Redox balance is key to explaining full vs. partial switching to low-yield metabolism

Hoek and Merks BMC Systems Biology Redox balance is key to explaining full vs. partial switching to low-yield metabolism Milan JA van Hoek 0 1 2 Roeland MH Merks 0 1 2 3 0 Centrum Wiskunde & Informatica, Life Sciences , Science Park 123, 1098 XG Amsterdam , The Netherlands 1 Netherlands Consortium for Systems Biology , Amsterdam , The Netherlands 2 Netherlands Institute for Systems Biology , Science Park 123, 1098 XG Amsterdam , The Netherlands 3 Mathematical Institute, Leiden University , P.O. Box 9512, 2300 RA Leiden , The Netherlands Background: Low-yield metabolism is a puzzling phenomenon in many unicellular and multicellular organisms. In abundance of glucose, many cells use a highly wasteful fermentation pathway despite the availability of a highyield pathway, producing many ATP molecules per glucose, e.g., oxidative phosphorylation. Some of these organisms, including the lactic acid bacterium Lactococcus lactis, downregulate their high-yield pathway in favor of the low-yield pathway. Other organisms, including Escherichia coli do not reduce the flux through the high-yield pathway, employing the low-yield pathway in parallel with a fully active high-yield pathway. For what reasons do some species use the high-yield and low-yield pathways concurrently and what makes others downregulate the high-yield pathway? A classic rationale for metabolic fermentation is overflow metabolism. Because the throughput of metabolic pathways is limited, influx of glucose exceeding the pathway's throughput capacity is thought to be redirected into an alternative, low-yield pathway. This overflow metabolism rationale suggests that cells would only use fermentation once the high-yield pathway runs at maximum rate, but it cannot explain why cells would decrease the flux through the high-yield pathway. Results: Using flux balance analysis with molecular crowding (FBAwMC), a recent extension to flux balance analysis (FBA) that assumes that the total flux through the metabolic network is limited, we investigate the differences between Saccharomyces cerevisiae and L. lactis that downregulate the high-yield pathway at increasing glucose concentrations, and E. coli, which keeps the high-yield pathway functioning at maximal rate. FBAwMC correctly predicts the metabolic switching mode in these three organisms, suggesting that metabolic network architecture is responsible for differences in metabolic switching mode. Based on our analysis, we expect gradual, overflow-like switching behavior in organisms that have an additional energy-yielding pathway that does not consume NADH (e. g., acetate production in E. coli). Flux decrease through the high-yield pathway is expected in organisms in which the high-yield and low-yield pathways compete for NADH. In support of this analysis, a simplified model of metabolic switching suggests that the extra energy generated during acetate production produces an additional optimal growth mode that smoothens the metabolic switch in E. coli. Conclusions: Maintaining redox balance is key to explaining why some microbes decrease the flux through the high-yield pathway, while other microbes use overflow-like low-yield metabolism. Metabolic switching; Genome-scale metabolic model; Flux Balance Analysis with Molecular Crowding; Overflow metabolism; Redox balance; Escherichia coli; Lactococcus lactis; Saccharomyces cerevisiae - Background One of the key steps in energy metabolism is to transfer the energy carried by sugars, including glucose, to the biological energy currency adenosine triphosphate (ATP). The number of ATP molecules generated by metabolizing one molecule of glucosethe ATP yield is one of the most basic measures of an organisms energy efficiency. One would perhaps expect that evolution has selected organisms for the ability to extract energy from their food at optimal efficiency by maximizing ATP yield. Yet surprisingly, many organisms switch between a high-yield pathway, e.g., aerobic respiration that yields more than thirty moles of ATP per mole glucose, and a highly inefficient, low-yield fermentation pathway that yields only two or three moles ATP per mole of glucose. This effect is known as the Crabtreeeffect in the bakers yeast Saccharomyces cerevisiae. S. cerevisiae turns glucose into CO2 in aerobic, glucoselimited conditions. But in abundance of glucose, glucose is converted into ethanol [1], even if oxygen levels do not limit aerobic metabolism. Many bacteria also use a high-yield metabolic pathway in glucose-limited conditions and a low-yield pathway in excess of glucose. Examples are Escherichia coli [2], Bacillus subtilis [3] and lactic acid bacteria, e.g., Lactobacillus plantarum and Lactococcus lactis [4,5]. The effect is also found in multicellular eukaryotes, including human cancer cells, where it is called the Warburg effect [6]. Muscle cells switch to low-yield metabolism during heavy exercise [7], fermenting glucose into lactic acid. Why cells would produce less ATP per glucose molecule than they can is a long-standing question in biology [8-12]. Microbial species show remarkable differences in their metabolic switching strategies. At low glucose concentrations and low growth rates, E. coli uses high-yield metabolism, aerobically converting glucose into CO2 and water. At higher glucose concentrations and fast growth rates, it redirects part of the glucose influx into a low-yield fermentation pathway, keeping oxidative phosphorylation fully active [2]. S. cerevisiae uses highyield, aerobic respiration at slow growth rates; at fast growth rates it ferments most glucose into ethanol, and downregulates aerobic respiration, keeping aerobic respiration active at a much lower rate. Although L. lactis does not have an aerobic respiration pathway, it still performs a metabolic switch. At fast growth rates it makes a full switch to lactic acid fermentation [4], which yields about 50% less ATP than the higher-yield mixed acid fermentation pathway, that produces formate, acetate and ethanol. A plausible explanation for metabolic switching is overflow metabolism. It assumes that organisms only switch to low-yield metabolism if the high-yield pathway is operating at maximum rate and cannot process any more molecules [13,14]. The remainder would then spill into the low-yield pathway. This explanation requires the low-yield pathway to operate at a faster rate than the high-yield pathway, which is likely the case [8,15,16]. Thus overflow metabolism plausibly explains concurrent use of high-yield and low-yield pathways, as in E. coli. However, a problem with overflow metabolism is that it does not explain why organisms like S. cerevisiae or L. lactis would partly switch off their high-yield pathways at high growth rates. Recent studies have suggested that the limited amount of metabolic enzymes fitting inside the cell may be key to low-yield metabolism [12,17,18]. Simply because cells can host only a finite number of metabolic enzymes, they (...truncated)