Aerobic and anaerobic central metabolism of Saccharomyces cerevisiae cells was explored in batch cultures on a minimal medium containing glucose as the sole carbon source, using biosynthetic fractional (13)C labeling of proteinogenic amino acids. This allowed, firstly, unravelling of the network of active central pathways in cytosol and mitochondria, secondly, determination of flux ratios characterizing glycolysis, pentose phosphate cycle, tricarboxylic acid cycle and C1-metabolism, and thirdly, assessment of intercompartmental transport fluxes of pyruvate, acetyl-CoA, oxaloacetate and glycine. The data also revealed that alanine aminotransferase is located in the mitochondria, and that amino acids are synthesized according to documented pathways. In both the aerobic and the anaerobic regime: (a) the mitochondrial glycine cleavage pathway is active, and efflux of glycine into the cytosol is observed; (b) the pentose phosphate pathways serve for biosynthesis only, i.e. phosphoenolpyruvate is entirely generated via glycolysis; (c) the majority of the cytosolic oxaloacetate is synthesized via anaplerotic carboxylation of pyruvate; (d) the malic enzyme plays a key role for mitochondrial pyruvate metabolism; (e) the transfer of oxaloacetate from the cytosol to the mitochondria is largely unidirectional, and the activity of the malate-aspartate shuttle and the succinate-fumarate carrier is low; (e) a large fraction of the mitochondrial pyruvate is imported from the cytosol; and (f) the glyoxylate cycle is inactive. In the aerobic regime, 75% of mitochondrial oxaloacetate arises from anaplerotic carboxylation of pyruvate, while in the anaerobic regime, the tricarboxylic acid cycle is operating in a branched fashion to fulfill biosynthetic demands only. The present study shows that fractional (13)C labeling of amino acids represents a powerful approach to study compartmented eukaryotic systems.

BackgroundYeasts are attractive expression platforms for many recombinant proteins, and there is evidence for an important interrelation between the protein secretion machinery and environmental stresses. While adaptive responses to such stresses are extensively studied in Saccharomyces cerevisiae, little is known about their impact on the physiology of Pichia pastoris. We have recently reported a beneficial effect of hypoxia on recombinant Fab secretion in P. pastoris chemostat cultivations. As a consequence, a systems biology approach was used to comprehensively identify cellular adaptations to low oxygen availability and the additional burden of protein production. Gene expression profiling was combined with proteomic analyses and the 13C isotope labelling based experimental determination of metabolic fluxes in the central carbon metabolism.ResultsThe physiological adaptation of P. pastoris to hypoxia showed distinct traits in relation to the model yeast S. cerevisiae. There was a positive correlation between the transcriptomic, proteomic and metabolic fluxes adaptation of P. pastoris core metabolism to hypoxia, yielding clear evidence of a strong transcriptional regulation component of key pathways such as glycolysis, pentose phosphate pathway and TCA cycle. In addition, the adaptation to reduced oxygen revealed important changes in lipid metabolism, stress responses, as well as protein folding and trafficking.ConclusionsThis systems level study helped to understand the physiological adaptations of cellular mechanisms to low oxygen availability in a recombinant P. pastoris strain. Remarkably, the integration of data from three different levels allowed for the identification of differences in the regulation of the core metabolism between P. pastoris and S. cerevisiae. Detailed comparative analysis of the transcriptomic data also led to new insights into the gene expression profiles of several cellular processes that are not only susceptible to low oxygen concentrations, but might also contribute to enhanced protein secretion.

The phytohormones jasmonates (JAs) constitute an important class of elicitors for many plant secondary metabolic pathways. However, JAs do not act independently but operate in complex networks with crosstalk to several other phytohormonal signaling pathways. Here, crosstalk was detected between the JA and abscisic acid (ABA) signaling pathways in the regulation of tobacco (Nicotiana tabacum) alkaloid biosynthesis. A tobacco gene from the PYR/PYL/RCAR family, NtPYL4, the expression of which is regulated by JAs, was found to encode a functional ABA receptor. NtPYL4 inhibited the type-2C protein phosphatases known to be key negative regulators of ABA signaling in an ABA-dependent manner. Overexpression of NtPYL4 in tobacco hairy roots caused a reprogramming of the cellular metabolism that resulted in a decreased alkaloid accumulation and conferred ABA sensitivity to the production of alkaloids. In contrast, the alkaloid biosynthetic pathway was not responsive to ABA in control tobacco roots. Functional analysis of the Arabidopsis (Arabidopsis thaliana) homologs of NtPYL4, PYL4 and PYL5, indicated that also in Arabidopsis altered PYL expression affected the JA response, both in terms of biomass and anthocyanin production. These findings define a connection between a component of the core ABA signaling pathway and the JA responses and contribute to the understanding of the role of JAs in balancing tradeoffs between growth and defense.nicotine | phenylpropanoid | primary metabolism | secondary metabolism | stress response

BackgroundThe methylotrophic yeast Pichia pastoris has emerged as one of the most promising yeast hosts for the production of heterologous proteins. Mixed feeds of methanol and a multicarbon source instead of methanol as sole carbon source have been shown to improve product productivities and alleviate metabolic burden derived from protein production. Nevertheless, systematic quantitative studies on the relationships between the central metabolism and recombinant protein production in P. pastoris are still rather limited, particularly when growing this yeast on mixed carbon sources, thus hampering future metabolic network engineering strategies for improved protein production.ResultsThe metabolic flux distribution in the central metabolism of P. pastoris growing on a mixed feed of glucose and methanol was analyzed by Metabolic Flux Analysis (MFA) using 13C-NMR-derived constraints. For this purpose, we defined new flux ratios for methanol assimilation pathways in P. pastoris cells growing on glucose:methanol mixtures. By using this experimental approach, the metabolic burden caused by the overexpression and secretion of a Rhizopus oryzae lipase (Rol) in P. pastoris was further analyzed. This protein has been previously shown to trigger the unfolded protein response in P. pastoris. A series of 13C-tracer experiments were performed on aerobic chemostat cultivations with a control and two different Rol producing strains growing at a dilution rate of 0.09 h−1 using a glucose:methanol 80:20 (w/w) mix as carbon source.The MFA performed in this study reveals a significant redistristribution of carbon fluxes in the central carbon metabolism when comparing the two recombinant strains vs the control strain, reflected in increased glycolytic, TCA cycle and NADH regeneration fluxes, as well as higher methanol dissimilation rates.ConclusionsOverall, a further 13C-based MFA development to characterise the central metabolism of methylotrophic yeasts when growing on mixed methanol:multicarbon sources has been implemented, thus providing a new tool for the investigation of the relationships between central metabolism and protein production. Specifically, the study points at a limited but significant impact of the conformational stress associated to secretion of recombinant proteins on the central metabolism, occurring even at modest production levels.

Background: The yeast Saccharomyces cerevisiae is able to adjust to external oxygen availability by utilizing both respirative and fermentative metabolic modes. Adjusting the metabolic mode involves alteration of the intracellular metabolic fluxes that are determined by the cell's multilevel regulatory network. Oxygen is a major determinant of the physiology of S. cerevisiae but understanding of the oxygen dependence of intracellular flux distributions is still scarce.

Saccharomyces cerevisiae CEN.PK113-1A was grown in glucose-limited chemostat culture with 0%, 0.5%, 1.0%, 2.8% or 20.9% O2 in the inlet gas (D=0.10 h(-1), pH 5, 30 degrees C) to determine the effects of oxygen on 17 metabolites and 69 genes related to central carbon metabolism. The concentrations of tricarboxylic acid cycle (TCA) metabolites and all glycolytic metabolites except 2-phosphoglycerate+3-phosphoglycerate and phosphoenolpyruvate were higher in anaerobic than in fully aerobic conditions. Provision of only 0.5-1% O2 reduced the concentrations of most metabolites, as compared with anaerobic conditions. Transcription of most genes analyzed was reduced in 0%, 0.5% or 1.0% O2 relative to cells grown in 2.8% or 20.9% O2. Ethanol production was observed with 2.8% or less O2. After steady-state analysis in defined oxygen concentrations, the conditions were switched from aerobic to anaerobic. Metabolite and transcript levels were monitored for up to 96 h after the transition, and this showed that more than 30 h was required for the cells to fully adapt to anaerobiosis. Levels of metabolites of upper glycolysis and the TCA cycle increased following the transition to anaerobic conditions, whereas those of metabolites of lower glycolysis generally decreased. Gene regulation was more complex, with some genes showing transient upregulation or downregulation during the adaptation to anaerobic conditions.

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