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 Esch erichia coli, the genes and enzymes involved in its biosynthesis have not been identified. Thus, we tried to isolate E. coli mutants deficie nt in the biosynthesis and their complementary genes by use of an arti ficial phenotypic screening system employing three carotenoid biosynth etic genes, crtE, crtB, and crtI, Cells were mutagenized with ethylmet hanesulfonate, then transformed with a plasmid for expression of the c arotenogenic genes. Mutants deficient in biosynthesis of isopentenyl d iphosphate were expected to form white colonies, because they are unab le to produce enough lycopene, whereas wild-type cells form red coloni es. Among large numbers of red colonies, we identified 117 white colon ies. Next, we transformed each mutant with an E. coli genomic library. Twenty-nine complementary genes that restore red color of host coloni es were isolated. A homology search and further complementation study using subcloned genes revealed that the true complementary genes encod e isopentenyl diphosphate isomerase, subunits of ATP synthase, enzymes of the Krebs cycle, some aldehyde dehydrogenases, phosphate acetyltra nsferase, and enzymes which relate to the biosynthesis of ubiquinones and menaquinones. Two unknown genes were also found, designated elb1 a nd 2, which may be involved in the early steps of isoprenoid biosynthe sis.