AbstractThe five‐carbon metabolic intermediate isopentenyl diphosphate constitutes the basic building block for the biosynthesis of all isoprenoids in all forms of life. Two distinct pathways lead from amphibolic intermediates to isopentenyl diphosphate. The Gram‐positive cocci and certain other pathogenic bacteria employ exclusively the mevalonate pathway, a set of six enzyme‐catalyzed reactions that convert 3 mol of acetyl‐CoA to 1 mol each of carbon dioxide and isopentenyl diphosphate. The survival of the Gram‐positive cocci requires a fully functional set of mevalonate pathway enzymes. These enzymes therefore represent potential targets of inhibitors that might be employed as antibiotics directed against multidrug‐resistant strains of certain bacterial pathogens. A rapid throughput, bioreactor‐based assay to assess the effects of potential inhibitors on several enzymes simultaneously should prove useful for the survey of candidate inhibitors. To approach this goal, and as a proof of concept, we employed enzymes from the Gram‐positive pathogen Enterococcus faecalis. Purified recombinant enzymes that catalyze the first three reactions of the mevalonate pathway were immobilized in two kinds of continuous flow enzyme bioreactors: a classical hollow fiber bioreactor and an immobilized plug flow bioreactor that exploited a novel method of enzyme immobilization. Both bioreactor types employed recombinant acetoacetyl‐CoA thiolase, HMG‐CoA synthase, and HMG‐CoA reductase from E. faecalis to convert acetyl‐CoA to mevalonate, the central intermediate of the mevalonate pathway. Reactor performance was monitored continuously by spectrophotometric measurement of the concentration of NADPH in the reactor effluent. Additional potential applications of an Ni++ affinity support bioreactor include using recombinant enzymes from extremophiles for biosynthetic applications. Finally, linking a Ni++ affinity support bioreactor to an HPLC‐mass spectrometer would provide an experimental and pedagogical tool for study of metabolite flux and pool sizes of intermediates to model regulation in intact cells. © 2004 Wiley Periodicals, Inc.