Establishment of an efficient isoprene fermentation process by adopting inorganic phosphate limitation as the trigger to direct metabolic flux to the isoprene synthetic pathway.,We constructed isoprene-producing strains of Pantoea ananatis (a member of the Enterobacteriaceae family) by integrating a heterologous mevalonate pathway and a metabolic switch that senses external inorganic phosphate (Pi) levels. This metabolic switch enabled dual-phase isoprene production, where the initial cell growth phase under Pi-saturating conditions was uncoupled from the subsequent isoprene production phase under Pi-limiting conditions. In fed-batch fermentation using our best strain (SWITCH-PphoC/pIspSM) in a 1-l bioreactor, isoprene concentration in the off-gas was maintained between 300 and 460?ppm during the production phase and at 20?ppm during the cell growth phase, respectively. The strain SWITCH-PphoC/pIspSM produced totally 2·5?g?l-1 of isoprene from glucose with a 1·8% volumetric yield in 48?h.,This proof-of-concept study demonstrated that our Pi-dependent dual-phase production system using a P. ananatis strain as a producer has potential for industrial-scale isoprene fermentation.,This Pi-dependent dual-phase fermentation process could be an attractive and economically viable option for the production of various commercially valuable isoprenoids.,? 2019 The Society for Applied Microbiology.