Strong Translation Elongation Factor 1-Alpha (tef1α) Promoter Reporter Systems for Aspergillus niger

Strong promoters are crucial for microbial metabolic engineering and synthetic biology, enabling high-level gene expression and pathway optimization. In this study, we have identified the translation elongation factor 1-α gene promoter (tef1p) with its first intron (tef1INp) as the strongest promoter reported to date in Aspergillus niger. It exhibited more than 20-fold higher activity in a methylumbelliferone reporter assay than the commonly used glyceraldehyde-3-phosphate dehydrogenase (gpdA) promoter. The tef1INp demonstrated consistent performance across multiple reporter genes, including EGFP, mCherry, mRFP1, and GUS, yielding strong fluorescence and staining signals. Its robustness was further validated in a coexpression system using P2A peptides, which enabled efficient bicistronic expression with a streamlined vector design. Additionally, we achieved precise nuclear localization and colocalization using H2B- and H3-based fusion constructs with EGFP and mRFP1. Using this system, we successfully identified the nuclear localization signals (NLS) of H2B and H3 in A. niger. Overall, this work establishes versatile and high-performance genetic tools for advanced applications in fungal synthetic biology.