Metabolic footprint analysis of recombinant Escherichia coli strains during fed-batch fermentations

Abstract

Metabolic footprinting has become a valuable analytical approach for the characterization of phenotypes and the distinction of specific metabolic states resulting from environmental and/or genetic alterations. The metabolic impact of heterologous protein production in Escherichia coli cells is of particular interest, since there are numerous cellular stresses triggered during this process that limit the overall productivity, e.g. the stringent response. Because the knowledge on the metabolic responses in recombinant bioprocesses is still scarce, metabolic footprinting can provide relevant information on the intrinsic metabolic adjustments. Thus, the metabolic footprints generated by E. coli W3110 and the ΔrelA mutant strain during recombinant fed-batch fermentations at different experimental conditions were measured and interpreted. The IPTG-induction of the heterologous protein expression resulted in the rapid accumulation of inhibitors of the glyoxylate shunt in the culture broth, suggesting the clearance of this anaplerotic route to replenish the TCA intermediaries withdrawn for the additional formation of the heterologous protein. Nutritional shifts were also critical in the recombinant cellular metabolism, indicating that cells employ diverse strategies to counteract imbalances in the cellular metabolism, including the secretion of certain metabolites that are, most likely, used as a metabolic relief to survival processes.

Influence of the RelA Activity on E. coli Metabolism by Metabolite Profiling of Glucose-Limited Chemostat Cultures

“…It was found that ppGpp-deficient strains can maintain a metabolically productive state longer than the parent strains [15]. Thus, reducing the intracellular ppGpp levels seems to attenuate the pleiotropic effects on the metabolism, which is beneficial for the synthesis of foreign proteins.…”

Untargeted metabolic footprinting reveals a surprising breadth of metabolite uptake and release by Synechococcus sp. PCC 7002

“…17 More recently, metabolic footprinting was used to study pleiotropic effects of specific mutations, 18 investigation of metabolic impact of heterologous protein production, 19 and optimization of amino acid composition of growth media to support microbial growth.…”

A Comparative Proteome Analysis of Escherichia coli ΔrelA Mutant Cells

“…As such, it is relevant to understand the influence of the RelA activity in the coordination of metabolic processes under different growth conditions. It is anticipated that the rel A gene mutation will affect central metabolic processes, as it was previously reported (Carneiro et al, 2011), but the mechanisms by which these processes are regulated and which other entities/processes are involved is still vague. A recent study (Kanjee et al, 2012) has identified direct targeted proteins by the ppGpp alarmone, which explains to a certain extent the global physiological changes in the cellular metabolism.…”

“…Samples from chemostat cultivations with E. coli strains W3110 and Δ rel A mutants at two dilution rates were tested. The dilution rates of 0.1 and 0.2 h −1 were chosen in this study because they present distinct metabolic phenotypes as previously verified in our laboratory (Carneiro et al, 2011, 2012). In particular, at a dilution rate of 0.2 h −1 , wild-type E. coli cells tend to accumulate acetate, which is characteristic of the overflow metabolism.…”