Contreras Angela, Ribbeck Magdalena, Gutiérrez Guillermo D, Cañon Pablo M, Mendoza Sebastián N, Agosin Eduardo
Department of Chemical and Bioprocess Engineering, School of Engineering, Pontificia Universidad Católica de Chile, Santiago, Chile.
Mathomics, Center for Mathematical Modeling, Universidad de Chile, Santiago, Chile.
Front Microbiol. 2018 Mar 1;9:291. doi: 10.3389/fmicb.2018.00291. eCollection 2018.
The effect of ethanol on the metabolism of , the bacterium responsible for the malolactic fermentation (MLF) of wine, is still scarcely understood. Here, we characterized the global metabolic response in PSU-1 to increasing ethanol contents, ranging from 0 to 12% (v/v). We first optimized a wine-like, defined culture medium, MaxOeno, to allow sufficient bacterial growth to be able to quantitate different metabolites in batch cultures of . Then, taking advantage of the recently reconstructed genome-scale metabolic model iSM454 for PSU-1 and the resulting experimental data, we determined the redistribution of intracellular metabolic fluxes, under the different ethanol conditions. Four growth phases were clearly identified during the batch cultivation of PSU-1 strain, according to the temporal consumption of malic and citric acids, sugar and amino acids uptake, and biosynthesis rates of metabolic products - biomass, erythritol, mannitol and acetic acid, among others. We showed that, under increasing ethanol conditions, favors anabolic reactions related with cell maintenance, as the requirements of NAD(P) and ATP increased with ethanol content. Specifically, cultures containing 9 and 12% ethanol required 10 and 17 times more NGAM (non-growth associated maintenance ATP) during phase I, respectively, than cultures without ethanol. MLF and citric acid consumption are vital at high ethanol concentrations, as they are the main source for proton extrusion, allowing higher ATP production by FF-ATPase, the main route of ATP synthesis under these conditions. Mannitol and erythritol synthesis are the main sources of NAD(P), countervailing for 51-57% of its usage, as predicted by the model. Finally, cysteine shows the fastest specific consumption rate among the amino acids, confirming its key role for bacterial survival under ethanol stress. As a whole, this study provides a global insight into how ethanol content exerts a differential physiological response in PSU-1 strain. It will help to design better strategies of nutrient addition to achieve a successful MLF of wine.
乙醇对葡萄酒苹果酸-乳酸发酵（MLF）的 responsible for 细菌代谢的影响仍鲜为人知。在此，我们表征了PSU-1在乙醇含量从0至12%（v/v）增加时的全局代谢响应。我们首先优化了一种类似葡萄酒的、确定的培养基MaxOeno，以使细菌能够充分生长，从而能够在PSU-1的分批培养物中定量不同代谢物。然后，利用最近重建的PSU-1基因组规模代谢模型iSM454以及所得实验数据，我们确定了不同乙醇条件下细胞内代谢通量的重新分布。根据苹果酸和柠檬酸的时间消耗、糖和氨基酸的摄取以及代谢产物（生物量、赤藓糖醇、甘露醇和乙酸等）的生物合成速率，在PSU-1菌株的分批培养过程中明确鉴定出四个生长阶段。我们表明，在乙醇含量增加的条件下，随着乙醇含量的增加，对NAD(P)和ATP的需求增加，PSU-1有利于与细胞维持相关的合成代谢反应。具体而言，含有9%和12%乙醇的培养物在第一阶段分别比不含乙醇的培养物需要多10倍和17倍的NGAM（非生长相关维持ATP）。在高乙醇浓度下，MLF和柠檬酸消耗至关重要，因为它们是质子外排的主要来源，允许FF-ATPase产生更高的ATP，这是这些条件下ATP合成的主要途径。如模型所预测，甘露醇和赤藓糖醇的合成是NAD(P)的主要来源，抵消了其51 - 57%的使用量。最后，半胱氨酸在氨基酸中显示出最快的比消耗速率，证实了其在乙醇胁迫下对细菌存活的关键作用。总体而言，本研究全面深入地了解了乙醇含量如何在PSU-1菌株中产生不同的生理反应。这将有助于设计更好的营养添加策略，以实现葡萄酒的成功MLF。
