Abstract
BackgroundRedox homeostasis is essential to sustain metabolism and growth. We recently reported that yeast cells meet a gradual increase in imposed NADPH demand by progressively increasing flux through the pentose phosphate (PP) and acetate pathways and by exchanging NADH for NADPH in the cytosol, via a transhydrogenase-like cycle. Here, we studied the mechanisms underlying this metabolic response, through a combination of gene expression profiling and analyses of extracellular and intracellular metabolites and 13 C-flux analysis.ResultsNADPH oxidation was increased by reducing acetoin to 2,3-butanediol in a strain overexpressing an engineered NADPH-dependent butanediol dehydrogenase cultured in the presence of acetoin. An increase in NADPH demand to 22 times the anabolic requirement for NADPH was accompanied by the intracellular accumulation of PP pathway metabolites consistent with an increase in flux through this pathway. Increases in NADPH demand were accompanied by the successive induction of several genes of the PP pathway. NADPH-consuming pathways, such as amino-acid biosynthesis, were upregulated as an indirect effect of the decrease in NADPH availability. Metabolomic analysis showed that the most extreme modification of NADPH demand resulted in an energetic problem. Our results also highlight the influence of redox status on aroma production.ConclusionsCombined 13 C-flux, intracellular metabolite levels and microarrays analyses revealed that NADPH homeostasis, in response to a progressive increase in NADPH demand, was achieved by the regulation, at several levels, of the PP pathway. This pathway is principally under metabolic control, but regulation of the transcription of PP pathway genes can exert a stronger effect, by redirecting larger amounts of carbon to this pathway to satisfy the demand for NADPH. No coordinated response of genes involved in NADPH metabolism was observed, suggesting that yeast has no system for sensing NADPH/NADP+ ratio. Instead, the induction of NADPH-consuming amino-acid pathways in conditions of NADPH limitation may indirectly trigger the transcription of a set of PP pathway genes.
Highlights
Redox homeostasis is essential to sustain metabolism and growth
Strains exposed to different degrees of change in NADPH demand differed in their acetate production (Figure 2B) [5]
Pathways involved in NADPH synthesis In our previous model-based data reconciliation analysis of the response to NADPH demand modulation, we showed that yeast cells responded to increases in NADPH demand by increasing flux through the PP and acetate pathways, which covered 80% and 20% of the NADPH demand, respectively [5]
Summary
Redox homeostasis is essential to sustain metabolism and growth. We recently reported that yeast cells meet a gradual increase in imposed NADPH demand by progressively increasing flux through the pentose phosphate (PP) and acetate pathways and by exchanging NADH for NADPH in the cytosol, via a transhydrogenaselike cycle. Intracellular redox potential is determined principally by the ratio of NADH/NAD+ and NADPH/NADP+ cofactors, which are involved in about 200 reactions in Saccharomyces cerevisiae [1]. These metabolites are interconnected by many different pathways. We recently investigated the response to increases in NADPH oxidation using a biological system in which NADPH demand can be and gradually modified [5]. This system involves the culture of a strain overexpressing an engineered NADPH-dependent Bdh1p enzyme in the presence of acetoin (Figure 1). If demand was increased still further (40 times the anabolic demand), the PP pathway was saturated and our model predicted a role for the glycerol-DHA cycle, which exchanges NADP+ and NADH for NAD+ and NADPH, at the expense of one ATP molecule (Figure 1)
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