Metabolic mechanisms underlying osmoprotectant-enhanced xylitol biosynthesis from glucose by Zygoascus hellenicus

Xylitol, a high-value sugar alcohol, holds significant potential for applications in the food and pharmaceutical industries. Biomanufacturing xylitol from low-cost, green feedstocks provides a sustainable alternative to the energy-intensive and highly polluting chemical synthesis process. Current research primarily focuses on xylose-to-xylitol bioconversion, whereas native glucose-to-xylitol converters remain underexplored. In particular, a systematic understanding of their osmotic stress adaptation and metabolic regulation is lacking, which directly impedes the scale-up of xylitol production from low-cost glucose. To address this gap, this study investigated Zygoascus hellenicus, an osmotolerant yeast capable of directly converting glucose to xylitol, and proposed a novel strategy to enhance xylitol production by supplementing exogenous osmoprotectants. Results showed that adding 80 mL/L glycerol or 80 g/L trehalose increased xylitol titers to 28.44 g/L and 23.63 g/L, representing improvements of 41.99% and 17.99%, respectively, compared with the control group. Mechanistic investigations revealed the dual regulatory roles of these osmoprotectants. Physiologically, they enhanced osmotic tolerance, preserved membrane integrity, and stabilized enzyme activity. Metabolically, they significantly activated key enzymes of the pentose phosphate pathway, including glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase (G6PDH and 6PGDH) and inhibited glycolytic enzymes, thereby redirecting carbon flux toward xylitol biosynthesis. Dynamic metabolic flux analysis (dMFA) further confirmed that osmoprotectant treatment substantially increased the metabolic flux through the xylitol synthesis pathway mediated by G6PDH and 6PGDH, while reducing carbon flow in glycolysis. This study provides mechanism-based insights into xylitol production from glucose in a native producer, revealing a green and alternative glucose-based biomanufacturing route.