Supply-demand analysis of anaerobic free-energy metabolism in Saccharomyces cerevisiae

[摘要] ENGLISH ABSTRACT: Scientists and biochemical engineers alike are very interested in the control and regulationof free-energy metabolism in micro-organisms, whether the findings purelysatisfy scientific curiosity or translate into the meeting of biotechnology companydeadlines. We used a rather fundamental approach to investigate experimentallythe control and regulation of yeast free-energy metabolism in anaerobic chemostatcultures using supply-demand analysis. This conceptually simple, quantitativeframework, however, may lead to insight into the control properties of variousmetabolic pathways to be used in biotechnological applications.Supply-demand analysis is based on the theoretical framework of metabolic controlanalysis (MCA).Sections (of arbitrary size) of a metabolic pathway are groupedtogether around a linking metabolite. Those steps that produce the intermediateare combined into the supply block while the reactions that remove/consume theintermediate are grouped together as the demand. The elasticity coefficients ofthe supply and demand blocks (with regard to the linking metabolite concentration)can be used to determine the flux and concentration control coefficients byusing the traditional MCAsummation and connectivity theorems. Supply and demandrate characteristics are a powerful visual approach to determine and displaythe control structure of the pathway under consideration and sets supply-demandanalysis apart from traditional top-down analysis.Our first tool of analysis was a structured kinetic model of yeast growing in a chemos tat, constructed by using methods developed in our research group formodelling systems with variable volumes. Independent perturbations of the linkingmetabolite concentration resulted in a control profile where the control residedmainly in the demand (flux control coefficient of 0.92), as a result of a large negativesupply elasticity. This elasticity, however, varied greatly under different conditions,leading to increased flux control by the supply in some cases.We extended our research to an experimental setup of Saccharomyces cerevisiaegrowing in a glucose-limited chemos tat supplemented with yeast extract asa source of carbon intermediates. This allowed glucose to act solely as the freeenergysource, as confirmed by balancing the glucose flux with the fluxes towardsthe fermentation products, ethanol and carbon dioxide. We obtained the supplyrate characteristic by perturbing the ATPdemand through the addition of benzoate,which uncouples the proton gradient across the cell membrane. The demand ratecharacteristic was obtained by perturbing the ATP supply through changes in thedilution rate and thus the residual glucose concentration in the fermentor. Theconcentrations of ATPand ADPwere measured using a luciferase bioluminescenceassay, while the fermentation products were measured with HPLCand C02 withan acoustic off-gas analyser. For our experimental conditions the flux-control ofenergy metabolism resided predominantly in the supply with respect to the linkingmetabolite [ATP]/[ADP](chosen as an indication of the free-energy state of the cell),i.e. a flux control coefficient of 0.90. Further, the [ATP]/[ADP]was under stronghomeostatic control, as evidenced by the low [ATP]/[ADP]control coefficients of ±0.12.We adjusted the structured kinetic model by varying strategic parameters, sothat the results resembled the experimental observations more closely. However,the kinetics of our core model seem to be too simplistic to capture fully the extentof regulation displayed by the experimental system. The model did, however, revealthe regulatory importance of glucose transport into the cell. We conclude that thecontrol and regulation of free energy metabolism in yeast strongly depend on the culturing conditions and on the steady state being analysed.

[发布机构] Stellenbosch University