For many years it was accepted that isopentenyl diphosphate, the common precursor of all isoprenoids, was synthesized through the well known acetate͞mevalonate pathway. However, recent studies have shown that some bacteria, including Escherichia coli, use a mevalonate-independent pathway for the synthesis of isopentenyl diphosphate. The occurrence of this alternative pathway has also been reported in green algae and higher plants. The first reaction of this pathway consists of the condensation of (hydroxyethyl)thiamin derived from pyruvate with the C1 aldehyde group of D-glyceraldehyde 3-phosphate to yield D-1-deoxyxylulose 5-phosphate. In E. coli, D-1-deoxyxylulose 5-phosphate is also a precursor for the biosynthesis of thiamin and pyridoxol. Here we report the molecular cloning and characterization of a gene from E. coli, designated dxs, that encodes D-1-deoxyxylulose-5-phosphate synthase. The dxs gene was identified as part of an operon that also contains ispA, the gene that encodes farnesyl-diphosphate synthase. D-1-Deoxyxylulose-5-phosphate synthase belongs to a family of transketolase-like proteins that are highly conserved in evolution.Isoprenoids are ubiquitous compounds found in all living organisms. Some isoprenoids play essential roles in particular cell functions such as sterols, contributing to eukaryotic membrane architecture, acyclic polyprenoids found in the side chain of ubiquinone, plastoquinone, and chlorophylls, sugar carriers for polysaccharide biosynthesis, or carotenoids in photosynthetic organisms. Although the physiological role of other isoprenoids is less evident, like that of the vast array of plant secondary metabolites, some are known to play key roles in the adaptative responses to different environmental challenges. In spite of the remarkable diversity of structure and function, all isoprenoids originate from a single metabolic precursor, isopentenyl diphosphate (1, 2).For many years, it was accepted that isopentenyl diphosphate was synthesized through the well known acetate͞mevalonate pathway. However, recent studies have demonstrated that the mevalonate-dependent pathway does not operate in all living organisms (3, 4). An alternative mevalonate-independent pathway for isopentenyl diphosphate biosynthesis was initially characterized in bacteria (4, 5) and later also in green algae (6) and higher plants (7-11). The first reaction of the novel mevalonateindependent pathway involves the condensation of (hydroxyethyl)thiamin derived from pyruvate with the C1 aldehyde group of D-glyceraldehyde 3-phosphate to yield D-1-deoxyxylulose 5-phosphate (5, 12). In Escherichia coli, D-1-deoxyxylulose (most likely in the form of D-1-deoxyxylulose 5-phosphate) is efficiently incorporated into the prenyl side chain of menaquinone and ubiquinone (12,13). In plants, the incorporation of D-1-deoxyxylulose into isoprenoids has also been reported (11,14). In addition, D-1-deoxyxylulose has also been described as a precursor for the biosynthesis of thiamin and pyridoxol. D-1-Deoxyxylulose is the...