Overflow metabolism in Escherichia coli during steady-state growth: transcriptional regulation and effect of the redox ratio

Abstract

Overflow metabolism in the form of aerobic acetate excretion by Escherichia coli is an important physiological characteristic of this common industrial microorganism. Although acetate formation occurs under conditions of high glucose consumption, the genetic mechanisms that trigger this phenomenon are not clearly understood. We report on the role of the NADH/NAD ratio (redox ratio) in overflow metabolism. We modulated the redox ratio in E. coli through the expression of Streptococcus pneumoniae (water-forming) NADH oxidase. Using steady-state chemostat cultures, we demonstrated a strong correlation between acetate formation and this redox ratio. We furthermore completed genome-wide transcription analyses of a control E. coli strain and an E. coli strain overexpressing NADH oxidase. The transcription results showed that in the control strain, several genes involved in the tricarboxylic acid (TCA) cycle and respiration were repressed as the glucose consumption rate increased. Moreover, the relative repression of these genes was alleviated by expression of NADH oxidase and the resulting reduced redox ratio. Analysis of a promoter binding site upstream of the genes which correlated with redox ratio revealed a degenerate sequence with strong homology with the binding site for ArcA. Deletion of arcA resulted in acetate reduction and increased the biomass yield due to the increased capacities of the TCA cycle and respiration. Acetate formation was completely eliminated by reducing the redox ratio through expression of NADH oxidase in the arcA mutant, even at a very high glucose consumption rate. The results provide a basis for studying new regulatory mechanisms prevalent at reduced NADH/NAD ratios, as well as for designing more efficient bioprocesses.

FIG. 1.

Steady-state physiological profiles of E. coli in the presence of heterologous NADH oxidase. Y_X/S (⋄, ⧫) and q_A (○, •) values are compared for NOX⁻ (open symbols and dashed lines) and NOX⁺ (solid symbols and lines) as functions of the specific glucose consumption rate. The highest dilution rate studied was about 80% of μ_max for both strains. The arrows indicate for each strain the critical specific glucose consumption rates at which acetate formation commenced.

FIG. 2.

Steady-state respiration for NOX⁻ (open symbols and dashed lines) and NOX⁺ (solid symbols and lines). The steady-state q_O2 (▵,▴) and q_CO2 (▿,▾) values are shown as functions of q_S.

FIG. 3.

In vivo molar concentration ratio of NADH/NAD for NOX⁻ (□) and NOX⁺ (▪) as functions of q_S. The critical value of the NADH/NAD ratio at which acetate formation commences is about 0.06 for both NOX⁻ and NOX⁺ (indicated by vertical lines). q_A values are also shown for NOX⁻ (○) and NOX⁺ (•) as functions of q_S.

FIG. 4.

Intracellular concentrations of key glycolysis metabolites glucose-6-phosphate (red), fructose-6-phosphate (green), PEP (yellow), pyruvate (blue), and acetyl-CoA (pink) were measured under steady-state conditions in NOX⁻ (A) and NOX⁺ (B). q_A values are also shown for NOX⁻ (open circles and dashed lines) and NOX⁺ (filled circles and solid lines) as functions of q_S.

FIG. 5.

Transcriptional profile of central metabolic pathways for NOX⁻ (dashed lines) and NOX⁺ (solid lines). The mean values of the expression ratios are shown for all genes involved in glycolysis (green), the TCA cycle (red), the pentose phosphate pathway (blue), and respiration (black) as functions of q_S. Vertical lines show the demarcation between respiratory and respirofermentative metabolism for NOX⁻ (dotted) and for NOX⁺ (solid). See Fig. S1 in the supplemental material for detailed expression profiles of individual genes.

FIG. 6.

(Left) Hierarchical clustering of genes (rows) that are correlated (R > 0.9 or R < −0.9) with the redox ratio (NADH/NAD) in NOX⁻ as a function of increasing q_S (columns). (Right) Significantly overrepresented functional categories are shown in the table, along with the number of genes in each category and the P value of its significance as calculated using a hypergeometric distribution. Several key genes involved in the TCA cycle, respiration, and biosynthesis exhibited a strong negative correlation with the redox ratio. A large portion of the genes negatively correlated to the redox ratio were partially classified, revealing redox-dependent regulation of many of these genes.

FIG. 7.

Physiological characterization of ARCA⁻ NOX⁻ (open symbols and dashed lines) and ARCA⁻ NOX⁺ (solid symbols and lines) in accelerostat cultures. Y_X/S (⋄,⧫) and q_A (○, •) values are compared as functions of specific glucose consumption rate. The steady-state values of these parameters, obtained for NOX⁻ (dashed lines without symbols) and NOX⁺ (solid lines without symbols) by using chemostats, are also shown.

FIG. 8.

Respiration of ARCA⁻ NOX⁻ (open symbols and dashed lines) and ARCA⁻ NOX⁺ (solid symbols and lines) in accelerostat cultures. q_O2 (▵,▴) and q_CO2 (▿,▾) values are compared as functions of specific glucose consumption rate. The steady-state values of these parameters, obtained for NOX⁻ (dashed lines without symbols) and NOX⁺ (solid lines without symbols) by using chemostats, are also shown.