Cuixia Zhou

Protein glutaminase (PG), a specific deaminase derived from C. proteolyticum, has been shown to hold significant potential for improving plant protein functionality. However, its industrial application is hindered by low expression level, inefficient activation from its proenzyme form (pro-PG), and high production costs in native or common heterologous hosts. To develop an effective industrial host for active expression of PG based on food-grade strain, the Bacillus licheniformis BL-LKT strain originated from enterprises was systematically modified and engineered. First, the extracellular polysaccharide (EPS) gene cluster was knocked out to alleviate cell aggregation, thereby enhancing cell growth and fermentation performance. Subsequently, a series of single and double knockout mutants of key extracellular protease genes (aprE, bpr, epr, vpr, wpr) were constructed to reduce the risk of PG hydrolysis by these proteases, while retaining sufficient proteolytic activity for pro-PG activation. The PG was then expressed in these engineered hosts using the shuttle vector pLY-3-PG. Among the constructed strains, the mutant EPE, which lacks eps, aprE, and epr, exhibited the highest PG activity of 0.537 U/mL, representing a 5.33-fold increase compared with the original strain. Fed-batch fermentation in a 5-L bioreactor ultimately yielded a PG activity of 2.35 U/mL, a 337.6% increase largely attributable to the activation by endogenous proteases. These findings provide a strong foundation for utilizing engineered B. licheniformis as an efficient and industrially viable chassis for the cost-effective production of active PG.