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

## TL;DR

This study identifies and characterizes two enzymes from different plants that can produce the valuable aroma compound β-ionone, offering new tools for its sustainable production.

## Contribution

The study provides new CCD1 enzymes from Olea europaea and Ipomoea nil with distinct substrate specificities and biochemical properties for β-ionone production.

## Key findings

- Both OeCCD1 and InCCD1 cleave β-carotene to produce β-ionone, but only OeCCD1 acts on zeaxanthin.
- OeCCD1 shows higher catalytic efficiency than InCCD1 based on kinetic parameters.
- The study reveals functional diversity in CCD1 enzymes across different plant species.

## Abstract

β-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 from Olea europaea and Ipomoea nil. By expressing these genes in E. 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 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: OeCCD1 from Olea europaea and InCCD1 from Ipomoea 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 for Escherichia coli 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 OeCCD1 exhibits detectable activity on zeaxanthin; InCCD1 shows no in vitro cleavage of zeaxanthin. Kinetic characterization using β-apo-8′-carotenal as substrate revealed, for OeCCD1, a Km of 0.82 mM, Vmax of 2.30 U/mg (kcat = 3.35 s−1), and kcat/Km of 4.09 mM−1·s−1, whereas InCCD1 displayed 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.