Development of an intron-optimized dual base editor enables enhanced echinocandin B tolerance and regulatory circuit...

The production of the antifungal precursor echinocandin B (ECB) in Aspergillus nidulans is hindered by limited understanding of regulatory mechanisms and product inhibition. Dual base editors (simultaneous C-to-T and A-to-G base editing, CABE) offer an evolutionary approach to address these limitations, yet their application in filamentous fungi remains underdeveloped. A major challenge in base editor development is plasmid instability, as leaky expression of deaminase-nCas9 fusion protein during amplification in Escherichia coli often introduces unintended mutations in the plasmid backbone. To overcome this, we implemented a fungal intron-based strategy to block translation in E. coli while preserving fungal transcription. By optimizing intron positioning and screening highly active deaminases, a PAM-less SpRY-CABE system was developed in A. nidulans, achieving efficient dual base editing (>40 % C-to-T and >30 % A-to-G) within a 7-bp window. Using CABE-mediated promoter mutagenesis, we deciphered the transcriptional logic of AniJ, the pathway-specific regulator of ECB biosynthesis, and identified a conserved RNGCTGAS motif as its upstream activating sequence. Modular amplification of this motif in the aniA promoter enhanced gene expression and increased ECB production by 64 % (to 2.05 g/L). Furthermore, to mitigate ECB inhibition, CABE was employed for in situ evolution of β-1,3-glucan synthase (FKS1, the target of ECB), isolating ECB-tolerant mutants FKS1_(S674P) and FKS1_(R1385Y) that shortened fermentation cycles by 24 h. Together, this optimized SpRY-CABE system enables versatile genetic manipulation in A. nidulans, establishing a robust platform for fungal synthetic biology and industrial applications.