Characterization of Carotenoid Cleavage Oxygenase Genes in Cerasus humilis and Functional Analysis of ChCCD1

Carotenoid cleavage oxygenases (CCOs) are key enzymes that function in degrading carotenoids into a variety of apocarotenoids and some other compounds. In this study, we performed genome-wide identification and characterization analysis of CCO genes in Cerasus humilis. Totally, nine CCO genes could be classified into six subfamilies, including carotenoid cleavage dioxygenase 1 (CCD1), CCD4, CCD7, CCD8, CCD-like and nine-cis-epoxycarotenoid dioxygenase (NCED), were identified. Results of gene expression analysis showed that ChCCOs exhibited diverse expression patterns in different organs and in fruits at different ripening stages. To investigate the roles of ChCCOs in carotenoids degradation, enzyme assays of the ChCCD1 and ChCCD4 were performed in Escerichia coli BL21(DE3) that can accumulate lycopene, beta-carotene and zeaxanthin. The prokaryotic expressed ChCCD1 resulted in obvious degradation of lycopene, beta-carotene and zeaxanthin, but ChCCD4 did not show similar functions. To further determine the cleaved volatile apocarotenoids of these two proteins, headspace gas chromatography/mass spectrometer analysis was performed. Results showed that ChCCD1 could cleave lycopene at 5, 6 and 5', 6' positions to produce 6-methy-5hepten-2-one and could catalyze beta-carotene at 9, 10 and 9', 10' positions to generate beta D-ionone. Our study will be helpful for clarifying the roles of CCO genes especially ChCCD1 in regulating carotenoid degradation and apocarotenoid production in C. humilis.