In Vivo Quantification of Parallel and Bidirectional Fluxes in the Anaplerosis of Corynebacterium glutamicum

Abstract

The C 3 -C 4 metabolite interconversion at the anaplerotic node in many microorganisms involves a complex set of reactions. C 3 carboxylation to oxaloacetate can originate from phosphoenolpyruvate and pyruvate, and at the same time multiple C 4 -decarboxylating enzymes may be present. The functions of such parallel reactions are not yet fully understood. Using a 13 C NMR-based strategy, we here quantify the individual fluxes at the anaplerotic node of Corynebacterium glutamicum, which is an example of a bacterium possessing multiple carboxylation and decarboxylation reactions. C. glutamicum was grown with a 13 C-labeled glucose isotopomer mixture as the main carbon source and 13 C-labeled lactate as a cosubstrate. 58 isotopomers as well as 15 positional labels of biomass compounds were quantified. Applying a generally applicable mathematical model to include metabolite mass and carbon labeling balances, it is shown that pyruvate carboxylase contributed 91 ؎ 7% to C 3 carboxylation. The total in vivo carboxylation rate of 1.28 ؎ 0.14 mmol/g dry weight/h exceeds the demand of carboxylated metabolites for biosyntheses 3-fold. Excess oxaloacetate was recycled to phosphoenolpyruvate by phosphoenolpyruvate carboxykinase. This shows that the reactions at the anaplerotic node might serve additional purposes other than only providing C 4 metabolites for biosynthesis.The interconversions between the C 3 metabolism and the C 4 metabolites of the tricarboxylic acid cycle function either as a replenishment of tricarboxylic acid cycle intermediates (anaplerosis) or as the initial steps of gluconeogenesis. These carboxylation and decarboxylation reactions are catalyzed by a number of enzymes (1, 2). Synthesis of oxaloacetate via carboxylation of C 3 metabolites may be catalyzed by phosphoenolpyruvate (PEP) 1 carboxylase, PEP carboxytransphosphorylase, or pyruvate carboxylase. The reverse reaction, decarboxylation of oxaloacetate, may analogously lead to PEP or pyruvate, catalyzed by PEP carboxykinase or oxaloacetate decarboxylase, respectively. NAD ϩ -or NADP ϩ -dependent malic enzyme catalyzes the reaction from malate to pyruvate. In some organisms, this enzyme is also thought to act in a pyruvate-carboxylating sense (3).To date, a full understanding of these enzymatic reactions and their functions has been hindered by a lack of knowledge about their activities in vivo. The occurrence of parallel reactions and the involvement of a set of metabolites in activity control of the enzymes prevents reliable estimations on the actual enzyme use. Moreover, it is not possible to derive quantitative data on in vivo flux rates by enzyme characterizations alone. Instead, carbon-13 labeling techniques, which employed NMR spectroscopy (for an overview, see e.g. Refs. 4 and 5) or mass spectrometry, as well as carbon-14 radiolabeling methods have been used to quantify in vivo intracellular fluxes in central metabolism including conversions between PEP, pyruvate, and oxaloacetate/malate. However, although these studies quantified the total C ...

Analysis and Engineering of Metabolic Pathway Fluxes in Corynebacterium glutamicum

“…As additional gluconeogenetic enzymes, oxaloacetate decarboxylase [24] and PEP synthetase [25] have been proposed. It has been proposed that cyclic cooperation of these carboxylating and decarboxylating enzymes around the pyruvate node is involved in the regeneration of excess ATP [22,26,27]. Different reactions are linked to the supply of NADPH.…”

“…This mode would require a metabolic cycle of pyruvate carboxylase, malate dehydrogenase, and malic enzyme acting as a transhydrogenase to convert NADH into NADPH. Biochemical evidence for such a cycle has not been obtained so far, but a similar cyclic pathway involving pyruvate carboxylase, malate dehydrogenase, and PEP carboxykinase has recently been shown to operate in vivo in C. glutamicum [22,27]. Omitting the transhydrogenase-like cycle from the network results in a slightly lower maximum lysine yield of 0.75 mol mol À1 [77].…”

“…4a). It could be shown that carboxylating and decarboxylating enzymes are active at the same time, forming a cyclic pathway for interconversion of C 4 metabolites of the TCA cycle and C 3 metabolites of glycolysis [27]. This cyclic pathway is highly flexible, as cells can redistribute the flux depending on the metabolic burden ( Fig.…”

“…In most cases this results in an apparent NADPH excess, pointing at so far unassigned reactions which consume NADPH. Possible, but not validated, candidates are NADPH oxidase [132] or cyclic fluxes around the pyruvate node involving malic enzyme [27,124]. Towards higher production, this apparent excess diminishes and turns into an apparent NADPH limitation [5].…”

“…7). Among the most striking findings was the identification of pyruvate carboxylase as the major anaplerotic enzyme in C. glutamicum [27]. Subsequently, overexpression of its gene has been shown to improve lysine production [150].…”