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Thraustochytrids are abundant and ubiquitous marine protists that are important in global elemental cycling and in supporting oceanic food webs through de novo omega-3 polyunsaturated fatty acid production. Thraustochytrids are also one of few heterotrophic eukaryotes with the capacity to synthesize carotenoids, a class of antioxidative pigments made up of carotenes (e.g., β-carotene) and xanthophylls (e.g., astaxanthin). Heterotrophic production of carotenoids is typically associated with protection against oxidative stress, yet the eco-physiological role and evolutionary origin of thraustochytrid carotenoid production remain elusive. A. limacinum encodes the first three carotenoid biosynthesis-specific enzymes (phytoene synthase, phytoene desaturase, and lycopene cyclase) in a single trifunctional gene, crtIBY. In exploring the evolutionary origins of crtIBY, the most similar proteins were consistently found in a diverse group of protists, including the apusomonad Thecamonas trahens and the dinoflagellates Oxyrrhis marina and Noctiluca scintillans. Phylogenetic analyses suggest that carotenoid biosynthesis in this cluster originated from a lineage containing Actinobacteria, Bacteroidetes, and Archaea (ABA) and that the Halobacteria (Archaea) are likely the source of horizontal gene transfer. These findings reveal the first example of eukaryotic carotenoid biosynthesis with origins from the ABA lineage. To address the functional significance of thraustochytrid carotenogenesis, a transformation system enabling genetic manipulation of A. limacinum was developed using a bleomycin resistance gene (BleoR, ShBle), with a native GAPDH promoter and terminator system. To create a non-carotenogenic A. limacinum strain, inactivation of crtIBY was achieved by targeted insertion of the bleomycin resistance cassette. The resulting non-pigmented strains were stable and did not show a consistent growth disadvantage, revealing that carotenoids are not essential under typical lab growth conditions. Wildtype and non-carotenogenic A. limacinum strains were grown under different conditions to gain insight into the regulation of carotenoid accumulation. Wildtype cells showed greater carotenoid accumulation in nutrient-rich media and in agar plates containing methylene blue, which produces singlet oxygen when exposed to light. I speculate that carotenoid accumulation in A. limacinum is regulated as part of an oxidative stress response experienced in nutrient-rich media and by singlet oxygen, such as what A. limacinum may experience in its native habitat of decomposing mangrove leaves.