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

Abstract

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.