Mapping the Physiological Response of Oenococcus oeni to Ethanol Stress Using an Extended Genome-Scale Metabolic Model

Abstract

The effect of ethanol on the metabolism of Oenococcus oeni, the bacterium responsible for the malolactic fermentation (MLF) of wine, is still scarcely understood. Here, we characterized the global metabolic response in O. oeni 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 O. oeni. Then, taking advantage of the recently reconstructed genome-scale metabolic model iSM454 for O. oeni 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 O. oeni 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, O. oeni 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 F0F1-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 O. oeni PSU-1 strain. It will help to design better strategies of nutrient addition to achieve a successful MLF of wine.

Selection and Validation of Reference Genes for Quantitative Real-Time PCR Normalization Under Ethanol Stress Conditions in Oenococcus oeni SD-2a

“…However, the harsh environmental conditions of wine such as high ethanol concentration, low pH, and SO 2 , can often delay the growth of O. oeni and MLF process (G-Alegría et al, 2004 ; Olguín et al, 2015 ; Betteridge et al, 2017 ). Ethanol stress is generally considered as one of the main inhibitors of O. oeni growth in wine (Mendoza et al, 2017 ; Contreras et al, 2018 ). The strain O. oeni SD-2a is able to survive and grow in high ethanol conditions and shows strong MLF ability (Liu, 2002 ), however its ethanol stress response mechanism was still obscure.…”

Quantitative proteomic analysis reveals the ethanologenic metabolism regulation of Ethanoligenens harbinense by exogenous ethanol addition

“…The results indicated that the requirements of NAD(P) + and ATP increased during ethanol stress. The strain required 10 and 17 times more non-growth associated maintenance ATP during growth in medium containing 9% and 12% ethanol, respectively [29]. Therefore, the altered distribution of end-product yields in strain YUAN-3 indicates the need to redirect energy flow to maintain an efficient balance between ethanol stress response and growth.…”

“…More recently, the effect of ethanol on the global metabolic response in Oenococcus oeni was carried out using an extended genome-scale metabolic model. The results indicated that the requirements of NAD(P) + and ATP increased in ethanol stress, and the strain required 10 and 17 times more ATP for non-growth associated maintenance during growth in medium containing 9% and 12% ethanol, respectively [29]. Therefore, these studies demonstrated that it is difficult to determine the microbial ethanol stress response mechanisms through limited proteins or pathways.…”

Oenococcus oeni, a species born and moulded in wine: a critical review of the stress impacts of wine and the physiological responses

“…The (F 0 F 1 ) H + -ATPases and MLF are closely linked and are essential in countering acid stress (Tourdot-Maréchal et al 1999). It has not been investigated which function of the (F 0 F 1 ) H + -ATPases dominate, but the use of a genome-scale metabolic model has facilitated the calculation of significant ATP production by ATPases as well as ATP consumption in non-growth associated maintenance, attributed to ATPases (Contreras et al 2018). In several studies it has been observed that MLF appears to start once the O. oeni population reaches approximately 1 × 10 6 colony-forming unit (CFU)/mL (Rice 1965, Rice and Mattick 1970, Silver and Leighton 1981, Liu and Gallander 1982, Versai et al 1999.…”