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Applied and Environmental Microbiology, April 2008, p. 2129-2134, Vol. 74, No. 7
0099-2240/08/$08.00+0     doi:10.1128/AEM.01840-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Construction of Sterile ime1-Transgenic Saccharomyces cerevisiae Wine Yeasts Unable To Disseminate in Nature

Manuel Ramírez^* and Jesús Ambrona

Departamento de Microbiología, Facultad de Ciencias, Universidad de Extremadura, 06071 Badajoz, Spain

Received 8 August 2007/ Accepted 24 January 2008

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The use of new transgenic yeasts in industry carries a potential environmental risk because their dispersal, introducing new artificial genetic combinations into nature, could have unpredictable consequences. This risk could be avoided by using sterile transgenic yeasts that are unable to sporulate and mate with wild yeasts. These sterile yeasts would not survive the annual cyclic harvesting periods, being condemned to disappear in the wineries and vineyards in less than a year. We have constructed new ime1 wine yeasts that are unable to sporulate and mate, bear easy-to-detect genetic markers, and quickly disappear in grape must fermentation immediately after sporulation of the yeast population. These sterile yeasts maintained the same biotechnological properties as their parent yeasts without any detectable deleterious effect of the ime1 mutation. These yeasts are therefore interesting biotechnologically for food industry applications and for genetically modified microorganism environmental monitoring studies.

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Since Saccharomyces cerevisiae is the main yeast involved in wine making, it is a major target for wine technology improvement. There has been interesting progress in the development of genetically engineered wine yeasts and their industrial applications over the last 16 years (6, 11, 13-17, 19, 22, 23, 26, 27, 33-35, 40). Most of these genetically modified microorganisms (GMOs), however, do not meet the requirements for genetic stability and for the absence of bacterial DNA and antibiotic resistance markers. However, new technologies are now available that allow these requirements to be met (10). Several research groups have developed genetic modification strategies that satisfy present and forthcoming GMO regulations. These strategies reduce the amount of nonyeast DNA integrated into the modified strains, avoid the use of antibiotic resistance markers, and obtain transgenic yeasts that are genetically stable in the absence of selective pressure and which would be more acceptable to consumers (1, 28). Apart from purely technical limitations, GMOs are not reaching consumers because of long and costly administrative procedures, consumer distrust, and activist opposition. In the case of wine GMOs, additional problems arise from the specific aspects of the wine market, including international (International Organization of Vine and Wine), national, and local ("appellation d'origine") regulations. In addition, it has been found that commercial yeasts inoculated into the fermenting must become resident in the wineries and can eventually disperse into nature (2, 4, 5, 12, 36, 37, 41). Therefore, future consumer acceptance of transgenic wine yeasts will be dependent on the ability to control the risk of their spread into natural ecosystems.

In summary, apart from the purely rational principal of precaution, GMOs have received such bad press that consumer and authority acceptance has been very negatively influenced. A side effect has been that some researchers have turned to traditional genetic methods to improve wine yeasts genetically (2, 4, 10, 25, 29, 30), methods which historically have not been used for this purpose. Now, however, the availability of new procedures for the efficient ecological control of GMOs in nature could give consumers and activists more confidence and allow researchers to turn back to genetically engineered wine yeasts.

Most wild S. cerevisiae wine yeasts isolated from must fermentation are diploid homothallic strains (24, 30). They sporulate in the absence of nutrients, giving haploid spores that are very resistant to adverse environments (long periods of nutrient depletion, extreme temperatures, radiation, dryness, etc.). Sporulation can take place every year at the end of the vintage, and the spores can survive the periods between vintages. At the beginning of the next vintage, the spores germinate, giving haploid yeasts on the new ripened grape or in its must, where there are abundant nutrients available. These haploid strains, which can have two different mating types (a and ), mate with each other, and restore the diploid status that predominates in spontaneous must fermentations (3), in which multiplication occurs by asexual budding. The sexual part of the yeast biological cycle (sporulation and mating) is not required for wine making, although it is needed for the yeast to survive the adverse conditions during the cyclic starvation periods between vintages. If a yeast is unable to undergo meiosis, sporulation, and mating, i.e., if it is sexually sterile, it will disappear from nature because it cannot survive in the winery-vineyard ecosystem. An exception is the film yeast living in the surface of wines such as sherry that undergo biological aging. These populations can survive for years in the winery without sporulation (20), although they do need to sporulate to survive outside the winery.

IME1 is a nonessential gene required for yeast to enter into meiosis (7-9, 38, 39, 42, 43). Diploid homozygous ime1 yeasts can multiply asexually by budding (mitosis) and perform their normal living metabolism, but they are unable to sporulate. We analyze here the use of new ime1 transgenic wine yeasts that are unable to start meiosis but demonstrate good must fermentation performance as a potential strategy for industrial applications of GMOs, while avoiding their dissemination in nature. These new sterile yeast strains have good fitness and appropriate genetic markers and could very much help to forestall the risk of genetic pollution due to the dissemination of GMOs into nature.

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Yeast strains.
SMR10-11D (MAT/MATa HO/HO SMR^R/SMR^R [k2⁺]) is a killer wine yeast (2). SMR10-11DNK (MAT/MATa HO/HO, SMR^R/SMR^R [k2°]) is a nonkiller yeast from SMR10-11D. The haploid laboratory yeast YJR094C (mata ho his3 leu2 met15 ura3 ime1::G418^R) was obtained from EUROSCARF (i.e., the European Saccharomyces cerevisiae Archive for Functional Analysis). E324 (MAT/MATa HO/HO ura30/ura30 ime1::G418^R/ime1::G418^R [k2°]) is a nonkiller homozygous spore clone from the genetic cross SMR10-11DNKxYJR094C. H74 (MAT/MATa HO/ho HIS3/his31 LEU2/leu20 MET15/met150 URA3/ura30 IME1/ime1::G418^R [k2⁺]) is a killer heterozygous hybrid from the cross EX85xYJR094C. EX85 is a K2 killer prototrophic and homothallic S. cerevisiae wine yeast previously isolated from Spanish wineries and selected for wine making (32). 85R is a spontaneous cyh2^R killer-sensitive mutant, free of ScV-M2 virus, from EX85 (25, 31). 85R4A is a cyh2^R/cyh2^R killer-sensitive spore clone from 85R (30, 31). H68 [MAT/MATa HO/ho ILV2(SMR^R)/ILV2(smr^S) HIS3/his31 LEU2/leu20 MET15 met150 URA3/ura30 IME1/ime1::G418^R (k2°)] is a nonkiller heterozygous hybrid from the cross SMR10-11DNKxYJR094C. All of these S. cerevisiae wine strains were developed to provide good fermentation performance and have appropriate genetic markers to easily analyze the frequency of the different yeasts present in the same fermenting must. All strains except the ime1/ime1 mutants sporulated very well, with more than 80% of tetrads after 7 days in sporulation media, and the spore viability was >91%. E324 (ime1/ime1) did not sporulate at all.

Culture media and phenotype tests.
Standard culture media were used for yeast growth and phenotype tests (18). YEPD-agar contained 1% Bacto yeast extract, 2% Bacto peptone, 2% glucose, and 2% Bacto agar. YEPD+G418 is YEPD-agar supplemented with G418 (which is the antibiotic Geneticin, from Sigma [catalog no. G7034], presented as a concentrated aqueous solution) to a final concentration of 200 µg/ml. YEPD+cyh is YEPD-agar supplemented with cycloheximide (cyh) to a final concentration of 2 µg/ml. Synthetic minimal medium (SD) contained 0.67% yeast nitrogen base (without amino acids, with ammonium sulfate; Difco, Detroit, MI), 2% glucose, and 2% Bacto agar. Uracil (20 mg/liter), L-leucine (30 mg/liter), L-histidine-HCl (20 mg/liter), and L-methionine (20 mg/liter) were added when necessary. SD+smr is standard SD-agar supplemented with sulfometuron (smr) to a 100-µg/ml final concentration. The smr was prepared in a concentrated dimethyl sulfoxide solution (1%) and added to the media just before they were poured into petri dishes.

Standard yeast genetic procedures were used for sporulation of cultures and dissection of asci (21). Cells were grown on YEPD plates for 2 days at 30°C, transferred to sporulation plates (1% potassium acetate, 0.1% Bacto yeast extract, 0.05% glucose, 2% Bacto agar), and incubated for 7 to 20 days at 25°C until more than 80% of the cells had sporulated. A total of 24 asci from each yeast were dissected on YEPD plates, followed by incubation for 5 days at 30°C to determine the percentage of viable spores.

Must fermentation.
Grape must fermentation was performed in 5 ml of sterile white Pardina juice (23 °Brix, pH 3.5) supplemented with uracil (20 mg/liter) to facilitate the growth of newly originated homozygous ura30/ura30 yeasts. In first-round must fermentations, yeast cells of mutants and parental strains were cultured in YEPD broth for 2 days at 30°C (vegetative cells) and inoculated in the must (200 µl of culture for single-yeast fermentations and 100 µl for the two-yeast strain [Mix] fermentations). These two strains of each Mix fermentation had the same killer phenotype to avoid any side effect on the fermenting yeast population. The yeast populations (100-µl samples) from each fermentation stage (beginning, tumultuous, and final) were washed with sterile water, inoculated onto sporulation plates, and incubated at 25°C until observation of a tetrad proportion greater than 80% of the population. In the second-round must fermentations, the sterile must was inoculated with a small piece of the YEPD-agar containing 40 of the tetrads previously isolated with the needle of the micromanipulator. The same procedure was repeated to obtain the tetrads from the second-round must fermentations and inoculate a third round of must fermentations. Fermentations were conducted at 25°C for up to 20 days without agitation. The °Brix values were monitored each day to follow the fermentation kinetics. T₁₅ is the time needed to ferment 15% of the total sugars present in the must, and T₁₀₀ is the time needed to ferment 100% of the total sugars (30). Suitably diluted samples from each fermentation were spread onto YEPD plates to obtain isolated colonies after 2 days at 30°C. The amount of viable yeasts (in CFU) was determined by colony counting. SMR^R, G418^R, and cyh2^R phenotypes were determined by replica plating on SD+smr, YEPD+G418, and YEPD+cyh media (2, 4, 25).

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Survival of homozygous ime1/ime1 and heterozygous IME1/ime1 mutants after consecutive cycles of must fermentation and sporulation: monitoring of transgenic yeasts by an ime1-linked marker.
Sterile grape must was inoculated with two transgenic wine yeasts bearing the G418^R genetic marker linked to the ime1 mutation: E324 (homozygous ime1::G418^R/ ime1::G418^R, sexually sterile) and H74 (heterozygous IME1/ ime1::G418^R). Inoculations were also performed with mixes of one of these transgenic yeasts and a nontransgenic one: Mix-1 (50% E324 plus 50% SMR10-11DNK) and Mix-2 (50% H74 plus 50% SMR10-11D). As the in-parallel fermentation controls, the same sterile must was inoculated separately with the two nontransgenic wine yeasts SMR10-11D (SMR^R/SMR^R [k2⁺]) and SMR10-11DNK (SMR^R/SMR^R [k°]). Each yeast population during must fermentation was monitored by detecting the marker G418^R or SMR^R. The fermentation kinetics (data not shown) and the T₁₅ and T₁₀₀ fermentation parameters were similar for all fermentations (Table 1). All must fermentations were completed by the inoculated yeasts. In all of the fermentations inoculated with only one yeast strain, 100% of the yeasts maintained the marker phenotype. In the Mix-1 fermentation, the initial proportion of the two inoculated yeasts, E324 (41%) and SMR10-11DNK (59%), was roughly maintained throughout the fermentation. In Mix-2, the fermentation started with roughly the same proportion of the two strains, which oscillated during the fermentation, to finish with a greater proportion (61%) of the transgenic H74 (Table 2).

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TABLE 1. Kinetics parameters (T15 and T100) of first-round fermentations inoculated with transgenic yeasts bearing the ime1-linked G418R marker

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TABLE 2. Analysis of the frequency of genetic markers during the first round of must fermentations inoculated with transgenic yeasts bearing the ime1-linked G418R markera

A second round of must fermentations were inoculated with spores from the yeast population present on day 7 of fermentation in Mix-1 and Mix-2. Both fermentations started later than usual (greater T₁₅ than in the previous fermentation) because of the time required for spore germination and adaptation to the new environment (Table 3). The G418^R marker (E324 strain) disappeared in the Mix-1 fermentation. However, both original strains, H74 and SMR10-11D, were present at the beginning of the Mix-2 fermentation roughly in the same proportions (G418^R = 30%, SMR^R = 70%, day 4, Table 3) as in the original population before sporulation (G418^R = 29%, SMR^R = 71%, day 7, Table 2). As had been the case during the first Mix-2 fermentation, the proportion of the transgenic H74 strain (G418^R) oscillated during the fermentation to end higher at 71% (Table 3). No new yeasts without genetic markers or yeasts bearing both the G418^R and SMR^R markers were detected.

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TABLE 3. Analysis of the frequency of genetic markers during the second round of must fermentations inoculated with tetrads from a sporulated culture of the yeasts from the seventh day of the Mix-1 (G418R = 45%, SMRR = 55%) and Mix-2 (G418R = 29%, SMRR = 71%) first-round fermentations of transgenic yeasts bearing the ime1-linked G418R markera

To further analyze the evolution of the ime1::G418^R/IME1 heterozygous yeasts, a third must fermentation was inoculated with spores from the yeast population present on day 14 in the second Mix-2 fermentation. This time, the initial proportions of each genetic marker (G418^R = 39%, SMR^R = 55%, none = 3%, both the G418^R and SMR^R markers = 3%, day 3, Table 4) were quite different from those in the original second Mix-2 fermentation before sporulation (G418^R = 82%, SMR^R = 10%, none = 8%, day 14, Table 3). New heterozygous G418^R plus SMR^R yeasts appeared, and the proportion of G418^R yeasts decreased. Despite this, after the second round of sporulation, the ime1-linked G418^R marker still remained in a major proportion (39 to 54%).

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TABLE 4. Analysis of the frequency of genetic markers during the third round of must fermentations inoculated with tetrads from a sporulated culture of the yeasts from day 14 of the Mix-2 (G418R = 82%, SMRR = 10%, none = 8%) second-round fermentations of transgenic yeasts bearing the ime1-linked G418R markera

Survival of heterozygous IME1/ime1 mutants after consecutive cycles of must fermentation and sporulation: monitoring of transgenic yeasts by an ime1-nonlinked marker.
Sterile grape must was inoculated with a transgenic wine yeast bearing the SMR^R marker nonlinked to ime1, H68 (heterozygous IME1/ime1::G418^R SMR^R/smr^S), and with a mix of this transgenic yeast and a nontransgenic one, Mix-3 (50% H68 plus 50% 85R4A). As the in-parallel fermentation control, the same sterile must was inoculated with the nontransgenic wine yeast 85R4A (cyh2^R/cyh2^R). Monitoring of each yeast population during must fermentation was done by detecting the marker SMR^R or cyh2^R. The fermentation kinetics and the T₁₅ and T₁₀₀ fermentation parameters were similar for all fermentations (data not shown). All must fermentations were properly performed by the inoculated yeasts. In all of the fermentations inoculated with only one yeast strain, 100% of the yeasts maintained the marker phenotype. In Mix-3, there was some displacement of 85R4A by the transgenic H68, which was present at a greater proportion (70.7%) at the end of fermentation (data not shown).

A second round of must fermentations were inoculated with spores from the yeast population present on day 8 of the Mix-3 fermentation. The fermentation kinetics were slower than for the first Mix-3 fermentation, similar to what had been the case in the second-round Mix fermentations reported in the previous section. The percentages of yeasts with the original markers, SMR^R (H68) and cyh2^R (85R4A), remained at the end of fermentation at 35 and 48.6%, respectively, although the proportion of SMR^R yeasts decreased with respect to the starting proportions before sporulation (SMR^R = 70%, cyh2^R = 30%). As stated above (in Table 4), new wild-type yeasts without genetic markers were detected, accounting for the remaining 16.4% at the end of fermentation. No yeasts bearing both the cyh2^R and SMR^R markers were detected (data not shown).

To further analyze the evolution of the ime1::G418^R/IME1, SMR^R/smr^S heterozygous yeasts, a third must fermentation was inoculated with spores from the yeast population present on day 15 in the second Mix-3 fermentation (SMR^R = 27, cyh2^R = 49%, none = 24%). Again, as described above, the initial proportion of SMR^R yeasts decreased from the original proportion existing before sporulation (data not shown). Despite this, 10 to 11.4% of transgenic yeasts bearing the ime1-nonlinked SMR^R marker still remained after the second round of sporulation, although this time the proportion was lower than that for the ime1-linked G418^R marker (39.4 to 54.4% [see above]). New wild-type yeasts without genetic markers were detected in a proportion of 4.6 to 14% during the fermentation.

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All of the transgenic yeasts, homozygous ime1/ime1 or heterozygous IME1/ime1 mutants, remained at the end of the Mix fermentations before sporulation. Their biological fitness is therefore good enough to succeed in nature in competition with wild IME1/IME1 yeasts. The homozygous ime1/ime1 disappeared after sporulation in the consecutive Mix-1 fermentations, whereas the heterozygous IME1/ime1 mutants still remained after sporulation in the consecutive Mix-2 and Mix-3 fermentations. The G418^R marker disappeared in the Mix-1 fermentations because the transgenic ime1/ime1 E324 yeast was sterile and unable to sporulate. No new heterozygous G418^R plus SMR^R yeasts (from mating of haploid cells from different tetrads) were detected in this fermentation; i.e., there was no detectable lateral marker transfer from the transgenic yeast to the wild yeast before sporulation, which makes the dissemination into nature of artificial genetic constructs from homozygous ime1/ime1 sterile yeasts improbable.

The heterozygous IME1/ime1 transgenic yeasts, H74 and H68, remained after sporulation in the second round of Mix-2 and Mix-3 fermentations because they can enter meiosis and sporulate. The new yeasts that arose without genetic markers came from the mating of IME1 haploid yeasts from H74 or H68 tetrads. No yeasts bearing both the cyh2^R and SMR^R markers, and only a low proportion of G418^R plus SMR^R yeasts, were detected because the frequency of mating of haploid homothallic yeasts from different tetrads is very low (1.5%) in these conditions (3).

In the third fermentation round, the initial proportions of each genetic marker were very different from those in the original preceding fermentation before sporulation. In Mix-2, new heterozygous G418^R plus SMR^R yeasts arose from mating of haploid cells from different tetrads, confirming the existence of lateral marker transfer from the transgenic to the wild yeasts after sporulation, which could also be quite possible for in vitro-engineered genes in nature. The proportion of G418^R and SMR^R transgenic yeasts decreased in Mix-2 and Mix-3, respectively, because part of these yeasts from the preceding fermentations were homozygous ime1/ime1 mutants unable to sporulate. Despite this, the ime1-linked G418^R and the nonlinked SMR^R marker still remained after the second round of sporulation. Moreover, surprisingly, at the end of the third round of fermentations, the proportion of the ime1-linked G418^R marker was higher rather than lower than the proportion of the nonlinked SMR^R marker.

In conclusion, linking the in vitro-engineered gene to the ime1 mutation in heterozygous IME1/ime1 yeasts would not be enough to abolish or decrease the dissemination of transgenic wine yeasts, or their modified genes, into nature. Fully sterile homozygous ime1/ime1 wine yeasts are required for this purpose (Fig. 1). Our proposal of such an approach would be based on two premises. First, if a wine yeast cannot sporulate (be able to perform the sexual part of its life cycle), it will not be able to survive the cyclic adverse conditions in nature (long periods of nutrient depletion, extreme temperatures, radiation, dryness, etc.). Second, such yeasts can nevertheless maintain their biotechnological properties in the absence of sexual reproduction. The approach might therefore not be appropriate for organisms (microorganisms, crops, or animals) not fulfilling these two premises. However, although generally speaking there is no such thing as zero risk with commercialized GMOs, the discovery of new mutations that abolish or severely reduce the competitive capabilities of GMOs in nature could very much help prevent their dissemination into the environment.

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FIG. 1. Scheme of the disappearance of homozygous ime1/ime1 (A) and the survival of heterozygous IME1/ime1 (B) transgenic yeasts after consecutive cycles of must fermentation and sporulation.

 
This study was funded by grants 2PR01B002 and 2PR04B003 from the Extremadura Regional Government of Spain.

 
* Corresponding author. Mailing address: Departamento de Microbiología (Antiguo Rectorado), Facultad de Ciencias, Universidad de Extremadura, 06071 Badajoz, Spain. Phone: 34 924289426. Fax: 34 924289427. E-mail: mramirez{at}unex.es

Published ahead of print on 1 February 2008.

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Applied and Environmental Microbiology, April 2008, p. 2129-2134, Vol. 74, No. 7
0099-2240/08/$08.00+0     doi:10.1128/AEM.01840-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

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