Single cell dynamics in bacterial populations for the production of heterologous proteins

Abstract

Rising healthcare costs and increased population longevity have urged research and the development of methods for the production of biopharmaceuticals. A third of the total sale of biopharma are microbial recombinant products. Recently, a central feature of the microbial populations in fermenters is that of heterogeneity at single cell level. Considering the cellular heterogeneity, population level analysis used for bioprocess optimisation and control is misleading. Individual biological entities, their properties and their interaction with one another and the environment determine the overall behaviour monitored at population level. As far as protein production, the cellular variability is important for industry, because the yield affects the bioprocess’ profitability and viability. To predict cellular behaviour, cells in balanced growth are required. The fermentation methods that give balanced growth are batch and continuous. The present thesis investigates heterologous protein production in E. coli at single cell level. Most measurements at single cell level to unravel heterogeneity use fluorescence, hence the expression system includes green fluorescent protein (GFP). The main areas of this thesis are fermentations characteristics at single cell level in host and recombinant E. coli at different growth rates on glucose or glycerol substrate. The techniques used to quantify heterogeneity are flow and image cytometry. This work gives new insights into the cellular heterogeneity that exists in bioreactors and the extent of which it affects the bioprocess. Focusing on cellular physiology and metabolism of cells used for recombinant protein production, this work may further facilitate the use of bacteria as cell factories. Keywords: Escherichia coli, Single Cell, Flow Cytometry, Microscopy