Overexpression and Characterization of a Novel Plant Carotenoid Cleavage Dioxygenase 1 from Morus notabilis.

Abstract

Synthesis of β-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 β-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 β-ionone, 3-hydroxy-4-oxo-β-ionone, 3-hydroxy-β-ionone, and 3-hydroxy-α-ionone in vitro. MnCCD1 could also cleave lycopene and β-carotene at the 9, 10 (9', 10') bind bond to produce pseudoionone and β-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 β-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.