Impact of CO ₂ /HCO ₃ ^– Availability on Anaplerotic Flux in Pyruvate Dehydrogenase Complex-Deficient Corynebacterium glutamicum Strains

Abstract

The pyruvate dehydrogenase complex (PDHC) catalyzes the oxidative decarboxylation of pyruvate, yielding acetyl coenzyme A (acetyl-CoA) and CO2. The PDHC-deficient Corynebacterium glutamicum ΔaceE strain therefore lacks an important decarboxylation step in its central metabolism. Additional inactivation of pyc, encoding pyruvate carboxylase, resulted in a >15-h lag phase in the presence of glucose, while no growth defect was observed on gluconeogenetic substrates, such as acetate. Growth was successfully restored by deletion of ptsG, encoding the glucose-specific permease of the phosphotransferase system (PTS), thereby linking the observed phenotype to the increased sensitivity of the ΔaceE Δpyc strain to glucose catabolism. In this work, the ΔaceE Δpyc strain was used to systematically study the impact of perturbations of the intracellular CO2/HCO3– pool on growth and anaplerotic flux. Remarkably, all measures leading to enhanced CO2/HCO3– levels, such as external addition of HCO3–, increasing the pH, or rerouting metabolic flux via the pentose phosphate pathway, at least partially eliminated the lag phase of the ΔaceE Δpyc strain on glucose medium. In accordance with these results, inactivation of the urease enzyme, lowering the intracellular CO2/HCO3– pool, led to an even longer lag phase, accompanied by the excretion of l-valine and l-alanine. Transcriptome analysis, as well as an adaptive laboratory evolution experiment with the ΔaceE Δpyc strain, revealed the reduction of glucose uptake as a key adaptive measure to enhance growth on glucose-acetate mixtures. Taken together, our results highlight the significant impact of the intracellular CO2/HCO3– pool on metabolic flux distribution, which becomes especially evident in engineered strains exhibiting low endogenous CO2 production rates, as exemplified by PDHC-deficient strains. IMPORTANCE CO2 is a ubiquitous product of cellular metabolism and an essential substrate for carboxylation reactions. The pyruvate dehydrogenase complex (PDHC) catalyzes a central metabolic reaction contributing to the intracellular CO2/HCO3– pool in many organisms. In this study, we used a PDHC-deficient strain of Corynebacterium glutamicum, which additionally lacked pyruvate carboxylase (ΔaceE Δpyc). This strain featured a >15-h lag phase during growth on glucose-acetate mixtures. We used this strain to systematically assess the impact of alterations in the intracellular CO2/HCO3– pool on growth in glucose-acetate medium. Remarkably, all measures enhancing CO2/HCO3– levels successfully restored growth. These results emphasize the strong impact of the intracellular CO2/HCO3– pool on metabolic flux, especially in strains exhibiting low endogenous CO2 production rates.

Microfluidic single‐cell scale‐down bioreactors: A proof‐of‐concept for the growth of Corynebacterium glutamicum at oscillating pH values

“…The lower growth rate for these reference bioreactor experiments compared to the literature values obtained from batch cultivations could be a consequence of the experimental design. At such lower inoculation densities, CO 2 availability could be a limiting factor for anaplerotic reactions in the transition from lag to growth phase (Bäumchen et al, 2007; Blombach et al, 2013; Krüger et al, 2019). This could have affected the maximum growth rate in the two‐CR control experiment.…”

“…One could argue, that these differences potentially arose as a consequence of shear forces or oxygen limitation in the tubing. Furthermore, the reduction in growth rate can be explained by the additional current disturbances of the pumping tubes in the reactor and stirring, which discharge CO 2 from the medium, which has been reported to negatively impact growth, especially at low cell densities (Bäumchen et al, 2007; Blombach et al, 2013; Krüger et al, 2019). Compared to the named studies, the biomass concentrations were comparatively low here and therefore the CO 2 effect could be particularly effective.…”

Microfluidic single-cell scale-down bioreactors: A proof-of-concept for the growth of Corynebacterium glutamicum at oscillating pH values

“…The growth rate in the two-CR system is lower than that described in the literature for typical batch cultivations performed in flasks or bench top bioreactors (Graf et al, 2018;Unthan et al, 2014), which may be due to the influence of the shear rates and mechanical burden originating from continuous pumping of the cells between both compartments. Furthermore, this can be explained by the additional current disturbances of the pumping tubes in the reactor and stirring, which discharge CO 2 from the medium, which has been reported to negatively impact growth, especially at low cell densities (Bäumchen et al, 2007;Blombach, Buchholz, Busche, Kalinowski, & Takors, 2013;Krüger et al, 2019). Furthermore, the "interrupted" oxygen supply in the tube distance between the reactors could affect the growth rate.…”