Data_Sheet_1_A Novel Approach to Isolating Improved Industrial Interspecific Wine Yeasts Using Chromosomal Mutations as Potential Markers for Increased Fitness.pdf

<p>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.</p

Grape juice fermentation profile of AWRI 838 and hybrid strains CxM1-CxM5.

<p><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0062053#pone-0062053-g005" target="_blank">Figure 5a</a>. (top) Cell growth during fermentation as determined by Optical Density. Data points are presented with error bars. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0062053#pone-0062053-g005" target="_blank">Figure 5b</a>. (bottom) Sugar utilisation during fermentation as determined by Refractive Index. Data points are presented with error bars.</p

Fermentation chemistry analysis of wines using HPLC.

<p>Detection Limit 0.1g/L * g/L, ≠ % v/v Levels not connected by same letter are significantly different (p<0.05).</p

Solvent-extractable volatile fermentation products of AWRI 838, CXM1 and CXM4 in Chardonnay wines.

<p>Levels not connected by same letter are significantly different (p<0.05).</p

Genetic stability of CxM4 fermentation isolates using chromosomal targeted PCR-RFLP.

<p>First gel Chromosome XIV left arm, second gel Chromosome XIV right arm, third gel Chromosome XVI left arm and fourth gel Chromosome XVI right arm. Fifth gel Chromosome XII left arm, sixth gel Chromosome XII right arm, seventh gel Chromosome XIV left arm. Lane 1 100 bp ladder, lane 2 AWRI838, Lane 3 NCYC2888, lane 4 DNA from both parents, lane 5 Hybrid CxM4, lanes 6 to 55 isolates 1 to 50. Arrows point to isolates with altered chromosomal content.</p

Polyphenolic analysis of Chardonnay wines made by AWRI 838, CxM1 and CxM4 using UV Scan data: an index of Phenolic content.

<p>Levels not connected by same letter are significantly different (p<0.05).</p

Primer sets and restriction endonucleases used to generate species-specific chromosomal markers.

<p>Primer sets and restriction endonucleases used to generate species-specific chromosomal markers.</p

Genetic confirmation of cell hybridization by rDNA ITS PCR-RFLP.

<p>Lane 1 100 bp ladder, lane 2 AWRI838, lane 3 NCYC2888, lane 4 DNA from both parents, lanes 5–9 Hybrids CxM1-5.</p

Phenotypic assessment assay plates.

<p>Top row plates left to right; YEPD at temperatures 22°C, 4°C and 37°C. Bottom row plates left to right; YEP 25% glucose, YEPD 14% ethanol. Strains are plated in columns at 10 fold serial dilutions from top to bottom; columns 1–5 CxM5-CxM1 in descending order, column 6 NCYC2888, column 7 AWRI838.</p

Genetic stability of <i>S. cerevisiae</i> x <i>S. mikatae</i> hybrids using rDNA ITS PCR-RFLP.

<p>Top gel, CxM1 fermentation isolates and bottom gel, CxM4 fermentation isolates. Lane 1 100 bp ladder, lane 2 AWRI838, lane 3 NCYC2888, lane 4 DNA from both parents, lane 5, Hybrid, lanes 6–55 isolates 1–50. Arrow points to isolate with loss of <i>S. mikatae</i> rDNA.</p