Strong promoters and stable mRNAs are essential for the overproduction of heterologous proteins in Bacillus subtilis. To improve the strength of natural promoters and ensure robust protein output, promoter and genetic insulator engineering have been used. A series of plasmids containing single and dual promoters and genetic insulators to express alt3796 were engineered, which encoded an unsaturated glucuronyl hydrolase (UGL). As a first step, we screened the host and deleted the signal peptide (SPALT) of alt3796, successfully expressed secreted ALT3796 from B. subtilis WB800. Subsequently, to improve expression, we screened the dual promoter PHag-spoVG from a collection of 22 promoters, which yielded higher enzymatic activity. Finally, using a recombinant strain carrying a plasmid with the PHag-spoVG dual promoter and a genetic insulator, we obtained 40.9 U/mL of activity. Purified recombinant ALT3796 exhibited good stability and specifically degraded ulvan. In conclusion, a system for the heterologous expression of ALT3796 was constructed, and the obtained protein exhibited favorable properties, suggesting its potential for preparing novel ulvan oligosaccharides.
Keywords: Bacillus subtilis; Heterologous expression; Unsaturated glucuronyl hydrolase.
Copyright © 2024 Elsevier B.V. All rights reserved.