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Background <p>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 <i>Gluconobacter oxydans</i>.</p> Results <p>To improve the catalytic performance, <i>Go</i>XDH was engineered by site-saturation mutagenesis combined with a high-throughput screening method, and a variant <i>Go</i>XDH<sub>M3</sub> (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 <i>G. oxydans</i>. Using resting cells of <i>G. oxydans</i>/XDH<sub>M3</sub>-GDH-VHb, a xylitol titer of 29.02&#xa0;g/L were achieved from 40&#xa0;g/L D-arabitol within 30&#xa0;h.</p> Conclusion <p>The findings suggest that boosting XDH activity through semi-rational engineering markedly improves xylitol productivity in <i>G. oxydans</i>.</p>

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

  • 期刊论文

  • 出版模式：

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  • 发表日期：

    2026年6月22日

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  • Jun-Hao Su,
  • Jin-Cheng Guo,
  • Lu Liu,
  • Hai-Ling Zhang,
  • Dong Liu

摘要

Background

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.

Results

To improve the catalytic performance, GoXDH was engineered by site-saturation mutagenesis combined with a high-throughput screening method, and a variant GoXDHM3 (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/XDHM3-GDH-VHb, a xylitol titer of 29.02 g/L were achieved from 40 g/L D-arabitol within 30 h.

Conclusion

The findings suggest that boosting XDH activity through semi-rational engineering markedly improves xylitol productivity in G. oxydans.