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

Abstract

The isolation of the isopentenyl diphosphate (IPP) isomerase (EC 5.3.3.2) encoding gene (idi) of Phaffia rhodozyma by a new and direct selection procedure in a carotenogenic Escherichia coli strain is described. Isopentenyl diphosphate (IPP) isomerase is a key enzyme in the isoprenoid biosynthetic pathway which catalyses the interconversion of the primary five—carbon diphosphate building blocks dimethylallyl diphosphate (DMAPP) and IPP. Our results imply that this method should be generally useful for the isolation of IPP isomerase encoding genes in both genomic and cDNA libraries from other organisms. The structural gene comprises 1294 nucleotides encoding a 28.7 kDa polypeptide of 251 amino acids. Analysis of the nucleotide and amino acid sequence indicates clear homology to IPP isomerases of non—carotenogenic yeasts, Schizosaccharomyces pombe (46%) and Saccharomyces cerevisiae (42%). By high level expression of the heterologous IPP isomerase gene in a recombinant E. colia 3-fold increase in lycopene production was achieved. This indicates the potentials of this idi gene to improve carotenoid biosynthesis by metabolic pathway engineering in homologous and heterologous hosts.