Scalable, two‐stage, autoinduction of recombinant protein expression in E. coli utilizing phosphate depletion

We report the scalable production of recombinant proteins in Escherichia coli, reliant on tightly controlled autoinduction, triggered by phosphate depletion in the stationary phase. The method, reliant on engineered strains and plasmids, enables improved protein expression across scales. Expression levels using this approach have reached as high as 55% of the total cellular protein. The initial use of the method in instrumented fed‐batch fermentations enables cell densities of ∼30 gCDW/L and protein titers up to 8.1 ± 0.7 g/L (∼270 mg/gCDW). The process has also been adapted to an optimized autoinduction media, enabling routine batch production at culture volumes of 20 μl (384‐well plates), 100 μl (96‐well plates), 20 ml, and 100 ml. In batch cultures, cell densities routinely reach ∼5–7 gCDW/L, offering protein titers above 2 g/L. The methodology has been validated with a set of diverse heterologous proteins and is of general use for the facile optimization of routine protein expression from high throughput screens to fed‐batch fermentation. Highlights Stationary phase protein expression results in high titers. Autoinduction by phosphate depletion enables protein titers from 2 to 8 g/L. Autoinduction has been validated from 384‐well plates to instrumented bioreactors. We report the scalable production of recombinant proteins in E. coli, reliant on autoinduction, triggered by phosphate depletion in stationary phase. In fed batch fermentations protein titers up to 8.1+/−0.7 g/L (˜270 mg/gCDW) are achieved. The process has been adapted to an optimized autoinduction media, enabling routine batch production at culture volumes of 20 μL (384 well plates), 100 μL (96 well plates), 20 mL and 100 mL, offering protein titers above 2 g/L at these smaller scales.