Escherichia coli expressing the Erwinia carotenoid biosynthesis genes, crtE, crtB, crtI and crtY, form yellow-coloured colonies due to the presence of β-carotene. This host was used as a visible marker for evaluating regulatory systems operating in isoprenoid biosynthesis of E. coli. cDNAs enhancing carotenoid levels were isolated from the yeast Phaffia rhodozyma and the green alga Haematococcus pluvialis. Nucleotide sequence analysis indicated that they coded for proteins similar to isopentenyl diphosphate (IPP) isomerase of the yeast Saccharomyces cerevisiae. Determination of enzymic activity confirmed the identity of the gene products as IPP isomerases. The corresponding gene was isolated from the genomic library of S. cerevisiae based on its nucleotide sequence, and was confirmed to have the same effect as the above two IPP isomerase genes when introduced into the E. coli transformant accumulating β-carotene. In the three E. coli strains carrying the individual exogenous IPP isomerase genes, the increases in carotenoid levels are comparable to the increases in IPP isomerase enzyme activity with reference to control strains possessing the endogenous gene alone. These results imply that IPP isomerase forms an influential step in isoprenoid biosynthesis of the prokaryote E. coli, with potential for the efficient production of industrially useful isoprenoids by metabolic engineering.
Isoprenoid biosynthesis in bacteria: a novel pathway for the early steps leading to isopentenyl diphosphate
