modification. However, inefficient secretion and the intracellular retention of recombinant proteins present major challenges. Therefore, we resorted to exploiting enhanced green fluorescent protein (EGFP) as a reporter to monitor the intracellular localization of heterologous proteins in A. niger . We used a recombinant strain, CSE3, expressing EGFP fused with a synthetic signal peptide and expressed the fusion construct using the constitutive PcitA promoter. Fluorescence microscopy did not detect secreted EGFP fluorescence in the culture supernatant but revealed distinct punctate structures within the fungal hyphae. Organelle-specific dyes targeting vacuoles, nuclei, mitochondria, and the endoplasmic reticulum were employed to investigate the intracellular localization of the EGFP fusion. Our observations suggest that the EGFP was localized in the endoplasmic reticulum. The study shows that fluorescence-based visualization combined with organelle-specific dyes provide a practical approach to monitor the intracellular trafficking of heterologous proteins in filamentous fungi. The identification of such secretion bottlenecks would help improve heterologous protein expression in filamentous fungal platforms.