The biosynthesis of the aromatic carotene isorenieratene is restricted to green photosynthetic bacteria and a few actinomycetes. Among them Streptomyces griseus has been used to study the genes involved in this pathway. Five genes out of seven of two adjacent operons in one cluster could be identified to be sufficient for the synthesis of isorenieratene. Stepwise deletions of these genes demonstrated their participation in phytoene synthesis, phytoene desaturation and lycopene cyclization. The novel gene crtU was assigned to encode a unique desaturase responsible for the conversion of beta-carotene via beta-isorenieratene to isorenieratene by a desaturation/methyltransferation mechanism. Sequence analysis of crtU revealed two conserved regions, one at the N-terminus and the other at the C-terminus of the protein which is universal to different types of carotene desaturases. In addition, the sequence comprises a motif typically found in methyltransferases. The deletion of the two remaining genes of the cluster left the carotenoid biosynthetic pathway unaffected.