Improving d-Allulose Production via Alditol Oxidase-Directed Evolution Assisted by an Artificial Genetic Circuit

文献类型： 外文期刊

第一作者： Lv, Yongkun

作者： Lv, Yongkun;Chen, Song;Zhu, Lijuan;Wang, Shilei;Shi, Mengzhuo;Xu, Jingliang;Lv, Yongkun;Guo, Zhouyan;Xu, Peng;Chen, Zhenhua;Zhao, Anqi;Wang, Weigao;Xu, Yameng;Xu, Peng

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关键词： laboratory-directed evolution; D-allulose; alditol oxidase; artificial genetic circuit; growthcoupling

期刊名称：JOURNAL OF AGRICULTURAL AND FOOD CHEMISTRY （ 影响因子：6.2； 五年影响因子：6.4 ）

ISSN： 0021-8561

年卷期： 2025 年 73 卷 22 期

页码：

收录情况： SCI

摘要： As a good sucrose substitute, d-allulose has been substantially used in the food industry. In a previous work, we developed a d-allulose synthetic pathway and revealed that the alditol oxidase (AldO) is the key rate-limiting node. The laboratory-directed evolution (LDE) has been largely used for improving enzyme performance but is commonly faced with time-consuming screening processes or expensive equipment demand. In this study, we tried to develop an easy approach to obtain higher-performing AldO mutants through continuous LDE. First, we designed and validated a d-allulose-responsive genetic circuit. Next, we tuned its sensitivity and responsive range to make it suitable for growth coupling. Subsequently, the chassis fitness was coupled to d-allulose production by controlling the expression of a tetracycline exporter. Finally, the chassis equipped with the genetic circuit was used to evolve the AldO mutant library. After 3 passages, a mutant M4-15 was picked out, which produced 19.97% more d-allulose. Structural analysis showed a widened entrance of the catalytic pocket. This study achieved the continuous LDE of AldO and provided an easy and simple approach to evolve the d-allulose synthetic pathway. This study also represented a proof of concept example of accelerating the LDE evolution using an artificial genetic circuit.

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