Kaspar Valgepea, Ranno Nahku, Petri-Jaan Lahtvee, Liisa Arike, Kaarel Adamberg, and Raivo Vilu. Competence Center of Food and Fermentation Technology, Akadeemia tee 15B, Tallinn, Estonia
Acetate is a major byproduct in aerobic fermentation of Escherichia coli growing on glucose. Accumulation of acetate decreases maximum specific growth rate (µ) and recombinant protein production, but the mechanism of action is still not completely understood. To gain better insights into overflow metabolism, a comprehensive study to characterize acetate metabolism at the level of transcriptome, proteome and metabolome was conducted. Accelerostats are powerful cultivation methods for identifying metabolic switch points. Experiments with smooth change of µ (A-stat) and continuous addition of acetate at constant glucose consumption and dilution rate (D-stat) were carried out. The overflow switch in batch is surmised to be regulated by carbon catabolite repression as supported also in this study – acetate consumption in D-stat at µ=0.2 h-1 and no utilization at above overflow switch conditions – µ=0.5 h-1. Based on intra- and extracellular component analysis there might be several switch points between turning from fully respirative to respirofermentative metabolism. Moreover, A-stat enabled to precisely link the onset of overflow metabolism to the down-regulation of TCA (decrease in CO2-yield and gene expression). Carbon repressor protein activity was directly measured by cAMP levels, which started to decrease at µ=0.37 h-1 while acetate excretion started at µ=0.27 h-1 showing more complicated control of overflow. Switch mechanisms in acetate metabolic pathways have been postulated. Well reproducible A-stat provided also good overlap in transcriptome and proteome towards increase in µ. Therefore, continuous monitoring of quasi steady state culture parameters is valuable for the characterization of metabolism regulation.
