Production of recombinant proteins is an industrially important technique in the biopharmaceutical sector. Many recombinant proteins are problematic to generate in a soluble form in bacteria as they readily form insoluble inclusion bodies. Recombinant protein solubility can be enhanced by minimising stress imposed on bacteria through decreasing growth temperature and the rate of recombinant protein production. In this study, we determined whether these stress-minimisation techniques can be successfully applied to industrially relevant high cell density Escherichia coli fermentations generating a recombinant protein prone to forming inclusion bodies, CheY-GFP. Flow cytometry was used as a routine technique to rapidly determine bacterial productivity and physiology at the single cell level, enabling determination of culture heterogeneity. We show that stress minimisation can be applied to high cell density fermentations (up to a dry cell weight of >70 g L(-1)) using semi-defined media and glucose or glycerol as carbon sources, and using early or late induction of recombinant protein production, to produce high yields (up to 6 g L(-1)) of aggregation-prone recombinant protein in a soluble form. These results clearly demonstrate that stress minimisation is a viable option for the optimisation of high cell density industrial fermentations for the production of high yields of difficult-to-produce recombinant proteins, and present a workflow for the application of stress-minimisation techniques in a variety of fermentation protocols.

Recombinant protein production in bacterial hosts is a commercially important process in the pharmaceutical industry. Optimisation of such processes is of critical importance for process productivity and reproducibility. Here, flow cytometry methods were developed to assess characteristics of bacteria during two process steps that are infrequently studied: agar plate culture and liquid culture set-up. During storage on agar plates, three discrete populations of varying green fluorescence intensity were observed along with a progressive shift of cells from the high green fluorescence population to an intermediate green fluorescence population, observed to be due formation of amyloid inclusion bodies. The dynamics of cellular fluorescence and scatter properties upon setup of liquid cultures were also assessed. These methods have the potential to improve the development of fermentation set-up, a currently little-understood area.