Perturbation Experiments: Approaches for Metabolic Pathway Analysis in Bioreactors

Abstract

In the last decades, targeted metabolic engineering of microbial cells has become one of the major tools in bioprocess design and optimization. For successful application, a detailed knowledge is necessary about the relevant metabolic pathways and their regulation inside the cells. Since in vitro experiments cannot display process conditions and behavior properly, process data about the cells' metabolic state have to be collected in vivo. For this purpose, special techniques and methods are necessary. Therefore, most techniques enabling in vivo characterization of metabolic pathways rely on perturbation experiments, which can be divided into dynamic and steady-state approaches. To avoid any process disturbance, approaches which enable perturbation of cell metabolism in parallel to the continuing production process are reasonable. Furthermore, the fast dynamics of microbial production processes amplifies the need of parallelized data generation. These points motivate the development of a parallelized approach for multiple metabolic perturbation experiments outside the operating production reactor. An appropriate approach for in vivo characterization of metabolic pathways is presented and applied exemplarily to a microbial L-phenylalanine production process on a 15 L-scale.

Keywords: Constraint-based approaches; Escherichia coli; Glycerol; L-phenylalanine; Metabolic flux analysis; Metabolome quantification; Perturbation experiments; Steady-state experiments.