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 pathway in cyanobacteria, It was intended to isolate the 1-deoxy-D-xylulose 5-phosphate (DXP) reductoisomerase gene (dxr), as this gene encodes the enzyme which catalyzes a pathway-specific, indicative step of this pathway, For this purpose, a segment of dsr was amplified from Synechococcus leopoliensis SAUG 1402-1 DKA via PCR using oligonucleotides for conserved regions, Subsequent hybridization screening of a genomic cosmid library of S, leopoliensis with the PCR segment led to the identification of a 26.5 kbp locus on which a dxr homologous gene and two adjacent open reading frames organized in one operon were localized by DNA sequencing, The functionality of the gene was demonstrated expressing the gene in Escherichia coli and using the purified gene product in a photometrical NADPH dependent test based on the substrate DXP generating system. While the content of one of the central intermediates of the isoprenoid biosynthesis (dimethylallyl diphosphate = DMADP) was significantly (P less than or equal to 0.001) increased in E, coli cells overexpressing the DXP synthase gene (dxs) of S, leopoliensis, overexpression of dxr does not lead to an elevated DMADP level. Since even in strains harboring an expression fusion of dxs the additional overexpression of dxr does not influence the DMADP content, it is concluded that Dxs but not Dxr catalyzes a rate limiting step of the non-mevalonate isoprenoid biosynthesis, (C) 2000 Federation of European Biochemical Societies. Published by Elsevier Science B.V. All rights reserved.