Quantitative analysis of the high temperature-induced glycolytic flux increase in Saccharomyces cerevisiae reveals dominant metabolic regulation

Abstract

A major challenge in systems biology lies in the integration of processes occurring at different levels, such as transcription, translation, and metabolism, to understand the functioning of a living cell in its environment. We studied the high temperature-induced glycolytic flux increase in Saccharomyces cerevisiae and investigated the regulatory mechanisms underlying this increase. We used glucose-limited chemostat cultures to separate regulatory effects of temperature from effects on growth rate. Growth at increased temperature (38 degrees C versus 30 degrees C) resulted in a strongly increased glycolytic flux, accompanied by a switch from respiration to a partially fermentative metabolism. We observed an increased flux through all enzymes, ranging from 5- to 10-fold. We quantified the contributions of direct temperature effects on enzyme activities, the gene expression cascade and shifts in the metabolic network, to the increased flux through each enzyme. To do this we adapted flux regulation analysis. We show that the direct effect of temperature on enzyme kinetics can be included as a separate term. Together with hierarchical regulation and metabolic regulation, this term explains the total flux change between two steady states. Surprisingly, the effect of the cultivation temperature on enzyme catalytic capacity, both directly through the Arrhenius effect and indirectly through adapted gene expression, is only a moderate contribution to the increased glycolytic flux for most enzymes. The changes in flux are therefore largely caused by changes in the interaction of the enzymes with substrates, products, and effectors.

FIGURE 1.

Effect of temperature on the maximal growth rate. Specific growth rate ± S.D. of at least three independent batch fermentors was measured at various temperatures in the range of 27–41 °C.

FIGURE 2.

Temperature effect on physiological characteristics of glucose limited aerobic chemostat cultures grown at various temperatures in the range of 30–39 °C. A, effect of temperature on dry weight (solid diamonds) and yield (open diamonds). B, effect of temperature on glucose flux (solid squares), carbon dioxide flux (solid circles), oxygen flux (open circles), ethanol flux (open triangles), and glycerol flux (solid triangles).

FIGURE 3.

Stoichiometry of the glycolytic, fermentative, and tricarboxylic acid cycle (TCA) pathway. We calculated the in vivo fluxes through glycolysis. In this simplified scheme enzymes with the same flux are boxed together. The numbers next to the boxed enzymes are the calculated in vivo fluxes through the enzymes of 30 °C (underlined) and 38 °C (normal numbers) cultivations. Many enzyme abbreviations are defined in the abbreviation footnote. The following abbreviations are used: SC, storage carbohydrates; PPP, pentose-phosphate pathway; FBA, fructose-1,6-bisphosphate aldolase; PYK, pyruvate kinase.