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

doi:10.1016/j.enzmictec.2023.110267

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 Microb Technol. 2023 Sep:169:110267. doi: 10.1016/j.enzmictec.2023.110267. Epub 2023 Jun 2.

Authors

Tian Gan¹, Jingyi Fang¹, Yuxin Wang¹, Kaiqiang Liu¹, Yumin Sang¹, Hanchi Chen¹, Yuele Lu¹, Linjiang Zhu², Xiaolong Chen³

Affiliations

¹ Institute of Fermentation Engineering, Zhejiang University of Technology, Hangzhou 310014, China; College of Biotechnology and Bioengineering, Zhejiang University of Technology, Hangzhou 310014, China.
² Institute of Fermentation Engineering, Zhejiang University of Technology, Hangzhou 310014, China; College of Biotechnology and Bioengineering, Zhejiang University of Technology, Hangzhou 310014, China. Electronic address: zhulinjiang@zjut.edu.cn.
³ Institute of Fermentation Engineering, Zhejiang University of Technology, Hangzhou 310014, China; College of Biotechnology and Bioengineering, Zhejiang University of Technology, Hangzhou 310014, China. Electronic address: richard_chen@zjut.edu.cn.

PMID: 37321017
DOI: 10.1016/j.enzmictec.2023.110267

Abstract

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 P_sigH-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.

Keywords: 2-O-α-d-glucopyranosyl-L-ascorbic acid; Bacillus subtilis; Dual promoter; Promoter engineering; Sucrose phosphorylase.

MeSH terms

Ascorbic Acid* / metabolism
Bacillus subtilis* / genetics
Bacillus subtilis* / metabolism
Glucosyltransferases / genetics
Glucosyltransferases / metabolism

Substances

ascorbic acid 2-O-glucoside
sucrose phosphorylase
Ascorbic Acid
Glucosyltransferases