Characterization of the Acetate-Producing Pathways in Escherichia coli

Abstract

Although the bacterium E. coli is chosen as the host in many bioprocesses, the accumulation of a common byproduct, acetate, is often problematic. Acetate, when present at high levels, will inhibit both cell growth and recombinant protein productivity. In addition, products derived from the central aerobic metabolic pathway often compete with the acetate-producing pathways poxB and ackA-pta for glucose as the substrate. As such, a significant portion of the glucose may be excreted as acetate, wasting substrate that otherwise could have been used for the desired product. We have created mutant E. coli strains with a deletion of either the poxB or the ackA-pta pathway. These two strains, along with the wild-type strain, have been studied in batch reactors over a 12 h time period, at pH 7.0 and 6.0. The wild-type strain has also been studied using glucose as the carbon source. Data were collected to correlate cellular growth, extracellular metabolite production, enzyme activity, and gene expression. Results show that the ackA-pta pathway dominates in exponential phase, and the poxB pathway dominates in stationary phase. The ackA-pta pathway is repressed in acidic environments, whereas the poxB pathway is activated.

Manipulation of the Anoxic Metabolism in Escherichia coli by ArcB Deletion Variants in the ArcBA Two-Component System

“…3A). These sharp differences at the transcriptional level did not translate into a similar pattern at the enzymatic activity level, probably because of the regulation exerted by the overall energy of the cell on the activity of AckA (16,19). Moreover, the activity of AckA was lower in all the arcB mutants than in E. coli K1060, which fit well with the observed fluxes through this pathway (Fig.…”

Metabolic Flux Analysis of Escherichia coli creB and arcA Mutants Reveals Shared Control of Carbon Catabolism under Microaerobic Growth Conditions

“…It is worth mentioning that conversion of Pyr to extracellular acetate is usually assumed to occur through the reactions catalyzed by PDH and/or Pfl, Pta, and AckA, but it may also arise from Pyr oxidase activity (PoxB) in one step. The latter reaction could be active in acetate-producing cultures (17). Both reaction sequences create the same 13 C-labeling pattern, so that our present analysis cannot differentiate between the two routes.…”

“…All compounds of the reaction mixture were pipetted into a cuvette of 1-cm light path, and the reaction was initiated by adding the cell extract or the substrate to give a final volume of 1 ml. Protocols for citrate synthase, isocitrate lyase, and succinate dehydrogenase (SDH) were performed essentially as described elsewhere (18,69,71); lactate dehydrogenase was measured as indicated by Bunch et al (7); and acetate kinase as detailed by Dittrich et al (17).…”

The Legacy of HfrH: Mutations in the Two-Component System CreBC Are Responsible for the Unusual Phenotype of an Escherichia coli arcA Mutant

“…The differences in O 2 consumption rates when acetate was used as a substrate could reflect an increased acetate kinase activity in mutant and wild-type strains. Table 1 shows the results obtained when acetate kinase activity was determined for exponential LB medium cultures according to the method described by Dittrich et al (8). AckA activity was fourfold higher for CT1061 than for the strains harboring wildtype creC.…”