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

Abstract

Background: The 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).

Results: A 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.

Conclusions: The 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.

Figure 1

Proteins involved in glucose transport and phosphorylation in E. coli. EI, enzyme I; HPr, phosphohistidine carrier protein; IICB^Glc, integral membrane glucose permease; IIABC^Bgl, components of the beta-glucoside PTS complex; IIAB^Man and IICD^Man components of the mannose PTS complex; IIBC^Mal, components of the maltose PTS complex; IIABC^Nag, components of the N-acetylglucosamine PTS complex; GalP, galactose:H⁺ symporter; MglA, MglB and MglC, components of the galactose/glucose high-affinity ABC transporter; LamB, OmpF and OmpC, outer membrane proteins.

Figure 2

Specific growth rate as a function of the specific glucose uptake rate for various E. coli strains generated in this study.

Figure 3

Specific acetate production rate as a function of the specific growth rate for various E. coli strains generated in this study.

Figure 4

Plasmid pHN yield from biomass as a function of the specific glucose uptake rate for various E. coli strains generated in this study.