The Functional Characteristics and Soluble Expression of Saffron CCD2.

Crocins are important natural products predominantly obtained from the stigma of saffron, and that can be utilized as a medicinal compound, spice, and colorant with significant promise in the pharmaceutical, food, and cosmetic industries. Carotenoid cleavage dioxygenase 2 (CCD2) is a crucial limiting enzyme that has been reported to be responsible for the cleavage of zeaxanthin in the crocin biosynthetic pathway. However, the catalytic activity of CCD2 on β-carotene/lycopene remains elusive, and the soluble expression of CCD2 remains a big challenge. In this study, we reported the functional characteristics of CCD2, that can catalyze not only zeaxanthin cleavage but also β-carotene and lycopene cleavage. The molecular basis of the divergent functionality of CCD2 was elucidated using bioinformatic analysis and truncation studies. The protein expression optimization results demonstrated that the use of a maltose-binding protein (MBP) tag and the optimization of the induction conditions resulted in the production of more soluble protein. Correspondingly, the catalytic efficiency of soluble CCD2 was higher than that of the insoluble one, and the results further validated its functional verification. This study not only broadened the substrate profile of CCD2, but also achieved the soluble expression of CCD2. It provides a firm platform for CCD2 crystal structure resolution and facilitates the synthesis of crocetin and crocins.