Efficient carbon flux allocation towards D-pantothenic acid production via growth-decoupled strategy in Escherichia coli

[Display omitted] •Integrated systematic bio-engineering with a temperature-sensitive switch to decouple cell growth from DPA production.•Enhanced pyruvate conservation by controlling glucose uptake, EMP, and TCA pathways.•Redirected carbon flux from the TCA cycle by lowering cultivation temperature from 37 °C to 30 °C.•Achieved DPA titer (97.2 g/L) and yield (0.64 g/g glucose) in 5-liter fed-batch fermentation, much higher than those in previous reports. For industrial strain construction, rational allocation of carbon flux is of paramount importance especially for decoupling cell growth and chemical productions to get maximum titer, rate, yield (TRY), which become Gordian Knot. Here, a temperature-sensitive switch and genetic circuits was used for effectively decoupling cell growth from D-pantothenic acid (DPA) production, along with systematically metabolic engineering including blocking redundant pathways of pyruvate and enhancing DPA driving force. Afterwards, rapid biomass accumulation only happened during growth stage, and subsequent high-efficient DPA production was initiated with reducing fermentation temperature. Finally, 97.20 g/L DPA and 0.64 g/g glucose conversion rate were achieved in 5-liter fed-batch fermentation. These undisputedly represent a milestone for the biosynthesis of DPA. With using strategies for decoupling cell growth from chemical productions, it would serve as “Alexander’s sword” to cut Gordian Knot to get industrial chassis cells with excellent TRY for de novo biosynthesis of valuable chemicals.