Accurate Measurement of the in vivo  Ammonium Concentration in Saccharomyces cerevisiae

Ammonium (NH4+) is the most common N-source for yeast fermentations, and N-limitation is frequently applied to reduce growth and increase product yields. While there is significant molecular knowledge on NH4 + transport and assimilation, there have been few attempts to measure the in vivo concentration of this metabolite. In this article, we...

Interaction of storage carbohydrates and other cyclic fluxes with central metabolism: A quantitative approach by non-stationary 13C metabolic flux analysis

13C labeling experiments in aerobic glucose limited cultures of Saccharomyces cerevisiae at four different growth rates (0.054; 0.101, 0.207, 0.307 h?1) are used for calculating fluxes that include intracellular cycles (e.g., storage carbohydrate cycles, exchange fluxes with amino acids), which are rearranged depending on the growth rate. At low...

In vivo analysis of NH₄ ⁺ transport and central nitrogen metabolism in Saccharomyces cerevisiae during aerobic nitrogen-limited growth

Ammonium is the most common N source for yeast fermentations. Although its transport and assimilation mechanisms are well documented, there have been only a few attempts to measure the in vivo intracellular concentration of ammonium and assess its impact on gene expression. Using an isotope dilution mass spectrometry (IDMS)-based method, we...

Determination of the Cytosolic NADPH/NADP Ratio in Saccharomyces cerevisiae using Shikimate Dehydrogenase as Sensor Reaction

Eukaryotic metabolism is organised in complex networks of enzyme catalysed reactions which are distributed over different organelles. To quantify the compartmentalised reactions, quantitative measurements of relevant physiological variables in different compartments are needed, especially of cofactors. NADP(H) are critical components in cellular...

Fast “Feast/Famine” Cycles for Studying Microbial Physiology Under Dynamic Conditions: A Case Study with Saccharomyces cerevisiae

Microorganisms are constantly exposed to rapidly changing conditions, under natural as well as industrial production scale environments, especially due to large-scale substrate mixing limitations. In this work, we present an experimental approach based on a dynamic feast/famine regime (400 s) that leads to repetitive cycles with moderate changes...

Glucose-methanol co-utilization in Pichia pastoris studied by metabolomics and instationary 13C flux analysis

Background Several studies have shown that the utilization of mixed carbon feeds instead of methanol as sole carbon source is beneficial for protein production with the methylotrophic yeast Pichia pastoris. In particular, growth under mixed feed conditions appears to alleviate the metabolic burden related to stress responses triggered by protein...

Similar temperature dependencies of glycolytic enzymes: An evolutionary adaptation to temperature dynamics?

Background Temperature strongly affects microbial growth, and many microorganisms have to deal with temperature fluctuations in their natural environment. To understand regulation strategies that underlie microbial temperature responses and adaptation, we studied glycolytic pathway kinetics in Saccharomyces cerevisiae during temperature changes....

Substrate cycles in Penicillium chrysogenum quantified by isotopic non-stationary flux analysis

Background Penicillium chrysogenum, the main production strain for penicillin-G, has a high content of intracellular carbohydrates, especially reduced sugars such as mannitol, arabitol, erythritol, as well as trehalose and glycogen. In previous steady state 13C wash-in experiments a delay of labeling enrichments in glycolytic intermediates was...