High-Level De Novo Biosynthesis of Apigenin Via Systematic Metabolic Engineering in Yarrowia lipolytica

Abstract Apigenin, a bioactive flavonoid with promising nutraceutical and industrial applications, faces challenges in production due to the unsustainability of plant extraction methods. In this study, we engineered Yarrowia lipolytica as a cell factory for de novo apigenin biosynthesis via systematic metabolic engineering. Initially, the most efficient type II flavone synthase from Erigeron breviscapus (EbFNSII) was identified using whole-cell biocatalysis. Subsequently, the apigenin biosynthetic pathway was constructed in Y. lipolytica Po1f (Δku70). Then, using an exogenous precursor feeding strategy, naringenin was identified as a crucial node in carbon metabolic flux. Additionally, we optimized the shikimic acid pathway by inhibiting competing pathways and overexpressing essential genes, thereby increasing the availability of L-tyrosine. Next, increasing the supply of malonyl-CoA further boosted carbon metabolic flux, which enhanced apigenin production by 2.41-fold, reaching 58.26 mg/L in shake flasks. Finally, an apigenin titer of 1236.25 mg/L was achieved by the engineered strain YLAPI-8 in a 2.4-L bioreactor under fed-batch fermentation, representing the highest titer reported to date. This study establishes an efficient platform for apigenin production and offers guidance for achieving high titers of flavonoids in Y. lipolytica.