Modification of glucose import capacity in Escherichia coli: physiologic consequences and utility for improving DNA vaccine production

Abstract

BackgroundThe bacterium Escherichia coli can be grown employing various carbohydrates as sole carbon and energy source. Among them, glucose affords the highest growth rate. This sugar is nowadays widely employed as raw material in industrial fermentations. When E. coli grows in a medium containing non-limiting concentrations of glucose, a metabolic imbalance occurs whose main consequence is acetate secretion. The production of this toxic organic acid reduces strain productivity and viability. Solutions to this problem include reducing glucose concentration by substrate feeding strategies or the generation of mutant strains with impaired glucose import capacity. In this work, a collection of E. coli strains with inactive genes encoding proteins involved in glucose transport where generated to determine the effects of reduced glucose import capacity on growth rate, biomass yield, acetate and production of an experimental plasmid DNA vaccine (pHN).ResultsA group of 15 isogenic derivatives of E. coli W3110 were generated with single and multiple deletions of genes encoding glucose, mannose, beta-glucoside, maltose and N-acetylglucosamine components of the phosphoenolpyruvate:sugar phosphotransferase system (PTS), as well as the galactose symporter and the Mgl galactose/glucose ABC transporter. These strains were characterized by growing them in mineral salts medium supplemented with 2.5 g/L glucose. Maximum specific rates of glucose consumption (qs) spanning from 1.33 to 0.32 g/g h were displayed by the group of mutants and W3110, which resulted in specific growth rates ranging from 0.65-0.18 h-1. Acetate accumulation was reduced or abolished in cultures with all mutant strains. W3110 and five selected mutant derivatives were transformed with pHN. A 3.2-fold increase in pHN yield on biomass was observed in cultures of a mutant strain with deletion of genes encoding the glucose and mannose PTS components, as well as Mgl.ConclusionsThe group of E. coli mutants generated in this study displayed a reduction or elimination of overflow metabolism and a linear correlation between qs and the maximum specific growth rate as well as the acetate production rate. By comparing DNA vaccine production parameters among some of these mutants, it was possible to identify a near-optimal glucose import rate value for this particular application. The strains employed in this study should be a useful resource for studying the effects of different predefined qs values on production capacity for various biotechnological products.

Process development of periplasmatically produced single chain fragment variable against epidermal growth factor receptor in Escherichia coli

“…Due to the open-loop controlled feeding rate too early induction led to an ostensive glucose overfeed situation, which is an indication for a decreased glucose uptake via the Phospho Transferase-System, PTS (Fuentes et al, 2013). It might be speculated that the increased pressure inside the periplasmic space is artificially product-induced, since the PTS is indirectly controlled by the cell turgor (Stock and Ninfa, 1989;Neidhardt et al, 1990).…”

Accumulation of d -Glucose from Pentoses by Metabolically Engineered Escherichia coli

“…Additionally, E. coli responds to knockouts in transport genes: the expression of the galP, mglB, and lamB permease genes increased as a result of a ptsHI crr knockout (37). The mechanisms of D-glucose uptake under D-glucose-limiting and -excess conditions have been reviewed (38), and the various proteins involved in D-glucose uptake have recently been examined collectively in E. coli in the context of overflow metabolism and vaccine production (39). Interestingly, GluP has been implicated in D-glucose export in Bacillus subtilis (40), but we found no similar E. coli protein.…”

Metabolic variability in bioprocessing: implications of microbial phenotypic heterogeneity

“…From a scale-up perspective, it can be assumed that cells with reduced metabolic flexibility to environmental fluctuations would be more robust for biotechnological applications. For instance, the successive deletion of genes involved in glucose transport of E. coli resulted in a variety of growth and acetate production rates [46]. Strains with reduced acetate production rates were also able to produce higher amounts of an experimental plasmid DNA vaccine [47].…”