Fast "Feast/Famine" Cycles for Studying Microbial Physiology Under Dynamic Conditions: A Case Study with Saccharomyces cerevisiae.
Fast "Feast/Famine" Cycles for Studying Microbial Physiology Under Dynamic Conditions: A Case Study with Saccharomyces cerevisiae.
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DOI：
10.3390/metabo4020347
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发表时间：
2014-05-15
期刊：
影响因子：
4.1
通讯作者：
Wahl A
中科院分区：
文献类型：
--
作者：
Suarez-Mendez CA;Sousa A;Heijnen JJ;Wahl A
微生物在自然和工业生产规模的环境下不断暴露于快速变化的条件下，特别是由于大规模底物混合的限制。在这项工作中，我们提出了一种实验方法的基础上的动态盛宴/饥荒制度（400秒），导致重复的周期与适度的变化，在有氧葡萄糖培养的酿酒酵母的底物可用性。几个循环后，盛宴/饥饿产生了稳定和重复的模式，并及时产生了可再现的代谢反应，从而为在动态条件下研究微生物的生理学提供了一个强大的平台。我们发现，在盛宴/饥荒制度下，生物量产量略有减少（-5%），而平均底物和氧气消耗以及二氧化碳生产率相当。细胞内代谢物的动态响应与其他动态实验（特别是刺激-响应实验，SRE）相比表现出特定的差异。值得注意的是，在单脉冲实验中观察到的频繁报道的ATP悖论在这里应用的重复扰动期间不存在。我们发现，细胞内动态积累导致底物摄取速率的解偶联（在20 s时高达9倍的变化）。此外，与盛宴/饥饿获得的细胞内代谢物的动态曲线表明，在延迟响应的调节机制的存在。利用盛宴饥饿设置，可以在高频率下测量许多细胞状态，给出了可重复循环的特征。因此，盛宴/饥饿制度是系统生物学方法的通用平台，可以帮助我们在现实条件下识别和研究代谢物调控（例如，大规模生物反应器或自然环境）。
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).