1
1
A new methodology to obtain wine yeast strains overproducing mannoproteins
2
3
MANUEL QUIRÓS 1*, DANIEL GONZALEZ-RAMOS 2, LAURA TABERA2, RAMON
4
GONZALEZ 1,2
5
6
1
7
Tecnológico, C/ Madre de Dios, 51. 26006 Logroño (La Rioja), Spain
Instituto de Ciencias de la Vid y del Vino (CSIC-UR-CAR), Complejo Científico
8
9
10
2
Instituto de Fermentaciones Industriales (CSIC), C/ Juan de la Cierva, 3. 28006 Madrid,
Spain
11
12
* To whom correspondence should be addressed:
13
Instituto de Ciencias de la Vid y del Vino (CSIC-UR-CAR), Complejo Científico
14
Tecnológico
15
C/ Madre de Dios, 51
16
26006 Logroño (La Rioja)
17
Spain
18
Tel: +34 941 299691
19
Fax: +34 941 299608
20
E-mail address: mquiros@icvv.es
21
22
23
24
25
Short running title: Mannoprotein overproducing yeasts
2
26
ABSTRACT
27
Yeast mannoproteins are highly glycosylated proteins that are covalently bound to the β-1,3-
28
glucan present in the yeast cell wall. Among their outstanding enological properties, yeast
29
mannoproteins contribute to several aspects of wine quality by protecting against protein
30
haze, reducing astringency, retaining aroma compounds and stimulating growth of lactic-acid
31
bacteria. The development of a non-recombinant method to obtain enological yeast strains
32
overproducing mannoproteins would therefore be very useful. Our previous experience on the
33
genetic determinants of the release of these molecules by Saccharomyces cerevisiae has
34
allowed us to propose a new methodology to isolate and characterize wine yeast that
35
overproduce mannoproteins. The described methodology is based on the resistance of the
36
killer 9 toxin produced by Williopsis saturnus, a feature linked to an altered biogenesis of the
37
yeast cell wall.
38
39
40
41
42
Keywords: wine yeast, mannoproteins, UV mutagenesis, Williopsis saturnus, killer 9 toxin
43
resistance
44
45
46
47
48
3
49
INTRODUCTION
50
Yeast mannoproteins are highly glycosylated proteins containing over 90% sugars, mainly
51
mannose, that are located in the outermost layer of the yeast cell wall, representing between
52
35 to 40% of its dry weight. Most of them act as structural components, giving the cell wall
53
its active properties and being partially responsible for its permeability (Cid et al., 1995; Klis
54
et al., 2002). Due to their high sugar content, mannoproteins behave like polysaccharides
55
rather than like proteins. In Saccharomyces cerevisiae, the glycan portion of mannoproteins is
56
mainly composed of mannose, with some neutral oligosaccharides that contain N-
57
acetylglucosamine and acidic sugars containing mannosylphosphate (Jigami and Odani 1999).
58
Over the past decades, yeast mannoproteins have gained ground as one of the most interesting
59
molecules involved in the improvement of both the technological processes during wine
60
making and the sensorial properties of the resulting product. Several authors have emphasized
61
the key role played by these molecules on the chemical stabilization of wine by protecting
62
against protein haze (Dupin et al., 2000a, b; Gonzalez-Ramos and Gonzalez 2006; Schmidt et
63
al., 2009; Waters et al., 1994, 2005) and tartaric instability (Feuillat et al., 1998). Though it is
64
well known that physicochemical procedures for the stabilization of wines are satisfactorily
65
employed (e.g. bentonite fining against protein haze, Blade and Boulton 1988; cold
66
stabilisation to avoid tartaric instability, Boulton et al., 1998), several studies support that
67
these can also negatively affect the quality of the fined wine as some texture, flavour and
68
aroma compounds will also be lost (Høj et al., 2000; Voilley et al., 1990). Additionally, the
69
use of bentonite will cause a significant loss in the volume of treated wine, estimated to range
70
between 3 to 10%. Brown et al. (2007) cloned and overexpressed YOL155c and YDR055w in
71
S. cerevisiae laboratory strains, respectively encoding Hpf1p and Hpf2p (from Haze
72
protecting factors), and showed that turbidity was reduced up to 40% when Hpf2p was added
73
to the wine, which would clearly allow to minimize these procedures.
4
74
Other appreciated enological features of mannoproteins are their ability to absorb ochratoxin
75
A and thus reduce its concentration in the final product (Ringot et al., 2005), stimulate the
76
growth of lactic-acid bacteria (Guilloux-Benattier et al., 1995) due to the adsorption of
77
medium chain fatty acids released by yeast strains present in the medium and contribute to
78
several organoleptic features by retaining aroma compounds (Lubbers et al., 1994), reducing
79
astringency, improving the foaming properties of sparkling wines (Núñez et al., 2006) and
80
increasing the sweetness, body, roundness and mouthfeel of the wine (Guadalupe et al., 2007;
81
Vidal et al., 2004, for a review of mannoprotein properties see Caridi 2006).
82
For all these positive contributions it would be desirable to obtain new yeast strains that
83
would naturally overproduce mannoproteins. Previous works have allowed the identification
84
of some genetic determinants of the release of mannoproteins in wine yeasts (Gonzalez-
85
Ramos and Gonzalez 2006; Gonzalez-Ramos et al., 2008) and proven that wines fermented
86
with recombinant mannoprotein overproducing strains presented a higher stability against
87
protein haze compared to those obtained after fermentation with the native strain. Among the
88
different recombinant genotypes constructed and studied, strains deleted for KNR4 (from
89
Killer Nine Resistant, also known as SMI1), a gene involved in β-1,3-glucan biosynthesis
90
identified by Hong et al. (1994), yielded optimal results (Gonzalez-Ramos et al., 2008). Two
91
additional gene targets were later identified to improve mannoprotein release and bentonite
92
responsiveness (Gonzalez-Ramos et al., 2009). In the present paper, a new methodology for
93
the selection of non-recombinant mannoprotein overproducing yeast strains based on their
94
phenotypic properties is developed.
95
96
MATERIALS AND METHODS
97
Strains and culture conditions
5
98
Twelve different S. cerevisiae strains were used in this work. EC1118 is a wine yeast strain
99
commercialised by Lallemand Inc. (Ontario, Canada). T73-4 is a uridine auxotroph derived
100
from the winemaking strain T73 (Puig et al., 1998) EFD-31, EGD-13 and EKD-13 are double
101
deletion mutants derived from EC1118 lacking FKS1, GPI7 and KNR4 genes, respectively
102
(González-Ramos et al., 2008, 2009). TGD-13, TGASD-31 and TKD-123 are knockout
103
strains derived from T73-4 lacking GPI7, GAS1 and KNR4 genes, respectively (González-
104
Ramos et al., 2008, 2009). Finally, BY4741 strain (MATa; his3Δ1; leu2Δ0; met15Δ0;
105
ura3Δ0) was obtained from the EUROSCARF consortium while IFI475 and IFI87, both
106
industrial winemaking strains, were obtained from the Instituto de Fermentaciones
107
Industriales culture collection (CSIC). Strains were routinely maintained on YPD plates,
108
containing 2% glucose, 2% peptone, 1% yeast extract and 2% agar. GCY medium, containing
109
2% glucose, 2% BactoTM Casamino Acids (BD, Sparks, USA) and 0.67% DifcoTM Yeast
110
Nitrogen Base (BD) was the medium used to assay the release of mannoproteins and
111
polysaccharides. When fermentations were performed with BY4741 and its derived strains,
112
the medium was supplemented with leucine and uridine (24,9 and 83,0 mg/L, respectively) so
113
that the auxotrophies of the strains were complemented.
114
Production of killer 9 toxin
115
In order to produce the killer 9 toxin, Williopsis saturnus var. mrakii CECT 11031 strain was
116
grown in YPD broth buffered with 50 mM sodium citrate (pH 4.3) during 3 days at 19 ºC and
117
150 rpm in an orbital shaker according to the conditions described by Hong et al. (1994). This
118
culture was subsequently centrifuged and the supernatant filtered through 0.22 μm pore size
119
membranes and stored at 4 ºC until used. For the preparation of the k9-containing YPD plates,
120
sodium citrate buffered YPD agar plates were overlayed with 5 mL of the filtered supernatant
121
containing the killer 9 toxin and left open in a laminar air flow bench until its complete
122
absorption.
6
123
UV mutagenesis
124
S. cerevisiae strains were grown in 10 mL YPD broth at 28 ºC and 150 rpm o/n. Cells were
125
washed twice and resuspended in 1 mL sterile distilled water. The number of cells in the
126
suspensions was determined by serial dilution and counting on a BlauBrand Thoma counting
127
chamber (Brand GMBH + CO KG, Wertheim, Germany). A 20 mL cell suspension
128
containing 107 cells/mL was then prepared, placed in a Petri dish and irradiated under a UV
129
lamp (Camag, Muttenz, Switzerland) at 254 nm. 1 mL aliquots of the cell suspension were
130
taken after 20, 40 and 60 seconds of exposure, placed in Eppendorf tubes and kept in darkness
131
for 30 minutes. Serial 10-fold dilutions of each of these aliquots (up to 10-5) were then
132
prepared in saline solution and 100 μL were plated in YPD agar plates and k9 toxin-
133
containing YPD plates. These were incubated for 48-72 h at 28 ºC and survival and resistance
134
rates for each of the exposure times were then determined. k9 toxin-resistant mutants were
135
picked and transferred to fresh YPD plates.
136
In order to check the k9 toxin resistant phenotype, cells were grown o/n in YPD, washed with
137
sterile water and resuspended to a final concentration of 107 cells/mL. Four 10-fold serial
138
dilutions were performed and 5 µL of each spotted on selective YPD plates, containing k9
139
toxin, as well as non-selective YPD plates as control.
140
Quantification of total mannoproteins and polysaccharides
141
Yeasts were inoculated in 5 mL of GCY medium, grown at 30 ºC o/n and used to inoculate
142
100 mL shakeflasks containing 50 mL of GCY broth to a final O.D. of 0.1. Growth was
143
monitored by O.D.600 nm measurement. After 24 hours of incubation at 30 ºC and 150 rpm,
144
cultures were centrifuged and the supernatant transferred to clean Falcon tubes.
145
Macromolecules in these supernatants were separated from monosaccharides by gel filtration
146
using
147
manufacturer’s
Econo-Pac
columns
(Bio-Rad
recommendations.
The
Laboratories,
concentration
Hercules,
of
total
CA)
following
the
mannoproteins
and
7
148
polysaccharides in the eluted fraction was determined by the phenol-sulfuric acid method
149
described by Segarra et al. (1995) using a standard curve of commercial mannan from S.
150
cerevisiae (Sigma-Aldrich, St. Louis, MO). Furthermore, glucose and mannose content of the
151
macromolecular fraction was estimated performing acid hydrolysis as described by François
152
(2006) followed by HPLC determination of the released polysaccharides. Four replicates for
153
each sample were performed and data were analysed by one-way ANOVA and the Dunnett
154
test for comparison of means. Bivariate correlation between the final biomass values of the
155
strains used and their corresponding polysaccharide production values was studied using
156
Pearson’s correlation coefficient. In all cases, SPSS 15.0 software (SPSS Inc., Chicago, USA)
157
was used.
158
Fermentation of synthetic must
159
Industrial strains showing an improved release of mannoproteins in GCY medium were tested
160
in a synthetic must (pH 3.5) containing 10% glucose, 10% fructose, 0.6% citric acid, 0.6%
161
malic acid, 0.17% YNB without aminoacids and ammonium sulphate (Difco), 306 mg/L
162
NH4Cl and 2/3 of the amount of aminoacids used in a control synthetic grape must (CNC) by
163
Beltran et al. (2004). Fermentations were performed in duplicate at 20 ºC using 100 mL
164
bottles capped with Müller valves containing 40 mL of must. Yeast strains were grown o/n in
165
YPD broth, washed twice in sterile distilled water and finally inoculated at a final
166
concentration of 106 cells/mL. Fermentation time course was monitored by determining the
167
production of CO2 expressed as weight loss until weight was constant.
168
HPLC analysis of musts and wines
169
Fermentation samples were analysed by HPLC in order to determine the concentration of
170
glucose, fructose, ethanol and glycerol. Prior to injection in duplicate, samples were
171
centrifuged at 10000 rpm for 5 minutes, supernatants filtered through 0.45 μm pore size nylon
172
filters (Symta, Madrid, Spain) and diluted 5 or 10-fold. A Surveyor Plus chromatograph
8
173
(Thermo Fisher Scientific, Waltham, MA) equipped with a refraction index detector
174
(Surveyor RI Plus Detector) was used. The column employed was a HyperREZTM XP
175
Carbohydrate H+ 8μm (Thermo Fisher Scientific) assembled to its correspondent guard. 1.5
176
mM H2SO4 was used as the mobile phase with a flux of 0.6 mL/min and a column
177
temperature of 50 ºC.
178
Stress tolerance assays
179
To confirm the suitability of the selected overproducing strains for industrial purposes,
180
tolerance to three different stress factors, i.e. pH, osmolarity and temperature, was assayed.
181
Serial 10-fold dilutions of cell suspensions containing 107 cells/mL were prepared for all the
182
strains and 3 µL of each plated in YPD medium with a pH of 2.8, 3.0 and 3.2 (pH stress),
183
YPD containing 200, 250 and 300 g/L of glucose (osmotic stress) and YPD plates that were
184
incubated at 13, 20, 28 and 32 °C.
185
186
RESULTS
187
Common phenotype for yeasts overproducing mannoproteins
188
Previous studies carried out in our group have identified four genes whose deletion resulted in
189
an increased mannoprotein release. In order to find a common phenotype suitable for the
190
positive selection of mannoprotein overproducing mutants, we turned our sight to the primary
191
description of yeast mutations affecting these four genes. Hong et al. (1994) observed that
192
yeast mutants deficient in the KNR4 gene activity, encoding a regulatory protein required for
193
the correct targeting of the Stl2p MAP kinase, showed resistance to the k9 toxin from
194
Hansenula mrakii coupled to a reduced level of both (1.3)-β-glucan synthase activity and
195
(1,3)-β-glucan content in the cell wall. Taking this into account, a k9 toxin sensitivity assay
196
was performed using the knockout strains previously constructed, using their wild type
197
counterparts as control (Fig. 1). As expected (Hong et al., 1994), deletion of KNR4 resulted in
9
198
an improved resistance to the killer 9 toxin in the two genetic backgrounds studied (see EKD-
199
13 and TKD-123 strains in Fig.1). Similar results were obtained for the deletion of FKS1, a
200
gene encoding a subunit of the β-1,3-glucan synthase, in EC1118 background (EFD-31 strain)
201
and for the deletion of GAS1, encoding a glycoprotein of the plasma membrane, in T73-4
202
background (TGASD-31 strain). In contrast, deletion of GPI7, encoding an enzyme required
203
for the synthesis of the GPI anchor, a structure mediating the linkage of some proteins to the
204
plasma membrane or to the cell wall, resulted in an increased sensitivity to the toxin in both
205
genetic backgrounds (EGD-13 and TGD-13 strains).
206
Considering that the modification of three out of the four genetic targets analysed that had
207
been previously shown to improve mannoprotein release also conferred yeast cells an
208
increased resistance to k9 toxin, we decided to test this feature as an enrichment criterion for
209
mannoprotein overproducing mutants in mutagenesis experiments.
210
Preliminary k9 toxin selection of UV mutants
211
As a proof of concept, two different mutagenesis experiments followed by k9 resistance
212
selection were performed on the haploid strain BY4741. Survival rates after the different
213
irradiation times were calculated by colony counting on YPD plates and results ranged from
214
33 to 12% for a 20 seconds exposure, from 1.1 to 2.1% for 40 seconds and from 0.25 to
215
0.18% for a UV exposure of 60 seconds. Ten colonies were randomly picked both from the
216
control YPD plates and the selective YPD plates containing k9 toxin in order to compare
217
polysaccharide and mannoprotein release with the original strain after fermentation in GCY
218
medium. In general, final O.D. values of k9-resistant strains were significantly lower than
219
those obtained for the strains picked from non-selective YPD plates and from the control
220
strain. So, while BY4741 presented a final O.D. value of 9.42 ± 0.7, the average O.D.
221
obtained for the k9-resistant mutants was 6.3 ± 1.5 while that obtained for the ten strains
222
picked from non-selective YPD was 9.2 ± 1.4.
10
223
The release of total polysaccharides and mannoproteins in all the k9-resistant strains tested is
224
shown in Fig. 2. Production values in mg/L are shown in panel A while values normalized by
225
the final biomass are depicted in panel B. As can be observed for the non-normalized values,
226
7 out of the 10 mutants selected in YPD+k9 plates significantly produced higher amounts of
227
total polysaccharides and mannoproteins than the control strain, BY4741 (p<0.01). The
228
increase in the production of polysaccharides and mannoproteins for the k9 resistant mutants
229
ranged from 30% for mutant Mut3 to 238% for Mut6. When the normalized values are
230
considered, 8 out of the 10 mutants significantly produced more polysaccharides than the
231
control strain. On the contrary, none of the colonies isolated from the non-selective medium
232
after the UV treatment overproduced mannoproteins and total polysaccharides (p<0.01, data
233
not shown).
234
Mannoprotein overproducing mutants from industrial wine yeast strains
235
These encouraging results made us test the described enrichment procedure for the genetic
236
improvement of three different wine making strains: IFI87, IFI475 and T73. The survival
237
rates observed in this case significantly differed between strains. So, for IFI87, IFI475 and
238
T73 the rates obtained were 29.6, 46.7 and 51,4% after 20 seconds of UV exposure, 2.7, 43.6
239
and 24.9% for 40 s and 0.6, 7.0 and 1.4% for 60 s, respectively. It is noteworthy to mention
240
that the percentage of k9 resistant cells, calculated as (no. of colonies on YPD+k9 / no. of
241
colonies on YPD) x 100 varied for every strain and UV treatment but ranged between 0.18%
242
and 0.09%.
243
Ten colonies were randomly picked for each of the strains from YPD+k9 plates
244
corresponding to different survival rates and fermentations in GCY performed as in the
245
previous cases. As no overproducing mutants were identified for strain IFI475, the screening
246
was extended in this case with 40 additional strains.
11
247
In the case of strain IFI87, the most relevant overproducing mutant was Mut10, releasing
248
126.5 ± 2.8 mg/L of polysaccharides. This value represents a 3.5 times increase in the release
249
of mannoproteins, as the wild type strain produced an average of 35.8 ± 2 mg/L. More
250
discrete improvements were observed for strains Mut 24 and 47 derived from IFI475, which
251
increased the release of mannoproteins 19% and 25% respectively when compared to their
252
control and for T73 Mut1, with an increase of 14%. Increased production values were also
253
confirmed after normalization with the final biomass values.
254
Fermentation of synthetic must
255
In order to prove that the selected strains would increase the release of mannoproteins during
256
winemaking, fermentations using a synthetic must were carried out in duplicates.
257
Fermentation profiles did not differ significantly from the wild type strain in the case of the
258
two mutants obtained from IFI475 while some differences were observed in the case of IFI87
259
Mut10 and T73 Mut1 (Fig. 3). However, final concentration of the metabolites analysed by
260
HPLC (i.e. glucose, fructose, glycerol and ethanol) did not significantly differ from those
261
obtained for the wild type strains. Values obtained for the release of total polysaccharides are
262
shown in Fig. 4. It is relevant to mention that, while the differences found in the values
263
between IFI87 Mut10 and IFI475 Mut24 and 47 compared to their wild type strains were very
264
similar to the ones obtained after fermentation in GCY, a 94% increase in the production of
265
total polysaccharides was observed for strain T73 Mut1 when compared to the original strain
266
while fermentation on GCY had just shown a slight increase of 14%.
267
Even these results demonstrated the suitability of the selected strains we decided to perform
268
stress tolerance assays, so that they were exposed to different winemaking related stresses
269
(pH, osmotic and temperature stress). After monitoring growth for three days, no significant
270
differences were observed between the overproducing mutant strains and their wild type
271
counterparts (see Supplementary data).
12
272
273
DISCUSSION
274
During the last decades, several studies have demonstrated the positive contribution of yeast
275
mannoproteins to the sensorial quality and chemical stability of wines (Waters et al., 1993,
276
1994; Moine-Ledoux and Dubourdieu 1999; Dupin et al., 2000a, b). Different studies
277
performed in our group have identified several genetic targets for the improved release of
278
mannoproteins by S. cerevisiae during wine fermentations (Gonzalez-Ramos and Gonzalez
279
2006, Gonzalez-Ramos et al., 2009) However, the use of GMOs in food applications is
280
strictly regulated in most countries, including the USA and those belonging to the EU. As
281
genetic engineering of microorganisms does not therefore represent a viable option, genetic
282
improvement of yeast has normally been achieved either by parasexual hybridization by
283
means of protoplast fusion or by consecutive cycles of random mutagenesis and selection,
284
using both chemical or physical agents.
285
The method for the selection of yeast strains overproducing mannoproteins described in the
286
current paper consists in a random mutagenesis using a physical agent such as 254 nm UV
287
light, followed by a direct selection on YPD plates containing killer 9 toxin from Williopsis
288
saturnus var. mrakii. When mutagenesis was applied to the haploid strain BY4741, optimal
289
results were obtained, as 70% of the resistant strains assayed overproduced polysaccharides.
290
On the contrary, as mentioned in the Results section, none of the strains isolated from non-
291
selective YPD plates after UV exposure showed an increased production of polysaccharides.
292
This fact proves that selection with Williopsis saturnus killer 9 toxin results in a clear
293
enrichment in polysaccharide overproducing mutants and that, as clearly observed in our
294
results, the number of strains needed to be screened drastically decreases.
295
It is necessary to indicate that, in most cases, strains isolated from the k9 containing YPD
296
plates yielded lower biomass values when grown on GCY than the native strain, BY4741.
13
297
Indeed we found a negative correlation between the amount of total polysaccharides released
298
and the final biomass values of the cultures performed in GCY medium (Pearson’s correlation
299
coefficient = -0.661, p<0.05). By using killer 9 toxin on the YPD plates, we are in some way
300
selecting for strains presenting an impaired cell wall biosynthesis. Such a defective cell wall
301
could make these strains more sensitive to external factors such as pH or osmotic stress and
302
could explain the differences found in the biomass values observed. This fact sets a clear limit
303
on the degree of overproduction that could be reached when this methodology is applied. So,
304
strains severely affected in the biogenesis of their cell wall would release a much higher
305
amount of polysaccharides but would certainly not resist the different stresses present in a
306
grape must and would therefore not represent an industrial viable option.
307
Once the methodology showed its usefulness using a haploid laboratory strain, our next goal
308
was to prove its utility with industrial strains. Results obtained in this case were not as
309
dazzling as the observed for the haploid strain as, in the case of IFI87 and T73, one out of the
310
ten mutants analysed overproduced polysaccharides while this proportion dropped to two out
311
of forty in the case of IFI475 mutants. This finding might be due to the fact that, as previously
312
observed for the deletion of FKS1, GAS1, GPI7 and KNR4 (Gonzalez-Ramos and Gonzalez
313
2006, Gonzalez-Ramos et al., 2009), mutations of genes that can influence mannoprotein
314
release have a recessive phenotype. The probability of obtaining a recessive homozygous
315
strain by random mutagenesis is very low compared to the probabilities of getting loss-of-
316
function mutants using haploid strains, as only one point mutation could be enough. It should
317
always be kept in mind that, though not analysed in this paper, there is a clear effect of the
318
genetic background of the strain on the results obtained after deletion or interruption of these
319
genes (Gonzalez-Ramos and Gonzalez 2006, Gonzalez-Ramos et al., 2008, 2009). Indeed,
320
one of the reasons that might explain the differences in the proportion of overproducing
321
mutants observed for the three strains used in this work could be their ploidy. So, strains with
14
322
a higher ploidy would hamper the process of obtaining mutants overproducing
323
mannoproteins, as probabilities of getting homozygous strains for a certain mutation would
324
considerably decrease. In fact, when the proposed methodology was applied to the BY4743
325
strain, a diploid yeast strain presenting the same genetic background as BY4741, the
326
percentage of mutants overproducing mannoproteins drastically decreased, as none out of the
327
30 mutants studied overproduced these compounds (data not shown).
328
Considering the limitations pointed out for the experiment performed with the lab strain
329
BY4741, we decided to test the behaviour of the industrial strains selected in a synthetic must
330
and to study their resistance to different stresses. In general, fermentations performed in this
331
medium confirmed the results observed for fermentations in GCY in terms of improvement of
332
the release of mannoproteins. Besides, none of the selected strains presented a significantly
333
sensitive phenotype to any of the different stresses tested with regards to their wild type
334
counterparts (Supplementary data).
335
Considering the results presented, the proposed methodology represents a fast and
336
straightforward
337
mannoproteins, which can clearly contribute to the chemical stabilization of wine and to the
338
improvement of its organoleptic properties. An increased number of interesting
339
overproducing mutants could also be obtained applying the described methodology to spores
340
isolated from the desired industrial strains.
approach
to
obtain
non-recombinant
yeast
strains
overproducing
341
342
ACKNOWLEDGEMENTS
343
This work was supported by the Spanish Ministerio de Ciencia e Innovación (grants
344
AGL2006-02558, and Consolider CSD2007-00063). D.G.-R was supported by a pre-doctoral
345
fellowship from the Government of The Basque Country (Gobierno Vasco-Eusko Jaurlaritza).
346
The authors are grateful to José María Barcenilla and Laura López Berges for their excellent
15
347
technical assistance and to Jytte Laursen for kindly reviewing the English expression in this
348
manuscript.
349
350
REFERENCES
351
- Beltran, G., Novo, M., Rozès. N., Mas, A., Guillamón, J.M., 2004. Nitrogen catabolite
352
repression in Saccharomyces cerevisiae during wine fermentations. FEMS Yeast Research 4,
353
625-632.
354
- Blade, W.H., Boulton, R., 1988. Adsorption of protein by bentonite in a model wine
355
solution. American Journal of Enology and Viticulture 39, 193-199.
356
- Boulton, R.B., Singleton, V.L., Bisson, L.F., Kunkee, R.E. 1998. Heating and cooling
357
applications. In: Principles and practices of winemaking. Kluwer Academic/Plenum
358
Publishers, New York, pp. 492-520.
359
- Brown S.L., Stockdale V.J., Pettolino F., Pocock K.F., de Barros Lopes M., Williams P.J.,
360
Bacic A., Fincher G.B., Hoj P.B., Waters E.J., 2007. Reducing haziness in white wine by
361
overexpression of Saccharomyces cerevisiae genes YOL155c and YDR055w Applied
362
Microbiology and Biotechnology 73, 1363-1376.
363
- Caridi, A., 2006. Enological function of parietal yeast mannoproteins. Antonie van
364
Leeuwenhoek 89, 417-422.
365
- Cebollero, E., Gonzalez-Ramos, D., Tabera, L., Gonzalez, R., 2007. Transgenic wine yeast
366
technology comes of age: is it time for transgenic wine? Biotechnology Letters 29, 191-200.
367
- Cid, V.J., Duran, A., del Rey, F., Snyder, M.P., Nombela, C., Sanchez, M., 1995. Molecular
368
basis of cell integrity and morphogenesis in Saccharomyces cerevisiae. Microbiological
369
Reviews 59, 345-386.
370
- Dupin, I.V.S., McKinnon B.M., Ryan, C., Boulay, M., Markides, A.J., Jones, G.P.,
371
Williams, P.J., Waters, E.J., 2000a. Saccharomyces cerevisiae mannoproteins that protect
16
372
wine from protein haze: their release during fermentation and lees contact and a proposal for
373
their mechanism of action. Journal of Agricultural and Food Chemistry 48, 3098-3105.
374
- Dupin, I.V.S., Stockdale, V.J., Williams, P.J., Jones, G.P., Markides, A.J., Waters, E.J.,
375
2000b. Saccharomyces cerevisiae mannoproteins that protect wine from protein haze:
376
evaluation of extraction methods and inmunolocalization. Journal of Agricultural and Food
377
Chemistry 48, 1086-1095.
378
- François, J.M., 2006. A simple method for quantitative determination of polysaccharides in
379
fungal cell walls. Nature Protocols 1, 2995-3000.
380
- Feuillat, M., Charpentier, C., Nguyen van Long, T., 1998. Yeast's mannoproteins: a new
381
possible enological adjuvant. Bulletin de l’OIV. 71, 944-967.
382
- Gonzalez-Ramos, D., Cebollero, E., Gonzalez, R., 2008. A recombinant Saccharomyces
383
cerevisiae strain overproducing mannoproteins stabilizes wine against protein haze. Applied
384
and Environmental Microbiology 74, 5533-5540.
385
- Gonzalez-Ramos, D., Gonzalez, R., 2006. Genetic determinants of the release of
386
mannoproteins of enological interest by Saccharomyces cerevisiae. Journal of Agricultural
387
Food Chemistry 54, 9411-9416.
388
- Gonzalez-Ramos, D., Quirós, M., Gonzalez, R., 2009. Three different targets for the genetic
389
modification of wine yeast strains resulting in improved effectiveness of bentonite fining.
390
Journal of Agricultural Food Chemistry 57, 8373-8378.
391
- Guadalupe, Z., Palacios, A., Ayestarán, B., 2007. Maceration enzymes and mannoproteins: a
392
possible strategy to increase colloidal stability and color extraction in red wines. Journal of
393
Agricultural and Food Chemistry 55, 4854-4862.
394
- Guilloux-Benattier, M., Guerreau, J., Feuillat, M., 1995. Influence of initial colloid content
395
on yeast macromolecule production and on the metabolism of wine microorganisms.
396
American Journal of Enology and Viticulture 46, 486-492.
17
397
- Hong, Z., Mann, P., Brown, N.H., Tran, L.E., Shaw, K.J., Hare, R.S., Didomenico, B., 1994.
398
Cloning and characterization of KNR4, a yeast gene involved in (1,3)-β-glucan synthesis.
399
Molecular and Cellular Biology 14, 1017-1025.
400
- Høj, P.B., Tattersall, D.B., Adams, K., Pocock, K.F., Hayasaka, Y., van Heeswijck, R. and
401
Waters, E., 2000. The ‘haze proteins’ of wine – a summary of properties, factors affecting
402
their accumulation in grapes, and the amount of bentonite required for their removal from
403
wine. Proceedings of the American Society of Enology and Viticulture (ASEV) 50th
404
Anniversary Meeting, Seattle, Washington, pp. 149-154.
405
- Jigami, Y, Odani, T., 1999. Mannosylphosphate transfer to yeast mannan. Biochimica et
406
Biophysica Acta 1426, 335-345.
407
- Klis, F.M., Mol, P., Hellingwerf, K., Brul, S., 2002. Dynamics of cell wall structure in
408
Saccharomyces cerevisiae. FEMS Microbiology Reviews 26, 239-256.
409
- Lubbers, S., Voilley, A., Feuillat, M. and Charpentier, C., 1994. Influence of mannoproteins
410
from yeast on the aroma intensity of a model wine. Lebensmittel-Wissenschaft &
411
Technologie 27, 108-114.
412
- Moine-Ledoux, V., Dubourdieu, D., 1999. An invertase fragment responsible for improving
413
the protein stability of dry white wines. Journal of the Science of Food and Agriculture 79,
414
537-543.
415
- Núñez, Y.P., Carrascosa, A.V., Gonzalez, R., Polo, M.C., Martínez-Rodríguez, A., 2006.
416
Isolation and characterization of a thermally extracted yeast cell wall fraction potentially
417
useful for improving the foaming properties of sparkling wine. Journal of Agricultural and
418
Food Chemistry 54, 7898-7903.
419
- Puig, S., Ramón, D., Pérez-Ortín, J.E., 1998. Optimized method to obtain stable food-safe
420
recombinant wine yeast strains. Journal of Agricultural and Food Chemistry 46, 1689-1693.
18
421
- Ringot, D., Lerzy, B., Bonhoure, J.P., Auclair, E., Oriol, E., Larondelle, Y., 2005. Effect of
422
temperature on in vitro ochratoxin A biosorption onto yeast cell wall derivatives. Process
423
Biochemistry 40, 3008-3016.
424
- Segarra, I., Lao, C., López-Tamames, E., de la Torre-Boronat, M.C., 1995.
425
Spectrophotometric methods for the analysis of the polysaccharide levels in winemaking
426
products. American Journal of Enology and Viticulture 46, 564-570.
427
- Schmidt, S.A., Tan E.L., Brown, S., Nasution, U.L., Pettolino, F., MacIntyre, O.J., de Barros
428
Lopes, M., Waters, E.J., Anderson, P.A., 2009. Hpf2 glycan structure is critical for protection
429
against protein haze formation in white wine. Journal of Agricultural and Food Chemistry 57,
430
3308-3315.
431
- Vidal, S., Francis, L., Williams, P., Kwiatkwoski, M., Gawel, R., Cheynier, V., Waters, E.,
432
2004. The mouth-feel properties of polysaccharides and anthocyaneins in a wine like medium.
433
Food Chemistry 85, 519-525.
434
- Voilley, A., Lamer, C., Dubois, P., Feuillat, M., 1990. Influence of macromolecules and
435
treatments on the behaviour of aroma compounds in a model wine. Journal of Agricultural
436
and Food Chemistry 38, 248-251.
437
- Waters, E.J. Alexander, G., Muhlack, R., Pocock, K.F., Colby, C., O’Neill, B.K., Høj, P.B.,
438
Jones, P., 2005. Preventing protein haze in bottled white wine. Australian Journal of Grape
439
and Wine Research 11, 215-225.
440
- Waters, E.J., Pellerin, P., Brillouet, J.M., 1994. A Saccharomyces mannoprotein that
441
protects wine from protein haze. Carbohydrate Polymers 23, 185-191.
442
- Waters, E.J., Wallace, W., Tate, M.E., Williams, P.J., 1993. Isolation and partial
443
characterization of a natural haze protective factor from wine. Journal of Agricultural and
444
Food Chemistry 41, 724-730.
19
445
Captions for Figures
446
447
Fig. 1. Williopsis saturnus k9 toxin sensitivity assay performed with EC1118 and T73-4
448
knockout strains. Images on the left-hand side represent growth of the strains in non-selective
449
buffered YPD plates. Images on the right-hand side depict growth on k9-containing buffered
450
YPD plates.
451
452
Fig. 2. A) Production of polysaccharides (mg/L) obtained for ten BY4741 mutant strains after
453
UV mutagenesis, selection on YPD+k9 plates and fermentation in GCY medium.
454
B) Production values of the same strains normalized using the final biomass. In both cases
455
strains releasing a significantly higher amount of mannoproteins than the wild type strain are
456
marked with an asterisk (p<0.01).
457
458
Fig. 3. Time course of fermentations of industrial wild type and mutant strains in synthetic
459
must.
460
461
Fig. 4. Total polysaccharides (mg/L) released in synthetic must by the industrial mutant
462
strains selected in this work and their wild type counterparts.
463
464
Supplementary data
465
Growth of the mutant industrial strains selected and their wild type counterparts in YPD-
466
based media representing three different stress factor (pH, high osmolarity and temperature)
467
after 72 h. of incubation.
468
469
20
470
471
472
473
474
475
476
477
478
479
480
481
482
483
484
485
486
487
488
489
490
491
492
493
494
Buf
EC1
EFD-31
118
EGD-13
(ΔFKS1)
EKD-13
(ΔGPI7)
T7
(ΔKNR4)
TGD-13
3-4
TGASD-13
(ΔGPI7)
TKD-123
(ΔGAS1)
(ΔKNR4)
Fig. 1.
Manuel Quirós
fered
YP
D
Buffe
red
YPD
+ k9
120
A
*
100
80
60
*
40
20
0
Fig. 2.
Manuel Quirós
*
*
*
*
*
[Polysaccharides]/Biomass
(mg/LxOD)
495
496
497
498
499
500
501
502
503
504
505
506
507
508
509
510
511
512
[Polysaccharides] (mg/L)
21
25
*
B
20
15
10
5
0
*
*
*
*
*
*
*
22
CO2 produced (g)
513
514
515
516
517
518
519
520
521
522
523
524
525
526
527
528
529
530
531
5
5
5
4
4
4
3
3
3
2
2
2
IFI475
1
IFI87
1
IFI475 Mut 24
IFI87 Mut10
0
0
0
5
10
Time (days)
Fig. 3.
Manuel Quirós
15
20
1
T73
IFI475 Mut 47
T73 Mut1
0
0
5
10
Time (days)
15
20
0
5
10
Time (days)
15
20
23
200
Total polysaccharides (mg/L)
160
120
80
40
Fig. 4.
Manuel Quirós
M
ut
1
3
3
T7
T7
M
ut
47
M
ut
24
47
5
IF
I
47
5
47
5
IF
I
IF
I
M
ut
10
87
IF
I
87
0
IF
I
532
533
534
535
536
537
538
539
540
541
542
543
544
545
546
547
548
549
550
551
552
553
554
555
24
556
557
Supplementary material