Functional involvement of a deoxy-D-xylulose 5-phosphate reductoisomerase gene harboring locus of Synechococcus leopoliensis in isoprenoid biosynthesis

The present work aimed to proof the functionality of the non-mevalonate pat hway in cyanobacteria, It was intended to isolate the 1-deoxy-D-xylulose 5- phosphate (DXP) reductoisomerase gene (dxr), as this gene encodes the enzym e which catalyzes a pathway-specific, indicative step of this pathway, For this purpose, a segment of dsr was amplified from Synechococcus leopoliensi s SAUG 1402-1 DKA via PCR using oligonucleotides for conserved regions, Sub sequent hybridization screening of a genomic cosmid library of S, leopolien sis with the PCR segment led to the identification of a 26.5 kbp locus on w hich a dxr homologous gene and two adjacent open reading frames organized i n one operon were localized by DNA sequencing, The functionality of the gen e was demonstrated expressing the gene in Escherichia coli and using the pu rified gene product in a photometrical NADPH dependent test based on the su bstrate DXP generating system. While the content of one of the central inte rmediates of the isoprenoid biosynthesis (dimethylallyl diphosphate = DMADP ) was significantly (P less than or equal to 0.001) increased in E, coli ce lls overexpressing the DXP synthase gene (dxs) of S, leopoliensis, overexpr ession of dxr does not lead to an elevated DMADP level. Since even in strai ns harboring an expression fusion of dxs the additional overexpression of d xr does not influence the DMADP content, it is concluded that Dxs but not D xr catalyzes a rate limiting step of the non-mevalonate isoprenoid biosynth esis, (C) 2000 Federation of European Biochemical Societies. Published by E lsevier Science B.V. All rights reserved.