Impact of phenotypic heterogeneity and metabolic specialisation on metabolic engineering strategies: case of study of E.coli as a representative microbial cell factory

The goal of this research is to directly highlight the simultaneous occurrence of several phenotypes with distinct metabolic function among clonal population of E. coli., often used for recombinant protein and pDNA production in bioprocesses. This phenotypic heterogeneity, first due to the noise, is reinforced by environmental heterogeneities occurring at large scale during fed-batch processes. This phenotypic heterogeneity has been tracked according to GFP reporter strains (biosensors) that circulate in the bioreactor and encounter environmental heterogeneities. First, we have highlighted the simultaneous occurrence of several phenotypes with distinct metabolic functions. Indeed, a diversity of glucose uptake strategies has recently been noticed with the PtsG and MglABC transporters. Moreover, when E.coli encounters zones of glucose excess, acetate is produced through the overflow metabolism. Only the sub-population with high acs expression (acetate transporter) could consume this acetate. GFP reporter strains have thus been constructed for PtsG, MgIABC and acs genes. In addition of the stable GFP, two destabilized GFP variants for each gene have been used to obtain more instantaneous responses. The response of these biosensors have been followed by on-line flow cytometry. In the end, this experimental strategy for direct phenotyping at the single cell level will also be used to investigate the impact of metabolic engineering strategies on phenotypic heterogeneity, robustness and fitness of microbial population in industrial conditions