Introduction of terpene-producing ability in a wine strain of Saccharomyces cerevisiae

of Biotechnology, 21(1991) 239-252 Elsevier Science Publishers B.V.

Journal

239

BIOTEC 00695

Introduction of terpene-producing ability in a wine strain of Saccharomyces cerevisiae Catherine

Javelot, Patrick Girard, Benoit Colonna-Ceccaldi and Barbu Vladescu Centre

de Recherche

Pemod-Ricard,

CGteil,

France

(Received 1 June 1991; revision accepted 13 July 1991)

Summary The terpene-producing ability due to mutation in the ERG 20 gene coding for the farnesyl diphosphate synthetase in Saccharomyces cerevisiae was introduced into a wine yeast by crossing its meiotic progeny with an erg20 laboratory strain. After three backcross generations, strains displaying improved technological properties and more typical terpenic profile were obtained. These strains, which contribute a strong muscatel-type aroma to processed grape juice, are henceforth available for industrial applications. Wine yeast; Terpenes; Farnesyl diphosphate synthetase mutants

Introduction Mutant strains of Saccharomyces cerevisiae affected in the ergosterol pathway were shown to produce significant amounts of monoterpenes as a result of a particular mutation in the erg20 locus coding for farnesyl-diphosphate synthetase (Chambon et al., 1990). The mutants accumulate geranyl-pyrophosphate, which, as in higher plants (Croteau, 1980, is converted to monoterpenes such as geraniol and linalool, the amounts of which largely exceed those found in Muscat grape juice (Javelot et al., 1990). These exceptional properties suggested the appealing idea to utilize these mutants in wine making, in order to produce flavoring monoterpenes during Correspondence to: B. Vladescu, Centre de Recherche Pernod-Ricard, 94000 CrCteil, France.

120 avenue du Mar&ha1 Foch,

240

alcohol formation. While geraniol and linalool, the major aroma compounds of muscatel-type wines originate solely from grape, the use of terpene-producing yeasts should allow the generation of this particular aroma profile by de novo synthesis in completely non-aromatic raw materials. As the original mutants were completely blocked auxotrophs for ergosterol, they were unsuitable candidates for technological applications. Further isolation of partially suppressed strains, still able to produce monoterpenes (Javelot et al., 1990) opened up new perspectives, though technological parameters like growth, sugar consumption, ethanol tolerance were not really improved by the suppression of auxotrophy. It was demonstrated that introduction of new properties into wine yeasts is possible by selective hybridization and back-breeding (Thornton, 1985; Eustace and Thornton, 1987). Therefore, introducing the terpene-synthesizing ability into a production wine yeast strain appears to be extremely promising. This paper reports the results of crosses between meiotic segregants of a wine yeast strain and a terpene-producing mutant, in terms of terpene profile and wine-making qualities. Materials

and Methods

Strains

L116 is a derivative of a commercial Saccharomyces cereuisiae strain (Fermivin) used for wine making, purchased from Rapidase, The Netherlands. CM592 (a, erg9, erg20, sas, reu) derived from S. cereuisiae FL200, is a partially suppressed ergosterol auxotroph which produces significant amounts of geraniol and linalool (Javelot et al., 1990). CM593 is a diploid strain homozygous for erg9, erg20, sas and rev (MahC-Javelot, 1989). Wild type strains FL100 (a) (A.T.C.C. 28383) and FL200 (a) (A.T.C.C. 32119) were used as tester strains for the determination of the mating-type of the spores. Media

Routine culture medium was YPG (1% yeast extract, 1% bactopeptone, 2% glucose). YPG erg contained, in addition, an ergosterol supplement as described by Javelot et al. (1990). Plates were solidified with 2% agar. For growth studies YPGlSO (1% yeast extract, 1% bactopeptone, 15% glucose) was used. Sporula tion

One loop-full of exponentially grown cells were inoculated in 1 ml of sporulation medium (0.3% potassium acetate, 0.02% raffinose; Pomper et al., 1954) in a 50 ml Erlenmeyer flask. Asci could be recovered after 3-5 d of incubation at 25 o C under shaking.

241

Genetic techniques and screening of phenotypes

These were as described by MahC-Javelot (1989). Growth parameters

Cells were propagated in 100 ml YPG (or YPG erg) in 500 ml flasks at 30 o C on a shaker to early stationary phase (approx. 2 x lo8 cells per ml), harvested by centrifugation and washed with sterile cold water. Approximately 10’ cells were inoculated in 150 ml flasks containing 100 ml of YPGlSO. Cultures were incubated at 30 “C without agitation, and the process was followed by weighing the flasks at different times over a period of 3 d (Christensen, 1987). Alcohol formation rate was defined as the mean decrease in weight per h between 16 and 40 h of growth. Two independent experiments were performed, each comprising duplicate tests for a given strain and including the corresponding parental strains as a control. Analyses

Cells were removed by centrifugation from 64-h cultures and volatile compounds in the medium analysed by gas chromatography after dichlormethane extraction, using an HP5880 chromatograph (Hewlett Packard) with a FFAP column (MahC-Javelot, 1989). Ethanol, glycerol and glucose were determined by HPLC with a BioRad Ion Exchange Column (300 X 7.8 mm) packed with Aminex HPX-87H. Pilot experiments

Strains were tested at pilot scale in grape juice (total acidity 4.5 g 1-l H,SO,, sugar 195 g 1-l) sterilized with 400 ~1 1-l of dimethyl dicarbonate (Bayer) Cells were first inoculated in grape juice supplemented with ergosterol (4 mg 1-i) and allowed to grow for 48 h at 28°C under agitation, then transferred at a concentration of lo6 cells per ml in 2 1 bioreactors Set 002M (Setric GCnie Industriel) containing 1.5 1 of grape juice. As pointed out by preliminary experiments, the best aromatic profiles are obtained in 3 d at 24 o C.

Results Dissection of the wine strain L116

This particular strain was retained from among several commercial wine strains because of its superior parameters, good sporulation and relatively high spore viability.

242

After 4 d in sporulation medium, a great number of asci occurred, among which 14% were four-spored, while 16, 51 and 19% contained three, two or one spore, respectively. We dissected 33 four spored-asci; none of them yielded four viable spores; in one ascus, three spores were viable, while in 10 and 17 asci, two spores and, respectively one were viable; the remaining five asci yielded no viable spore. As no complete tetrad was obtained, 18 random spores were further examined for mating-type and sporulation ability. We found four a and seven (Y maters, none of which could sporulate; the remaining seven spores could not be classified with respect to mating-type, while four of them sporulated. Test experiments with these monosporic cultures did not show any significant difference between them in alcohol formation efficiency and, as expected, neither L116 nor its spores produced terpenes as revealed by GC analysis. As our purpose was to improve technological properties of the terpene-producing mutant by crossing it with a meiotic segregant of the wine strain, we looked in the progeny of L116 for an a-mating-type spore most similar to L116 with respect to growth rate and ethanol production and able to yield a great number of zygotes in crosses with CM592: the monosporic culture L116-7a was found to meet these requirements fairly well. Hybridization

with the terpene-producing mutant

Zygotes from the cross L116-7a x CM592 were isolated by micromanipulation and after 48 h of growth on YPG, transferred onto sporulation medium. Random spores were recovered on YPG erg plates after ether killing and a preliminary screening was performed according to colony size and color. Indeed, as CM592 grows slower than wild-type strains even in ergosterol supplemented media, it usually forms small colonies which, in addition, display a characteristic brownish color of yet unexplained origin. Therefore, among random spore colonies, the smallest ones were picked up and tested for ergosterol auxotrophy. Among 48 small brownish colonies, 21 turned out to be complete ergosterol auxotrophs (i.e. they were no more suppressed) while the remaining 27 were partially suppressed, like CM592. This is in good agreement with our previous work which demonstrated the partial suppression of ergosterol auxotrophy to be due to mutation in a single locus (sas) unlinked to erg9 or erg20 (Javelot et al., 1990). The 27 partially suppressed auxotrophs were further examined for terpene production by GC analysis: seven of them (Table 1) were found to be effective terpene producers. They were further examined for alcohol formation kinetics and efficiency (Fig. lA, Table 21, and terpene production (Table 3). The process was followed in liquid YPG150 medium without ergosterol; four spore-derived clones displayed alcohol formation rates higher than CM592 (6, 13, 14, 26) while the four others were not significantly different from CM592 (7, 17, 20, 26). Like the growth pattern, terpene production was largely affected by this first cross with the wild wine yeast. As a rule, higher amounts of total terpenes were obtained, while their relative proportions changed: citronellol and/or linalool

243 TABLE 1 Backcross products investigated in this study

cross

Hybrid spores *

Inbreeding products

Backcross generation I L116-7a x CM592 5 (ff) 6 (a) 13 (a) 14 (nm) 17 (a) 20 (a) 26 (a)

II L116-5b x13 120 (a) 121 (a) 122 (a) 123 (a) 124 (a) 125 (a)

26x20 26x13 13x17 13x 6 13x20

III L116-7a x 17 140 (a) 141 (nm) 143 (a) 144 f(Y) 145 (a) 147 (a) 149 (a) 100 x 140x 104x 145 x 147 x 145 x

L116-7a x26 100 (aI 101 (a) 102 (a) 103 (Lx) 104 (a) 106 (a) 107 f(Y) 108 (a)

L116-7a x 145 200 ((2) 201 (ff’) 202 (a) 203 (a) 204 (a)

L116-7a x103 210 (a) 211 (a) 212 (a) 213 (a) 214 (a)

103 103 103 147 103 100

26x 140 13x 120 13 x 103 * Selection for erg- sas; in brackets the mating type of the spores; nm = non-mater.

amounts increased more than geraniol. Small amounts of a-terpineol ( < 1 mg I-‘) were also observed with the best linalool-producing hybrid strains, but not with the original mutant (data not shown). In order to further reduce the genetic input of the laboratory strain, two additional backcrosses were performed: first generation spores 26, 13 and 17 and thereafter second generation spores 103 and 145 were crossed with spores of L116 (either L116-7a or L116-5b depending on the mating type required). The resulting progeny was then screened as described for the first generation. Thereby, 21 new terpene-producing hybrid monosporic clones were obtained in the 2nd backcross, and ten in the 3rd (Table 1); they were examined for process parameters (Fig. 1B and C; Table 2) and terpene production (Table 3) and appeared to be significantly altered as compared to the first generation backcross products. In parallel, crosses between meiotic segregants of the first two backcrosses were also performed whenever possible (Table 1). The resulting hybrids, homozygous for the mutations originating from CM592, as seen from their phenotypes, were compared to the homozygous laboratory diploid strain CM593 for alcohol formation efficiency (Fig. 2, Table 4) and terpene production (Table 5). Theoretically, after three backcross generations, the genetic contribution of the laboratory strain, deleterious with respect to technological properties, was reduced

C 17

17 5

14a

20 13 6 28

145

140 141 143 147 144 14s

211 213 210 214 212

146 012

3 Wslght

4

Lll6-7A

IOU (g)

CM502 CM592 26 28 103 107 102

204 203 200 202 201

103 100 101 108 108 104

LllB-7A

L116-7A

I 0

i 12

I

Weight Ion

I 3

I 4

(g)

121 122 120 125 124 123

I 012

I Wdght

!

I 3

I 4

IOU (0)

Fig. 1. Test of backcross products: weight loss after 40 h (mean of two duplicate experiments and standard deviation); values for the progeny of each backcross generation are represented together with those for the parent strains and for the previous generation products. (A) First backcross generation (cross CM592X L116-7a); (B) second generation (crosses 17 X L116-7a, 26 X L116-7a and 13 X L116-5b); (C) third generation (crosses 145 x L116-7a and 103 x L116-7a).

245 TABLE

2

Glucose cultures

left, ethanol and glycerol production by parental in YPG150; mean of two duplicate experiments)

Backcross generation

I

III

strains

and

backcross

products

Strain

Ethanol formation rate

Glucose gl-’

Glycerol gl-’

Ethanol gl-’

CM592 L116-7a

0.05 0.12

81 18

2.6 3.6

24 55

5 6 13 14 17 20 26

0.04 0.06 0.06 0.07 0.05 0.05 0.06

89 70 62 62 84 76 71

3.4 3.4 2.9 2.9 2.5 2.8 2.9

29 29 31 36 21 28 32

100 101 102 103 104 106 107 108 120 121 122 123 124 125 140 141 143 144 145 147 149

0.06 0.07 0.04 0.05 0.11 0.07 0.04 0.07 0.05 0.05 0.05 0.07 0.08 0.04 0.12 0.07 0.04 0.11 0.06 0.05 0.06

63 64 86 71 44 63 75 45 69 79 71 63 38 88 21 57 84 31 65 77 69

3.1 2.9 2.9 3.0 3.5 2.4 2.5 3.1 2.8 3.1 3.8 4.1 3.9 2.4 3.3 3.3 2.4 3.1 3.2 2.5 2.6

34 33 25 29 45 32 28 40 29 26 28 32 35 21 48 32 24 44 31 26 31

200 201 202 203 204 210 211 212 213 214

0.07 0.06 0.06 0.07 0.04 0.07 0.06 0.07 0.06 0.07

49 64 68 51 85 60 69 51 63 48

2.5 2.6 2.6 2.4 1.9 2.6 2.1 2.4 2.4 2.5

30 29 29 36 21 33 29 36 32 38

(64-h-old

246 TABLE Effect

3 of backcrossing

on the terpene

production;

values

in mg I-‘;

64-h-old

cultures

in YPGl50

Strain

Citronellol

Linalool

Geraniol

Total

CM592 L116-7a

0.41 0

0.17 0

2.24 0

2.82 0

5 6 13 14 17 20 26

1.1 3.5 2.12 4.8 4.9 0.9 5.46

0 3.1 1.97 3.2 2.1 0 0.82

3.6 7.7 5.1 5.1 9.3 3 4.42

4.7 14.3 9.19 13.1 16.3 3.9 10.7

100 101 102 103 104 106 107 108 120 121 122 123 124 125 140 141 143 144 145 147 149

3.58 3.77 4.22 6.03 4.27 2.55 2.27 2.94 5.09 0.4 2.1 0.8 0.9 3.2 2.82 3.09 3.12 4.21 4.71 3.24 3.31

0.6 0.49 0.67 0.49 0.62 0.64 0.47 0.42 2.24 0.5 1.38 0.95 0.6 2.14 0.86 1.33 0.25 0.34 0.57 0.42 0.8

4.97 5.50 4.62 6.43 3.06 3.6 3.43 3.59 2.77 0.75 2.6 1.32 1.43 2.72 3.14 4.11 2.82 3.61 3.91 2.74 4.31

9.15 9.76 9.51 12.95 7.95 6.79 6.17 6.95 10.1 1.65 6.08 3.07 2.93 8.06 6.82 8.53 6.19 8.16 9.19 6.4 8.42

200 201 202 203 204 210 211 212 213 214

7.51 4.26 4.32 3.59 2.85 4.18 4.19 4.6 4.57 3.33

1.09 0.6 0.65 0.59 2.1 5.12 4.86 0.29 3.88 0.27

5.01 4.63 4.17 3.83 3.86 4.53 4.74 4.58 4.44 2.6

13.61 9.49 9.14 8.01 8.8 13.83 13.78 9.47 12.89 6.2

to 6.25%; indeed, we recovered a set of terpene-producing strains with better performances and different terpene profiles than the original 592 mutant. In Fig. 3, typical progress of alcohol formation for the diploid mutant strain CM593, the wine strain L116 and two of the best inbred products of the backcross

247

CM593 26X20 147X103 13x17 140x103 13X120 100x146 145x147 26X140 13x103 20X13 104x103 13X20 13X6 100x103

Lll6

7

t

I I

I I

I

I I

0

1

2

3

4

Weight loss (g) Fig. 2. Ethanol formation efficiency of brother-sister mating products: weight loss in 40-h-old cultures in YPGlSO without ergosterol (mean of two duplicate experiments and standard deviation). TABLE 4 Efficiency with brother-sister mating products: ethanol formation rate, glucose left, ethanol and glycerol production (64-h-old cultures in YPGISO; mean of two duplicate experiments) Strain

L116 CM593 13x

6

13x 13x 13x 26x 13x103 13x120 26x140

100x 104 x 140 x 145 x 147 x 100 x

20 17 26 20

14.5 103 103 147 103 103

Ethanol formation rate 0.11

Glucose gl-’

Glycerol gl-’

Ethanol gl-’

19

0.06

65

4.4 2.0

57 29

0.08 0.08 0.06 0.07 0.06 0.07 0.06 0.07 0.06 0.07 0.06 0.07 0.06

45 50 66 54 65 56 66 55 65

2.3 2.3 2.1 2.8 2.2 2.5 2.3 2.3 2.6 2.5 2.3 2.2 2.3 2.6

31 37 31 35 30 33 28 35 31 39 32 31 32 49

0.10

51 64 59 61 30

TABLE 5 Terpene production with inbred hybrids corresponding to the first two backcross generations, compared to the homozygous laboratory diploid CM593; values in mg I -I, 64-h-old cultures in YPG150 Hybrid

Citronellol

Lll6 CM593 13X 6 13x 20 13x 17 13x 26 26x 20 13x103 13x 120 26x 140 100x 145 104 x 103 140x 103 145 x 147 147x 103 100 x 103

Linalool

Geraniol

0 2.3

0 0.63

0 4

3.9 5.3 6.4 5.3 10.0 6.9 5.3 1.7 3.2 8.9 2.2 1.7 2.7 5.6

3.0 1.6 2.7 2.3 6.0 2.9 1.6 0.6 0.5 0.6 0.5 0.4 0.5 4.4

5.7 1.9 10.1 3.7 6.2 1.5 1.9 2.9 5.8 2.3 4.0 3.2 4.3 7.3

Total 0 6.93 12.6 8.8 19.2 11.3 22. I 11.3 8.8 5.2 9.5 11.8 6.7 5.3 7.5 17.3

are compared. Although glucose consumption was not significantly improved, it was worth testing the new strains in grape must in small-scale pilot experiments. Pilot experiments

Four hybrids of each of the first two backcross generations and three brothersister matings were tested in grape juice in 2 1 reactors and compared to the wine

L116 100x103 104x103 CM593

0

10

20

JO

40

50

60

70

80

h

Fig. 3. Wild-type strain L116, diploid laboratory mutant strain CM593 and two brother-sister mating products of the backcross, 104 X 103 and 100 X 103.

249 TABLE

6

Pilot-scale

processing

Strain

Ethanol (g I--‘)

L116-7a CM592 6 13 17 26 100 101 104 108 CM593 13x 100x 104x

26 145 103

of grape Glucose (g I-‘)

must

by backcross

products

and parental

strains

(after

72 h) terpenes

Linalool

Citronellol

Geraniol

Total

(ms I-‘)

(mg I-‘)

(ms I-‘)

(ms I-‘)

0

0

0

78.4

13

0

6.4

168

0.2

0

1.2

1.4

5.6 12.0 16.8 21.6 22.4 26.4 36.8 22.4

176 150 162 148 129 120 110 137

0.2 3.1 2.2 1.3 2.6 4.4 1.9 I.8

0 0.3 1.5 0.6 1.0 0.6 0.3 0.6

1.0 4.0 5.3 4.3 5.0 4.4 1.5 3.9

1.2 7.6 9.0 6.3 8.9 9.7 4.2 6.7

5.6

159

1.1

0

3.3

4.5

19.2 27.2 34.4

136 124 106

2.2 5.0 4.8

0.4 0.7 0.3

4.6 5.8 2.7

7.4 12 8.3

strain and to the mutant laboratory strains for glucose consumption, ethanol and terpene production (Table 6). Although the hybrid strains resulting from the backcrosses were not as efficient as the wine strain, these small-scale pilot experiments showed that, as in synthetic medium, efficiency has been largely improved in hybrid strains compared to laboratory mutants (up to a 6-fold increase in ethanol production). Total terpene production also increased, as did the ratio linalool/geraniol, which resulted in wine of higher aromatic quality.

Discussion

The aim of this backcrossing program was to change as much as ‘possible the genetic background of the laboratory mutant strain CM592 in order to improve technological characteristics, such as alcohol formation efficiency, while retaining the three recessive mutations essential for terpene production. It was demonstrated (Thornton, 1985) that a character controlled by a single chromosomal gene can be readily introduced into a wine yeast by selective hybridization. Most winemaking characteristics are, however, under multigenic control and backcrossing often gives rather unpredictable results. This report shows that characters controlled by several unlinked recessive mutations, like the terpene-producing ability can be introduced into a wine yeast as well.

250

As a rule, wine yeasts display poor sporulation and low spore viability (Kusewicz and Johnston, 1980). In this respect, L116 which was selected from among a dozen commercial wine strains, is rather unusual: it sporulated well and yielded a relatively high proportion of viable spores: 30% of the spores in four-spored asci were viable after microdissection, while some of the spores expressed mating-type, some others did not do so but instead could sporulate like the original strain. Obviously, this wine strain is of a rather complex genetic constitution. Nevertheless, spore L116-7a chosen as wild-type parent for three generations of backcross seems to be of a more regular constitution: most of the hybrids constructed with it sporulated fairly well and the resulting spores expressed mating type (data not shown). The outcome of three generations of backcross was a set of terpene-producing hybrids with significantly higher ethanol formation efficiencies compared to the initial laboratory mutant CM592; however, none of them was actually as good as a wild type strain. This might be due to the considerable pressure we imposed by exerting simultaneous selection for the three independent mutations erg9, erg20 and sas. On the other hand, as ergosterol is known to be essential for ethanol tolerance (D’Amore and Stewart, 1987), there is an apparent contradiction in looking for a strain displaying higher ethanol production while bearing two nmtations in the sterol pathway, which tend to reduce the ergosterol content of the cell. Surprisingly, since the first generation, terpene production was greatly affected by the backcross. A general increase in total terpene production (up to 5 times) could be noted, probably due to the change of the genetic background of the mutant strain. In addition, the terpene profile itself was also altered, citronellol and linalool increasing more than geraniol. As described by Gramatica et al. (1982), citronellol results from the bioconversion of geraniol by the yeast. One unexpected effect of the backcross was to enhance this bioconversion, as the wild-type parent L116-7a was found itself to convert geraniol into citronellol at a higher rate than the wild-type laboratory strain FL200, from which CM592 originated (data not shown). The origin of linalool remains unknown. Recent attempts to elucidate this question revealed that in vitro, geranyl pyrophosphate can give linalool by direct chemical transformation. As dephosphorylation of geranyl pyrophosphate prior to its transformation in linalool yields geraniol, linalool formation must compete with this process for the pool of geranyl pyrophosphate. The properties of the phosphatase(s) involved in these alternative routes might be responsible for changes in the ratio of these two monoterpenes. Whatever their origin, such modifications in the linalool and citronellol contents are beneficial to the aromatic profile of the product obtained. The best strains selected in this backcross program seem henceforth promisingly suitable for industrial applications.

Acknowledgements

We wish to express our gratitude to F. Karst for stimulating and helpful discussions and for the gift of the original terpene-producing mutant. Thanks are also due to Prof. H. Heslot for critical reading of the manuscript, advice and fruitful discussions.

References Chambon, C., Ladeveze, V., Oulmouden, A., Servouse, M. and Karst, F. (1990) Isolation and properties of yeast mutants affected in farnesyl diphosphate synthetase. Curr. Genet. 18, 41-46. Christensen, B.E. (1987) Cross breeding of distillers’ yeast by hybridization of spore derived clones. Carlsberg Res. Commun. 52, 253-262. Croteau, R. (1981) Biosynthesis of monoterpenes. In: Porter, J.W. and Spurgeon, S.L. (Eds.), Biosynthesis of Isoprenoid Compounds, Wiley, New-York, Vol. 1, pp. 225-282. D’Amore, T. and Stewart, G.G. (1987) Ethanol tolerance of yeast. Enzyme Microb. Technol. 9, 322-329. Eustace, R. and Thornton, R.J. (1987) Selective hybridization of wine yeast for higher yields of glycerol. Can. J. Microbial. 33, 112-117. Gramatica, P., Manitto, P., Maria Ranzi, B., Delbianco, A. and Francavilla, M. (1982) Stereospecific reduction of geraniol to R-c + J-citronellol by Saccharomyces cereuisiae. Experientia 38, 775. Javelot, C., Karst, F., Ladeveze, V., Chambon, C. and Vladescu, B. (1990) Production of monoterpenes by yeast mutants defective in sterol biosynthesis. In: Nga, B.H. and Lee, Y.K. (Eds.), Microbiology Applications in Food Biotechnology, pp. 101-122. Kusewicz, D. and Johnston, J. (1980) Genetic analysis of cryophilic and mesophilic wine yeasts. J. Inst. Brew. 86, 25-27. Mahe-Javelot, C. (1989) Mutants de Saccharomyces cereuisiae producteurs de terpbnes. These de Doctorat, Institut National Agronomique Paris-Grignon. Pomper, S., Daniels, K.M. and McKee, D.W. (1954) Genetic analysis of polyploid yeast. Genetics 39, 343-355. Thornton, R.J. (1985) The introduction of flocculation into a homothallic wine yeast. A practical example of the modification of winemaking properties by the use of genetic techniques. Am. J. Enol. Vitic. 36, 47-49.