Control of the Glycolytic Flux in Saccharomyces cerevisiae Grown at Low Temperature

Abstract

Growth temperature has a profound impact on the kinetic properties of enzymes in microbial metabolic networks. Activities of glycolytic enzymes in Saccharomyces cerevisiae were up to 7.5-fold lower when assayed at 12°C than at 30°C. Nevertheless, the in vivo glycolytic flux in chemostat cultures (dilution rate: 0.03 h ؊1 ) grown at these two temperatures was essentially the same. To investigate how yeast maintained a constant glycolytic flux despite the kinetic challenge imposed by a lower growth temperature, a systems approach was applied that involved metabolic flux analysis, transcript analysis, enzyme activity assays, and metabolite analysis. Expression of hexosetransporter genes was affected by the growth temperature, as indicated by differential transcription of five HXT genes and changed zero trans-influx kinetics of [ 14 C]glucose transport. No such significant changes in gene expression were observed for any of the glycolytic enzymes. Fermentative capacity (assayed off-line at 30°C), which was 2-fold higher in cells grown at 12°C, was therefore probably controlled predominantly by glucose transport. Massive differences in the intracellular concentrations of nucleotides (resulting in an increased adenylate energy charge at low temperature) and glycolytic intermediates indicated a dominant role of metabolic control as opposed to gene expression in the adaptation of glycolytic enzyme activity to different temperatures. In evolutionary terms, this predominant reliance on metabolic control of a central pathway, which represents a significant fraction of the cellular protein of the organism, may be advantageous to limit the need for protein synthesis and degradation during adaptation to diurnal temperature cycles.Changing ambient temperature, for example, as a result of diurnal temperature cycling, is among the most common environmental changes that microorganisms have to contend with in nature. Temperature effects on microbial physiology are also relevant for the industrial exploitation of microorganisms. For example, the temperature in industrial processes for production of alcoholic beverages with the yeast Saccharomyces cerevisiae (commonly 8 -20°C) is much lower than the optimum temperature range for growth (25-30°C) (1).Much of the current knowledge on adaptation of the model eukaryote S. cerevisiae to suboptimal temperatures has been derived from studies on cold shock. These studies have identified cold-induced changes in membrane lipid composition (2), transport functions, translational efficiency, protein folding, and nucleic acid structure (for a review, see Ref.3). Transcriptome analysis of cold adaptation in S. cerevisiae has revealed several types of responses that depend on the temperature range applied, namely "cold shock" (between 20 and 10°C) (4, 5) and "near freezing" (Ͻ10°C) (6) conditions. Responses to low temperature are also affected by the exposure period to low temperature (early phase and late phase responses (4, 6)). The early response in cold shock experiments encompasses upregulati...

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