Atmospheric room-temperature plasma mutagenesis and heterologous expression of dextranase in Aspergillus niger

Dextranase (DEX) is a high-value biocatalyst with substantial potential in oral hygiene and diverse industrial bioprocesses, yet its practical application is frequently constrained by the insufficient yields of wild-type strains. In this study, a high-efficiency DEX production system was established through an integrated strategy comprising Atmospheric and Room Temperature Plasma (ARTP) mutagenesis, molecular mechanism elucidation, and host-specific promoter engineering. A stable mutant, Chaetomium gracile B12, was isolated, exhibiting a 30.6-fold increase in DEX activity (16.23 U/mL) compared to the parental strain. Sequence analysis and multi-scale computational simulations, including AlphaFold2-based modeling and 100 ns molecular dynamics, generated a testable hypothesis that this mutation may contribute this enhancement. This substitution was found to augment structural rigidity and refine the spatial orientation of the substrate within the active pocket, facilitating a more "productive alignment" for catalysis. Furthermore, heterologous expression optimization uncovered a distinct host-specific promoter preference: the heterologous Pcdna1 promoter was most effective in C. gracile, whereas the endogenous inducible PgpdA proved superior in Aspergillus niger. Notably, this pioneering heterologous expression of DEX in A. niger streamlined the production cycle, slashing the fermentation period from 6 days to 48 h. Following systematic optimization of carbon/nitrogen sources and aeration intensity, the engineered A. niger strain H achieved a peak DEX activity of 36.00 U/mL. Biochemical characterization revealed that the recombinant DEX possessed a favorable near-neutral optimal pH (6.0-7.0) and a temperature optimum of 50 °C. Overall, this study reports the first heterologous expression of a dextranase in A. niger, reducing the fermentation period from 6 days to 48 h, and provides a comprehensive strategy for the overproduction of dextranase and offers molecular insights into enzymatic "fine-tuning", yielding a high-titer, neutral-pH compatible biocatalyst with significant promise for oral care and industrial applications.