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.Escherichia coli accumulates acetic acid when growing at a high rate of glucose consumption even in the presence of ample oxygen (2,11,30). This phenomenon is known as overflow metabolism. Acetate is generated when carbon flux from acetyl-coenzyme A (CoA) is directed to acetate instead of entering the tricarboxylic acid (TCA) cycle (11). This by-product induces a stress response even at extremely low concentrations (21), hinders growth (26), and reduces the production of recombinant proteins (37). Overflow metabolism has been attributed to an enzymatic limitation in the TCA cycle (27). In E. coli the complete oxidation of 1 mol of glucose in glycolysis and the TCA cycle generates 10 mol of NAD(P)H and 2 mol of FADH 2 (31):If the rate of oxygen utilization is sufficiently high, the reduced cofactors generated by glucose consumption are reoxidized in the electron transport chain, which serves the dual purpose of maintaining an optimal redox environment and generating energy by oxidative phosphorylation. In the absence of oxygen glucose cannot be completely oxidized, and metabolic intermediates accumulate to maintain the redox balance. Even in the presence of oxygen, if the rate of glucose consumption is greater than the capacity to reoxidize the reduced equivalents generated...

Transcriptional Regulation of the Respiratory Genes in the CyanobacteriumSynechocystissp. PCC 6803 during the Early Response to Glucose Feeding

“…In addition to carbohydrate catabolism-related genes, the genes coding for oxidative phosphorylation components, such as NDH-I, CtaI, and ATP synthase, are also regulated at the transcription level during the light-to-dark transition (Gill et al, 2002;Kucho et al, 2005). The coordinated regulation of carbohydrate catabolism and oxidative phosphorylation would ensure a close connection between the generation of cellular reducing power in the form of NAD(P)H during Glc catabolism and its subsequent utilization via the respiratory electron transport chain for the production of ATP: This has been reported previously for other prokaryotes, including Bacillus subtilis and Escherichia coli (Blencke et al, 2003;Vemuri et al, 2006). Despite the circumstantial evidence, a clear result demonstrating the relationship between respiratory activity and the coordinated regulation of respiratory genes at the transcriptional level in Synechocystis is still lacking.…”

NADH Availability Limits Asymmetric Biocatalytic Epoxidation in a Growing Recombinant Escherichia coli Strain

“…The reduced acetate formation at high glucose uptake rates in NADH oxidase-containing E. coli correlated with a reduction of the NADH/NAD ratio, which was proposed to contribute to the regulation of the TCA cycle and acetate formation (71). During styrene epoxidation, however, the high biocatalysisrelated NADH consumption rate did not prevent acetate formation.…”

“…Additional energy may be required for associated reactions such as uncoupling and 2-phenylethanol formation and for sustaining the presence of heterologous StyAB and toxic compounds. Moreover, the carbon source can be channeled into acetate when the carbon flux into the central metabolic pathways exceeds the activity of the TCA cycle during aerobic growth on glucose (56,70,71).…”

“…5]). Similarly, Vemuri et al observed increased specific glucose uptake as well as CO 2 and thus NAD(P)H formation rates and decreased biomass yields when an active NADH oxidase catalyzing the reduction of O 2 to H 2 O was present in recombinant E. coli MG1655 (71). However, during glucose-limited growth, this strain showed reduced acetate formation, whereas E. coli JM101(pSPZ10) showed increasing acetate formation at styrene epoxidation rates of above 15 U (g CDW) Ϫ1 (Fig.…”

“…Interestingly, Vemuri et al found that knockout of ArcA, a component of the ArcB/ArcA signal transduction system regulating gene expression in response to redox conditions (22,36,39), also led to an increase of the critical glucose uptake rate (71) and that the cumulative effect of NADH oxidase expression and arcA knockout can be used to improve recombinant protein production, which often is acetate sensitive (73). The reduced acetate formation at high glucose uptake rates in NADH oxidase-containing E. coli correlated with a reduction of the NADH/NAD ratio, which was proposed to contribute to the regulation of the TCA cycle and acetate formation (71).…”