The impact of CO2/HCO3- availability on anaplerotic flux in PDHC-deficient Corynebacterium glutamicum strains

The pyruvate dehydrogenase complex (PDHC) catalyzes the oxidative decarboxylation of pyruvate yielding acetyl-CoA and CO2. The PDHC-deficient Corynebacterium glutamicum strain {Delta}aceE 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 {Delta}aceE {Delta}pyc to glucose catabolism. In the following, strain {Delta}aceE {Delta}pyc 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 {Delta}aceE {Delta}pyc 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 {Delta}aceE {Delta}pyc 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.\n\nImportanceCO2 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 ({Delta}aceE {Delta}pyc). 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.