Identification, Cloning, and Functional Characterization of Carotenoid Cleavage Dioxygenase (CCD) from <i>Olea europaea</i> and <i>Ipomoea nil</i>
The aromatic C<sub>13</sub> 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....
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2025-06-01
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| author | Kaixuan Ke Yufeng Zhang Xinyi Wang Zhaoyan Luo Yangyang Chen Xianying Fang Linguo Zhao |
| author_facet | Kaixuan Ke Yufeng Zhang Xinyi Wang Zhaoyan Luo Yangyang Chen Xianying Fang Linguo Zhao |
| author_sort | Kaixuan Ke |
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| description | The aromatic C<sub>13</sub> 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<sub>m</sub> of 0.82 mM, V<sub>max</sub> of 2.30 U/mg (k<sub>cat</sub> = 3.35 s<sup>−1</sup>), and k<sub>cat</sub>/K<sub>m</sub> of 4.09 mM<sup>−1</sup>·s<sup>−1</sup>, whereas <i>InCCD1</i> displayed K<sub>m</sub> = 0.69 mM, V<sub>max</sub> = 1.22 U/mg (k<sub>cat</sub> = 1.82 s<sup>−1</sup>), and k<sub>cat</sub>/K<sub>m</sub> = 2.64 mM<sup>−1</sup>·s<sup>−1</sup>. 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. |
| format | Article |
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| spelling | doaj-art-d2eb50477a374a87b407f40863a5e6a02025-08-20T03:13:41ZengMDPI AGBiology2079-77372025-06-0114775210.3390/biology14070752Identification, Cloning, and Functional Characterization of Carotenoid Cleavage Dioxygenase (CCD) from <i>Olea europaea</i> and <i>Ipomoea nil</i>Kaixuan Ke0Yufeng Zhang1Xinyi Wang2Zhaoyan Luo3Yangyang Chen4Xianying Fang5Linguo Zhao6National Key Laboratory for the Development and Utilization of Forest Food Resources, Nanjing Forestry University, Nanjing 210037, ChinaNational Key Laboratory for the Development and Utilization of Forest Food Resources, Nanjing Forestry University, Nanjing 210037, ChinaNational Key Laboratory for the Development and Utilization of Forest Food Resources, Nanjing Forestry University, Nanjing 210037, ChinaNational Key Laboratory for the Development and Utilization of Forest Food Resources, Nanjing Forestry University, Nanjing 210037, ChinaNational Key Laboratory for the Development and Utilization of Forest Food Resources, Nanjing Forestry University, Nanjing 210037, ChinaNational Key Laboratory for the Development and Utilization of Forest Food Resources, Nanjing Forestry University, Nanjing 210037, ChinaNational Key Laboratory for the Development and Utilization of Forest Food Resources, Nanjing Forestry University, Nanjing 210037, ChinaThe aromatic C<sub>13</sub> 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<sub>m</sub> of 0.82 mM, V<sub>max</sub> of 2.30 U/mg (k<sub>cat</sub> = 3.35 s<sup>−1</sup>), and k<sub>cat</sub>/K<sub>m</sub> of 4.09 mM<sup>−1</sup>·s<sup>−1</sup>, whereas <i>InCCD1</i> displayed K<sub>m</sub> = 0.69 mM, V<sub>max</sub> = 1.22 U/mg (k<sub>cat</sub> = 1.82 s<sup>−1</sup>), and k<sub>cat</sub>/K<sub>m</sub> = 2.64 mM<sup>−1</sup>·s<sup>−1</sup>. 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.https://www.mdpi.com/2079-7737/14/7/752carotenoid cleavage dioxygenasesCCD1 familyenzyme characterizationβ-ionone |
| spellingShingle | Kaixuan Ke Yufeng Zhang Xinyi Wang Zhaoyan Luo Yangyang Chen Xianying Fang Linguo Zhao Identification, Cloning, and Functional Characterization of Carotenoid Cleavage Dioxygenase (CCD) from <i>Olea europaea</i> and <i>Ipomoea nil</i> Biology carotenoid cleavage dioxygenases CCD1 family enzyme characterization β-ionone |
| title | Identification, Cloning, and Functional Characterization of Carotenoid Cleavage Dioxygenase (CCD) from <i>Olea europaea</i> and <i>Ipomoea nil</i> |
| title_full | Identification, Cloning, and Functional Characterization of Carotenoid Cleavage Dioxygenase (CCD) from <i>Olea europaea</i> and <i>Ipomoea nil</i> |
| title_fullStr | Identification, Cloning, and Functional Characterization of Carotenoid Cleavage Dioxygenase (CCD) from <i>Olea europaea</i> and <i>Ipomoea nil</i> |
| title_full_unstemmed | Identification, Cloning, and Functional Characterization of Carotenoid Cleavage Dioxygenase (CCD) from <i>Olea europaea</i> and <i>Ipomoea nil</i> |
| title_short | Identification, Cloning, and Functional Characterization of Carotenoid Cleavage Dioxygenase (CCD) from <i>Olea europaea</i> and <i>Ipomoea nil</i> |
| title_sort | identification cloning and functional characterization of carotenoid cleavage dioxygenase ccd from i olea europaea i and i ipomoea nil i |
| topic | carotenoid cleavage dioxygenases CCD1 family enzyme characterization β-ionone |
| url | https://www.mdpi.com/2079-7737/14/7/752 |
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