Identification, Cloning, and Functional Characterization of Carotenoid Cleavage Dioxygenase (CCD) from Olea europaea and Ipomoea nil

The aromatic C₁₃ apocarotenoid β-ionone is a high-value natural-flavor and -fragrance compound derived from the oxidative cleavage of carotenoids. Carotenoid cleavage dioxygenases (CCDs) play a pivotal role in the biosynthesis of volatile apocarotenoids, particularly β-ionone. In this study, we report the identification, cloning, and functional characterization of two CCD1 homologs: <i>OeCCD1</i> from <i>Olea europaea</i> and <i>InCCD1</i> from <i>Ipomoea nil</i>. These two species, which, respectively, represent a woody perennial and a herbaceous annual, were selected to explore the potential functional divergence of CCD1 enzymes across different plant growth forms. These CCD1 genes were synthesized using codon optimization for <i>Escherichia coli</i> expression, followed by heterologous expression and purification using a GST-fusion system. In vitro assays confirmed that both enzymes cleave β-carotene at the 9,10 (9',10') double bond to yield β-ionone, but only <i>OeCCD1</i> exhibits detectable activity on zeaxanthin; <i>InCCD1</i> shows no in vitro cleavage of zeaxanthin. Kinetic characterization using β-apo-8'-carotenal as substrate revealed, for <i>OeCCD1</i>, a K_m of 0.82 mM, V_max of 2.30 U/mg (k_cat = 3.35 s^-1), and k_cat/K_m of 4.09 mM^-1·s^-1, whereas <i>InCCD1</i> displayed K_m = 0.69 mM, V_max = 1.22 U/mg (k_cat = 1.82 s^-1), and k_cat/K_m = 2.64 mM^-1·s^-1. The optimization of expression parameters, as well as the systematic evaluation of temperature, pH, solvent, and metal ion effects, provided further insights into the stability and functional diversity within the plant CCD1 family. Overall, these findings offer promising enzymatic tools for the sustainable production of β-ionone and related apocarotenoids in engineered microbial cell factories.