Isorenieratene biosynthesis in green sulfur bacteria requires the cooperative actions of two carotenoid cyclases

The cyclization of lycopene to gamma- or beta-carotene is a major branch point in the biosynthesis of carotenoids in photosynthetic bacteria. Four families of carotenoid cyclases are known, and each family includes both mono- and dicyclases, which catalyze the formation of gamma- and beta-carotene, respectively. Green sulfur bacteria (GSB) synthesize aromatic carotenoids, of which the most commonly occurring types are the monocyclic chlorobactene and the dicyclic isorenieratene. Recently, the cruA gene, encoding a conserved hypothetical protein found in the genomes of all GSB and some cyanobacteria, was identified as a lycopene cyclase. Further genomic analyses have found that all available fully sequenced genomes of GSB encode an ortholog of cruA. Additionally, the genomes of all isorenieratene-producing species of GSB encode a cruA paralog, now named cruB. The cruA gene from the chlorobactene-producing GSB species Chlorobaculum tepidum and both cruA and cruB from the brown-colored, isorenieratene-producing GSB species Chlorobium phaeobacteroides strain DSM 266(T) were heterologously expressed in lycopene-and neurosporene-producing strains of Escherichia coli, and the cruB gene of Chlorobium clathratiforme strain DSM 5477(T) was also heterologously expressed in C. tepidum by inserting the gene at the bchU locus. The results show that CruA is probably a lycopene monocyclase in all GSB and that CruB is a gamma-carotene cyclase in isorenieratene-producing species. Consequently, the branch point for the synthesis of mono-and dicyclic carotenoids in GSB seems to be the modification of gamma-carotene, rather than the cyclization of lycopene as occurs in cyanobacteria.