EARLY STEPS IN CAROTENOID BIOSYNTHESIS - SEQUENCES AND TRANSCRIPTIONAL ANALYSIS OF THE CRTI AND CRTB GENES OF RHODOBACTER-SPHAEROIDES AND OVEREXPRESSION AND REACTIVATION OF CRTI IN ESCHERICHIA-COLI AND R-SPHAEROIDES

In the purple photosynthetic bacterium Rhodobacter sphaeroides, the de saturation of phytoene has already been implicated in the assembly of the light-harvesting 2 complex (H. P. Lang and C. N. Hunter, Biochem. J. 298:197-205, 1994). The phytoene synthase and desaturase enzymes me diate the first steps specific for carotenoid biosynthesis up to and i ncluding the synthesis of the colored carotenoid neurosporene. In this report,we present the DNA and deduced amino acid sequences of the gen es encoding these proteins, namely, crtB and crtl, from R. sphaeroides and present evidence for the existence of a crtIB operon. Both genes have been shown to possess putative puc and puf operon-like promoter s equences, and oxygen regulation anal the point of initiation of the cr tl transcript have been demonstrated. The complete crtl gene has been overexpressed in Escherichia coli and R. sphaeroides and shown to cata lyze three desaturations of phytoene to give neurosporene. This activi ty was shown to be ATP dependent, and the cofactor requirement was inv estigated by using a spectroscopic assay for in vitro carotenogenic ac tivity. Although the crtl and crtB genes have been sequenced from a nu mber of different organisms, the transcriptional organization and regu lation of these genes have not been analyzed in detail. In this report , we have located the transcription initiation point and have shown th at R. sphaeroides possesses an oxygen-regulated CrtI-type phytoene,,de saturase gene that forms a transcriptional operon with crtB.