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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.
Plants produce a vast repertoire of lowmolecular-mass natural products used in the food, cosmetic, and pharmaceutical industries. The molecular basis of this diversity is largely unknown, as many of the enzymes and the genes regulating their biosynthesis have not been identified. It has been estimated that 15 to 25% of the plant genome specifies pathways of natural product biosynthesis (1). One such pathway leads to bixin, also known as annatto, a pigment synthesized by a single terrestrial plant, Bixa orellana, which is native to tropical America. Bixin is a dicarboxylic monomethyl ester apocarotenoid that confers red color to seeds of B. orellana (Fig. 1A) from which it has been extracted and used in foods and cosmetics as a soluble color additive (2-4) since pre-Colombian times. From an economic point of view, bixin ranks second among natural color additives used in industry (3).
The mechanism of bixin biosynthesis remains unresolved. However, it has been suggested that a C^sub 40^ carotenoid (5), most probably lycopene, may be the precursor of bixin (6, 7). On the basis of structural similarity between bixin and the saffron pigment crocetin, the reaction could implicate a dioxygenase, an aldehyde dehydrogenase, and a methyltransferase in a series of reactions proceeding sequentially from lycopene (Fig. 1B). This putative pathway is supported by analysis of lycopene cleavage derivatives that accumulate in trace amounts in Bixa seeds (8) and also by the recent characterization of plant dioxygenases that cleave carotenoid chromophore at defined sites. Maize VP14, the first characterized of this series, catalyzes the cleavage of 9-cis-violaxanthin and 9'-cisneoxanthin into xanthoxin, the precursor of abscisic acid (9). Along the same lines, Arabidopis thaliana carotenoid cleavage dioxygenase 1 (AtCCDI) (10) and Crocus sativus zeaxanthin cleavage dioxygenase (CsZCD) (11) catalyze the initial steps of mycorradicin and...