Genome-wide identification of the Fermentome; genes required for successful and timely completion of wine-like fermentation by Saccharomyces cerevisiae

BACKGROUND: Wine fermentation is a harsh ecological niche to which wine yeast are well adapted. The initial high osmotic pressure and acidity of grape juice is followed by nutrient depletion and increasing concentrations of ethanol as the fermentation progresses. Yeast's adaptation to these and many other environmental stresses, enables successful completion of high-sugar fermentations. Earlier transcriptomic and growth studies have tentatively identified genes important for high-sugar fermentation. Whilst useful, such studies did not consider extended growth (>5 days) in a temporally dynamic multi-stressor environment such as that found in many industrial fermentation processes. Here, we identify genes whose deletion has minimal or no effect on growth, but results in failure to achieve timely completion of the fermentation of a chemically defined grape juice with 200 g L-1 total sugar. RESULTS: Micro- and laboratory-scale experimental fermentations were conducted to identify 72 clones from ~5,100 homozygous diploid single-gene yeast deletants, which exhibited protracted fermentation in a high-sugar medium. Another 21 clones (related by gene function, but initially eliminated from the screen because of possible growth defects) were also included. Clustering and numerical enrichment of genes annotated to specific Gene Ontology (GO) terms highlighted the vacuole's role in ion homeostasis and pH regulation, through vacuole acidification. CONCLUSION: We have identified 93 genes whose deletion resulted in the duration of fermentation being at least 20% longer than the wild type. An extreme phenotype, 'stuck' fermentation, was also observed when DOA4, NPT1, PLC1, PTK2, SIN3, SSQ1, TPS1, TPS2 or ZAP1 were deleted. These 93 Fermentation Essential Genes (FEG) are required to complete an extended high-sugar (wine-like) fermentation. Their importance is highlighted in our Fermentation Relevant Yeast Genes (FRYG) database, generated from literature and the fermentation-relevant phenotypic characteristics of null mutants described in the Saccharomyces Genome Database. The 93-gene set is collectively referred to as the 'Fermentome'. The fact that 10 genes highlighted in this study have not previously been linked to fermentation-related stresses, supports our experimental rationale. These findings, together with investigations of the genetic diversity of industrial strains, are crucial for understanding the mechanisms behind yeast's response and adaptation to stresses imposed during high-sugar fermentations.Michelle E Walker, Trung D Nguyen, Tommaso Liccioli, Frank Schmid, Nicholas Kalatzis, Joanna F Sundstrom, Jennifer M Gardner and Vladimir Jirane

Parasite spread at the domestic animal - wildlife interface: anthropogenic habitat use, phylogeny and body mass drive risk of cat and dog flea (Ctenocephalides spp.) infestation in wild mammals

Spillover of parasites at the domestic animal - wildlife interface is a pervasive threat to animal health. Cat and dog fleas (Ctenocephalides felis and C. canis) are among the world's most invasive and economically important ectoparasites. Although both species are presumed to infest a diversity of host species across the globe, knowledge on their distributions in wildlife is poor. We built a global dataset of wild mammal host associations for cat and dog fleas, and used Bayesian hierarchical models to identify traits that predict wildlife infestation probability. We complemented this by calculating functional-phylogenetic host specificity to assess whether fleas are restricted to hosts with similar evolutionary histories, diet or habitat niches.Over 130 wildlife species have been found to harbour cat fleas, representing nearly 20% of all mammal species sampled for fleas. Phylogenetic models indicate cat fleas are capable of infesting a broad diversity of wild mammal species through ecological fitting. Those that use anthropogenic habitats are at highest risk. Dog fleas, by contrast, have been recorded in 31 mammal species that are primarily restricted to certain phylogenetic clades, including canids, felids and murids. Both flea species are commonly reported infesting mammals that are feral (free-roaming cats and dogs) or introduced (red foxes, black rats and brown rats), suggesting the breakdown of barriers between wildlife and invasive reservoir species will increase spillover at the domestic animal - wildlife interface.Our empirical evidence shows that cat fleas are incredibly host-generalist, likely exhibiting a host range that is among the broadest of all ectoparasites. Reducing wild species' contact rates with domestic animals across natural and anthropogenic habitats, together with mitigating impacts of invasive reservoir hosts, will be crucial for reducing invasive flea infestations in wild mammals

Adelaide researchers head 'over the ditch' for Australasian yeast meeting

University of Adelaide Research Fellows Michelle Walker, Joanna Sundstrom and Tommaso Liccioli together with students Simon Dillon, Danfeng Long, Trung Nguyen, Ee Lin Tek and Jin Zhang report on the "Yeasts: Products, Discovery and more".Michelle Walker, Joanna Sundstrom, Tommaso Liccioli, Simon Dillon, Danfeng Long, Trung Nguyen, Ee Lin Tek and Jin Zhan

A novel methodology independent of fermentation rate for assessment of the fructophilic character of wine yeast strains

The yeast Saccharomyces cerevisiae has a fundamental role in fermenting grape juice to wine. During alcoholic fermentation its catabolic activity converts sugars (which in grape juice are a near equal ratio of glucose and fructose) and other grape compounds into ethanol, carbon dioxide and sensorily important metabolites. However, S. cerevisiae typically utilises glucose and fructose with different efficiency: glucose is preferred and is consumed at a higher rate than fructose. This results in an increasing difference between the concentrations of glucose and fructose during fermentation. In this study 20 commercially available strains were investigated to determine their relative abilities to utilise glucose and fructose. Parameters measured included fermentation duration and the kinetics of utilisation of fructose when supplied as sole carbon source or in an equimolar mix with glucose. The data were then analysed using mathematical calculations in an effort to identify fermentation attributes which were indicative of overall fructose utilisation and fermentation performance. Fermentation durations ranged from 74.6 to over 150 h, with clear differences in the degree to which glucose utilisation was preferential. Given this variability we sought to gain a more holistic indication of strain performance that was independent of fermentation rate and therefore utilized the area under the curve (AUC) of fermentation of individual or combined sugars. In this way it was possible to rank the 20 strains for their ability to consume fructose relative to glucose. Moreover, it was shown that fermentations performed in media containing fructose as sole carbon source did not predict the fructophilicity of strains in wine-like conditions (equimolar mixture of glucose and fructose). This work provides important information for programs which seek to generate strains that are faster or more reliable fermenters.T. Liccioli, P. J. Chambers, V. Jirane

Novel wine yeast for improved performance in fermentation

University of Adelaide researchers are using directed evolution techniques as an alternative, non-GMO method for wine yeast strain improvement, with the project ultimately aimed at helping winemakers avoid "sticky" fermentation situations.T. Liccioli, M.E. Walker, J.F. Sundstrom, J.M. Gardner and V. Jirane

Grociana piccola: A rare example of Republican military fortifications in Italy

Recent investigations at Grociana piccola, a site in northeastern Italy consisting of two sub-rectangular fortifications, offer the rare opportunity to investigate Early Roman military architecture outside the Iberian peninsula. Excavations have revealed an inner rubble masonry rampart dated to the 2nd c. BCE by associated pottery, mainly amphora remains. This date suggests that the fortification was in use during the first Roman conquest and/or later campaigns of the 2nd c. BCE, providing one of the earliest and smallest examples of a military fort. The fort's ramparts were built using the same building technique as much larger 2nd-c. BCE military camps. Another trench uncovered the northeastern corner of the outer rampart and a probable tower or artillery platform which can be connected to a temporary camp built during the mid-1st c. BCE

Genome wide screening for the identification of genes influencing colour in red wine fermentations

Abstract of talk presented at meeting of the Australasian Yeast Network and the New Zealand Microbiological Society Eukaryotic Special Interest GroupTommaso Liccioli, Michelle Walker, Frank Schmid, Nicholas Kalatzis, Jennie Gardner, Paul Grbin and Vladimir Jirane

Generation and characterisation of improved wine microbes

Actes de colloque du 10e symposium international d'oenologie de BordeauxV. Jiranek, T. Liccioli, A. Betteridge, K. Sumby, M. Walker, J. Gardner, J. Sundstro

Overexpression studies – an approach to understand the mechanisms behind successful alcoholic fermentation.

Abstract of talk presented at meeting of the Australasian Yeast Network and the New Zealand Microbiological Society Eukaryotic Special Interest GroupPredictable and reliable alcoholic fermentation by wine yeast Saccharomyces cerevisiae at specified temperatures as well as production of desirable flavour and aroma compounds, although not essential to the yeast, are sought-after winemaking attributes. The cellular mechanisms that allow yeast to grow in and respond to the harsh wine fermentation environment and successfully adapt to changing chemical stresses incurred during fermentation are largely unclear. A genome-wide screening approach utilising collections of yeast mutants with individual known gene deletions, is one paradigm being used to address this gap. Our research has focused on screening approaches collections of yeast with individual gene deletants or gene overexpressers under fermentation conditions, to identify genes that are required for fermentation and which can modulate fermentation outcome. Such genes are representative of what we term the ‘fermentome’. We have previously reported on the identification of 93 genes which lead to fermentation protraction upon deletion, a dataset referred to as the Fermentation Essential Genes (FEG). In this study we report on the construction of an over-expression library in a haploid wine yeast background, which is suitable for fermentation studies in high sugar medium, specifically chemically defined grape juice (CDGJM). This library was screened on a micro-scale (1.8 mL) with the aim of identifying those genes whose over-expression resulted in enhanced or protracted fermentation performance, and lastly, affected the fermentation profile of 36 known yeast-derived aroma compounds. This presentation will focus on preliminary findings on a subset of overexpression strains which are currently being evaluated for fermentation performance at a laboratory (100 mL) scale. Aroma data will not be discussed. It is envisaged that data collated from this study will expand on the FEG dataset as well as other datasets which together, comprise of the yeast ‘Fermentome’. Through collation of gene datasets relevant to fermentation, we are able to identify and understand which genes (and their products) and cellular processes enable yeast to adapt and grow in grape juice and undergo reliable alcoholic fermentation as in winemaking. We have already constructed a number of gene modifications in haploid wine yeast, with the view of producing robust yeast which exhibit robust fermentation under nitrogen limited or high sugar containing CDGJM under laboratory scale conditions. We are also using the knowledge gained from our research to guide selection strategies in directed evolution approaches to generate new ‘industry ready’ wine yeast with improved fermentation attributes.Michelle Walker, Jade Haggerty, Jin Zhang, Trung D. Nguyen, Joanna Sundstrom, Tommaso Liccioli, Jennie Gardner and Vladimir Jirane