Abstract
Filamentous fungi such as Aspergillus oryzae and Aspergillus niger 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 A. oryzae and A. niger 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 Aspergillus to zhongshengmycin. Through an attB/attP-mediated integration system, we achieved heterologous expression of β-glucuronidase in A. oryzae with a selection efficiency of 85.71 % (6/7). Additionally, recombination of megfp-sed5 in A. niger 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.