Abstract: Aspergillus niger is an important production strain in food industry, widely used in production of enzymes and organic acids. To improve the genetic efficiency of A. niger, the transformation and recombinant strain screening strategies were optimized. According to the reported optimal transformation conditions, A. niger AG11 was transformed via electroporation, Agrobacterium-mediated and PEG-mediated transformation (PMT), and a hygromycin transformation plate yield 0, 30, and 6 transformants, respectively. PMT achieved the highest positive rate of transformants. Based on the optimized protoplast preparation condition: 0.8-1 g loose mycelial pellets were incubated in 10 mL Yatalase solution (0.8 mg/mL, pH 5.5) for 2.5 h to obtain protoplasts and used to PMT, the transformation efficiency was increased by 14-fold than that before optimization. To improve the screening efficiency of transformants, the asparaginase (ASN) gene fused with green fluorescent protein (GFP) was integrated into the glycosylase locus of A. niger AG11, and those spores expressing ASN were sorted by flow cytometry. The results showed that 0.3% of the spores could express the GFP (a total of 300 000 spores were screened). Enzyme activity analysis indicated that 65% of the spores expressing GFP could produce ASN. These results provide an important methodological basis for gene editing of A. niger.
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