Comparison of Two Alternative Dominant Selectable Markers for Wine Yeast Transformation

Abstract

Genetic improvement of industrial yeast strains is restricted by the availability of selectable transformation markers. Antibiotic resistance markers have to be avoided for public health reasons, while auxotrophy markers are generally not useful for wine yeast strain transformation because most industrial Saccharomyces cerevisiae strains are prototrophic. For this work, we performed a comparative study of the usefulness of two alternative dominant selectable markers in both episomic and centromeric plasmids. Even though the selection for sulfite resistance conferred by FZF1-4 resulted in a larger number of transformants for a laboratory strain, the p-fluoro-DL-phenylalanine resistance conferred by ARO4-OFP resulted in a more suitable selection marker for all industrial strains tested. Both episomic and centromeric constructions carrying this marker resulted in transformation frequencies close to or above 10 3 transformants per g of DNA for the three wine yeast strains tested.During the past century, the selection of microbial strains has been one of the main sources of improvement for traditional fermentation processes, including the production of fermented foods such as wine or dairy products. For enology, the natural variability of Saccharomyces cerevisiae strains has been exploited by means of the isolation of wild yeast strains from grapes, musts, or successful fermentations; characterization of the isolates; and selection of the most suitable strains for each particular wine-making process or production area. Classical genetic methods such as the isolation of spontaneous or induced mutants or sexual crosses have been seldom used for the improvement of S. cerevisiae strains used for wine making (2,19,23,26,37), probably because of the difficulties in the design of selection procedures and because of the fact that, very often, the progeny of an excellent wine-making strain performs poorer than the parent strain (18). During the last 15 years, several attempts to construct genetically engineered yeast strains have been published, and very interesting improvements in the wine-making process or the quality of the wine obtained have been reported, including improved primary and secondary flavors, malic acid decarboxylation by yeast, increased resveratrol, lactic acid, or glycerol contents, and improved survival properties under technological conditions (6,11,16,17,20,27,32,34,38,43,46,47). However, because wild yeast strains are usually prototrophic, most of the strains obtained for these works contained antibiotic resistance genes which were used for selection of the recombinant strains and would be unacceptable for marketing. Puig et al. (35) published an interesting method for obtaining recombinant wine yeast strains that are free of antibiotic selection markers. In this method, a ura3⌬ strain is first constructed by sequential gene replacement of the wild-type URA3 gene, and the kanamycin resistance cassette used to this end is removed by homologous recombination. The resulting auxotrophic strain can then be ...

A RecombinantSaccharomyces cerevisiaeStrain Overproducing Mannoproteins Stabilizes Wine against Protein Haze

“…To compensate for this, we decided to use long flanking regions (about 500 bp) to increase the frequency of homologous recombination at the KNR4 locus rather than gene conversion at the markers' loci. A high frequency of homologous recombination was also desirable to counteract the low frequency of spontaneous mutation in ARO4 that could also lead to the selection of false positives (6). The use of three different selection markers and long homologous flanking regions turned out to be very convenient, especially in the case of the T73-4 strain carrying three alleles of KNR4.…”

“…For the ARO4-OFP marker, after the transformation experiment, cells were diluted 10-fold in YPD broth and incubated for 17 h at 30°C and 200 rpm to allow expression of the resistance allele before the selective pressure was applied. Transformants were selected on SD-PFP plates after 5 days of incubation at 30°C (6). For the KanMX4 marker, cells were diluted twofold in YPD broth and incubated for 1 h at 30°C and 200 rpm to allow expression of the resistance allele.…”

The role ofGAP1gene in the nitrogen metabolism ofSaccharomyces cerevisiaeduring wine fermentation

“…Because industrial strains are usually prototrophic, dominant selectable markers such as antibiotic resistance are mainly used. The introduction of antibiotic-resistant genes into industrial wine yeasts would be absolutely unacceptable for marketing (Cebollero and Gonzalez 2004). The construction of auxotrophic wine strains based on nutritional markers like URA3, LEU2 or LYS2 have been proposed as alternative markers to antibiotic resistance (Puig et al 1998).…”

Construction of a recombinant autolytic wine yeast strain overexpressing the csc1‐1 allele

“…Transformation of S. cerevisiae EC1118 industrial strain was carried out using the lithium acetate method described by Ito et al, (1983). 35 Specific modifications for transformation of industrial strains were considered in accordance with Cebollero and Gonzalez, (2004). 36 Briefly, yeast strains were transformed with 20 lg of DNA, and before the selection of recombinant cells, yeast were diluted 10 times in YPD medium and incubated during 17 h at 30 C and 200 rpm to allow the expression of the selection marker ARO4-OFP.…”

“…35 Specific modifications for transformation of industrial strains were considered in accordance with Cebollero and Gonzalez, (2004). 36 Briefly, yeast strains were transformed with 20 lg of DNA, and before the selection of recombinant cells, yeast were diluted 10 times in YPD medium and incubated during 17 h at 30 C and 200 rpm to allow the expression of the selection marker ARO4-OFP. Transformed cells were selected on SD þ Tyr plates with 2 g/L of PFP after 5 days of incubation at 30 C.…”