Strains and growth conditions

Saccharomyces cerevisiae Zymaflore® FX10 (Laffort), a homozygous and homothallic commercial wine yeast strain was used as host strain for genetic modification. The heterozygous mutants were constructed using the short flanking homology method [38], by transforming FX10 using the lithium acetate procedure [39] with a PCR fragment obtained by amplification of the kanMX4 cassette and flanking regions from the appropriate homozygous deletion strain in the BY4743 background (Open Biosystems, Huntsville, USA). Selection of heterozygous mutants was performed in YPD solid media plates supplemented with 200 mg/L of geneticin (G418). Correct insertion of the kanMX4 cassette was verified by PCR using primers upstream and downstream of the deleted region combined with primers inside kanMX4. Primers used for the construction and verification of recombinant strains are shown in Additional file 3: Table S1. Amplification strategies are summarized in Additional file 4: Figure S3. The homozygous mutants were constructed by sporulating the heterozygous mutants in a medium supplemented with geneticin. The geneticin resistance feature segregated 2:2 as expected. Since the original strain was homothallic, strains recovered from the segregation analysis plates were spontaneous autodiploids, homozygous for the corresponding gene deletion, as verified by PCR (see above).

Yeast strains were grown at 28 °C and maintained at 4 °C on yeast peptone dextrose (YPD) plates (2 % glucose, 2 % peptone, 1 % yeast extract, and 2 % agar), as well as in glycerol stocks at −80 °C. Fermentation experiments were performed in natural grape must (ca. 200 g/L sugars).