Carotenoid cleavage oxygenases are nonheme iron enzymes that specifically cleave carbon-carbon double bonds of carotenoids. Their apocarotenoid cleavage products serve as important signaling molecules that are involved in various biological processes. A database search revealed the presence of putative carotenoid cleavage oxygenase genes in the genomes of Sphingopyxis alaskensis RB2256 and Plesiocystis pacifica SIR-1. The four genes sala_1698, sala_1008, ppsir1_15490 and ppsir1_17230 were cloned and heterologously expressed in carotenoid-producing Escherichia coli JM109 strains. Two of the four encoded proteins exhibited carotenoid cleavage activity. S. alaskensis RB2256 carotenoid cleavage oxygenase (SaCCO), which is encoded by sala_1698, was shown to cleave acyclic and monocyclic substrates. Coexpression of sala_1698 in carotenoid-producing E. coli JM109 strains revealed cleavage activity for lycopene, hydroxylycopene, and dihydroxylycopene. The monocyclic substrate apo-8'-carotenal was cleaved in vitro by purified SaCCO at the 9'/10' and 11'/12' double bonds. The second enzyme, P. pacifica SIR-1 carotenoid cleavage oxygenase (PpCCO), is encoded by ppsir1_15490. PpCCO-mediated carotenoid cleavage requires the presence of either hydroxy or keto groups. PpCCO cleaved zeaxanthin, hydroxylycopene, and dihydroxylycopene, and also the C(50) carotenoids decaprenoxanthin, sarprenoxanthin and sarcinaxanthin, in carotenoid-producing E. coli JM109 strains. Whole cells of E. coli JM109 overexpressing ppsir1_15490mut, a mutant of ppsir1_15490 with enhanced gene expression, were applied for the conversion of carotenoids. Analysis of the carotenoid cleavage products revealed a single cleavage site at the 13'/14' double bond for astaxanthin, and two cleavage sites at the 11'/12' or 13'/14' double bond for zeaxanthin, nostoxanthin, and canthaxanthin.