Tunable recombinant protein production with E. coli in a mixed feed environment

Abstract

Escherichia coli (E. coli) is an organism intensely used for recombinant protein expression. However, to get industrial relevant applications intensive strain development work and vector optimization is needed. In E. coli with pBAD promoter L-Arabinose is working as inducer and typically D-Glucose as sole substrate, because mainly strains, not able to metabolize L-Arabinose, are used. L-Arabinose metabolizing strains allow a "mixed feed" approach meaning that both substrates are administered and metabolized simultaneously. "Mixed feed" bioprocesses were already investigated with Pichia Pastoris and concluded in immense improvement of the processes; however mixed feed approaches are entirely unexplored for recombinant bacterial expression systems. Using pBAD expression system in an L-Arabinose metabolizing strain with a mixed feed approach promises widespread application in expression of complex recombinant products as transcription could be regulated by varying specific L-arabinose uptake rates. So this should give the opportunity to control transcription and specific growth independently and consequently the metabolic burden would be reduced. Using green fluorescent protein (GFP) as model product, the system will be thoroughly characterized: "All or none" induction (subpopulations of producing and non-producing cells) is a potential problem. Detections of subpopulations of producing and non producing cells will be done by flow cytometric measurements. The impact of the specific D-glucose and L-arabinose uptake rates on productivity, titer and time space yield will be investigated. This will be done using a multivariate study with specific D-Glucose (only substrate) and specific L-arabinose (substrate and inducer) uptake rates as factors. Experiments with E. coli expressing the industrial relevant enzyme pyranose 2-oxidase (P2O) are going to be carried out to deepen the system knowledge and especially find out, how process parameters influence inclusion bodies (IBs) formation and further on if the formation can be prevented. The thorough investigation of the pBAD mixed feed bioprocess across different products will lead to useful extrapolations of optimal process parameters for other products. Hence, the thesis will provide the basis for the establishment of pBAD mixed feed bioprocesses as versatile and highly efficient platform expression system for a wide spectrum of recombinant products.