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

Simple Summaryβ-Ionone is a valuable aroma compound widely used in the fragrance, food, and pharmaceutical industries. In this study, we identify and characterize two carotenoid cleavage dioxygenase 1 (CCD1) genes fromOlea europaeaandIpomoea nil. By expressing these genes inE. coli, we evaluate their ability to produce β-ionone and analyze their biochemical properties. Our findings not only provide insights into the functional diversity of CCD1 enzymes across plant species, but also offer new genetic resources for the microbial biosynthesis of natural aroma compounds.The aromatic C13apocarotenoid 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:OeCCD1fromOlea europaeaandInCCD1fromIpomoea nil. 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 forEscherichia coliexpression, 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 onlyOeCCD1exhibits detectable activity on zeaxanthin;InCCD1shows no in vitro cleavage of zeaxanthin. Kinetic characterization using β-apo-8′-carotenal as substrate revealed, forOeCCD1, a Kmof 0.82 mM, Vmaxof 2.30 U/mg (kcat= 3.35 s−1), and kcat/Kmof 4.09 mM−1·s−1, whereasInCCD1displayed Km= 0.69 mM, Vmax= 1.22 U/mg (kcat= 1.82 s−1), and kcat/Km= 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.