No abstract

No abstract

Overflow metabolism is well known for yeast, bacteria and mammalian cells. It typically occurs under glucose excess conditions and is characterized by excretions of by-products such as ethanol, acetate or lactate. This phenomenon, also denoted the short-term Crabtree effect, has been extensively studied over the past few decades, however, its basic regulatory mechanism and functional role in metabolism is still unknown. Here we present a comprehensive quantitative and time-dependent analysis of the exometabolome of Escherichia coli, Corynebacterium glutamicum, Bacillus licheniformis, and Saccharomyces cerevisiae during well-controlled bioreactor cultivations. Most surprisingly, in all cases a great diversity of central metabolic intermediates and amino acids is found in the culture medium with extracellular concentrations varying in the micromolar range. Different hypotheses for these observations are formulated and experimentally tested. As a result, the intermediates in the culture medium during batch growth must originate from passive or active transportation due to a new phenomenon termed “extended” overflow metabolism. Moreover, we provide broad evidence that this could be a common feature of all microorganism species when cultivated under conditions of carbon excess and non-inhibited carbon uptake. In turn, this finding has consequences for metabolite balancing and, particularly, for intracellular metabolite quantification and 13C-metabolic flux analysis.

To determine the in vivo fluxes of the central metabolism we have developed a comprehensive approach exclusively based on the fundamental enzyme reactions known to be present, the fate of the carbon atoms of individual reactions, and the metabolite balance of the culture. No information on the energy balance is required, nor information on enzyme activities, or the directionalities of reactions. Our approach combines the power of 'H-detected 13C nuclear magnetic resonance spectroscopy t o follow individual carbons with the simplicity of establishing carbon balances of bacterial cultures. We grew a lysine-producing strain of Corynebacterium glutamicum to the metabolic and isotopic steady state with [l-'3Clglucose and determined the fractional enrichments in 27 carbon atoms of 11 amino acids isolated from the cell. Since precursor metabolites of the central metabolism are incorporated in an exactly defined manner in the carbon skeleton of amino acids, the fractional enrichments i n carbons of precursor metabolites (oxaloacetate, glyceraldehyde 3-phosphate, erythrose 4-phosphate, etc.) became directly accessible. A concise and generally applicable mathematical model was established using matrix calculus to express all metabolite mass and carbon labeling balances. An appropriate all-purpose software for the iterative solution of the equations is supplied. Applying this comprehensive methodology t o C. glutamicum, all major fluxes within the central metabolism were determined. The result is that the flux through the pentose phosphate pathway is 66.4% (relative to the glucose input flux of 1.49 mmol/g dry weight h), that of entry into the tricarboxylic acid cycle 62.2%, and the contribution of the succinylase pathway of lysine synthesis 73.7%. Due t o the large amount and high quality of measured data in vivo exchange reactions could also be quantitated with particularly high exchange rates within the pentose phosphate pathway for the ribose 5-phosphate transketolase reaction. Moreover, the total net flux of the anaplerotic reactions was quantitated as 38.0%. Most importantly, we found that in vivo one component within these anaplerotic reactions is a back flux from the carbon 4 units of the tricarboxylic acid cycle to the carbon 3 units of glycolysis of

SummaryWhereas intracellular carbon metabolism has emerged as an attractive drug target, the carbon sources of intracellularly replicating pathogens, such as the tuberculosis bacillus Mycobacterium tuberculosis, which causes long-term infections in one-third of the world’s population, remain mostly unknown. We used a systems-based approach—13C-flux spectral analysis (FSA) complemented with manual analysis—to measure the metabolic interaction between M. tuberculosis and its macrophage host cell. 13C-FSA analysis of experimental data showed that M. tuberculosis obtains a mixture of amino acids, C1 and C2 substrates from its host cell. We experimentally confirmed that the C1 substrate was derived from CO2. 13C labeling experiments performed on a phosphoenolpyruvate carboxykinase mutant revealed that intracellular M. tuberculosis has access to glycolytic C3 substrates. These findings provide constraints for developing novel chemotherapeutics.