Establishment of an efficient CRISPR-dCas9-SunTag transcriptional activation system in Aspergillus nidulans for enhancing emodin production

The intricate regulatory networks in filamentous fungi severely constrain the production of high-value natural products. Here, we developed an efficient CRISPR-dCas9-SunTag activation system to overcome transcriptional repression in Aspergillus nidulans . Through systematic optimization of GCN4 repeat numbers and implementation of P2A-mediated dCas9-SunTag/GCN4-VP64 tandem expression, this platform showed 20-fold higher activation efficiency compared to dCas9-VPR system. When deployed to regulate the emodin biosynthetic pathway, multiplexed sgRNA targeting enabled coordinated upregulation of three key biosynthetic genes mdpF , mdpG , and mdpH . This precise regulation approach minimized byproduct accumulation while boosting emodin titer to 101 mg/L, achieving 71 % higher production than the natural transcription factor overexpression. Furthermore, this optimized CRISPRa system demonstrated consistent stability during liquid fermentation in 1 L bioreactors (emodin 1.3 g/L) and solid-state fermentation (emodin 1.5 mg/g cell dry weight), highlighting its potential for scalable fungal metabolic engineering. This study establishes a modular and precise genetic toolkit for reprogramming fungal secondary metabolism, offering broad potential for the engineered biosynthesis of high-value compounds.