Optimizing oleaginous yeast cell factories for flavonoids and hydroxylated flavonoids biosynthesis

Abstract Plants possess myriads of secondary metabolites with a broad spectrum of health-promoting benefits. Up to date, plant extraction is still the primary route to produce high-value natural products, which inherently suffers from economics and scalability issues. Heterologous production in microbial host is considered as a feasible approach to overcoming these limitations. Flavonoid and its hydroxylated derivatives represent a diversified family of bioactive compounds, most prominently known as antioxidant and anti-aging agents. Oleaginous yeast is rich in hydrophobic lipid bodies and spatially-organized organelles, which provides the ideal environment for the regioselectivity and stereoselectivity of many plant-specific enzymes. In this report, we validated that Y. lipolytica is a superior platform for heterologous production of high-value flavonoids and hydroxylated flavonoids. By modular construction and characterization, we determined the rate-limiting steps for efficient flavonoids biosynthesis in Y. lipolytica . We evaluated various precursor pathways and unleashed the metabolic potential of Y. lipolytica to produce flavonoids, including the supply of acetyl-CoA, malonyl-CoA and chorismate. Coupled with the optimized chalcone synthase module and the hydroxylation module, our engineered strain produced 252.4 mg/L naringenin, 134.2 mg/L eriodictyol and 110.5 mg/L taxifolin from glucose. Collectively, these findings demonstrate our ability to harness oleaginous yeast as microbial workhorse to expand nature’s biosynthetic potential, enabling us to bridge the gap between drug discovery and natural product manufacturing.