Functional Analysis of Carotenoid Biosynthetic Genes and Pathway of the Filamentous Anoxygenic Phototrophic Bacterium Chloroflexus aurantiacus

Background/Objectives: Carotenoids play essential roles in light harvesting, photoprotection, and oxidative stress defense in photosynthetic organisms. The filamentous anoxygenic phototroph Chloroflexus aurantiacus synthesizes diverse carotenoids; however, the biosynthetic basis and regulation of these pigments are unclear. Methods: Carotenoid compositions of Cfx. aurantiacus grown under various conditions were analyzed by HPLC. Furthermore, candidate carotenoid biosynthesis genes from Cfx. aurantiacus were hetero-logously expressed in Escherichia coli to analyze their functions. Results: We examined carotenoid composition in cells grown under anaerobic light, aerobic dark, and aerobic light conditions, and found that the major carotenoid species changed markedly in response to oxygen availability. Under anaerobic photosynthetic conditions, γ-carotene, β-carotene, and hydroxylated γ-carotene derivatives were predominant, whereas aerobic growth induced the accumulation of keto-carotenoids, including canthaxanthin and echinenone-related compounds. Genome mining revealed candidate carotenoid biosynthesis genes, including three crtI paralogs and genes encoding enzymes involved in cyclization, hydroxylation, glucosylation, esterification, and ketolation. Heterologous expression in E. coli revealed that CrtI1 acts as a phytoene desaturase, CrtY catalyzes cyclization of lycopene to β-carotene via γ-carotene, CruF functions as a 1,2-hydratase, CruC as a carotenoid glucosyltransferase, CruD as an acyltransferase involved in esterification, and CrtO as a β-carotene ketolase. Conclusions: These results establish a carotenoid biosynthetic pathway in Cfx. aurantiacus and show that oxygen-dependent transcriptional regulation, particularly of CrtO, contributes to the selective production of keto-carotenoids under aerobic conditions. Our findings provide new insight into carotenoid metabolism in filamentous anoxygenic phototrophs and the adaptive physiological strategies that support growth under contrasting environmental conditions.