A new methodology to obtain wine yeast strains
overproducing mannoproteins
Quirós, M.1, Gonzalez-Ramos, D.2, Tabera, L.2, Morales, P. 1, Pérez-Través, L.3, Barcenilla, J.M.2 and
Gonzalez. R.1
1
Instituto de Ciencias de la Vid y del Vino (CSIC-UR-CAR), Logroño, Spain.
2 Instituto de Fermentaciones Industriales (CSIC), Madrid, Spain.
3 Departamento de Biotecnología. Instituto de Agroquímica y Tecnología de los Alimentos (CSIC), Burjassot, Spain.
Introduction
Yeast mannoproteins are highly glycosylated proteins that are covalently bound to the β-1,3-glucan present in the yeast cell wall. These complex macromolecules are
structurally composed of polymers of sugars, mainly mannose, that are covalently linked to peptides and represent around 40% of the cell wall dry weight. Yeast
mannoproteins contribute to several aspects of wine quality by protecting against protein haze, reducing astringency, retaining aroma and other undesired compounds
such as OTA and stimulating the growth of lactic-acid bacteria. Therefore, the development of a non-recombinant method to obtain enological yeast strains overproducing
mannoproteins would be very useful. Previous works developed in our group have allowed the identification of several genetic determinants for the release of wine yeast
mannoproteins (Gonzalez-Ramos and Gonzalez, 2006; Gonzalez-Ramos et al., 2008). Among the different recombinant genotypes constructed and studied, strains deleted
for KNR4 (from Killer Nine Resistant, also known as SMI1), a gene involved in β-1,3-glucan biosynthesis yielded optimal results (Gonzalez-Ramos et al., 2008). In the present
paper, a new methodology for the selection of non-recombinant mannoprotein overproducing yeast strains based on their resistance to the killer 9 toxin of Williopsis
saturnus is developed.
Materials and methods
5
CO2 produced (g)
UV Mutagenesis
One haploid laboratory strainis BY4741 and three industrial winemaking strains
IFI87, IFI475 and T73 were used in this study. Overnight cultures were washed
twice and suspensions containing 107 cells/mL were then prepared, placed in a
Petri dish and irradiated under a UV lamp at 254 nm. 1 mL aliquots were taken
after 20, 40 and 60 s of exposure and kept in darkness for 30 min. Serial 10-fold
dilutions of each of these aliquots (up to 10−5) were prepared in saline solution and
100 μL were plated in YPD agar plates and k9 toxin-containing YPD plates. These
were incubated for 48–72 h at 28 °C and survival and resistance rates for each of
the exposure times were then determined. k9 toxin-resistant mutants were picked
and transferred to fresh YPD plates. Ten colonies were randomly picked both from
the control YPD plates and the selective YPD plates containing k9 toxin in order to
compare polysaccharide and mannoprotein release with the original strain after
fermentation in GCY medium
The percentage of overproducing mutants obtained for the industrial strains was
not as stunning as the observed for the haploid strain. However, mutants
producing promising results in GCY broth were tested in synthetic must. Their
fermentation performance and the release of total polysaccharides and
mannoproteins compared to their wild type counterparts are depicted in Figs. 2A
and B respectively.
5
5
4
4
4
3
3
3
2
2
2
A)
IFI475
IFI87
1
IFI87 Mut10
Quantification of total mannoproteins and polysaccharides
The concentration of total polysaccharides in the macromolecular fraction of the
supernantants was determined by the phenol-sulfuric acid method described by
Segarra et al. (1995) using a standard curve of commercial mannan from S.
cerevisiae and the concentration of mannoproteins determined by a acid hydrolysisHPLC method recently developed in our lab (Quirós et al., In Press).
5
10
15
Total polysaccharides (mg/L)
T73
0
20
T73 Mut1
0
5
10
15
20
0
5
Time (days)
Time (days)
200
1
IFI475 Mut 47
0
0
0
Fermentation of GCY and synthetic must
In order to measure the improvement in the release of mannoproteins, selected
resistant strains were inoculated into 100 mL shakeflasks containing 50 mL of GCY
broth to a final OD600nm of 0.1 and grown for 24 h at 30 °C and 150 rpm. Cultures
were centrifuged and the supernatant transferred to clean Falcon tubes.
Macromolecules in these supernatants were separated from monosaccharides by
gel filtration using Econo-Pac columns (Bio-Rad).
Industrial strains showing an improved release of mannoproteins in GCY medium
were tested in a synthetic must (pH 3.5) containing 10% glucose, 10% fructose,
0.6% citric acid, 0.6%malic acid, 0.17% YNB without aminoacids and ammonium
sulphate (Difco), 306 mg/L NH4Cl and 2/3 of the amount of aminoacids used in a
control synthetic grape must (CNC) by Beltran et al. (2004). Fermentations were
performed in duplicate at 20 °C using 100 mL bottles capped with Müller valves
containing 40 mL of must. Fermentation time course was monitored by determining
the production of CO2 expressed as weight loss until weight was constant.
IFI475 Mut 24
1
10
15
Time (days)
B)
160
Fig. 2. A) Time course of fermentations of industrial
wild type and mutant strains in synthetic must.
B) Total polysaccharides (mg/L) released in synthetic
must by the industrial mutant strains selected in this
work and their wild type counterparts.
120
80
40
0
Once tested the fermentation performance and the improved release of
mannoproteins, we decided to study their resistance to different stresses. As seen
in Fig. 3, none of the selected strains presented a significantly sensitive
phenotype to any of the different stresses tested with regards to their wild type
counterparts.
pH
Fig. 3
Glucose
IFI87
IFI 87 Mut10
IFI475
IFI475 Mut24
IFI475 Mut47
T73
T73 Mut1
Results
pH 2.8
[Polysaccharides]/Biomass
(mg/LxOD)
*
25
20
15
10
5
0
*
*
*
*
*
*
*
pH 3.2
200 g/L
250 g/L
300 g/L
Temperature
The normalized release of total polysaccharides and mannoproteins in all the k9resistant strains tested is shown in Fig. 1 in mg/(L∙OD). Eight out of the ten mutants
significantly produced more polysaccharides than the control strain (p<0.01). The
increase in the production of polysaccharides and mannoproteins ranged from 30%
for mutant Mut3 to 238% for Mut6. However, none of the colonies isolated from
the non-selective medium after the UV treatment overproduced mannoproteins
and total polysaccharides (p<0.01, data not shown)
Fig. 1. Normalized production of
polysaccharides and mannoproteins
(mg/L∙OD) obtained for ten BY4741
mutant strains after UV mutagenesis,
selection on YPD+k9 plates and
fermentation in GCY medium. Strains
releasing a significantly higher amount
of mannoproteins than the wild type
strain are marked with an asterisk
(p<0.01)
pH 3.0
IFI87
IFI 87 Mut10
IFI475
IFI475 Mut24
IFI475 Mut47
T73
T73 Mut1
13 °C
20 °C
28 °C
32 °C
Conclusions
The proposed methodology represents a fast and straightforward
approach to obtain non-recombinant yeast strains overproducing
mannoproteins. The overproducing mutants present similar fermentative
performance to those of their wild type counterparts and do not present a hypersensitive phenotype to any of the stress factors tested. An increased number of
interesting overproducing mutants could also be obtained applying the described
methodology to spores isolated from the desired industrial strains.
References
- Gonzalez-Ramos and Gonzalez (2006) J. Agric. Food Chem. 54, 9411-9416.
- Gonzalez-Ramos et al. (2008) Appl. Environ. Microbiol. 74, 5533-5540.
- Beltrán et al. (2004) FEMS Yeast Res. 4, 625-632.
- Quirós et al. Food Chem. doi:10.1016/j.foodchem.2010.08.066
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