Design of 5′-UTR to Enhance Keratinase Activity in <i>Bacillus subtilis</i>

Keratinase is an important industrial enzyme, but its application performance is limited by its low activity. A rational design of 5′-UTRs that increases translation efficiency is an important approach to enhance protein expression. Herein, we optimized the 5′-UTR of the recombinant keratinase KerZ1 expression element to enhance its secretory activity in Bacillus subtilis WB600 through Spacer design, RBS screening, and sequence simplification. First, the A/U content in Spacer was increased by the site-directed saturation mutation of G/C bases, and the activity of keratinase secreted by mutant strain B. subtilis WB600-SP was 7.94 times higher than that of KerZ1. Subsequently, the keratinase activity secreted by the mutant strain B. subtilis WB600-SP-R was further increased to 13.45 times that of KerZ1 based on the prediction of RBS translation efficiency and the multi-site saturation mutation screening. Finally, the keratinase activity secreted by the mutant strain B. subtilis WB600-SP-R-D reached 204.44 KU mL−1 by reducing the length of the 5′ end of the 5′-UTR, which was 19.70 times that of KerZ1. In a 5 L fermenter, the keratinase activity secreted by B. subtilis WB600-SP-R-D after 25 h fermentation was 797.05 KU mL−1, which indicated its high production intensity. Overall, the strategy of this study and the obtained keratinase mutants will provide a good reference for the expression regulation of keratinase and other industrial enzymes