Development of a novel transformation system using zhongshengmycin in Aspergillus oryzae and Aspergillus niger

Filamentous fungi such as <i>Aspergillus oryzae</i> and <i>Aspergillus niger</i> serve as valuable microbial cell factories with diverse applications. However, genetic manipulation in these fungi is often hindered by the lack of efficient resistance-based transformation systems. To address this challenge, we developed a novel transformation system for both <i>A. oryzae</i> and <i>A. niger</i> based on zhongshengmycin, a cost-effective streptothricin-class antibiotic, used in combination with the resistance gene encoding nourseothricin acetyltransferase (NAT). Our tests revealed that sensitization with SDS and EDTA-Na₂ disrupts cell membrane integrity, thereby enhancing the susceptibility of <i>Aspergillus</i> to zhongshengmycin. Through an attB/attP-mediated integration system, we achieved heterologous expression of β-glucuronidase in <i>A. oryzae</i> with a selection efficiency of 85.71 % (6/7). Additionally, recombination of <i>megfp-sed5</i> in <i>A. niger</i> achieved a transformation efficiency of 83.33 % (10/12), comparable to that of the PyrG system, thus establishing a versatile platform for fungal biotechnology. This system enables precise genomic integration for metabolic rewiring, offering new strategies for the synthetic biology-driven improvement of industrial filamentous fungi.