A Compact, Universal Fusion Tag Enables Robust Expression of Challenging Enzymes for Starch Bioconversion and...

Against the backdrop of the growing global demand for green and sustainable biotechnology, efficiently and cost-effectively expressing enzyme proteins in multi-enzyme systems remains challenging. Drawing inspiration from the high-level expression of glucose-6-phosphate isomerase from Escherichia coli in Bacillus subtilis, this study employed a region truncation analysis strategy based on three-dimensional structures to identify and develop a 30-amino acid N-terminal microtag, N30. When applied to enzyme expression in the artificial starch anabolic pathway (ASAP), the N30 tag enabled secretion levels of around 10 g/L for eight key enzymes. Significantly, N30(M22D)-TpiA reached 15 g/L in fed-batch fermentation, establishing an extremely efficient secretory expression system for ASAP enzymes. Furthermore, N30 was found to achieve detectable expression of multiple eukaryotic proteins that were previously unexpressible in Bacillus subtilis. Meanwhile, the fusion expression of rate-limiting enzymes in the synthetic pathway increased the titers of riboflavin and menaquinone-7 by 2.4- and 2.3-fold, respectively demonstrating the versatility and efficiency of N30 in protein synthesis and metabolic engineering applications. N30 provides a compact, activity-preserving, and scalable solution for the synthesis of enzymes for sustainable CO₂ conversion, starch bioconversion, and other applications.