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. 2018 Mar 1:9:291.
doi: 10.3389/fmicb.2018.00291. eCollection 2018.

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

Angela Contreras  1 , Magdalena Ribbeck  1 , Guillermo D Gutiérrez  1 , Pablo M Cañon  1 , Sebastián N Mendoza  2   3 , Eduardo Agosin  1
Affiliations

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

Angela Contreras et al. Front Microbiol. .

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.

Keywords: Oenococcus oeni; genome-scale metabolic model; lactic acid bacteria; malolactic fermentation; physiological ethanol response; wine-like defined culture medium.

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Figures

FIGURE 1
FIGURE 1
Framework for the incorporation of the experimental data. (1) General approach used to model exponential phase in GEMS. (2) Our experimental results showed four phases: three growth phases and a fourth phase, corresponding to stationary phase. Growth phases couldn’t be modeled individually as there was accumulation, as observed in the experimental results. (3) These accumulation compounds were: malate and mannitol, and the amino acids valine, phenylalanine, cysteine and threonine. Accumulation was observed between phases I to II, and II to III, and not from III to IV. (4) The extended model was constructed, which was able to simulate simultaneously the three phases identified for each ethanol level, and the accumulation observed experimentally. This matrix includes the following components: a matrix “Z” that contains three “S” matrix, one per phase, and 2 “a” vectors (1 × 6), where “a” represents the accumulation reactions that allows interaction between phases; thus, matrix sigma has a size (3n × d), where d = 3m + 2∗6. The extended model also includes vector “u” (1 × d) of internal fluxes, limited by vector “l” and “p” (1 × d), and a vector “k” of weights (1 × 3n). (5) To simplify the analysis, the extended matrix can be divided into three independent problems, one corresponding to each phase, and thus each one includes an S matrix, and a set of accumulation reactions as input of the system (accumulation in the previous phase) and/or output of the system (accumulation in the consecutive phase). (6) The span of these accumulation reactions was determined with flux variability analysis in the extended model, with the experimental data fixed in the model. (7) Estimations and further calculations were carried out in the independent model described in (5), with the span determined in (6) for the accumulation reactions.
FIGURE 2
FIGURE 2
Effect of ethanol concentration on the growth of O. oeni PSU-1 strain cultivated in MaxOeno, a defined wine-like culture medium. Each growth phase is delimited by discontinued vertical lines. Phases I, II III, and IV last between 0 to 48 h, 48 to 104 h and 104 to168 h and 168 to 264 h of cultivation, respectively.
FIGURE 3
FIGURE 3
Specific consumption of glucose (A), and fructose (B) and concomitant specific production of the related products, erythritol (C) and mannitol (D). during cultivation of O. oeni PSU-1 under increasing ethanol contents. Statistical analysis only was performed in phase I and shared letters indicate no significant difference (Mood test, p < 0.05).
FIGURE 4
FIGURE 4
Specific consumption of L-malate (A) and citrate (B) and specific production of acetate (C) L-lactate (D) and D-lactate (E) during growth of O. oeni PSU-1, under increasing ethanol contents. Statistical analysis only was performed in phase I and shared letters indicate no significant difference (Mood test, p < 0.05).
FIGURE 5
FIGURE 5
Specific consumption rates of amino acids during cultivation of O. oeni PSU-1 under increasing ethanol contents. (A) Cysteine. (B) Aspartic acid. (C) Threonine. (D) Histidine. (E) Serine. (F) Proline. (G) Lysine. (H) Alanine. Statistical analysis was only performed for phase I and shared letters indicate no significant difference (Mood test, p < 0.05).
FIGURE 6
FIGURE 6
Non-growth associated maintenance (NGAM) and in silico determined specific production rates of key metabolites of O. oeni. (A) NGAM, (B) NAD(P)+/NAD(P)H, (C) ATP produced by F0F1-ATPase, (D) ATP produced by phosphoketolase pathway, (E) Total ATP, (i.e.) ATP produced by both F0F1-ATPase and phosphoketolase pathways.
FIGURE 7
FIGURE 7
Metabolic flux redistribution of the central carbon metabolic pathways of O. oeni PSU-1 upon cultivation in a culture medium without and with 3, 6, 9, and 12% (yellow boxes, from top to bottom) ethanol concentration, during growth phase I. Polygons with colors orange, blue and green indicate consumption (or production) of ATP, NADH and NADPH, respectively.
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