Heterologous Production of Cyprosin B in Nicotiana benthamiana: Unveiling the Role of the Plant-Specific Insert Domain in Protein Function and Subcellular Localization

The aqueous extract of Cynara cardunculus flowers is traditionally used in cheese production across Mediterranean countries. To meet the growing industrial demand for plant-based milk-clotting enzymes and to explore potential biotechnological applications, we initiated a study to heterologously produce cyprosin B (CYPB), a key milk-clotting enzyme from C. cardunculus, in Nicotiana benthamiana. We also investigated the role of its plant-specific insert (PSI) domain in the CYPBs activity and its localization. In this study, full-length CYPB and a PSI domain deleted CYPB (CYPBΔPSI) were transiently expressed in N. benthamiana leaves using Agrobacterium-mediated infiltration. The leaves were harvested nine days post-infiltration, and proteins were purified, yielding approximately 81 mg/kg (CYPB) and 60 mg/kg (CYPBΔPSI) fresh weight. CYPBΔPSI showed significantly higher proteolytic activity (156.72 IU/mg) than CYPB (57.2 IU/mg), indicating that the PSI domain is not essential for enzymatic activity and that its removal results in enhanced enzymatic efficiency. In the milk-clotting activity assay, CYPBΔPSI demonstrated a significantly faster clotting time than full-length CYPB, indicating enhanced milk-clotting efficiency for CYPBΔPSI. Subcellular localization studies revealed that CYPB and PSI were localized in the vacuole and endocytic vesicles. In contrast, CYPBΔPSI was primarily localized in the endoplasmic reticulum (ER) and the tonoplast, suggesting that the PSI domain is critical for vacuolar targeting and membrane permeabilization that affects overall protein yield. This study demonstrates the feasibility of using N. benthamiana as a platform for the scalable production of more efficient recombinant CYPB. It highlights the multifunctional role of the PSI domain in vacuolar sorting without impairing its functionality. These results underscore the potential of plant-based expression systems as a viable alternative for the industrial production of plant milk-clotting enzymes, with significant implications for sustainable cheese production.