Regulation of proenzyme activation and metabolic engineering for protein-glutaminase production in Bacillus subtilis

The protein-glutaminase (PG, EC 3.5.1.44) specifically targets glutamine residues in proteins and peptides, and has significant potential for enhancing the functional characteristics and processing efficiency of plant proteins. However, natural PG production faces challenges such as low enzymatic yield and difficult genetic manipulation. To address these challenges, a novel self-activating PG expression system was developed in Bacillus subtilis. First, pro-PG (PPG)-activated proteases were identified in B. subtilis by constructing a series of engineered strains. Second, the co-expression of PPG and PPG-activated protease in B. subtilis WB800 for mature PG (mPG) production was analyzed, and it was found that the supply and activation of PPG during fermentation was insufficient. Therefore, the gene expression components of PPG and protease, including the promoter and RBS, were further optimized. In addition, the key genes of the maltose metabolic pathway were knocked out, and the engineered strain W8ΔM2-AE-Pmal380 showed the highest capacity for PG production. Finally, a 53.0 U/mL mPG yield was achieved in a 5-L bioreactor within 64 h. This study establishes an efficient platform for industrial PG production and provides a reference for the expression and activation of other proenzymes.