Phylogeny-guided discovery of a crocetin dialdehyde-producing carotenoid cleavage dioxygenase from Paulownia tomentosa

[EN] Carotenoid cleavage dioxygenases (CCDs) mediate the oxidative cleavage of carotenoids, producing apocarotenoids with diverse biological functions and industrial relevance. Phylogenetic analysis of CCDs from Paulownia tomentosa identified CCD4-2 as a candidate enzyme potentially involved in crocetin biosynthesis. CCD4-2 clustered closely with functionally characterized CCD4 enzymes from Buddleja and Verbascum (73.7% and 70.0% identity, respectively), both of which cleave carotenoids at the C7-C8 and C7 '-C8 ' double bonds to produce crocetin dialdehyde. In silico predictions and subcellular localization studies, supported that P. tomentosa CCD4-2 is plastid-targeted and likely interacts with membrane-bound carotenoid pools. Functional assays demonstrated that CCD4-2 cleaves specifically beta-carotene at C7-C8 and C7 '-C8 ' but does not act on linear or asymmetric carotenoids. Although beta-carotene was abundant in P. tomentosa leaves and flowers, crocins were not detected, likely due to substrate competition and the high expression of other CCD enzymes, particularly CCD1 and CCD4-4, which may divert beta-carotene toward alternative cleavage pathways or products. To verify the catalytic function in planta, CCD4-2 was expressed using a viral vector in Nicotiana benthamiana. In this lowcompetition background, crocins accumulation reached values close to 4 mg/g dry weight, confirming the cleavage activity of CCD4-2. These findings underscore the presence of catalytically valuable CCD enzymes within plant genomes and demonstrate how prior knowledge of CCD enzymatic activity and phylogenetic relationships can facilitate the identification of novel candidates for the biotechnological production of high-value apocarotenoids.