Bixin, also known as annatto, is a seed-specific pigment widely used in foods and cosmetics since pre-Columbian times. We show that three genes from Bixa orellana, native to tropical America, govern bixin biosynthesis. These genes code for lycopene cleavage dioxygenase, bixin aldehyde dehydrogenase, and norbixin carboxyl methyltransferase, which catalyze the sequential conversion of lycopene into bixin. Introduction of these three genes in Escherichia coli engineered to produce lycopene induced bixin synthesis, thus expanding the supply of this economically important plant product.

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Phytoene is the frst C40 intermediate in the biogenesis of carotenoids. It is formed by two enzyme activities, catalyzing (i) the coupling of two molecules of geranylgeranyl diphosphate to yield prephytoene diphosphate and (i) the conversion of prephytoene diphosphate into phytoene. We show now, with Capsicum chromoplast stroma, that the overall activity resides in a single protein, which has been purified to homogeneity by affinity chromatography. The monomeric structure and the molecular size (Mr 47,500) were demonstrated by NaDodSO4/PAGE and glycerol gradient centrifugation. Further characterization was achieved by using specific antibodies which allowed immunofractionation and immunoprecipitation of the enzymatic activity from chromoplast stroma. The two reactions followed conventional MichaelisMenten kinetics, with Km values of 0.30 ,.M and 0.27 IAM, respectively, for geranylgeranyl diphosphate and prephytoene diphosphate. The activity of the enzyme depends strictly upon the presence ofMn2. This selectivity may be one of the factors regulating the competition with potentially rival enzymes converting geranylgeranyl diphosphate into other plastid terpenoids. The two enzymatic reactions were inhibited by inorganic pyrophosphate and by the arginine-specific reagent hydroxyphenylglyoxal. In no instance were the two reactions kinetically uncoupled. These properties strongly suggest that the same enzyme catalyzes the two consecutive reactions, and we propose to name it phytoene synthase.Carotenoids are required for the normal differentiation and function of plastids. Phytoene, the first C40 intermediate in the carotenoid pathway, is synthesized by a soluble enzymatic complex localized in plastid stroma (1)(2)(3)(4)(5), by the multistep process outlined in Scheme I. The early steps, for which the individual enzymes were recently isolated and purified (6), are the isomerization of isopentenyl diphosphate (1) into dimethylallyl diphosphate (2), followed by three prenylations forming successively geranyl diphosphate (3), farnesyl diphosphate (4), and geranylgeranyl diphosphate (5). This sequence of reactions is common to all terpenoids, including other plastid terpenoids (chlorophylls, plastoquinones, tocopherols, phylloquinones, and polyterpenes). On the other hand, the next two steps, in which prephytoene diphosphate (6) and phytoene (7) are formed, are catalyzed by the first carotenoid-specific enzymes in the pathway, tentatively termed "geranylgeranyl pyrophosphate geranylgeranyltransferase" and "phytoene synthase" (7) since they have been neither isolated nor characterized previously from any other source (5,8,9). Therefore, the identification of the enzyme components catalyzing these two steps is crucial for our understanding of the molecular mechanism of carotenoid biosynthesis and of its regulation; a particularly interesting The present paper answers this question. We show, with Capsicum chromoplasts, previously selected for studies of carotenogenesis (3,(10)(11)(12), that a single monomeric protein ...