The complex interactions between yeasts and their environment are brought sharply into focus when wine fermentations fail to complete. Retrospective analyses of such failures are difficult or impossible. The relationship between yeast strain genetics and its interaction with the grape juice environment is the subject of this work. Two hundred commercially available wine yeasts, or yeasts isolated from wine, were sequenced to evaluate wine yeast genomic diversity. A representative subset of those sequenced yeasts was used to evaluate the fitness impacts of grape juice variables commonly associated with poor fermentation performance and the genetic determinants of some traits identified.
The complex interactions between yeasts and their environment are brought sharply into focus when wine fermentations fail to complete. Retrospective analyses of such failures are difficult or impossible because of the many combinations of factors that may lead to this undesirable outcome. These factors include choice of yeast strain, of which there are many, grape juice composition and winemaking interventions.
Comparative genomics of yeast
The relationship between yeast strain genetics and a yeast’s interaction with the grape juice environment is the subject of this work. Two hundred commercially available wine yeasts, or yeasts isolated from wine, were sequenced to evaluate wine yeast genomic diversity. Whole genome sequence data is now available for 200 wine yeast via the National Center for Biotechnology Information short read archive under the accession number SRP066835. This data archive contains genomic sequence information on wine, ale and cider yeasts of commercial and environmental origin, including species of Saccharomyces cerevisiae, Saccharomyces uvarum and hybrids of S. cerevisiae with non-cerevisiae Saccharomyces yeast including S. eubayanus, S. paradoxus and S. kudriazevii. The comparative analysis of this data has been published in the journal Genes|Genomes|Genetics (Borneman et al. 2016).
Yeast response to environment - comparative analysis of molecularly barcoded yeast
A fundamental property of any commercial yeast is the degree to which it can compete for resources in their working environment. Yeast competitiveness was assessed directly in this project using a representative collection of barcoded wine yeasts. Strain selection for this representative subset of 94 yeasts was informed by the results of comparative genomics. Each strain was tagged with a unique DNA barcode introduced into a phenotypically neutral location. The tags were required to identify strains in mixed cultures, thus enabling the parallel determination of fitness profiles in a range of industrially relevant media formulations. This was achieved through competition experiments in which differential fitness was assessed in response to environmental challenges.
Environmental variables commonly associated with poor fermentation performance, such as sugar concentration and temperature, were not discriminating factors of yeast strain fitness. Copper concentration and nitrogen availability were, however, powerful contributors to fitness variations between wine yeast strains. Fitness-based predictions of performance were validated using single inoculum fermentations. These experiments showed a high concordance between pooled culture fitness and individual strain performance profiles.
Following initial explorations of yeast and environment interactions that were assessed in defined medium, later stages of the project progressed to fitness profiling experiments using freshly prepared unfiltered juice. This enabled a comparison with defined medium results (fitness validation) using an environment complete with the rich microbial ecology that comprises a standard grape juice. Fitness of strains was evaluated in grape juice with different additives (modified nitrogen, SO2, increased sugar concentration etc.) and against a panel of juices. This work showed that juice
preferences exist for some strains and lends weight to the idea that strain/must matching may offer tangible benefits when selecting strains for fermentation.
Uncovering the genetic basis of performance
To uncover the genetic basis of performance in specific environmental conditions, classical genetics (mating and screening of progeny) and modern genomics (whole genome sequencing of progeny) have been combined to identify regions of the genome that positively or negatively contribute to the performance of different strains. The primary focus has been on sulfite and copper tolerance, both of which are highly discriminatory for wine yeasts. The genetic basis of resistance to both of these stresses is well researched and much is already known about the evolutionary strategies that yeast use to tolerate these powerful selective agents. However, this work is uncovering an unlikely interdependence between these strategies, the exact nature of which is still being explored.
The project benefitted substantially from the involvement of several industry partners including Yalumba, Treasury Wine Estates through their Wolf Blass winery, and Pernod Ricard through their Orlando winery at Rowland Flat. The project also benefitted through access to sequencing resources at the Ramaciotti Centre for Genomics (UNSW Sydney, Australia).