RT Journal Article SR Electronic T1 The impact of CO2/HCO3- availability on anaplerotic flux in PDHC-deficient Corynebacterium glutamicum strains JF bioRxiv FD Cold Spring Harbor Laboratory SP 663856 DO 10.1101/663856 A1 Krüger, Aileen A1 Wiechert, Johanna A1 Gätgens, Cornelia A1 Polen, Tino A1 Mahr, Regina A1 Frunzke, Julia YR 2019 UL http://biorxiv.org/content/early/2019/06/09/663856.abstract AB The pyruvate dehydrogenase complex (PDHC) catalyzes the oxidative decarboxylation of pyruvate yielding acetyl-CoA and CO2. The PDHC-deficient Corynebacterium glutamicum strain ΔaceE is therefore lacking an important decarboxylation step in central metabolism. Additional inactivation of pyc, encoding pyruvate carboxylase, resulted in a >15 hour lag phase in the presence of glucose, while no growth defect was observed on gluconeogenetic substrates like acetate. Growth was successfully restored by deletion of ptsG encoding the glucose-specific permease of the PTS system, thereby linking the observed phenotype to the increased sensitivity of strain ΔaceE Δpyc to glucose catabolism. In the following, strain ΔaceE Δpyc 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 pentose phosphate pathway, at least partially eliminated the lag phase of strain ΔaceE Δpyc on glucose medium. In accordance, inactivation of the urease enzyme, lowering the intracellular CO2/HCO3- pool, led to an even longer lag phase accompanied with the excretion of L-valine and L-alanine. Transcriptome analysis as well as an adaptive laboratory evolution experiment of strain ΔaceE Δpyc revealed the reduction of glucose uptake as a key adaptive measure to enhance growth on glucose/acetate mixtures. Altogether, our results highlight the significant impact of the intracellular CO2/HCO3- pool on metabolic flux distribution, which becomes especially evident in engineered strains suffering from 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 was additionally lacking 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 on glucose-containing medium. Remarkably, all measures enhancing the CO2/HCO3- levels successfully restored growth emphasizing the strong impact of the intracellular CO2/HCO3- pool on metabolic flux especially in strains suffering from low endogenous CO2 production rates.