EC1118EC1118

EKDEKD--1313

BY4743BY4743

ΔΔKNR4KNR4

5mM H5mM H220022 10 10 mMmM HH220022

YPDYPD

EC1118EC1118

EKDEKD--1313

BY4743BY4743

ΔΔKNR4KNR4

YPGYPG

EC1118EC1118

EKDEKD--1313

BY4743BY4743

ΔΔKNR4KNR4

5mM H5mM H220022 10 10 mMmM HH220022

YPDYPD

EC1118EC1118

EKDEKD--1313

BY4743BY4743

ΔΔKNR4KNR4

YPGYPG

Technological properties and transcriptomic profile Technological properties and transcriptomic profile of a genetically engineered of a genetically engineered Saccharomyces cerevisiaeSaccharomyces cerevisiae

wine yeast strain overproducing mannoproteinswine yeast strain overproducing mannoproteins

AcknowledgmentsAcknowledgments: This work was supported by a predoctoral fellowship from INIA (grant 18-BOE208 31-08-2006), the project INIA (RTA2005-00169-00-00) and the Picasso program (HF2006-0066) and are grateful to Pierre Delobel and Isabelle Sanchez for technical assistance and data analysis in Montpellier and to Manuel Quirós for the quantification of mannoproteins in Logroño.

Penacho, VanessaPenacho, Vanessa11; Valero, Eva; Valero, Eva22; Blondin, Bruno; Blondin, Bruno33 and González Ramón and González Ramón11

1 Instituto de Ciencias de la Vid y del Vino (CSIC-UR-CAR), Lab. Enología, Complejo Científico Tecnológico, C/ Madre de Dios, 51, 26006 Logroño (La Rioja), Spain.

2 Universidad Pablo de Olavide, Dpto. de Biología Molecular e Ingeniería Bioquímica, Ctra. Utrera, km 1 - 41013 Sevilla, Spain.3 INRA, Montpellier SupAgro, UMR Sci Oenol 1083, F-34060 Montpellier, France.

INTRODUCTIONINTRODUCTION

The cell wall of Saccharomyces cerevisiae is a highly dynamic structure which functions in morphogenesis, bud emergence, conjugation by mating, adherence and flocculation. It is composed in a 40% by mannoproteins, highly glycosylated proteins that are released during wine fermentation and aging contributing to wine quality. Several recent papers investigated the effect of deletions of genes related to cell wall biogenesis on the release of mannoproteins, resulting in recombinant strains improved in this feature.

MATERIALS & METHODSMATERIALS & METHODS

Saccharomyces cerevisiae Saccharomyces cerevisiae strains strains

EC1118 (wine industrial strain); EKD-13 (recombinant strain; deletion KNR4 gene: β-1,3-glucan biosynthesis; EC1118 background); BY4743 (yeast deletion parental strain; MATa/ MATα his3∆1/his3∆1 leu2∆0/leu2∆0 ura3∆0/ura3∆0 met15∆0/MET15); S. cerevisiae YKO Homozygous Diploid Strain – KNR4 gene deletion.

Assay conditionsAssay conditions

Monitored batch fermentation: Performed in bioreactors (1.1 L working volume, anaerobiosis, constant stirring, 28ºC). Media: synthetic must MS300-G (200 g/L glucose, 6 g/L citric acid, 6 g/L DL-malic acid, mineral salts, vitamins, anaerobic growth factors, 300 mg/L assimilable nitrogen). Sampling at 1, 10, 70 and 90 g/L of CO2 released as representative stages of early, mid exponential and stationary growth phase, and end of fermentation, respectively. Variable analyzed: quantification of mannoproteins by the acid hydrolysis/HPLC method. Microarray analysis: LOWESS function method normalization (limmaGUI package, software R). Calculated log2 ratios comparing EKD-13 strain to the reference strain EC1118 (significantly differential expression: p-value<0.05). Enrichment of functional categories: FunSpec interpreter, YEASTRACT.

Aging on lees: Performed in flasks (300 mL working volume, anaerobiosis, static conditions, 28ºC). Media: synthetic must MS300-GA modified (no malic acid, no amino acids). Sampling at stationary growth phase and weekly along 8 weeks. Variable analyzed: quantification of mannoproteins by the acid hydrolysis/HPLC; free amino nitrogen fraction released as the result of autolysis evolution by the OPA.

Flocculation: Cellular deflocculating in 50 mM Na-citrate (pH 3.0) - 5 mM EDTA buffer. Induced flocculation with 20 mM CaCl2. Spectrophotometric measure (OD600nm) in a 60 minutes-time course. Flocculation was expressed in % as the decrease in absorbance after 50 minutes. Media: synthetic must MS300-GA. YPD, as control.

Oxidative stress/glycerol utilization: Cultures in stationary growth phase (108 cells/mL) were treated with H2O2 at a final concentration of 5 and 10 mM. Serial 10-fold dilutions of cell suspensions were spotted onto YPD, YPG plates (in glycerol utilization).

Statistical analysis of dataStatistical analysis of data

Reproducibility: one-way ANOVA; Dunnett test (comparison of means); significantly differences: p-value<0.05. SPSS 15.0 software.

Since Since PAUPAU genes are usually induced by anaerobiosis, the genetic modification seems to have two genes are usually induced by anaerobiosis, the genetic modification seems to have two main consequences on transcription during fermentation, impairment of oxidative stress response, main consequences on transcription during fermentation, impairment of oxidative stress response, and an anticipated or overdone adaptation to anaerobiosis, as illustrated by the enrichment in and an anticipated or overdone adaptation to anaerobiosis, as illustrated by the enrichment in genes induced by anaerobiosis among the genes overexpressed in EKD-13; and enrichment in genes genes induced by anaerobiosis among the genes overexpressed in EKD-13; and enrichment in genes repressed by anaerobiosis or induced by aerobiosis, among the genes underexpressed in EKD-13repressed by anaerobiosis or induced by aerobiosis, among the genes underexpressed in EKD-13 . . The shift towards anaerobic adaptation is in agreement with the increased sensitivity to The shift towards anaerobic adaptation is in agreement with the increased sensitivity to oxidative stress, and impaired growth glycerol (below).oxidative stress, and impaired growth glycerol (below).

MONITORED FERMENTATIONMONITORED FERMENTATION

AGING ON LEESAGING ON LEES

DEGREE OF FLOCCULATIONDEGREE OF FLOCCULATION

OXIDATIVE STRESS/GLYCEROL UTILIZATIONOXIDATIVE STRESS/GLYCEROL UTILIZATION

CONCLUSIONSCONCLUSIONS

Considering the pattern and the level of mannoprotein release and the Considering the pattern and the level of mannoprotein release and the flocculation behavior, EKD-13 or equivalent non-recombinant strains flocculation behavior, EKD-13 or equivalent non-recombinant strains would be perfect in order to increase mannoprotein content of wines, would be perfect in order to increase mannoprotein content of wines, as well as shortening the aging period of either still wines aged o lees or as well as shortening the aging period of either still wines aged o lees or sparkling wines. The apparently overdone transcriptional adaptation to sparkling wines. The apparently overdone transcriptional adaptation to anaerobiosis does not seem to involve detrimental effects on anaerobiosis does not seem to involve detrimental effects on fermentation performance.fermentation performance.

Time course fermentation was monitored by measuring the amount of COTime course fermentation was monitored by measuring the amount of CO22 released, expressed released, expressed as weight loss over successive 20-min periods. The rate of COas weight loss over successive 20-min periods. The rate of CO22 production was calculated production was calculated automatically by polynomial smoothing of COautomatically by polynomial smoothing of CO22 evolution. evolution.

RESULTSRESULTS

The recombinant strain shows increased release The recombinant strain shows increased release of mannoproteins just during the fermentation of mannoproteins just during the fermentation step. In contrast, amino acids are released also step. In contrast, amino acids are released also during the aging period. Increased release of during the aging period. Increased release of mannoproteins by EKD-13 does not seem to be mannoproteins by EKD-13 does not seem to be related to increased autolysis. Net release of related to increased autolysis. Net release of either mannoproteins or amino acids is always either mannoproteins or amino acids is always higher for the recombinant strain.higher for the recombinant strain.

This flocculation phenotype is in agreement This flocculation phenotype is in agreement with the enrichment in Flo1p targets among with the enrichment in Flo1p targets among genes whose expression is affected by the genes whose expression is affected by the genetic modification (according to genetic modification (according to YEASTRACT; data not shown).YEASTRACT; data not shown).

0

10

20

30

40

50

60

70

2 4 5 6 7 8

Time (weeks)

am

ino

ac

ids

(mg

/L)

EC1118EKD·13

REFERENCESREFERENCES

Gonzalez-Ramos et al. 2008. Appl. Environ. Microbiol. 74:5533–5540

Quirós et al. DOI:10.1016/j.foodchem.2010.08.0660