Synergistic strategies for high production of <i>Geobacillus stearothermophilus</i> α-amylase in <i>Bacillus subtilis</i>

Although the α-amylase (AmyS) from Geobacillus stearothermophilus exhibits high thermostability, its low enzymatic activity (44.36 ± 2.02 U/mL) severely hinders industrial applications. Given its strong protein secretion capability and GRAS (generally recognized as safe) status, this study employed Bacillus subtilis as the host to enhance AmyS production. Through error-prone PCR and high-throughput screening, a triple mutant T151A/K178E/T458A (AmySM) was generated, showing a 17.67% increase in activity (51.34 ± 1.11 U/mL). AmySM activity was further increased by 29.32% to 65.23 ± 2.33 U/mL using the signal peptide SPykwD, selected from a comprehensive library for superior secretion efficiency. The promoter PgsiB enhanced activity by 37.61% to 89.54 ± 2.95 U/mL. Optimizing the ribosome binding site (RBS) resulted in an additional 48.83% increase in activity, yielding a final activity of 134.02 ± 3.54 U/mL, which corresponds to a 3.02-fold improvement over the initial strain WBSW (44.36 ± 2.02 U/mL). Ultimately, scale-up fermentation in a 5-L bioreactor yielded a maximum extracellular activity of 1244.17 ± 48.66 U/mL at 72 hours, a 2.84-fold increase over the control. This multi-level strategy provides a rational framework for high-efficiency AmySM production, paving the way for extracellular production of high-value proteins in the GRAS host B. subtilis.