Metabolic Engineering of Yarrowia lipolytica for High-Level de novo Biosynthesis of Xylitol from Glucose.
Xylitol is a highly functional sweetener with extensive applications. Sustainable biosynthesis from glucose is desirable yet metabolically challenging. Here, we engineered Yarrowia lipolytica as a cell factory by constructing a core biosynthetic route via combinatorial screening and multicopy integration of d-arabitol dehydrogenases (ArDH) and an NADPH-dependent xylitol dehydrogenase (XDH) in the robust chassis NBRC1631. To further drive the metabolic flux and alleviate bottlenecks, we employed a synergistic push-and-pull strategy: overexpressing glucose transporters (YH3 and YH4), while upregulating pentose phosphate pathway enzymes (ZWF1 and GND1) to enhance NADPH regeneration, matching the redox demand of the synthetic cascade. Following two-stage pH-controlled fed-batch fermentation in a 3 L bioreactor, the final engineered strain achieved a record-high xylitol titer of 39.0 g/L with a yield of 0.09 g/g glucose. This study establishes a productive platform for microbial de novo xylitol biosynthesis from glucose, offering a green and economically viable route for industrial production.