Genetic context-dependent regulation of FlbE enhances morphology modulation and citric acid production in Aspergillus niger.

Aspergillus niger is a prominent industrial filamentous fungus widely utilized in industrial-scale biomanufacturing of organic acids and enzymes, where mycelial morphology critically influences fermentation performance. FlbE, a key developmental regulator in filamentous fungi, is traditionally known for primarily controlling the differentiation of vegetative hyphae into conidiophores. Intriguingly, deletion of flbE in the protein-producing strain MA234.1 resulted in normal conidiation, indicating potential strain-specific functions beyond its canonical developmental role. To explore this, we employed CRISPR/Cas9-mediated in situ Tet-on promoter replacement to construct titratable flbE expression mutants in two genetically distinct strains: the protein-producing MA70.15 and the citric acid-producing D353.8. Phenotypic analyses revealed that flbE coordinated conidiation, hyphal growth, and pellet morphology in a strain-dependent manner. Notably, flbE repression in D353.8 led to a significant reduction in mycelial pellet size and a 22.7% increase in citric acid production. Transcriptomic profiling under titratable flbE induction revealed the activation of developmental regulators (flbB, flbD) and central metabolic genes, while genes involved in cell wall remodeling (exsG, eglB), glucose uptake (mstH), citrate export (cexA), and extracellular hydrolases (glaA, amyA, pepA, lipA) were downregulated, accompanied by the morphological shift and metabolic enhancement. These findings unveiled that FlbE plays a pleiotropic and strain-specific role in integrating fungal development with primary metabolism. Moreover, this study identifies flbE as a promising genetic target for morphology engineering and metabolic optimization in A. niger, offering a new strategy for enhancing citric acid biomanufacturing.