Engineering dispersed mycelium morphology in Aspergillus niger for enhanced mycoprotein production via CRISPR/Cas9-mediated genome editing.

Filamentous fungi are widely utilized in industrial fermentation processes due to their high productivity, with mycelial morphology directly influencing fermentation broth viscosity and target product yield, which is a critical parameter for process optimization. Aspergillus niger, an FDA-approved safe filamentous fungus, typically forms tightly packed mycelial pellets in submerged cultures, which severely restricts its industrial application potential by limiting mass transfer efficiency. To address this challenge, CRISPR/Cas9 mediated genome editing coupled with fermentation optimization enhanced microbial protein production in A. niger. Endogenous α-1,3-glucan synthase genes (agsA, agsB) and galactosaminogalactan (GAG) synthase genes (sph3, uge3) were disrupted using CRISPR/Cas9, achieving complete dispersion of filamentous pellets in liquid media. This morphological engineering strategy resulted in a 77.52 % increase in biomass and 39.98 % enhancement in mycelial protein content compared to the wild-type strain (A. niger Li2). Transcriptomic analysis revealed that the engineered strain (A. niger AnΔABSU) exhibited upregulated transporter proteins (ABC transporters, MFS transporters, sugar transporters), accelerating nutrient uptake and energy metabolism; altered cell wall integrity pathways, including activation of the MAPK signaling cascade and increased sensitivity to cell wall stressors; enhanced amino acid biosynthesis, driven by upregulated gene expression in key metabolic pathways. Furthermore, response surface methodology (RSM) with Box-Behnken design optimized the fermentation medium, yielding 16.67 g/L biomass and 45.91 % protein content, representing 115.37 % and 67.01 % improvements over the unoptimized wild-type control. This study establishes a novel paradigm for constructing high-efficiency microbial protein cell factories via integrated morphological-engineering and fermentation optimization.