Selection of wine yeast strains with enhancedfermentative fitness and glutathione production

by a multi-phase approachLuciana De Vero1, Tommaso Bonciani2, Paolo Giudici3

1Unimore Microbial Culture Collection, Department of Life Sciences, University of Modena and Reggio Emilia, Reggio Emilia, Italy;2AEB group, Brescia, Italy; 3Acetaia Giudici, Reggio Emilia, Italy.

Email: luciana.devero@unimore.it

Focus of the workWe exploited the random genetic recombination associated to a library of 69 monosporic clones ofSaccharomyces cerevisiae strain 21T2 (=UMCC 855) to search for new candidate strains possessing both primaryand secondary traits. This was achieved by consecutively applying three directional selective criteria: molybdateresistance (1), fermentative aptitude (2) and glutathione production (3).The strains used in this study are deposited in the University of Modena and Reggio Emilia (Unimore) MicrobialCulture Collection (UMCC - www.umcc.unimore.it).

ResultsGenotyping of the selected monosporic clonesThe screening of the 69 MCs on YNB plates supplemented with 2.5 mM Mo(VI) confirmed the phenotype already assessed in the work by Mezzetti et al.(2017). Only nine MCs were able to grow, showing a phenotypic similarity to the high-GSH producing strain UMCC 2581 (growth + dark blue color). TheseMCs were included in two PCR amplifications aimed at differentiating the set under the molecular point of view and at assessing the actual divergence ofthe sibling monosporic clones (Fig. 1).Growth curves at 15 mM ammonium molybdateThe assessment of growth for the nine MCs in YPD supplemented with 15 mM Mo(VI) selected the strains which were highly resistant to Mo(VI).The yeast set was split into two groups (Fig. 2), one which yielded a significant growth and one which was inhibited by molybdate. The strains belonging tothe first group were thus selected for the next investigation steps, as they were the most likely to feature a high production of GSH according to thefindings of Mezzetti et al. (2014).Phenotypic screening on platesThe tests assessed the growth ability of the strains in many different environments and stress conditions. All MCs showed a behavior similar to that ofstrain UMCC 855 concerning most of the growth conditions (Table 1).Fermentative trialsThe strains belonging to the resistant group were applied to fermentative trials in synthetic must alongside parental strain UMCC 855. Two strains, 21T2-D41 and 21T2-D58 showed a strong fermentative behavior similar to that of UMCC 855 (Fig. 3). The remaining two strains were discarded as theirfermentative performance did not serve the purpose of our genetic improvement strategy.GSH Production21T2-D41 and 21T2-D58 strains produced a high concentration of glutathione (GSH) and showed a good fermentative aptitude. In particular, 21T2-D58strain was able to produce 4.22 mg/L of GSH, comparable to that of UMCC 2581 strain, which was the highest GSH-producer (4.71 mg/L). Both of these laststrains yielded a GSH concentration, which was significantly higher than the one produced by the parental strain UMCC 855 (2.11 mg/L).

ConclusionsAdaptive evolution strategies are especially apt for dealing with complex phenotypes, as they allow the free recombination of allelic variants, followed by aselective step aimed at retrieving only the individuals showing the trait of interest (De Vero et al. 2011, 2017). Our three-phase strategy allowed us to couplethe GSH over-production trait with a robust and reliable fermentative profile. The results confirm the effectiveness of meiotic segregation/recombination inproviding clones, which can even be improved in certain traits compared to their parents (Bonciani et al. 2018).

References• Bonciani T, De Vero L, Mezzetti F, Fay JC, Giudici P (2018). A multi-phase approach to select new wine yeast strains with enhanced fermentative fitness and glutathione production. Appl Microbiol Biotechnol. 2018

Mar;102(5):2269-2278. doi: 10.1007/s00253-018-8773-3. Epub 2018 Jan 22.• De Vero L, Bonciani T, Verspohl A, Mezzetti F, Giudici P (2017). High-glutathione producing yeasts obtained by genetic improvement strategies: a focus on adaptive evolution approaches for novel wine strains. AIMS Microbiol

3:155–170. doi:10.3934/microbiol.2017.2.155• De Vero L, Solieri L, Giudici P (2011). Evolution-based strategy to generate non-genetically modified organisms Saccharomyces cerevisiae strains impaired in sulfate assimilation pathway. Lett Appl Microbiol 53:572–575.

doi:10.1111/j.1472-765X.2011.03140.x• Mezzetti, F, De Vero L, Giudici P (2014). Evolved Saccharomyces cerevisiae wine strains with enhanced glutathione production obtained by an evolution-based strategy. FEMS Yeast Res 14:977-987. doi:10.1111/1567-

1364.12186• Mezzetti F, Fay JC, Giudici P, De Vero L (2017). Genetic variation and expression changes associated with molybdate resistance from a glutathione producing wine strain of Saccharomyces cerevisiae. PLoS One12(7):e0180814.

doi: 10.1371/journal.pone.0180814

IntroductionWinemaking is a complex process in which many factors, including grape variety, must quality, technologicalprocedures and involved microorganisms, contribute to obtain the final product. Due to this wide range ofoenological stress-factors, it is necessary to apply an integrated approach for the rapid constitution of yeastsexpressing several complex phenotypes. In this work, we applied a multi-step and strongly targeted geneticimprovement strategy aimed at combining a strong fermentative aptitude (primary trait) with an enhancedproduction of glutathione (secondary trait).

Resistant Cluster

Parent-like cluster

Sensitive Cluster

SCHEME OF THE MULTI-STEP STRATEGY

Step 1. Further selection with ahigher concentration of molybdate.

Step 2.Selection of the strains with the best fermentative aptitude.

Step 3. Selection of the best Glutathioneproducers.

Table 2 Phenotypic screening of the selected strains

Strain Cell/mL EthOH

12%

EthOH

16%

Temp.

4 °C

Temp.

27 °C

Temp.

37 °C

Sucrose

200 g/L

Sucrose

250 g/L

Sucrose

300 g/L

YAN

req.

21T2-D2

1 x 106

5 x 105

1 x 105

5 x 104

21T2-D5

1 x 106

5 x 105

1 x 105

5 x 104

21T2-D41

1 x 106

5 x 105

1 x 105

5 x 104

21T2-D58

1 x 106

5 x 105

1 x 105

5 x 104

UMCC

855

1 x 106

5 x 105

1 x 105

5 x 104

*Color-Code

Growth Color

+++

++

+

+/-

-

*The increasingly darker gray strands for a more vigorous growth of the spots on the plates according the color-code

reported in the legend.

Fig. 1 Similarity tree comparing both Interdelta and (GTG)5-PCR profiles associated to the nine Mo-resistant monosporic clones. The clustering was performed via GelCompare software, applying the criterion of mean similarity with the band-based Dice method. The similarity scale is also displayed above the similarity tree. On the right part of the figure the matching UMCC code is indicated for each strain

Fig. 2 Growth curves of the nine Mo-resistant monosporic clones in YPD supplemented with 15 mM ammonium molybdate

Fig. 3 Loss of CO2 over the course of 21-days microfermentative trials in synthetic must. In this trials the four monosporic clones resistant to 15 mM ammonium molybdate were included alongside parental strain UMCC 855 and evolved strain UMCC 2581

Fig. 4 Total glutathione concentration after microfermentative trials. Graph shows means with SD (n=3) of the synthetic must (CTRL), the UMCC 855 parental strain, the evolved strain UMCC 2581 and four monosporic clones. Means with different uppercase letters are significantly different from each other according to the Duncan Post-Hoc test

Table 2 Phenotypic screening of the selected strains

Strain Cell/mL EthOH

12%

EthOH

16%

Temp.

4 °C

Temp.

27 °C

Temp.

37 °C

Sucrose

200 g/L

Sucrose

250 g/L

Sucrose

300 g/L

YAN

req.

21T2-D2

1 x 106

5 x 105

1 x 105

5 x 104

21T2-D5

1 x 106

5 x 105

1 x 105

5 x 104

21T2-D41

1 x 106

5 x 105

1 x 105

5 x 104

21T2-D58

1 x 106

5 x 105

1 x 105

5 x 104

UMCC

855

1 x 106

5 x 105

1 x 105

5 x 104

*Color-Code

Growth Color

+++

++

+

+/-

-

*The increasingly darker gray strands for a more vigorous growth of the spots on the plates according the color-code

reported in the legend.

Step 1

Yeast Nitrogen Base medium (YNB) + Mo(VI)

[1, 2.5 and 5mM]

UMCC 855Parental

strain

Dissection of UMCC 855

tetrads

69 Mono Sporic Cultures (MSC)

Test on Mo(VI) and identification of the

phenotype for each MSC

Step 3

Step 2

Creation of clusters according to the phenotype

Bulk Segregant Analysis and creation of clusters by Mezzetti et al. (2017).

Table 1 Phenotypic screening of the selected strains

*The increasingly darker grey strands for a more vigorous growth of the spots on the plates according the color-code reported in the legend