Enhancement of the d‑Allulose 3‑Epimerase Expression in Bacillus subtilis through Both Transcriptional and Translational Regulations

• Published in: Journal of agricultural and food chemistry Vol. 72; no. 14; pp. 8052 - 8059
• Main Authors: Zhang, Wenli, Ren, Hu, Chen, JiaJun, Ni, Dawei, Xu, Wei, Mu, Wanmeng
• Format: Journal Article
• Language: English
• Published: United States American Chemical Society 10.04.2024
• Subjects: Bacillus subtilis - genetics; Bacillus subtilis - metabolism; Biotechnology and Biological Transformations; Fructose - metabolism; Ketoses; Racemases and Epimerases - genetics; Racemases and Epimerases - metabolism; transcriptional regulation; translational regulation; d-allulose 3-epimerase
• ISSN: 0021-8561; 1520-5118
• DOI: 10.1021/acs.jafc.4c01122

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.