Integrated Multi‐Omics Analysis Elucidates the Metabolic Basis of Enhanced Echinocandin B Biosynthesis and Guides Targeted Engineering in Aspergillus nidulans

Echinocandin B (ECB) serves as a crucial precursor for the antifungal drug anidulafungin, yet its production cost remains high due to low fermentation titers. We previously screened a highly efficient ECB producer Aspergillus nidulans ZJB16068, but its further metabolic engineering is hindered by unclear physiological and regulatory mechanisms responsible for high production. To address this, we conducted time-resolved transcriptomic and proteomic analyses across three fermentation phases (days 4, 7, and 10). Our results reveal that during the transition from growth to production, primary metabolism is systematically downregulated, while pathways supplying precursor amino acids (L-Thr, L-Pro, L-Orn) are upregulated in the later production phase. Notably, acetyl-CoA flux is redirected toward secondary metabolism through citrate synthase downregulation, accompanied by enhanced NADPH generation via the pentose phosphate pathway to support the heightened demand for reducing power. Unexpectedly, ECB biosynthetic genes are not induced, whereas the competing sterigmatocystin pathway acts as a major acetyl-CoA sink. Further analysis identified limited fatty acid β-oxidation as a constraint on acetyl-CoA availability. Overexpression of β-oxidation genes (FOX1, FOX2, POT1) increased ECB titers by 25.1%-33.5%. This study delineates the physiological phenotype underlying high ECB production and demonstrates how multi-omics-driven target identification guides effective metabolic engineering for strain enhancement.