Profiling heterologous 3' terminator-dependent expression variation in Aspergillus oryzae

Transcriptional terminators are key to defining transcript boundaries, ensuring mRNA maturation, and maintaining expression stability, yet they remain underexplored in filamentous fungi. Here, we systematically benchmarked 15 heterologous 3' terminators in Aspergillus oryzae by measuring protein output via mCherry fluorescence and transcriptional readthrough based on a quantitative readthrough index (RTI) derived from RT-qPCR. Across the tested terminators, mCherry expression varied more than 5-fold and RTI spanned over 50-fold, indicating substantial functional diversity. Expression strength and termination efficiency were only weakly correlated (Spearman ρ = -0.39, p = 0.165), indicating that these two properties are largely independent and can be optimized separately. Strong terminators -TactA, Txyn1, TmutA and TtrpC combined good mCherry expression (>ΔT) and small readthrough (RT < 0.15). In contrast, short synthetic sequences (≤70 bp) showed low mCherry output accompanied by transcriptional leakage. Interestingly, widely used toolkit elements such as Ttef1 and Tcyc1 performed poorly in A. oryzae, highlighting the importance of empirical characterization over assumed transferability. Polyadenylation site mapping by 3' rapid amplification of cDNA ends (3'RACE) reveals high-performing terminators that possess focused cleavage sites, continuous poly(A) tails, and recognizable A[AT]TAAA-like motifs with moderately AU-rich upstream regions, whereas weak terminators exhibited dispersed cleavage and sparse upstream sequence elements. Targeted disruption of the canonical AATAAA hexamer in TactA did not significantly impair expression or termination efficiency, demonstrating that the canonical PAS is not strictly required when AU-rich upstream sequence elements are present. Terminator performance rankings were robust across three carbon sources and were confirmed using a secreted NanoLuc luciferase reporter, demonstrating that functional behavior is largely sequence intrinsic. These findings demonstrate a functional terminator architecture and suggest a practical guideline for terminator selection in A. oryzae.