Promoter engineering for efficient production of sucrose phosphorylase in Bacillus subtilis and its application in enzymatic synthesis of 2-O-α-D-glucopyranosyl-L-ascorbic acid.

Tian Gan, Jingyi Fang, Yuxin Wang, Kaiqiang Liu, Yumin Sang, Hanchi Chen, Yuele Lu, Linjiang Zhu, Xiaolong Chen

Enzyme and microbial technology 2023 Sep

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2-O-α-D-glucopyranosyl-L-ascorbic acid (AA-2G), a stable glucoside derivative of L-ascorbic acid (L-AA), can be one-step synthesized by sucrose phosphorylase (SPase). In this study, we attempted to produce extracellular SPase in Bacillus subtilis WB800 for the food-grade production of AA-2G. The results showed that the secretion of SPases did not require signal peptide. Promoter and its compatibility to target SPase gene were proved to be the key factors for high-level secretion. The strong promoter P43 and synthetic SPase gene derived from Bifidobacterium longum (BloSPase) were selected due to generate a relatively high extracellular activity (0.94 U/mL) for L-AA glycosylation. A highly active dual-promoter system PsigH-100-P43 was further constructed, which produced the highest extracellular and intracellular activity were 5.53 U/mL and 6.85 U/mL in fed-batch fermentation, respectively. Up to 113.58 g/L of AA-2G could be achieved by the supernatant of fermentation broth and a higher yield of 146.42 g/L was obtained by whole-cells biotransformation. Therefore, the optimal dual-promoter system in B. subtilis is suitable for the food-grade scale-up production of AA-2G. Copyright © 2023 Elsevier Inc. All rights reserved.

Citation

Tian Gan, Jingyi Fang, Yuxin Wang, Kaiqiang Liu, Yumin Sang, Hanchi Chen, Yuele Lu, Linjiang Zhu, Xiaolong Chen. Promoter engineering for efficient production of sucrose phosphorylase in Bacillus subtilis and its application in enzymatic synthesis of 2-O-α-D-glucopyranosyl-L-ascorbic acid. Enzyme and microbial technology. 2023 Sep;169:110267