Abstract
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 in substrate availability in an aerobic glucose cultivation of Saccharomyces cerevisiae. After a few cycles, the feast/famine produced a stable and repetitive pattern with a reproducible metabolic response in time, thus providing a robust platform for studying the microorganism’s physiology under dynamic conditions. We found that the biomass yield was slightly reduced (−5%) under the feast/famine regime, while the averaged substrate and oxygen consumption as well as the carbon dioxide production rates were comparable. The dynamic response of the intracellular metabolites showed specific differences in comparison to other dynamic experiments (especially stimulus-response experiments, SRE). Remarkably, the frequently reported ATP paradox observed in single pulse experiments was not present during the repetitive perturbations applied here. We found that intracellular dynamic accumulations led to an uncoupling of the substrate uptake rate (up to 9-fold change at 20 s.) Moreover, the dynamic profiles of the intracellular metabolites obtained with the feast/famine suggest the presence of regulatory mechanisms that resulted in a delayed response. With the feast famine setup many cellular states can be measured at high frequency given the feature of reproducible cycles. The feast/famine regime is thus a versatile platform for systems biology approaches, which can help us to identify and investigate metabolite regulations under realistic conditions (e.g., large-scale bioreactors or natural environments).
Highlights
In the natural environment, and at large scale industrial cultivation, organisms are exposed to rapid dynamic conditions [1], which are repetitive [2]
While in Mashego et al [9] and Theobald et al [6] it was observed that FBP increased right after the pulse and remained constant at high levels, in our observations we found that FBP followed the pattern of the extracellular glucose
We propose a platform that uses a block-wise feeding regime for the identification of metabolic states under conditions similar to those encountered in nature as well as industrial large scale cultivations
Summary
At large scale industrial cultivation, organisms are exposed to rapid dynamic conditions [1], which are repetitive [2]. We present a complete description of the yeast’s metabolic response to a stimulus-response experiment referred to as feast/famine by which, the microorganisms are exposed to repetitive changes in substrate availability as a result of a block-wise feeding regime in a time window of seconds to minutes. With this setup, we focus on the short-term response by monitoring the in vivo metabolic activity (intra and extracellular concentrations and fluxes) during cycles of 400 s. At this short time-scale, it can be assumed that the metabolic fluxes are mainly controlled by metabolite interactions, while enzyme concentrations remain constant over the cycles [13,14]
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