A Novel Approach to Isolating Improved Industrial Interspecific Wine Yeasts Using Chromosomal Mutations as Potential Markers for Increased Fitness

Abstract

Wine yeast breeding programs utilizing interspecific hybridization deliver cost-effective tools to winemakers looking to differentiate their wines through the development of new wine styles. The addition of a non-Saccharomyces cerevisiae genome to a commercial wine yeast can generate novel phenotypes ranging from wine flavor and aroma diversity to improvements in targeted fermentation traits. In the current study we utilized a novel approach to screen isolates from an evolving population for increased fitness in a S. cerevisiae × S. uvarum interspecific hybrid previously generated to incorporate the targeted phenotype of lower volatile acidity production. Sequential grape-juice fermentations provided a selective environment from which to screen isolates. Chromosomal markers were used in a novel approach to identify isolates with potential increased fitness. A strain with increased fitness relative to its parents was isolated from an early timepoint in the evolving population, thereby minimizing the risk of introducing collateral mutations and potentially undesirable phenotypes. The evolved strain retained the desirable fermentation trait of reduced volatile acidity production, along with other winemaking traits of importance while exhibiting improved fermentation kinetics.

Keywords: chromosomal mutations; evolving populations; increased fitness; interspecific wine yeast hybrids; retain desired phenotype.

FIGURE 1

Design of adaptive evolution experiment to generate mutants with increased fitness in a winemaking context. Serial batch passaging of S. cerevisiae × S. uvarum interspecific hybrid AWRI1572 in Chardonnay juice.

FIGURE 2

Heat map depicting AWRI1572 chromosomal loss in adaptive evolution experiment. (A) Blue box displays S. uvarum chromosomal loss with darkness of hue linked to increased frequency. (B) Red box displays S. cerevisiae chromosomal loss with darkness of hue linked to increased frequency. Scale 0–20 reflects loss per 20 isolates screened from triplicate ferments designated a, b, and c. Roman symbols I, III, and V refer to fermentation Stages 1, 3, and 5, respectively.

FIGURE 3

Saccharomyces uvarum Chromosome 14 loss from AWRI1572 during adaptive evolution experiment. Red ‘C’ box depicts only S. cerevisiae chromosome retained. Blue ‘H’ box depicts both S. cerevisiae and S. uvarum chromosome retained. (A) Stage 2 fermentation; (B) Stage 3 fermentation; (C) Stage 5 fermentation.

FIGURE 4

Ploidy levels of AWRI1572 during adaptive evolution experiment. Analyses of 60 isolates from each ferment series with mean ploidy values and whiskers at 10–90 percentile.

FIGURE 5

Chardonnay juice fermentation progression as determined by weight loss in grams of CO₂. Data points are represented with standard deviation error bars: (A) 225 g/L sugar Chardonnay juice; (B) 350 g/L sugar Chardonnay juice.

FIGURE 6

Ethanol tolerance assay plates. Plates left to right; YEPD, YEPD with 12% ethanol, 14% ethanol, or 16% ethanol. Strains were plated in columns at 10-fold serial dilutions from top to bottom; column1 AWRI838 (S. cerevisiae), column 2 AWRI1176 (S. uvarum), column 3 AWRI1572 (ancestral hybrid), column 4 AWRI2530 (evolved hybrid).

FIGURE 7

Competition assay of AWRI1572 vs AWRI2530. Data points are represented with error bars showing 95% Confidence Intervals. 100 isolates analyzed for presence/absence of S. uvarum Chromosome 14 from each triplicate fermentation. Student’s t-test: Series #1 8.13E-04, Series #2 1.64E-05.