Identification of genes affecting lycopene formation in Escherichia coli transformed with carotenoid biosynthetic genes: candidates for early genes in isoprenoid biosynthesis.

Although isopentenyl diphosphate is a precursor of isoprenoids in Escherichia coli, the genes and enzymes involved in its biosynthesis have not been identified. Thus, we tried to isolate E. coli mutants deficient in the biosynthesis and their complementary genes by use of an artificial phenotypic screening system employing three carotenoid biosynthetic genes, crtE, crtB, and crtI. Cells were mutagenized with ethylmethanesulfonate, then transformed with a plasmid for expression of the carotenogenic genes. Mutants deficient in biosynthesis of isopentenyl diphosphate were expected to form white colonies, because they are unable to produce enough lycopene, whereas wild-type cells form red colonies. Among large numbers of red colonies, we identified 117 white colonies. Next, we transformed each mutant with an E. coli genomic library. Twenty-nine complementary genes that restore red color of host colonies were isolated. A homology search and further complementation study using subcloned genes revealed that the true complementary genes encode isopentenyl diphosphate isomerase, subunits of ATP synthase, enzymes of the Krebs cycle, some aldehyde dehydrogenases, phosphate acetyltransferase, and enzymes which relate to the biosynthesis of ubiquinones and menaquinones. Two unknown genes were also found, designated elb1 and 2, which may be involved in the early steps of isoprenoid biosynthesis.