Regulatory Effects and Mechanisms of Dissolved Oxygen on Xylitol Biosynthesis from Glucose by Zygoascus hellenicus B11.

To overcome the bottleneck of high pollution associated with using xylose as a raw material for xylitol biosynthesis, this study focused on a yeast strain, Zygoascus hellenicus B11, which can directly utilize glucose as a carbon source. We investigated the regulatory effects and underlying mechanisms of different oxygen conditions on xylitol biosynthesis. Through systematic scale-up studies from shake flasks to bioreactors, we characterized the dynamic profiles of cell growth, substrate consumption, and xylitol synthesis under various agitation speeds and aeration conditions. Furthermore, by constructing an unsteady-state metabolic kinetic model for this strain and measuring key enzyme activities and intracellular cofactor levels, we quantitatively analyzed the distribution of carbon metabolic flux in the network for xylitol synthesis from glucose. Xylitol synthesis was significantly regulated by oxygen levels. A moderate increase in oxygen promoted xylitol production, with the highest concentration of 51.49 g/L achieved under the optimal aeration condition (3 NL/min). However, excessively high oxygen levels promoted the reverse oxidation of xylitol to xylulose, catalyzed by xylitol dehydrogenase (XDH), diverting more metabolic flux towards cell growth and thereby weakening xylitol synthesis. Based on enzyme activity assays, cofactor measurements, and metabolic kinetic model simulations, we propose the following mechanistic interpretation: optimal oxygen levels not only redirect more carbon flux into the pentose phosphate pathway, enhancing precursor supply, but also maintain a high NADPH/NADP⁺ ratio. Based on these combined experimental and modeling data, we infer that this cofactor balance shifts the reversible XDH-catalyzed reaction equilibrium toward net xylitol synthesis. The study provides key strategies and theoretical support for improving xylitol concentration through dynamic oxygen supply.