@article{Dong2026, 
author = {Chen Dong and Yu Zhang and Zifu Ni and Mengxin Shen and Faming Lin and Xiaoran Wang and Yongchun Shi and Yuchen Miao and Jinggong Guo and Ran Wang},
title = {Optimizing carotenoid cleavage dioxygenase 4 (CCD4) for enhanced β-ionone production in Nicotiana tabacum},
year = {2026},
journal = {The Crop Journal},
volume = {14},
number = {1},
pages = {214-223},
keywords = {β-Ionone, β-Carotene, Genome editing, Semi rational design},
url = {https://www.sciopen.com/article/10.1016/j.cj.2025.03.011},
doi = {10.1016/j.cj.2025.03.011},
abstract = {Carotenoid cleavage dioxygenase 4 (CCD4) controls the rate-limiting step of β-ionone biosynthesis, making it a valuable target for healthcare and pharmaceutical applications. Nicotiana tabacum, a carotenoid-richd crop species, is a promising source for β-ionone production. This study aimed to modify CCD4 activity to increase β-ionone yield in tobacco. We identified two isoforms of CCD4 in N. tabacum, NtCCD4a and NtCCD4b, with NtCCD4a exhibiting significantly higher expression levels than NtCCD4b. Using solid-phase microextraction gas chromatography-mass spectrometry (SPME-GC–MS), we demonstrated that NtCCD4a effectively catalyzes the cleavage of β-carotene to produce β-ionone. To improve its enzymatic activity, we applied structure-based rational design to reconstruct the active pocket of NtCCD4a, followed by high-throughput screening of mutant variants. Three single base mutants, F181G, F184L, and F337M, in NtCCD4a showed enhanced β-ionone production compared to the wild-type, with F337M yielding the highest amount. No synergistic effects were observed among the three mutants. Transgenic tobacco plants expressing the F181G, F184L, and F337M mutations had accelerated β-carotene cleavage and increased β-ionone production relative to the wild-type NtCCD4a. Our results establish a framework for the design of CCD4 in major crop species through genome editing technology.}
}