The impact of CO₂/HCO₃^-availability on anaplerotic flux in PDHC-deficientCorynebacterium glutamicumstrains

Abstract

18The pyruvate dehydrogenase complex (PDHC) catalyzes the oxidative 19 decarboxylation of pyruvate yielding acetyl-CoA and CO 2 . The PDHC-deficient 20 Corynebacterium glutamicum strain ΔaceE is therefore lacking an important 21 decarboxylation step in central metabolism. Additional inactivation of pyc, encoding 22 pyruvate carboxylase, resulted in a >15 hour lag phase in the presence of glucose, 23 while no growth defect was observed on gluconeogenetic substrates like acetate. 24 Growth was successfully restored by deletion of ptsG encoding the glucose-specific 25 permease of the PTS system, thereby linking the observed phenotype to the 26 increased sensitivity of strain ΔaceE Δpyc to glucose catabolism. In the following, 27 strain ΔaceE Δpyc was used to systematically study the impact of perturbations of 28 the intracellular CO 2 /HCO 3 pool on growth and anaplerotic flux. Remarkably, all 29 measures leading to enhanced CO 2 /HCO 3 levels, such as external addition of HCO 3 -30 , increasing the pH, or rerouting metabolic flux via pentose phosphate pathway, at 31 least partially eliminated the lag phase of strain ΔaceE Δpyc on glucose medium. In 32 accordance, inactivation of the urease enzyme, lowering the intracellular CO 2 /HCO 3 -33 pool, led to an even longer lag phase accompanied with the excretion of L-valine and 34 L-alanine. Transcriptome analysis as well as an adaptive laboratory evolution 35 experiment of strain ΔaceE Δpyc revealed the reduction of glucose uptake as a key 36adaptive measure to enhance growth on glucose/acetate mixtures. Altogether, our 37 results highlight the significant impact of the intracellular CO 2 /HCO 3 pool on 38 metabolic flux distribution, which becomes especially evident in engineered strains 39 suffering from low endogenous CO 2 production rates as exemplified by PDHC-40 deficient strains. 41 42 3 Importance: CO 2 is a ubiquitous product of cellular metabolism and an essential 43 substrate for carboxylation reactions. The pyruvate dehydrogenase complex (PDHC) 44 catalyzes a central metabolic reaction contributing to the intracellular CO 2 /HCO 3 pool 45 in many organisms. In this study, we used a PDHC-deficient strain of 46 Corynebacterium glutamicum, which was additionally lacking pyruvate carboxylase 47 52 metabolic flux especially in strains suffering from low endogenous CO 2 production 53 rates. 54 55 56 2-, which is influenced 59 by the pH of the medium (1). As a product of decarboxylating reactions and substrate 60 for carboxylations, CO 2 and bicarbonate (HCO 3 -) are involved in various metabolic 61 processes. Consequently, the intracellular CO 2 /HCO 3 pool has a significant impact 62 on metabolic fluxes like e.g. anaplerosis, especially in the early phase of cultivations 63 when cell density is low (2). 64 During aerobic growth, the pyruvate dehydrogenase complex (PDHC), which is 65 conserved in various microbial species, catalyzes an important reaction contributing 66 to the intracellular CO 2 /HCO 3 pool (3, 4). The PDHC is a multienzyme complex 67 be...