Table_1_Oenococcus oeni Exopolysaccharide Biosynthesis, a Tool to Improve Malolactic Starter Performance.docx

<p>Oenococcus oeni is the lactic acid bacterium that most commonly drives malolactic fermentation (MLF) in wine. Though the importance of MLF in terms of wine microbial stability and sensory improvement is well established, it remains a winemaking step not so easy to control. O. oeni displays many adaptation tools to resist the harsh wine conditions which explain its natural dominance at this stage of winemaking. Previous findings showed that capsular polysaccharides and endogenous produced dextran increased the survival rate and the conservation time of malolactic starters. In this paper, we showed that exopolysaccharides specific production rates were increased in the presence of single stressors relevant to wine (pH, ethanol). The transcription of the associated genes was investigated in distinct O. oeni strains. The conditions in which eps genes and EPS synthesis were most stimulated were then evaluated for the production of freeze dried malolactic starters, for acclimation procedures and for MLF efficiency. Sensory analysis tests on the resulting wines were finally performed.</p

Image_3_Oenococcus oeni Exopolysaccharide Biosynthesis, a Tool to Improve Malolactic Starter Performance.TIF

<p>Oenococcus oeni is the lactic acid bacterium that most commonly drives malolactic fermentation (MLF) in wine. Though the importance of MLF in terms of wine microbial stability and sensory improvement is well established, it remains a winemaking step not so easy to control. O. oeni displays many adaptation tools to resist the harsh wine conditions which explain its natural dominance at this stage of winemaking. Previous findings showed that capsular polysaccharides and endogenous produced dextran increased the survival rate and the conservation time of malolactic starters. In this paper, we showed that exopolysaccharides specific production rates were increased in the presence of single stressors relevant to wine (pH, ethanol). The transcription of the associated genes was investigated in distinct O. oeni strains. The conditions in which eps genes and EPS synthesis were most stimulated were then evaluated for the production of freeze dried malolactic starters, for acclimation procedures and for MLF efficiency. Sensory analysis tests on the resulting wines were finally performed.</p

Image_2_Oenococcus oeni Exopolysaccharide Biosynthesis, a Tool to Improve Malolactic Starter Performance.TIF

<p>Oenococcus oeni is the lactic acid bacterium that most commonly drives malolactic fermentation (MLF) in wine. Though the importance of MLF in terms of wine microbial stability and sensory improvement is well established, it remains a winemaking step not so easy to control. O. oeni displays many adaptation tools to resist the harsh wine conditions which explain its natural dominance at this stage of winemaking. Previous findings showed that capsular polysaccharides and endogenous produced dextran increased the survival rate and the conservation time of malolactic starters. In this paper, we showed that exopolysaccharides specific production rates were increased in the presence of single stressors relevant to wine (pH, ethanol). The transcription of the associated genes was investigated in distinct O. oeni strains. The conditions in which eps genes and EPS synthesis were most stimulated were then evaluated for the production of freeze dried malolactic starters, for acclimation procedures and for MLF efficiency. Sensory analysis tests on the resulting wines were finally performed.</p

Observation of <i>O. oeni</i> capsules by transmission electron microscopy.

<p>The black arrow indicates the place where the capsule may appear as a dark halo/layer when present. The strain <i>L. lactis</i> IL1403, which displays a thin polysaccharide pellicle as demonstrated by Chapot Chartier et al. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0098898#pone.0098898-ChapotChartier1" target="_blank">[70]</a>, serves as a reference. Strains <i>O. oeni</i> S28 and 0607 are clearly encapsulated, while strain 0205 has no dense area beyond the peptidoglycan layer (light gray layer).</p

Structural analysis of the soluble exopolysaccharides produced by selected strains.

a<p>All the strains in the Table also displayed <i>eps1</i> and <i>eps2</i> clusters. None displayed <i>gtf.</i></p>b<p>The EPS concentration was determined by the anthrone sulfuric method. The number between brackets indicates the number of chromatographic peaks after gel permeation on superdex 30 column. The peak at 5500 Da was always present. The second peak, when present indicates the presence of polymers with molecular weight higher than 1 000 000 Da.</p>c<p>ND: not determined, no high molecular weight EPS produced.</p

Protein sequence identity in <i>eps1</i> clusters.

a<p>NC: No Cazy number.</p>b<p>Identity (%) between proteins of selected strains representative of each model :<i>O. oeni</i> PSU-1 (model A) is used as a reference, and ortholog proteins of strain <i>O. oeni</i> B429 (model B) and <i>O. oeni</i> B422 (model C) are compared to <i>O. oeni</i> PSU-1 ones, except for WoaF, for which the sequence found in <i>O. oeni</i> B-429 is used as the reference. When two strains display the same model of cluster <i>eps1</i>, the identity between related proteins is higher than 98%. Abs: protein absent.</p

Genetic organization of <i>O oeni</i> chromosome regions harboring <i>dsrO</i> and <i>dsrV</i> genes.

<p>Example of strains <i>O. oeni</i> PSU-1, BAA-1163, 0607 and 277. The strain 277 also diplays a <i>dsrO</i> gene, similar to that found in <i>O. oeni</i> PSU-1.</p

Schematic representation of the <i>eps loci</i> on the chromosome of <i>O. oeni</i>.

<p>The chromosome of <i>O. oeni</i> PSU-1 is represented with its own <i>eps genes or loci</i> (black). The position of the adjacent regions of the additional <i>loci</i> found in other <i>O. oeni</i> strains are presented in gray: <i>eps1</i> and <i>eps2</i>: heteropolysaccharide clusters; <i>gtf:</i> β-glucan synthase gene; <i>it3</i> and <i>it4</i>: priming glycosyltransferase isolated genes; <i>dsrO</i> and <i>dsrV</i>: dextransucrase genes; <i>levO</i>: levansucrase gene.</p

Putative precursor biosynthetic pathways active in <i>O. oeni</i> deduced from genome analysis.

<p>The enzyme full names and the accession numbers of reference proteins are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0098898#pone.0098898.s001" target="_blank">Table S1</a> (panel precurors). The solid arrows indicate the central pathways (glucose 6-P to xylulose-5-P and PEP and acetyl-CoA) and the pathways potentially active in all the strains studied, as the associated enzymes are encoded by the 50 genomes studied. The dashed arrows indicate pathways putatively active in a smaller number of strains. The EPS monomer precursors potentially available in all the strains studied are boxed in solid lines, while the precursors putatively available in a limited number of strains are boxed with dotted lines. “?” indicate metabolic steps for which no enzyme was identified from the genome analyses. <i>P: phosphate, CoA : coenzyme-A, NDP : nucleotidyl-diphosphate, CDP : cytidyl-diphosphate, UDP : uridine-diphosphate; GDP: guanosine-diphosphate, dTDP : desoxythymidine diphosphate, Glc : glucose, Fru : fructose, GlcA : glucuronic acid, Gal : galactose, Galp : galactopyranose, Galf : galactofuranose</i>, LicA: choline kinase, LicC: choline cytidyltransferase <i>LRha, L-rhamnose, GlN : glucosamine, N-Ac-Glc : N-acetyl glucosamine, N-Ac-Gal : N-acetyl-galactosamine, N-Ac-Man : N-acetyl-mannosamine, G-A-P : glyceraldehyde 3-phosphate, DHAP: dihydroxyacetone phosphate, PEP : phosphoenolpyruvate.</i></p