High-efficiency production of recombinant sucrose phosphorylase in Bacillus subtilis through combinatorial optimization of genetic elements

Sucrose phosphorylase (SPase) is widely used for the preparation of functional glycosides like 2-O-α-d-glucopyranosyl glycerol (2-αGG). Its heterologous expression in Bacillus subtilis has attracted wide interest. Here, the expression of LreSP-MT derived from Limosilactobacillus reuteri was tested through a combinatorial strategy. The promoter adjacent to gene was found to be a key factor in achieving high-level expression, and the best promoter P_yvyD increased extracellular enzyme activity by 310 % compared to the strong promoter P₄₃. The dual promoter P_ydjO-P_yvyD further enhanced enzymatic activity by 22 %. Optimal RBS increased enzymatic activity by 14 % after RBS screening and engineering. The optimal recombinant strain B. subtilis WB800 (pP_ydjO-P_yvyD-RBS_B15-LreSP-MT) produced a high extracellular enzyme activity of 9.1 U/mL and extracellular recombinant protein yield of up to 8.0 g/L in a fed-batch fermentation. The 10-fold diluted fermentation broth yielded up to 322.6 g/L of 2-αGG through a sucrose-feeding biotransformation strategy, suggesting it is a high-efficiency bioprocess to produce 2-αGG. The results also highlight the critical role of genetic element compatibility for heterologous gene expression in B. subtilis.