Optimizing carotenoid cleavage dioxygenase 4 (CCD4) for enhanced β-ionone production in Nicotiana tabacum

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.