Conclusions

1. A new methodologytoobtainwineyeaststrainsoverproducing mannoproteins Quirós, M.1, Gonzalez-Ramos, D.2, Tabera, L.2, Morales, P. 1, Pérez-Través, L.3, Barcenilla, J.M.2 and Gonzalez. R.1 1 Instituto de Ciencias de la Vid y del Vino (CSIC-UR-CAR), Logroño, Spain. 2 Instituto de Fermentaciones Industriales (CSIC), Madrid, Spain. 3 Departamento de Biotecnología. Instituto de Agroquímica y Tecnología de los Alimentos (CSIC), Burjassot, Spain. Introduction Yeast mannoproteins are highly glycosylated proteins that are covalently bound to the β-1,3-glucan present in the yeast cell wall. These complex macromolecules are structurally composed of polymers of sugars, mainly mannose, that are covalently linked to peptides and represent around 40% of the cell wall dry weight. Yeast mannoproteins contribute to several aspects of wine quality by protecting against protein haze, reducing astringency, retaining aroma and other undesired compounds such as OTA and stimulating the growth of lactic-acid bacteria. Therefore, the development of a non-recombinant method to obtain enological yeast strains overproducing mannoproteins would be very useful. Previous works developed in our group have allowed the identification of several genetic determinants for the release of wine yeast mannoproteins (Gonzalez-Ramos and Gonzalez, 2006; Gonzalez-Ramos et al., 2008). Among the different recombinant genotypes constructed and studied, strains deleted for KNR4 (from Killer Nine Resistant, also known as SMI1), a gene involved in β-1,3-glucan biosynthesis yielded optimal results (Gonzalez-Ramos et al., 2008). In the present paper, a new methodology for the selection of non-recombinant mannoprotein overproducing yeast strains based on their resistance to the killer 9 toxin of Williopsissaturnus is developed. Materials and methods UV Mutagenesis Onehaploidlaboratorystrainis BY4741 and three industrial winemakingstrains IFI87, IFI475 and T73 wereused in thisstudy. Overnight cultureswere washed twice and suspensions containing 107 cells/mL were then prepared, placed in a Petri dish and irradiated under a UV lamp at 254 nm. 1 mL aliquots were taken after 20, 40 and 60 s of exposure and kept in darkness for 30 min. Serial 10-fold dilutions of each of these aliquots (up to 10−5) were prepared in saline solution and 100 μL were plated in YPD agar plates and k9 toxin-containing YPD plates. These were incubated for 48–72 h at 28 °C and survival and resistance rates for each of the exposure times were then determined. k9 toxin-resistant mutants were picked and transferred to fresh YPD plates. Ten colonies were randomly picked both from the control YPD plates and the selective YPD plates containing k9 toxin in order to compare polysaccharide and mannoprotein release with the original strain after fermentation in GCY medium The percentage of overproducing mutants obtained for the industrial strains was not as stunning as the observed for the haploid strain. However, mutants producing promising results in GCY broth were tested in synthetic must. Their fermentation performance and the release of total polysaccharides and mannoproteins compared to their wild type counterparts are depicted in Figs. 2A and B respectively. A) Fermentation of GCY and syntheticmust In order to measure the improvement in the release of mannoproteins, selected resistant strains were inoculated into 100 mL shakeflasks containing 50 mL of GCY broth to a final OD600nm of 0.1 and grown for 24 h at 30 °C and 150 rpm. Cultures were centrifuged and the supernatant transferred to clean Falcon tubes. Macromolecules in these supernatants were separated from monosaccharides by gel filtration using Econo-Pac columns (Bio-Rad). Industrial strains showing an improved release of mannoproteins in GCY medium were tested in a synthetic must (pH 3.5) containing 10% glucose, 10% fructose, 0.6% citric acid, 0.6%malic acid, 0.17% YNB without aminoacids and ammonium sulphate (Difco), 306 mg/L NH4Cl and 2/3 of the amount of aminoacids used in a control synthetic grape must (CNC) by Beltran et al. (2004). Fermentations were performed in duplicate at 20 °C using 100 mL bottles capped with Müller valves containing 40 mL of must. Fermentation time course was monitored by determining the production of CO2 expressed as weight loss until weight was constant. B) Fig. 2. A) Time course of fermentations of industrial wild type and mutant strains in synthetic must. B) Total polysaccharides (mg/L) released in synthetic must by the industrial mutant strains selected in this work and their wild type counterparts. Once tested the fermentation performance and the improved release of mannoproteins, we decided to study their resistance to different stresses. As seen in Fig. 3, none of the selected strains presented a significantly sensitive phenotype to any of the different stresses tested with regards to their wild type counterparts. Quantification of total mannoproteins and polysaccharides The concentration of total polysaccharides in the macromolecular fraction of the supernantants was determined by the phenol-sulfuric acid method described by Segarra et al. (1995) using a standard curve of commercial mannan from S. cerevisiae and the concentration of mannoproteins determined by a acid hydrolysis-HPLC method recently developed in our lab (Quirós et al., In Press). pH Glucose Fig. 3 Results The normalized release of total polysaccharides and mannoproteins in all the k9-resistant strains tested is shown in Fig. 1 in mg/(L∙OD). Eight out of the ten mutants significantly produced more polysaccharides than the control strain(p<0.01). The increase in the production of polysaccharides and mannoproteins ranged from 30% for mutant Mut3 to 238% for Mut6. However, none of the colonies isolated from the non-selective medium after the UV treatment overproduced mannoproteins and total polysaccharides (p<0.01, data not shown) 200 g/L 250 g/L 300 g/L Temperature Conclusions The proposed methodology represents a fast and straightforward approach to obtain non-recombinant yeast strains overproducing mannoproteins. The overproducing mutants present similar fermentative performance to those of their wild type counterparts and do not present a hyper-sensitive phenotype to any of the stress factors tested. An increased number of interesting overproducing mutants could also be obtained applying the described methodology to spores isolated from the desired industrial strains. Fig. 1. Normalized production of polysaccharides and mannoproteins (mg/L∙OD) obtained for ten BY4741 mutant strains after UV mutagenesis, selection on YPD+k9 plates and fermentation in GCY medium. Strains releasing a significantly higher amount of mannoproteins than the wild type strain are marked with an asterisk (p<0.01) • References • Gonzalez-Ramos and Gonzalez (2006) J. Agric. FoodChem. 54, 9411-9416. • Gonzalez-Ramos et al. (2008) Appl. Environ. Microbiol. 74, 5533-5540. • Beltrán et al. (2004) FEMS Yeast Res. 4, 625-632. • Quirós et al. FoodChem. doi:10.1016/j.foodchem.2010.08.066