Improving xylitol production by semi-rational engineering of xylitol dehydrogenase and optimizing cofactor regeneration in Gluconobacter oxydans

Xylitol, a widely used sugar substitute in food and medicine, can be produced through microbial bioconversion using glucose as the primary substrate. In this process, a critical factor limiting xylitol production is the relatively low activity of xylitol dehydrogenase (XDH) during the biotransformation of D-arabitol to xylitol by resting cells of Gluconobacter oxydans . To improve the catalytic performance, Go XDH was engineered by site-saturation mutagenesis combined with a high-throughput screening method, and a variant Go XDH M3 (S77C/S106N/A110N) with high activity was obtained, showing a 2.49-fold increase in catalytic activity. The structural analysis revealed that the ‌S77C/S106N/A110N‌ mutations enhanced proton and electron transfer rates while stabilizing the hydrophilic substrate-binding pocket and the tetrameric structure. Additionally, by optimizing the coenzyme regeneration system and enhancing the oxygen transfer efficiency, we developed an efficient biotransformation of D-arabitol to xylitol in G. oxydans . Using resting cells of G. oxydans /XDH M3 -GDH-VHb, a xylitol titer of 29.02 g/L were achieved from 40 g/L D-arabitol within 30 h. The findings suggest that boosting XDH activity through semi-rational engineering markedly improves xylitol productivity in G. oxydans .