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Technological properties and transcriptomic profile
of a genetically engineered Saccharomyces cerevisiae
wine yeast strain overproducing mannoproteins
Penacho, Vanessa1; Valero, Eva2; Blondin, Bruno3 and González Ramón1
1
2
3
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.
Universidad Pablo de Olavide, Dpto. de Biología Molecular e Ingeniería Bioquímica, Ctra. Utrera, km 1 - 41013 Sevilla, Spain.
INRA, Montpellier SupAgro, UMR Sci Oenol 1083, F-34060 Montpellier, France.
INTRODUCTION
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.
RESULTS
MATERIALS & METHODS
Saccharomyces cerevisiae 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.
MONITORED FERMENTATION
Time course fermentation was monitored by measuring the amount of CO2 released, expressed
as weight loss over successive 20-min periods. The rate of CO2 production was calculated
automatically by polynomial smoothing of CO2 evolution.
Assay 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: pvalue<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.
Since PAU genes are usually induced by anaerobiosis, the genetic modification seems to have two
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 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-13.
The shift towards anaerobic adaptation is in agreement with the increased sensitivity to
oxidative stress, and impaired growth glycerol (below).
AGING ON LEES
10 mM H202
ΔKNR4
70
amino acids (mg/L)
EC1118
EC1118
EKD·13
EKD-13
50
40
BY4743
30
20
ΔKNR4
10
0
4
5
6
7
8
Time (weeks)
Considering the pattern and the level of mannoprotein release and the
flocculation behavior, EKD-13 or equivalent non-recombinant strains
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
sparkling wines. The apparently overdone transcriptional adaptation to
anaerobiosis does not seem to involve detrimental effects on
fermentation performance.
5mM H202
BY4743
2
CONCLUSIONS
YPG
EKD-13
60
Reproducibility: one-way ANOVA; Dunnett test (comparison of means);
significantly differences: p-value<0.05. SPSS 15.0 software.
YPD
EC1118
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 data
OXIDATIVE STRESS/GLYCEROL UTILIZATION
DEGREE OF FLOCCULATION
The recombinant strain shows increased release
of mannoproteins just during the fermentation
step. In contrast, amino acids are released also
during the aging period. Increased release of
mannoproteins by EKD-13 does not seem to be
related to increased autolysis. Net release of
either mannoproteins or amino acids is always
higher for the recombinant strain.
Acknowledgments: 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.
This flocculation phenotype is in agreement
with the enrichment in Flo1p targets among
genes whose expression is affected by the
genetic
modification
(according
to
YEASTRACT; data not shown).
REFERENCES
Gonzalez-Ramos et al. 2008. Appl. Environ. Microbiol. 74:5533–5540
Quirós et al. DOI:10.1016/j.foodchem.2010.08.0660