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

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.