Metabolic-Flux Profiling of the Yeasts Saccharomyces cerevisiae and Pichia stipitis

Abstract

The so far largely uncharacterized central carbon metabolism of the yeast Pichia stipitis was explored in batch and glucose-limited chemostat cultures using metabolic-flux ratio analysis by nuclear magnetic resonance. The concomitantly characterized network of active metabolic pathways was compared to those identified in Saccharomyces cerevisiae, which led to the following conclusions. (i) There is a remarkably low use of the non-oxidative pentose phosphate (PP) pathway for glucose catabolism in S. cerevisiae when compared to P. stipitis batch cultures. (ii) Metabolism of P. stipitis batch cultures is fully respirative, which contrasts with the predominantly respiro-fermentative metabolic state of S. cerevisiae. (iii) Glucose catabolism in chemostat cultures of both yeasts is primarily oxidative. (iv) In both yeasts there is significant in vivo malic enzyme activity during growth on glucose. (v) The amino acid biosynthesis pathways are identical in both yeasts. The present investigation thus demonstrates the power of metabolic-flux ratio analysis for comparative profiling of central carbon metabolism in lower eukaryotes. Although not used for glucose catabolism in batch culture, we demonstrate that the PP pathway in S. cerevisiae has a generally high catabolic capacity by overexpressing the Escherichia coli transhydrogenase UdhA in phosphoglucose isomerase-deficient S. cerevisiae.The two yeasts Saccharomyces cerevisiae and Pichia stipitis exhibit fundamentally different modes of metabolic regulation in glucose-containing media. At high extracellular concentrations of glucose, one observes simultaneous fermentation and respiration (respiro-fermentative metabolism) in S. cerevisiae at high growth rates even under fully aerobic conditions (11,34,53). In Crabtree-positive yeasts, such as S. cerevisiae, elevated glucose concentrations induce the carbon catabolite repression response (16), resulting in low levels of transcription of genes involved in respiration and in the tricarboxylic acid (TCA) cycle. In contrast, the Crabtree-negative P. stipitis exhibits predominantly respirative metabolism even at high glucose concentrations (33). These obvious differences in metabolic regulation between the two yeasts provide motivation for investigation of potential differences in the central carbon pathways. Stable isotope labeling experiments appear to be a promising approach, since it reveals in vivo activity of pathways and reactions (10,43). Biosynthetically directed fractional (BDF) 13 C labeling of amino acids can provide comprehensive insight into central carbon metabolism, yielding a network of active pathways and quantification of intracellular flux ratios (36,37,44,46); its use in the present study promises to expand on the results of earlier labeling studies in yeast that focused on intermediates or products of individual pathways or reactions (14,17,20,22,41,57). BDF 13 C labeling is achieved by growing cells on mixtures of unlabeled and uniformly 13 C-labeled [U-13 C 6 ]glucose (40, 44). Using two-dimensional (...

The Soluble and Membrane-bound Transhydrogenases UdhA and PntAB Have Divergent Functions in NADPH Metabolism of Escherichia coli

“…Under metabolic conditions that led to excess NADPH formation, however, the soluble transhydrogenase UdhA was essential for growth. Such conditions were growth on acetate or in phosphoglucose isomerase mutants that catabolized glucose almost exclusively through the PP pathway, thereby producing a vast excess of NADPH, as was also described for S. cerevisiae (20). Another metabolic condition with excess NADPH formation in E. coli is slow growth in glucose-limited chemostat cultures, because fully respiratory growth with high TCA cycle fluxes of about 80% of the specific glucose uptake rate in these cultures produced much NADPH in the isocitrate dehydrogenase reaction (27,45).…”

“…Indeed, organisms lacking transhydrogenases, such as the yeast Saccharomyces cerevisiae, cannot tolerate imbalances between catabolic NADPH production and anabolic NADPH consumption (18,19), unless a soluble isoform is expressed (19,20). Further means of NADPH reoxidation must exist, however, because isotopic tracer-based carbon flux analysis revealed that at least some bacteria without transhydrogenase homologues exhibit a similar uncoupling (21)(22)(23)(24)(25).…”

Responses of theCentral Metabolism in Escherichia coli to PhosphoglucoseIsomerase and Glucose-6-Phosphate DehydrogenaseKnockouts

“…Thus, it appears that the transhydrogenase reaction plays an important role in maintaining the NADPH balance in phosphoglucose isomerase-deficient E. coli. Consistently, phosphoglucose isomerase knockout mutants of Saccharomyces cerevisiae, in which the transhydrogenase is absent, are blocked for growth on glucose (15). The reaction converting NADPH to NADH may be catalyzed by the soluble transhydrogenase UdhA, since a physiological role of this enzyme is the reoxidation of NADPH (1, 2).…”

Different Biochemical Mechanisms Ensure Network-Wide Balancing of Reducing Equivalents in Microbial Metabolism

“…In contrast, glucose-limited chemostat growth with extensive TCA cycle fluxes results in significant overproduction of NADPH (16,68), which is presumably balanced through the soluble transhydrogenase UdhA, shown previously to carry out this function in E. coli cells growing on acetate and in mutants with high PP pathway fluxes (55). This counterbalancing of NADPH overproduction can be readily transferred to other microbes through heterologous expression of the soluble transhydrogenase (17). Second, many bacteria (34,35,54,60,71) and yeast (49) contain NAD(H) kinases that can directly convert NAD(H) into NAD(P)H at the expense of ATP.…”