Isolation of the isopentenyl diphosphate isomerase encoding gene of Phaffia rhodozyma; improved carotenoid production in Escherichia coli

The isolation of the isopentenyl diphosphate (IPP) isomerase (EC 5.3.3.2) e ncoding gene (idi) of Phaffia rhodozyma by a new and direct selection proce dure in a carotenogenic Escherichia coli strain is described. Isopentenyl d iphosphate (IPP) isomerase is a key enzyme in the isoprenoid biosynthetic p athway which catalyses the interconversion of the primary five-carbon dipho sphate building blocks dimethylallyl diphosphate (DMAPP) and IPP. Our resul ts imply that this method should be generally useful for the isolation of I PP isomerase encoding genes in both genomic and cDNA libraries from other o rganisms. The structural gene comprises 1294 nucleotides encoding a 28.7 kD a polypeptide of 251 amino acids. Analysis of the nucleotide and amino acid sequence indicates clear homology to IPP isomerases of non-carotenogenic y easts, Schizosaccharomyces pombe (46%) and Saccharomyces cerevisiae (42%). By high level expression of the heterologous IPP isomerase gene in a recomb inant E. coli a 3-fold increase in lycopene production was achieved. This i ndicates the potentials of this idi gene to improve carotenoid biosynthesis by metabolic pathway engineering in homologous and heterologous hosts.