Journal of Biotechnology 92 (2001) 27 – 35

Multidrug resistance as a dominant molecular marker in

transformation of wine yeast

Z. Kozovska

a

, A. Maraz

b

, I. Magyar

c

, J. Subik

a,

*

a

Department of Microbiology and Virology, Faculty of Natural Sciences, Comenius Uni

6ersity, Mlynska dolina B-

2

,

84215

Bratisla

6a, Slo6akia

b

Department of Microbiology and Biotechnology, Faculty of Food Sciences, Szent Ist

6an Uni6ersity, Somloi ut

14

-

16

,

1118

Budapest, Hungary

c

Department of Enology, Faculty of Food Sciences, Szent Ist

6an Uni6ersity, Somloi ut

14

-

16

,

1118

Budapest, Hungary

Received 2 February 2001; received in revised form 7 June 2001; accepted 20 June 2001

Abstract

Pure wine yeast cultures are increasingly used in winemaking to perform controlled fermentations and produce wine

of reproducible quality. For the genetic manipulation of natural wine yeast strains dominant selective markers are
obviously useful. Here we demonstrate the successful use of the mutated PDR

3 gene as a dominant molecular marker

for the selection of transformants of prototrophic wine yeast Saccharomyces cere

6isiae. The selected transformants

displayed a multidrug resistance phenotype that was resistant to strobilurin derivatives and azoles used to control
pathogenic fungi in agriculture and medicine, respectively. Random amplification of DNA sequences and elec-
trophoretic karyotyping of the host and transformed strains after microvinification experiments resulted in the same
gel electrophoresis patterns. The chemical and sensory analysis of experimental wines proved that the used
transformants preserved all their useful winemaking properties indicating that the pdr

3-9 allele does not deteriorate

the technological properties of the transformed wine yeast strain. © 2001 Elsevier Science B.V. All rights reserved.

Keywords

:

Dominant marker; PDR

3

gene; Multidrug resistance; Transformation; Wine yeast

www.elsevier.com/locate/jbiotec

1. Introduction

Saccharomyces cere

6isiae strains belong to the

most widely used yeast in winemaking. They rep-
resent a dominant part of the microflora of grape
juices and young wines. Nowadays, many wine-
makers use the pure wine yeast cultures to per-

form controlled must fermentation resulting in
production of wines of reproducible quality
(Snow, 1983; Malik et al., 1990; Nemecek et al.,
1990; Querol et al., 1992). The yeast is selected for
desirable characteristics like ethanol and osmotol-
erance, high fermentation activity or cold and
chemoresistance (Snow, 1983; Moreno et al.,
1991). The improvement of the traits that play a
role in must fermentation is one of the most
important biotechnological challenges in wine
yeast.

* Corresponding author. Tel.: + 42-12-6029-6631; fax: +

42-12-6542-9064.

E-mail address

:

[e-mail] (J. Subik).

0168-1656/01/$ - see front matter © 2001 Elsevier Science B.V. All rights reserved.

PII: S 0 1 6 8 - 1 6 5 6 ( 0 1 ) 0 0 3 4 6 - 7

Z. Kozo

6ska et al.

/

Journal of Biotechnology

92 (2001) 27 – 35

28

The advent of molecular techniques and their

applications to the study of wine fermentation
provide a useful tool for genetic improvement of
wine yeast strains. However, they are pro-
totrophic and homothallic with a higher degree of
ploidy. Therefore, dominant selectable markers
are required for a successful transformation of
such strains. Several dominant drug resistance
markers have been developed for use in S. cere-
6isiae (reviewed in Van den Berg and Steensma,
1997; Lackova and Subik, 1998) from which the
G418 resistance (Wach et al., 1994) have achieved
a wide-spread use.

Recent studies carried out in our laboratory

showed that both multicopy and centromeric vec-
tors bearing the mutant pdr

3

-

9

allele provide a

tool for the direct selection of transformants of
laboratory and polyploid industrial strains of bak-
er’s yeast (Nourani et al., 1997; Lackova and
Subik, 1999). The Pdr3p mutant protein acts as a
gain-of-function transcriptional activator respon-
sible for the establishment of multidrug resistance
phenotype due to the overexpression of several
membrane drug efflux pumps (Nourani et al.,
1997; Michalkova-Papajova et al., 2000; DeRisi et
al., 2000).

In this work, the pdr

3

-

9

gene was used as the

dominant selectable marker for the transforma-
tion of a natural wine strain of S. cere

6isiae.

2. Materials and methods

2

.

1

. Strains and culture media

The ethanol tolerant S. cere

6isiae wine yeast

strains of the FV series-originated from the vine-
growing regions in Slovakia (Malik et al., 1996)
were used for transformation and microvinifica-
tion experiments. Escherichia coli strain XL1 Blue
was used for plasmid amplification. Yeast cells
were grown at 30 °C in complex glucose (YPD)
and complex glycerol (YEG) medium (1% yeast
extract, 2% bacteriological peptone, 2% glucose or
2% glycerol) or in minimal (YNB) medium (0.67%
yeast nitrogen base without amino acids, 2% glu-
cose or 2% glycerol plus 1% ethanol). E. coli cells
were grown at 37 °C in the LB medium (1%

tryptone, 1% NaCl, 0.5% yeast extract, pH 7.5).
The plasmid propagation medium was supple-
mented with 100

mg ml

− 1

of ampicillin. The me-

dia were solidified with 2% bactoagar.

2

.

2

. Plasmid and transformation protocols

The mutant pdr

3

-

9

allele under the control of

its promoter was cloned on centromeric plasmid
pFL38/pdr3-9 (ARS

1

CEN

4

URA

3

ori amp

R

)

(Lackova and Subik, 1999). E. coli were trans-
formed and all DNA manipulations were carried
out as described in Sambrook et al. (1989). The
yeast strain S. cere

6isiae FV1 was transformed by

electroporation (Thompson et al., 1998) and the
resulting transformants were selected for drug re-
sistance. Plasmid DNA was extracted from yeast
cells as described in Alister and Ward (1990).

2

.

3

. Drug sensiti

6ity assays

The sensitivity of yeast cells to drugs was as-

sayed by determination of growth inhibition zones
(Michalkova-Papajova et al., 2000) using antifun-
gal susceptibility discs (ITEST plus Ltd, Czech
Republic) and by measuring the minimal in-
hibitory concentrations (MIC) on the solid
medium containing different drug concentrations
(Nourani et al., 1997). In the case of mitochon-
drial

inhibitors

(chloramphenicol,

mucidin,

azoxystrobin and kresoxim-methyl) the media
contained 2% glycerol and 1% ethanol instead of
glucose. Drug resistance was scored after 6 days
of growth at 30 °C.

2

.

4

. Determination of plasmid loss

Transformants were grown in liquid YPD

medium and re-inoculated to fresh medium to
initial concentration of 1 × 10

5

cell ml

− 1

each 24

h. Appropriate dilutions of the cell suspension
were plated onto solid YPD medium. After 2 days
of growth at 30 °C, colonies were replica-plated
onto YPD medium containing cycloheximide (1
mg ml

− 1

) and two YPG media containing chlo-

ramphenicol (3 mg ml

− 1

) and mucidin (0.2

mg

ml

− 1

), respectively, for the evaluation of the plas-

mid-containing fraction.

Z. Kozo

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Journal of Biotechnology

92 (2001) 27 – 35

29

2

.

5

. RAPD-PCR analysis

2

.

5

.

1

. DNA isolation

About 1.5 ml of the overnight shaken culture in

YPD medium was centrifuged and the cells were
re-suspended in 200

ml breaking buffer (2% Tri-

ton-X 100, 1% SDS, 100 mM NaCl, 10 mM
Tris – HCl, 1 mM EDTA, pH 8). After addition of
0.3 g glass beads (0.5 mm diameter) and 200

ml

PCIA

(phenol:chloroform:isoamylalcohol =

25:24:1) the mixture was vortexed for 3 min. 200
ml TE buffer (10 mM Tris–HCl, 1 mM EDTA,
pH 8) was added by gently mixing and the tube
was centrifuged for 5 min. DNA in supernatant
was precipitated with ethanol and treated by
RNase (10

mg ml

− 1

) in 100

ml TE buffer.

2

.

5

.

2

. PCR amplification by random primers

The PCR cocktail (30

ml) contained 50 pM

primer (P1: 5

%-GTGGTGGTG-3%, P2: 5%-CGCGT-

GCCCA-3

%, OPE7: 5%-AGATGCAGCC-3%), 25

mM MgCl

2

, 20 mM dNTPs, 1

mg genomic DNA

and 1.5 U Taq polymerase. Amplification was
performed as follows, 2 min at 95 °C; 35 cycles at
95 °C for 30 s; at 40 °C (P1) or 38 °C (P2 and
OPE7) for 30 s and 72 °C for 3 min. Final
extension, 72 °C for 7 min.

2

.

6

. Electrophoretic karyotyping

Intact chromosomes were prepared by lysis of

protoplasts embedded into low melting point
agarose according to the method of Carle and
Olson (1985). Separation of chromosomes was
performed by Rotaphor Type IV (Biometra) in
0.9% agarose according to the instruction of the
producer.

2

.

7

. Micro

6inification experiments

White must of Furmint grape from Tokaj re-

gion, with 258 g l

− 1

reducing sugar, 9.2 g l

− 1

titratable acid, 4/62 g l

− 1

free/total sulphur diox-

ide, pH 3.04 was sterilised by filtration and inocu-
lated with the yeast strains at a concentration of
10

6

cells/ml. Fermentation was performed in 600

ml must volume (in 700 ml bottle), three parallels
at 18 °C. Progress of fermentation was monitored

by measuring the refraction of the must (Abbe-
type Refractometer, Zeiss). At the end of the
fermentation, wines were centrifuged at 6000 rpm
for 10 min to remove yeast cells and kept at 4 °C
prior analysis. Enological parameters of the wines
were determined as described in Querol et al.
(1990). Sensory analyses were done by profes-
sional judges.

3. Results

3

.

1

. Transformation for drug resistance of natural

wine yeast

The wine yeast strain S. cere

6isiae FV1 used in

the transformation experiments was isolated from
Mu¨ller – Thurgau wine originating from the vine-
growing region Skalica – Zahorie. It was selected
from the five strains of the FV series (Malik et al.,
1996) due to its highest susceptibility to mucidin
and chloramphenicol, the antibiotics inhibiting
the respiration and protein synthesis in mitochon-
dria, respectively, and also to cycloheximide,
which is a potent inhibitor of cytoplasmic protein
synthesis.

After subcloning, the prototrophic strain FV1

was transformed by electroporation with plasmid
pFL38/pdr3-9 carrying the gain-of-function allele
of the PDR

3

gene as the dominant selectable

marker (Lackova and Subik, 1999). Transfor-
mants were selected for resistance to either cyclo-
heximide (1

mg ml

− 1

) or chloramphenicol (3 mg

ml

− 1

). In the transformed strain the frequency of

resistance of spontaneous mutants to chloram-
phenicol or cycloheximide was at least six times
lower (Table 1). The range of transformation
efficiencies in repeated experiments was between 1
and 4 × 10

3

per

mg DNA. Transformants were

distinguished from the spontaneous mutants on
their ability to display multidrug resistance and
provide 9.3 kb plasmid DNA conferring ampi-
cillin resistance in E. coli and giving the expected
DNA fragments after restriction with HindIII or
HpaI (Nourani et al., 1997). In isolated transfor-
mants the plasmid conferred a high level of resis-
tance to different drugs (Table 1) corroborating
the results obtained by industrial strains of bak-
er’s yeast.

Z. Kozo

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Journal of Biotechnology

92 (2001) 27 – 35

30

Table 1
Transformation of natural wine yeast strain with a centromeric plasmid carrying the pdr

3

-

9

mutant allele

S. cere

6isiae

Plasmid

Frequency of spontaneous mutants Minimal inhibitory concentration
and transformants selected as

Cyh

R

Cmp

R

CYH (

mg ml

−1

)

CMP (mg ml

−1

)

MUC (

mg ml

−1

)

3.2×10

−7

2.5×10

−6

FV1

0.6

No

2

0.1

pFL38/pdr3-9

FV1

1.1×10

−5

1.5×10

−5

2.5

4

0.6

Abbreviations: Cyh

R

, cycloheximide resistant; Cmp

R

, chloramphenicol resistant; CYH, cycloheximide; CMP, chloramphenicol;

MUC, mucidin.

In spite of the present centromere, moderate

loss of plasmid from transformants was observed
during mitotic growth in the absence of any drug
in complex medium containing glucose (Fig. 1).
However, 50% of yeast population still showed
multidrug resistance (Cyh

R

Cmp

R

Muc

R

) even

after 40 cell generations indicating a sufficiently
high stability of plasmid in transformants of the
natural wine yeast strain.

3

.

2

. Drug resistance pattern, experimental wine

production and molecular fingerprinting

The PDR

3

gene is known to regulate subsets of

genes involved in multidrug resistance and other
biological processes (DeRisi et al., 2000). There-
fore, we determined the susceptibility of the natu-
ral wine yeast strain S. cere

6isiae FV1 and its

transformants to several antifungal agents used in
agriculture and medicine, and assessed the enolog-
ical parameters of the wines obtained by both
yeast strains.

As is shown in Table 2, transformants carrying

the pdr

3

-

9

allele exhibited differential degrees of

susceptibility to fungicides used to control the
filamentous fungi in the vineyards. Clear resis-
tance was observed to the derivatives of strobil-
urin (Mucidin, Discus, Quadris) and certain
azoles (Hattrick, Topas). On the other hand,
transformants were found to be more sensitive to
Anvil 5SC and Vectra 10SC, or exhibited no
changes to other commercial fungicides tested. An
increased level of resistance of transformants was
also observed for several antimycotics, that in the
case of imidazoles was more pronounced on com-
plex than on minimal medium (Table 3).

To show how the natural vinification ability of

the FV1 strain is conserved in its transformants
bearing the pdr

3

-

9

allele we carried out mi-

crovinification experiments with two yeast strains.
The analytic parameters of the wines obtained
and their sensory analysis are shown in Table 4
and Table 5, respectively. The results from three
experiments clearly show that no significant dif-
ferences were observed between the natural yeast
strain and its pdr

3

-

9

transformants.

Possible changes in the length of chromosomes

as the consequence of the interaction of recombi-
nant plasmids and chromosomes during vinifica-

Fig. 1. Stability of plasmid pFL38/pdr3-9 in the population of
the wine yeast transformants growing in complex medium with
glucose under non-selective conditions.

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Journal of Biotechnology

92 (2001) 27 – 35

31

Table 2
Susceptibility to different fungicides of the S. cere

6isiae strain FV1 and its transformants TFV1

Fungicide

Commercial name

Minimal inhibitory concentration (

mg ml

−1

)

FV1

Active compounds

TFV1

Kresoxim-methyl

Discus

0.05

0.6

0.04

Quadris

0.4

Azoxystrobin

0.10

0.6

Strobilurin A

Mucidin

400

Anvil 5SC

200

Hexaconazole

Bumper 25C

Propiconazole

200

200

50

75

Hattrick

Tebuconazole plus tolyfluanide

200

Penconazole

600

Topas

Bromuconazole

Vectra 10SC

75

50

100

Acrobat

100

Dimethomorh plus mancozeb

8000

Fosethyl-Al

8000

Aliette

Folpet

Folpan 80WG

50

50

50

50

Novozir MN80

Mancozeb

100

Metiram

100

Polyram WG

Metalaxyl plus mercury oxychloride

Ridomyl Plus 48WP

5000

5000

\16 000

\16 000

Sumilex 50WP

Procymidone

16 000

16 000

Sulphur

Thiovit

Table 3
Susceptibility to antimycotics of the S. cere

6isiae strain FV1 and its transformant TFV1 determined on complex and minimal

medium containing glucose

Medium

Saccharomyces

Diameter of inhibition zone (mm)

cere

6isiae strain

Azoles

Polyenes

ECO

MCZ

CLO

KET

BIF

FLU

ITR

PIM

AMB

NYS

5-FC

27

19

18

13

10

YPD

0

FV1

0

20

0

19

0

15

12

0

0

0

0

0

18

0

19

0

TFV1

28

28

20

8

17

0

FV1

11

YNB

22

12

27

30

26

25

10

0

11

0

0

24

10

25

40

TFV1

Abbreviations: ECO, econazole; MCZ, miconazole; CLO, clotrimazole; KET, ketoconazole; BIF, bifonazole; FLU, fluconazole;
ITR, itraconazole; PIM, pimaricin; AMB, amphotericin B; NYS, nystatin; 5-FC, 5-fluorocytosin.

tion was checked by electrophoretic separation of
the intact chromosomes. Results shown in Fig. 2
indicate that no detectable changes occurred dur-
ing propagation of the cells, either they lost the
cycloheximid resistance (TFV1/S isolates) or they
retained it (TFV1/R isolates). Random amplifica-
tion of the genomic DNA sequences by three
different primers also showed that no alteration in

the length or number of the amplified fragments
took place (Fig. 3).

The data obtained here indicate that multiple

drug resistance conferred by mutant pdr

3

-

9

allele

is a valuable dominant selectable marker that can
be successfully used to transform natural S. cere-
6isiae wine strains without deterioration of their
enological properties.

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Journal of Biotechnology

92 (2001) 27 – 35

32

Table 4
Chemical analysis of experimental wines after completion of
alcoholic fermentation

Enological parameter

Value

FV1

TFV1

1.67

90.13

4.63

93.71

Reducing sugar (g l

−1

)

Ethanol (%, v/v)

15.05

90.13

14.92

90.11

49.7

918.67

61.3

93.97

Acetaldehyde (mg l

−1

)

29.70

90.34

Total extract (g l

−1

)

32.80

94.33

8.27

90.13

8.70

90.11

Total acidity (g l

−1

)

Volatile acidity (g l

−1

)

0.62

90.06

0.67

90.09

3.17

90.01

3.14

90.01

pH
Colour (A

420

)

0.17

90.01

0.12

90.02

The main values of triplicates and their confidence intervals at
P = 0.05.

Fig. 2. Electrophoretic karyograms of the host (FV1) and the
transformant (TFV1) strains isolated at the end of the mi-
crovinification. Lane 1, marker strain (S288c); lane 2, FV1;
lanes 3 – 8, TFV1/S1 – TFV1/S6; lane 9, TFV1; lanes 10 – 16,
TFV1/R1 – TFV1/R7.

4. Discussion

In this study we successfully used the pdr

3

-

9

mutant allele on the centromeric plasmid for di-
rect selection of transformants of the natural wine
yeast S. cere

6isiae strain FV1 for resistance to

chloramphenicol and cycloheximide, respectively.
Equally effective as pdr

3

-

9

were the other gain-of-

function pdr

3

alleles, like the pdr

3

-

7

(Nourani et

al., 1997) and pdr

3

-

20

(Simonics et al., 2000)

(data not shown). The pdr

3

-

9

dominant selectable

marker encodes the mutant transcription factor
Pdr3p, with Y276H amino acid substitution,
which is responsible for an increased expression of
several drug efflux pumps (Nourani et al., 1997;
DeRisi et al., 2000). The substrate specificity of

these membrane transporters is relatively broad
(Kolaczkowski et al., 1998; Bauer et al., 2000).
Therefore, the prototrophic wine yeast transfor-
mants were expected to possess an increased level
of resistance to many structurally and functionally
unrelated drugs. In fact, resistance of transfor-
mants to several antifungal agents used against
phytopathogenic fungi and human fungal patho-
gens was clearly demonstrated (Tables 2 and 3)
even in the presence of at least two copies of the
chromosomal wild-type PDR

3

gene in the strain

FV1 (Malik et al., 1996). Therefore, after the
efficient transformation using the improved proto-
col for the preparation of yeast cells for electropo-
ration (Thompson et al., 1998), transformants

Table 5
Sensory analysis of experimental wines produced by the S. cere

6isiae strain FV1 and its transformants TFV1

Flavour/taste

Odour

Total scores

Colour

Wines

3.0

5.0

FV1

3.4

11.4
11.7

4.9

3.1

3.5

4.7

3.2

3.2

11.1

TFV1

4.9

3.3

3.6

11.7

4.9

3.3

11.8

3.6

3.0

3.4

3.8

12.1

0.7136

0.7800

0.4046

Level of significance (P)

0.6765

The six wine samples (2 strains×3 parallels) were examined by five judges in one trial. The identity of the wines examined in
randomised order was not known by the judges. Data were evaluated by one-way analysis of variance where the individual scores
of the judges were considered ‘repetition’.

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Journal of Biotechnology

92 (2001) 27 – 35

33

may be directly selected for resistance to any drug
known to be actively pumped out of the cells
carrying a gain-of-function mutation in either
PDR

1

or PDR

3

gene.

Since the prototrophic strain FV1 sporulate

with the frequency of about 50% (Malik et al.,
1996), at least half of the resulting spores are
expected to harbour the centromeric plasmid and

Fig. 3. RAPD-PCR patterns of the transformants isolated at the end of the microvinification. A-primer P1, B-primer P2, C-primer
OPE7. Lane 1, DNA size marker; lane 2, TFV1; lanes 3 – 14, TFV1/R1 – TFV1/R12; lanes 15 – 20, TFV1/S1 – TFV1/S6.

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Journal of Biotechnology

92 (2001) 27 – 35

34

express multidrug resistance phenotype. This cre-
ates an opportunity for wine yeast strain im-
provement by genetic hybridisation using the
mitochondrial (rho

−

) respiratory deficient pdr

3

-

9

spores (induced by ethidium bromide) and res-
piratory competent (rho

+

) drug sensitive partner

cells. After mass mating of spore suspensions the
resulting hybrids can be selected as a respiratory
competent and drug resistant cell population that
will be able to sporulate.

The PDR

3

gene controls (directly or indirectly)

the transcription of numerous genes involved not
only in multidrug resistance phenomenon but
also in sugar transport, organelle function, phos-
pholipid metabolism, cell wall biosynthesis and
resistance to physical stresses (DeRisi et al.,
2000). The changes in the expression of such a
large set of genes could also modify some tech-
nological properties of the transformed yeast
strains. However, as shown in Tables 4 and 5,
transformants of wine yeast carrying centromeric
plasmids with pdr

3

-

9

mutant allele produced ex-

perimental wines of the same quality as their
parental natural strain FV1. This opens the way
to construct recombinant wine yeast strains con-
taining various enologically interesting enzymes
(Pretorius, 2000). Nevertheless, single gain-of-
function mutant alleles of the PDR

3

gene may

also have a beneficial effects for transformants of
industrial strains. This has already been shown
for the mutant alleles of homologous PDR

1

gene

in clotrimazole-resistant sake yeast that showed
improved fermentation activity in sake mash
even in the final fermentation stage (Mizoguchi
et al., 1999).

Acknowledgements

We thank Professor F. Malik for the wine

yeast strains, Dr J. Markovic for the samples of
commercial fungicides and Dr D. Hanson for
critical reading of the manuscript. This work was
supported by grants from the Slovak Grant
Agency of Science (VEGA), Slovak Ministry of
Education and Howard Hughes Medical Institute
(USA).

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