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

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 (<i>tef1</i>p) with its first intron (<i>tef1IN</i>p) as the strongest promoter reported to date in <i>Aspergillus niger</i>. It exhibited more than 20-fold higher activity in a methylumbelliferone reporter assay than the commonly used glyceraldehyde-3-phosphate dehydrogenase (<i>gpdA</i>) promoter. The <i>t</i><i>ef1</i>INp 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 <i>A. niger</i>. Overall, this work establishes versatile and high-performance genetic tools for advanced applications in fungal synthetic biology.