Enhancement of the <scp>d</scp>-Allulose 3-Epimerase Expression in <i>Bacillus subtilis</i> through Both Transcriptional and Translational Regulations
Wenli Zhang, Hu Ren, Jiajun Chen, Dawei Ni, Wei Xu, Wanmeng Mu
Abstract
d -Allulose, a functional bulk sweetener, has recently attracted increasing attention because of its low-caloric-ness properties and diverse health effects. d -Allulose is industrially produced by the enzymatic epimerization of d -fructose, which is catalyzed by ketose 3-epimerase (KEase). In this study, the food-grade expression of KEase was studied using Bacillus subtills as the host. Clostridium sp. d -allulose 3-epimerase (Clsp-DAEase) was screened from nine d -allulose-producing KEases, showing better potential for expression in B. subtills WB600. Promoter-based transcriptional regulation and N-terminal coding sequence (NCS)-based translational regulation were studied to enhance the DAEase expression level. In addition, the synergistic effect of promoter and NCS on the Clsp-DAEase expression was studied. Finally, the strain with the combination of a P HapII promoter and gln A -Up NCS was selected as the best Clsp-DAEase-producing strain. It efficiently produced Clsp-DAEase with a total activity of 333.2 and 1860.6 U/mL by shake-flask and fed-batch cultivations, respectively.