A modular secretion platform in Bacillus subtilis enables scalable production of protein-glutamine glutaminase

Abstract

Protein-glutamine glutaminase (PGG) is a promising enzyme for improving the functionality of plant proteins, but its industrial production is limited by low native yields and the need for proteolytic activation. Here, we developed a standardized modular Golden Gate-based secretion platform in Bacillus subtilis to screen promoter-signal peptide combinations for extracellular production of Chryseobacterium proteolyticum PGG ( Cp PGG) within a common construct architecture. Reporter and enzyme-based screening identified Pgrac100-amyQ* as the configuration that consistently supported robust secretion and functional Cp PGG production in B. subtilis 168, where the secreted proenzyme underwent host-mediated maturation. In 3-L batch bioreactor cultivation in rich medium, this construct reached 2.9 U/mL of supernatant (SN), whereas fed-batch cultivation in defined medium revealed extracellular proteolysis as a major limitation under high-cell-density conditions. To decouple secretion from activation, the construct was transferred to the protease-deficient strain KO7-S which enabled stable accumulation of non-processed Cp PGG. Subsequent controlled in vitro activation with a food-grade neutral protease yielded 12.8 ± 0.8 U/mL SN. Together, these results show that efficient PGG production in B. subtilis requires coordinated control of secretion, extracellular stability and proenzyme activation. • Modular screening identified Pgrac100-amyQ* for CpPGG secretion in B. subtilis. • Proteolysis limited high-cell-density fed-batch production in B. subtilis 168. • In Bs KO7-S, secretion and activation were decoupled yielding 12.8 ± 0.8 U/mL SN.