To Örebro University

Plant systems have gained increased attention as an alternative platform for producing heterologous proteins, particularly for industrially relevant proteins. The Cynara cardunculus L. flower extract is traditionally used in cheese production across Mediterranean countries due to its milk-clotting properties. To address the growing demand for plant-based milk-clotting enzymes, we investigated the heterologous production of cyprosin B (CYPB), a key milk-clotting enzyme, in Nicotiana benthamiana. We also examined the role of its plant-specific insert (PSI) domain in enzymatic activity, protein yield, and subcellular localization. Full-length CYPB and a PSI domain-deleted variant (CYPBΔPSI) were transiently expressed in N. benthamiana leaves using agroinfiltration. Proteins were purified nine days post-infiltration, yielding ~81 mg/kg (CYPB) and ~60 mg/kg (CYPBΔPSI) fresh weight. CYPBΔPSI showed higher proteolytic activity (~168 IU/mg) than CYPB (~57 IU/mg) and exhibited faster milk clotting times, suggesting that PSI removal may contribute to enhanced enzymatic efficiency. However, additional factors such as altered glycosylation or localization may also play a role. Subcellular localization indicated that CYPB and its PSI domain targeted the vacuole and endocytic vesicles, while CYPBΔPSI predominantly localized to the endoplasmic reticulum and tonoplast. This suggests that the PSI domain’s vital role in vacuolar targeting and membrane permeabilization ultimately influences protein yield. Our study shows N. benthamiana as a scalable platform for producing recombinant CYPB variants with improved enzymatic activity. It highlights the PSI domain's role in vacuolar sorting without impairing 21 function. These findings contribute to the development of plant-based systems for milk-clotting 22 enzymes for cheese-making.