Escherichia coli engineered to synthesize isopentenyl diphosphate and dimethylallyl diphosphate from mevalonate: a novel system for the genetic analysis of the 2-C-methyl-d-erythritol 4-phosphate pathway for isoprenoid biosynthesis

Abstract

Isopentenyl diphosphate (IPP) and its isomer dimethylallyl diphosphate (DMAPP) constitute the basic building block of isoprenoids, a family of compounds that is extraordinarily diverse in structure and function. IPP and DMAPP can be synthesized by two independent pathways: the mevalonate pathway and the recently discovered 2-C-methyl-d-erythritol 4-phosphate (MEP) pathway. Although the MEP pathway is essential in most eubacteria, algae and plants and has enormous biotechnological interest, only some of its steps have been determined. We devised a system suitable for the genetic analysis of the MEP pathway in Escherichia coli. A synthetic operon coding for yeast 5-diphosphomevalonate decarboxylase, human 5-phosphomevalonate kinase, yeast mevalonate kinase and E. coli isopentenyl diphosphate isomerase was incorporated in the chromosome of this bacterium. The expression of this operon allowed the synthesis of IPP and DMAPP from mevalonate added exogenously and complementation of lethal mutants of the MEP pathway. We used this system to show that the ygbP, ychB and ygbB genes are essential in E. coli and that the steps catalysed by the products of these genes belong to the trunk line of the MEP pathway.

YgbQ, a cell division protein in Escherichia coli and Vibrio cholerae , localizes in codependent fashion with FtsL to the division site

“…The gene is in a gene cluster, including the downstream genes ygbP, ygbB, and ygbO, found in many Gram-negative and Gram-positive bacteria. Both ygbP and ygbB are essential genes involved in the pathway for isoprenoid biosynthesis in E. coli (17).…”

“…The genomic organization of this region in V. cholerae and E. coli is similar. In E. coli it is known that the two genes downstream of ygbQ, ygbP, and ygbB are essential (17). To determine if the inability to grow in the absence of arabinose was caused by ygbQ or the downstream genes, a plasmid containing only ygbQ, pNJ25, was constructed.…”

“…Depletion of ygbQ in a strain expressing ygbP, B, and O from a different promoter results in a filamentous phenotype, which indicates that in the ygbQ null, expression of the ygbP, B, and O genes was sufficient for growth (data not shown). Furthermore, the ygbP and ygbB depletion strains described by Campos et al (17) have a depletion phenotype different from the ygbQ depletion strain; they stop growing immediately when the complementing pathway is inactivated, and cells do not filament. These results show that, as in V. cholerae, YgbQ in E. coli is essential, and its depletion results in inhibition of cell division.…”

Structure of 2C-methyl- d -erythritol 2,4- cyclodiphosphate synthase: An essential enzyme for isoprenoid biosynthesis and target for antimicrobial drug development

“…In the fifth stage, directly relevant to the present study, CDP-ME2P is converted to MECP and CMP ( Fig. 1) by MECP synthase, an enzyme that, in E. coli, is encoded by the ispF gene (9,10,(19)(20)(21). The stages by which MECP is converted to IPP are not yet clear, but further details of the DOXP͞MEP pathway are expected to be unveiled in due course.…”

“…Five distinct steps of the DOXP͞MEP pathway have been elucidated (6)(7)(8)(9)(10). The pathway starts with the condensation of pyruvate and glyceraldehyde 3-phosphate to produce DOXP, which is then converted to MEP in reactions catalyzed by DOXP synthase (11,12) and DOXP reductoisomerase (13)(14)(15), respectively.…”