A Novel Metabolic Cycle Catalyzes Glucose Oxidation and Anaplerosis in Hungry Escherichia coli

Abstract

Complete oxidation of carbohydrates to CO 2 is considered to be the exclusive property of the ubiquitous tricarboxylic acid cycle, the central process in cellular energy metabolism of aerobic organisms. Based on metabolism-wide in vivo quantification of intracellular carbon fluxes, we describe here complete oxidation of carbohydrates via the novel P-enolpyruvate (PEP)-glyoxylate cycle, in which two PEP molecules are oxidized by means of acetyl coenzyme A, citrate, glyoxylate, and oxaloacetate to CO 2 , and one PEP is regenerated. Key reactions are the constituents of the glyoxylate shunt and PEP carboxykinase, whose conjoint operation in this bi-functional catabolic and anabolic cycle is in sharp contrast to their generally recognized functions in anaplerosis and gluconeogenesis, respectively. Parallel operation of the PEP-glyoxylate cycle and the tricarboxylic acid cycle was identified in the bacterium Escherichia coli under conditions of glucose hunger in a slow-growing continuous culture. Because the PEPglyoxylate cycle was also active in glucose excess batch cultures of an NADPH-overproducing phosphoglucose isomerase mutant, one function of this new central pathway may be the decoupling of catabolism from NADPH formation that would otherwise occur in the tricarboxylic acid cycle.Structuring of cellular networks into pathways with distinct functions is pivotal for comprehension of "textbook" biochemistry. The ability of existing model pathways to portray flux through complex metabolic networks, however, is an open question that is just beginning to be addressed theoretically (1-3) and experimentally (4, 5). Microbial growth on the most abundant carbon-source glucose represses transcription of metabolic functions that are required on alternative carbon sources. This universal phenomenon is referred to as catabolite repression and includes a number of mechanistically distinguishable but physiologically related regulation mechanisms (6, 7). Although catabolite repression is strong under conditions of feast with excess glucose, microbes typically thrive under conditions of starvation (absence of nutrients) or hunger (suboptimal supply of nutrients) in their natural environments (8,9). This metabolic state of hunger, between optimal growth and starvation, can be studied in glucose-limited continuous (chemostat) cultures with very low glucose concentrations at a rate of growth that is controlled by the experimenter. Catabolite repression is absent under the severe glucose limitation in slow growing chemostat cultures (9 -11), and, as a consequence, increased in vivo activity of repressed metabolic enzymes is often observed with advanced methods of metabolic flux analyses based on 13 C-labeling experiments (4, 5, 12, 13).Here we elucidate metabolic impacts of severe and absent catabolite repression during growth of Escherichia coli in glucose-excess batch cultures and glucose-limited chemostat cultures. Direct analytical interpretation of 13 C-labeling patterns in proteinogenic amino acids was used to establish the ...

The Soluble and Membrane-bound Transhydrogenases UdhA and PntAB Have Divergent Functions in NADPH Metabolism of Escherichia coli

“…4). Only a relatively small fraction of the consumed glucose was oxidized to CO 2 within the TCA cycle, as was described previously for batch cultures of the same strain using a comprehensive isotopomer model (37). Consistent with the flux ratios (Fig.…”

“…Moreover, the Pgi mutant was the only strain with an exceptional low fraction of oxaloacetate originating from PEP and a significant fraction of oxaloacetate originating from glyoxylate (Fig. 3), as was described before (37). The resulting absolute flux distribution reveals significant activity of the recently identified PEP-glyoxylate cycle (37), i.e.…”

“…Metabolic Flux Ratio Analysis by GC-MS-Direct analytical interpretation of GC-MS-derived 13 C-labeling pattern in proteinogenic amino acids was used to quantify independent flux partitioning ratios (36,37). Biomass pellets harvested from 13 C-labeling experiments were resuspended and hydrolyzed in 1.5 ml of 6 M HCl at 105°C for 24 h in sealed 2.2-ml Eppendorf tubes.…”

“…3 and 6; Refs. 26,36,37). The resulting flux rerouting caused a metabolic condition with more than 100% excess NADPH formation (Figs.…”

“…By comparing 13 C-labeling pattern in proteinogenic amino acids, this strictly local analysis quantifies ratios of converging fluxes at the junction of two or more pathways or reactions (43,44). Different from global data fitting in the net flux analysis described below, this approach yields ratios of fluxes that are independent of each other (23,27,37,45). In separate batch cultures that were grown either on 100% [1-13 C]glucose or on a mixture of 20% [U- 13 C]glucose and 80% unlabeled glucose, the overall flux profiles were very similar in the UdhA mutant and the wild type (Fig.…”