Overexpression and Characterization of a Novel Plant Carotenoid Cleavage Dioxygenase 1 from Morus notabilis
Synthesis of ??ionone in microbial cell factories is limited by the efficiency of carotenoid cleavage dioxygenases (CCDs). To obtain genes responsible for specific cleavage of carotenoids generating ??ionone, a novel carotenoid cleavage dioxygenase 1 from Morus notabilis was cloned and overexpressed in Escherichia coli. The MnCCD1 protein was able to cleave a variety of carotenoids at the positions 9, 10 (9?, 10?) to produce ??ionone, 3?hydroxy?4?oxo???ionone, 3?hydroxy???ionone, and 3?hydroxy???ionone in?vitro. MnCCD1 could also cleave lycopene and ??carotene at the 9, 10 (9?, 10?) bind bond to produce pseudoionone and ??ionone, respectively, in E.?coli accumulating carotenoids. The enzyme activity of MnCCD1 was reached 2.98?U/mL at optimized conditions (temperature 28?°C, IPTG 0.1?mM, induction time 24?h). The biochemical characterization of MnCCD1 revealed the optimal activities were at pH?8.4 and 35?°C. The addition of 10?% ethanol could increase enzyme activity at above 15?%. However, an obvious decline was observed on enzyme activity as the concentration of Fe2+ increased (0–1?mM). The Vmax for ??apo?8??carotenal was 72.5?U/mg, while the Km was 0.83?mM. The results provide a foundation for developing the application of carotenoid cleavage dioxygenases as biocatalysis and synthetic biology platforms to produce volatile aroma components from carotenoids.