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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.

Extending the time before harvest—and therefore increasing grape maturity—enhances ripe fruit wine flavours and wine colour intensity, and also decreases unripe green and vegetal wine flavours. Greater maturity, however, increases sugar concentration, which in turn leads to wines with elevated ethanol concentration. High ethanol concentration can reduce the complexity of a wine by suppressing aroma intensity, while also increasing the perception of ‘hotness’ and ‘bitterness’. In addition, health and economic considerations combined with market demand have the wine industry actively seeking ways to facilitate the production of wines with lower alcohol concentration. This review summarises the latest research aimed at reducing wine alcohol concentration, including viticultural practices, pre-fermentation and winemaking practices, microbiological strategies, and post-fermentation approaches and processing technologies. The effect of these practices on wine flavour is also discussed.

Yeast starter cultures for winemaking have traditionally been developed from successful fermentation isolates. Over the past 10–20 years there has been a move to harness genetic techniques to improve production strains. The approaches to achieve this have been similar to those applied in the agricultural sector for crop and livestock development. Thus, domestication of wine yeasts now involves hybridisation, mutagenesis, selection and screening. There is also a growing interest in tapping into isolates of non-Saccharomyces yeasts to develop novel starter cultures that bring complexity and potentially enhance regionality of wines. This review will cover these developments and will look to the future opportunities arising from the application of genetic engineering and synthetic biology approaches for the introduction of novel phenotypes that are outside of the reach of traditional methods.

Deacidification of wines by malolactic fermentation (MLF) is an essential step in the production of most red, many white and some sparkling base wines. While this secondary fermentation can occur spontaneously, the majority of winemakers, particularly in new world winemaking regions, prefer to minimise the risk of a failed or sluggish MLF by inoculating with a reliable, commercially available starter culture. This review focuses on the scientific literature underpinning growing trends in the application of MLF starters. It considers the literature on co-inoculation regimes, where MLF bacteria are inoculated into a ferment prior to completion of alcoholic fermentation and what benefits this might bring relative to sequential inoculation, when bacteria are added after completion of primary fermentation. It also considers the benefits of the growing trend of using bacteria other than the traditional Oenococcus oeni for induction of MLF, and of developing starter cultures from regional isolates of MLF bacteria to enhance regional identity of wines.

We report the first whole transcriptome RNAseq analysis of the wine-associated lactic acid bacterium Oenococcus oeni using a combination of reference-based mapping and de novo transcript assembly in three distinct strains during malolactic fermentation in Cabernet Sauvignon wine. Two of the strains (AWRIB551 and AWRIB552) exhibited similar transcriptomes relative to the third strain (AWRIB419) which was dissimilar by comparison. Significant intra-specific variation for genes related to glycolysis/gluconeogenesis, purine metabolism, aminoacyl-tRNA biosynthesis, ABC transporters and phosphotransferase systems was observed. Importantly, thirteen genes associated with the production of diacetyl, a commercially valuable aroma and flavour compound, were also found to be differentially expressed between the strains in a manner that correlated positively with total diacetyl production. This included a key strain-specific gene that is predicted to encode a l-lactate dehydrogenase that may enable l-lactic acid to be utilised as a precursor for the production of diacetyl. In conjunction with previous comparative genomic studies of O. oeni, this study progresses the understanding of genetic variations which contribute to the phenotypes of this industrially-important bacterium.

Over recent decades, the average ethanol concentration of wine has increased, largely due to consumer preference for wine styles associated with increased grape maturity; sugar content increases with grape maturity, and this translates into increased alcohol content in wine. However, high ethanol content impacts wine sensory properties, reducing the perceived complexity of flavors and aromas. In addition, for health and economic reasons, the wine sector is actively seeking technologies to facilitate the production of wines with lower ethanol content. Nonconventional yeast species, in particular, non- Saccharomyces yeasts, have shown potential for producing wines with lower alcohol content. These yeast species, which are largely associated with grapes preharvest, are present in the early stages of fermentation but, in general, are not capable of completing alcoholic fermentation. We have evaluated 50 different non- Saccharomyces isolates belonging to 24 different genera for their capacity to produce wine with a lower ethanol concentration when used in sequential inoculation regimes with a Saccharomyces cerevisiae wine strain. A sequential inoculation of Metschnikowia pulcherrima AWRI1149 followed by an S. cerevisiae wine strain was best able to produce wine with an ethanol concentration lower than that achieved with the single-inoculum, wine yeast control. Sequential fermentations utilizing AWRI1149 produced wines with 0.9% (vol/vol) and 1.6% (vol/vol) (corresponding to 7.1 g/liter and 12.6 g/liter, respectively) lower ethanol concentrations in Chardonnay and Shiraz wines, respectively. In Chardonnay wine, the total concentration of esters and higher alcohols was higher for wines generated from sequential inoculations, whereas the total concentration of volatile acids was significantly lower. In sequentially inoculated Shiraz wines, the total concentration of higher alcohols was higher and the total concentration of volatile acids was reduced compared with those in control S. cerevisiae wines, whereas the total concentrations of esters were not significantly different.

The application of Next Generation sequencing to comparative genomics is enabling in-depth characterization of genetic variation between wine yeast strains used in fermentation starter cultures. Knowledge from this work will be harnessed in strain development programs. As a result, winemakers will soon have at their disposal novel, improved yeast starter cultures displaying increased reliability and providing a means of tailoring wine sensory characteristics for new and ever-changing markets. Refereed/Peer-reviewed

Saccharomyces cerevisiae has evolved a highly efficient strategy for energy generation which maximizes ATP energy production from sugar. This adaptation enables efficient energy generation under anaerobic conditions and limits competition from other microorganisms by producing toxic metabolites, such as ethanol and CO 2 . Yeast fermentative and flavor capacity forms the biotechnological basis of a wide range of alcohol-containing beverages. Largely as a result of consumer demand for improved flavor, the alcohol content of some beverages like wine has increased. However, a global trend has recently emerged toward lowering the ethanol content of alcoholic beverages. One option for decreasing ethanol concentration is to use yeast strains able to divert some carbon away from ethanol production. In the case of wine, we have generated and evaluated a large number of gene modifications that were predicted, or known, to impact ethanol formation. Using the same yeast genetic background, 41 modifications were assessed. Enhancing glycerol production by increasing expression of the glyceraldehyde-3-phosphate dehydrogenase gene, GPD1 , was the most efficient strategy to lower ethanol concentration. However, additional modifications were needed to avoid negatively affecting wine quality. Two strains carrying several stable, chromosomally integrated modifications showed significantly lower ethanol production in fermenting grape juice. Strain AWRI2531 was able to decrease ethanol concentrations from 15.6% (vol/vol) to 13.2% (vol/vol), whereas AWRI2532 lowered ethanol content from 15.6% (vol/vol) to 12% (vol/vol) in both Chardonnay and Cabernet Sauvignon juices. Both strains, however, produced high concentrations of acetaldehyde and acetoin, which negatively affect wine flavor. Further modifications of these strains allowed reduction of these metabolites.

There are a variety of methods available for determining ethanol tolerance phenotypes in Saccharomyces cerevisiae. Many of these methods are limited in through-put and/or application. We describe here a microtiter-plate, growth-based, liquid-culture, rapid ethanol tolerance assay (RETA) that overcomes these limitations. Determinations of ethanol tolerance gave results that were comparable to shake-flask cultures, and there was a high level of intra- and inter-plate reproducibility. We report the successful application of this assay to determining the segregation pattern of ethanol tolerance in backcrosses of two ethanol-tolerant mutants (SM and CM) to a non-tolerant parent (W303-1A); RETA was used to determine ethanol tolerance in numerous progeny resulting from these crosses. This assay, or variations thereof, will prove be of great value for anyone attempting to develop quantitative, high-throughput growth assays for yeast or other microorganisms.

Chardonnay, being the predominant white wine-grape cultivar in the Australian wine sector, is subject to widely varying winemaking processes with the aim of producing a variety of wine styles. Therefore, juice composition might not always be ideal for optimal fermentation outcomes. Our aim was to better understand the composition of Chardonnay juice and how compositional parameters impact on fermentation outcomes. This was achieved through a survey of 96 commercially prepared Chardonnay juices during the 2009 vintage. Common juice variables were estimated using near infrared spectroscopy, and elemental composition was determined using radial view inductively coupled plasma optical emission spectrometry. The influence of elemental composition on fermentation outcomes was assessed by fermentation of a defined medium formulated to reflect the composition and range of concentrations as determined by the juice survey. Yeast (Saccharomyces cerevisiae) strain effects were also assessed. Key parameters influencing fermentation outcomes were verified by laboratory scale fermentation of Chardonnay juice. This exploration of Chardonnay juice identified interactions between juice pH and potassium concentration as key factors impacting on fermentation performance and wine quality. Outcomes differed depending on yeast strain.

Saccharomyces cerevisiae is traditionally used for alcoholic beverage and bioethanol production; however, its performance during fermentation is compromised by the impact of ethanol accumulation on cell vitality. This article reviews studies into the molecular basis of the ethanol stress response and ethanol tolerance of S. cerevisiae; such knowledge can facilitate the development of genetic engineering strategies for improving cell performance during ethanol stress. Previous studies have used a variety of strains and conditions, which is problematic, because the impact of ethanol stress on gene expression is influenced by the environment. There is however some commonality in Gene Ontology categories affected by ethanol assault that suggests that the ethanol stress response of S. cerevisiae is compromised by constraints on energy production, leading to increased expression of genes associated with glycolysis and mitochondrial function, and decreased gene expression in energy-demanding growth-related processes. Studies using genome-wide screens suggest that the maintenance of vacuole function is important for ethanol tolerance, possibly because of the roles of this organelle in protein turnover and maintaining ion homoeostasis. Accumulation of Asr1 and Rat8 in the nucleus specifically during ethanol stress suggests S. cerevisiae has a specific response to ethanol stress although this supposition remains controversial.

There is increasing demand by the wine industry for technologies to reduce the alcohol content of wine without compromising product quality. This review describes biological-based approaches that influence ethanol yield during fermentation, most of which have attempted to partially redirect yeast metabolism away from ethanol production. Genetic engineering approaches have been used to develop yeast strains that produce less ethanol, and this has provided insight into how yeast respond to metabolic rerouting. However, public attitudes towards the use of GMOs (Genetically Modified Organisms) in food products suggests that novel yeast strains will have to be generated using non-GM approaches, such as evolutionary engineering.

Many bacteria display substantial intra-specific genomic diversity that produces significant phenotypic variation between strains of the same species. Understanding the genetic basis of these strain-specific phenotypes is especially important for industrial microorganisms where these characters match individual strains to specific industrial processes. Oenococcus oeni, a bacterium used during winemaking, is one such industrial species where large numbers of strains show significant differences in commercially important industrial phenotypes. To ascertain the basis of these phenotypic differences, the genomic content of ten wine strains of O. oeni were mapped by array-based comparative genome hybridization (aCGH). These strains comprised a genomically diverse group in which large sections of the reference genome were often absent from individual strains. To place the aCGH results in context, whole genome sequence was obtained for one of these strains and compared with two previously sequenced, unrelated strains. While the three strains shared a core group of conserved ORFs, up to 10% of the coding potential of any one strain was specific to that isolate. The genome of O. oeni is therefore likely to be much larger than that present in any single strain and it is these strain-specific regions that are likely to be responsible for differences in industrial phenotypes.

Trehalose is known to protect cells from various environmental assaults; however, its role in the ethanol tolerance of Saccharomyces cerevisiae remains controversial. Many previous studies report correlations between trehalose levels and ethanol tolerance across a variety of strains, yet variations in genetic background make it difficult to separate the impact of trehalose from other stress response factors. In the current study, investigations were conducted on the ethanol tolerance of S. cerevisiae BY4742 and BY4742 deletion strains, tsl1Delta and nth1Delta, across a range of ethanol concentrations. It was found that trehalose does play a role in ethanol tolerance at lethal ethanol concentrations, but not at sublethal ethanol concentrations; differences of 20-40% in the intracellular trehalose concentration did not provide any growth advantage for cells incubated in the presence of sublethal ethanol concentrations. It was speculated that the ethanol concentration-dependent nature of the trehalose effect supports a mechanism for trehalose in protecting cellular proteins from the damaging effects of ethanol.

Many industrial strains of Saccharomyces cerevisiae have been selected primarily for their ability to convert sugars into ethanol efficiently despite exposure to a variety of stresses. To begin investigation of the genetic basis of phenotypic variation in industrial strains of S. cerevisiae, we have sequenced the genome of a wine yeast, AWRI1631, and have compared this sequence with both the laboratory strain S288c and the human pathogenic isolate YJM789. AWRI1631 was found to be substantially different from S288c and YJM789, especially at the level of single-nucleotide polymorphisms, which were present, on average, every 150 bp between all three strains. In addition, there were major differences in the arrangement and number of Ty elements between the strains, as well as several regions of DNA that were specific to AWRI1631 and that were predicted to encode proteins that are unique to this industrial strain.

Yeast research represents an important nexus between fundamental and applied research. Just as fundamental yeast research transitioned from classical, reductionist strategies to whole-genome techniques, whole-genome studies are advancing to the next level of biological research, referred to as systems biology. Industries that rely on high-performing yeast, such as the wine industry, are therefore poised to reap the many benefits that systems biology can provide. This includes the promise of strain development at speeds and costs which are unobtainable using current techniques. This article reviews the current state of whole-genome techniques available to yeast researchers and outlines how these processes can be used to obtain 'systems-level' information to provide insights into winemaking.

To remain competitive in increasingly overcrowded markets, yeast strain development programmes are crucial for fermentation-based food and beverage industries. In a winemaking context, there are many yeast phenotypes that stand to be improved. For example, winemakers endeavouring to produce sweet dessert wines wrestle with fermentation challenges particular to fermenting high-sugar juices, which can lead to elevated volatile acidity levels and extended fermentation times. In the current study, we used natural yeast breeding techniques to generate Saccharomyces spp. interspecific hybrids as a non-genetically modified (GM) strategy to introduce targeted improvements in important, wine-relevant traits. The hybrids were generated by mating a robust wine strain of Saccharomyces cerevisiae with a wine isolate of Saccharomyces bayanus, a species previously reported to produce wines with low concentrations of acetic acid. Two hybrids generated from the cross showed robust fermentation properties in high-sugar grape juice and produced botrytised Riesling wines with much lower concentrations of acetic acid relative to the industrial wine yeast parent. The hybrids also displayed suitability for icewine production when bench-marked against an industry standard icewine yeast, by delivering icewines with lower levels of acetic acid. Additionally, the hybrid yeast produced wines with novel aroma and flavour profiles and established that choice of yeast strain impacts on wine colour. These new hybrid yeasts display the desired targeted fermentation phenotypes from both parents, robust fermentation in high-sugar juice and the production of wines with low volatile acidity, thus establishing their suitability for wine styles that are traditionally troubled by excessive volatile acidity levels.

Saccharomyces cerevisiae yeast is integral to crafting well-balanced, flavoursome wines from simple, sugar-rich grape juice. Any improvements to wine yeast fermentation performance or the sensory properties it imparts to wine will therefore benefit winemakers and consumers. Frontier technologies, particularly the burgeoning “omics”-based fields of systems and synthetic biology, should therefore be of interest to the producer of high-quality wine. This chapter discusses the nexus between yeast research and winemaking. It addresses how winemakers and scientists face up to the challenges of consumer perceptions and opinions regarding the intervention of science and technology, and it raises important questions about the direction of yeast research, its contribution to science, and the future of winemaking.

Drinking wine is a manifestly sensuous experience. Wine stimulates most of our senses; particularly smell (olfaction) and taste, and, to a lesser extent, sight and touch. These sensory inputs interact with the limbic system in our brain which is associated with emotions and memory. Agreeable smells can thus evoke feelings of enjoyment and nostalgia. But how are tastants and aromas detected? Over the past few years a great deal has been learnt about how small tastant and odorant molecules are detected by specific protein receptors located in our mouth and in nasal cavities, respectively. Indeed, we have a vast array of olfactory receptors encoded by a group of genes that represent a significant part of the human genome. Interestingly, many more aroma compounds can be detected and discriminated than can be accounted for by the number of olfactory receptors that are encoded by our genes. Such disparity implies a level of complexity in this system which is not yet fully understood. Sophistication in olfaction is something that winemakers (and drinkers!) have long appreciated, and now scientists are beginning to unravel some of the underlying mysteries. Discovering why some individuals are more receptive to different tastes and smells than others will help wine producers understand variation in consumer preferences between different parts of the world, and possibly capture new opportunities in a changing global marketplace. Given such prospects, this present review offers a timely summary of some key developments in our understanding of odorant and tastant detection. We also consider how genetic components in human olfaction might be utilised to develop a ‘biosensor’ for aroma detection and discrimination.

The yeast Dekkera bruxellensis is a major contaminant of industrial fermentations, such as those used for the production of biofuel and wine, where it outlasts and, under some conditions, outcompetes the major industrial yeast Saccharomyces cerevisiae. In order to investigate the level of inter-strain variation that is present within this economically important species, the genomes of four diverse D. bruxellensis isolates were compared. While each of the four strains was shown to contain a core diploid genome, which is clearly sufficient for survival, two of the four isolates have a third haploid complement of chromosomes. The sequences of these additional haploid genomes were both highly divergent from those comprising the diploid core and divergent between the two triploid strains. Similar to examples in the Saccharomyces spp. clade, where some allotriploids have arisen on the basis of enhanced ability to survive a range of environmental conditions, it is likely these strains are products of two independent hybridisation events that may have involved multiple species or distinct sub-species of Dekkera. Interestingly these triploid strains represent the vast majority (92%) of isolates from across the Australian wine industry, suggesting that the additional set of chromosomes may confer a selective advantage in winery environments that has resulted in these hybrid strains all-but replacing their diploid counterparts in Australian winery settings. In addition to the apparent inter-specific hybridisation events, chromosomal aberrations such as strain-specific insertions and deletions and loss-of-heterozygosity by gene conversion were also commonplace. While these events are likely to have affected many phenotypes across these strains, we have been able to link a specific deletion to the inability to utilise nitrate by some strains of D. bruxellensis, a phenotype that may have direct impacts in the ability for these strains to compete with S. cerevisiae., Author Summary The yeast D. bruxellensis is of great importance in biofuel and fermented beverage industries, largely as a contaminant and/or spoilage organism. Its lifestyle is not unlike that of the wine/brewing/baking yeast S. cerevisiae, with independent evolutionary pathways having led to this convergence; these species are phylogenetically very distant. Unlike S. cerevisiae, D. bruxellensis is highly intractable in the laboratory; it is difficult to mate and to transform, making even the most basic genetic analysis very difficult. Thus we still have a great deal to learn about this economically important yeast. The latest gene sequencing technologies are, however, providing a means of addressing these limitations. The current manuscript describes a comparative genomics approach to providing insights into inter-strain variations that shape the genomic landscape of D. bruxellensis. Like other industrial yeasts, it has a diploid core genome, but there are also triploid isolates which possess the core diploid complement with an additional, more distantly related, full set of chromosomes. Evidence presented in this paper suggests that this form of triploidy has arisen more than once in the evolutionary history of D. bruxellensis, and it confers a selective advantage for strains of this yeast isolated from wineries.

Poly(ADP-ribose) polymerase-1 (PARP-1) is a nuclear enzyme involved in a range of activities associated with DNA metabolism and plays a key role in maintaining the integrity of DNA and chromatin structure. As such, this enzyme is likely to provide a useful target when using a rational drug design approach to develop pharmaceutical reagents, including cancer therapeutics. However, there is still a great deal to learn about the mode of action of PARP-1 and therefore efforts are being directed at gaining a better understanding of the relationship between its structure and function. To this end we have developed a rapid and relatively simple approach to producing and purifying PARP-1. Unlike traditional PARP-1 purification protocols, the method described here requires only one chromatography step thus minimizing losses of the enzyme and also avoids the use of a competitive inhibitor-based affinity chromatography step, which is common to several other protocols in the literature. The product of the method described here is high-quality native PARP-1 with a high specific activity and K(m) and V(max) values similar to what is reported by other workers in the field. This protocol is particularly well suited to making PARP-1 in a quantity and of a quality suitable for structure-function studies.

Plasmid DNA (pCSL17) isolated from Corynebacterium glutamicum transformed recipient McrBC+ strains of Escherichia coli with lower efficiency than McrBC- strains, confirming a previous report by Tauch et al. (FEMS Microbiol. Lett. 123 (1994) 343-348) which inferred that C. glutamicum DNA contains methylcytidine. Analysis of nucleotides in C. glutamicum-derived chromosomal and plasmid DNA failed to detect significant levels of methylated adenosine, but methylated cytidine was readily detected. Restriction enzymes which are inhibited by the presence of methylcytidine in their recognition sequence failed to cut pCSL17 from C. glutamicum, whereas enzymes which require methylation at adenosine in GATC sequences failed to cut. Failure of HaeIII to cut two specific sites of C. glutamicum-derived pCSL17 identified the first cytidine in the sequence GGCCGC as one target of methylation in this species, which contains the methyltransferase recognition sequence. Although Brevibacterium lactofermentum-derived DNA showed a similar methylation pattern by HPLC analysis, HaeIII cleaved these GGCCGC sites, suggesting differences in the specificity of methylation between these two species. Results for all analyses of B. flavum DNA were identical to those for C. glutamicum.

Despite its industrial importance, the yeast species Dekkera (Brettanomyces) bruxellensis has remained poorly understood at the genetic level. In this study we describe whole genome sequencing and analysis for a prevalent wine spoilage strain, AWRI1499. The 12.7 Mb assembly, consisting of 324 contigs in 99 scaffolds (super-contigs) at 26-fold coverage, exhibits a relatively high density of single nucleotide polymorphisms (SNPs). Haplotype sampling for 1.2% of open reading frames suggested that the D. bruxellensis AWRI1499 genome is comprised of a moderately heterozygous diploid genome, in combination with a divergent haploid genome. Gene content analysis revealed enrichment in membrane proteins, particularly transporters, along with oxidoreductase enzymes. Availability of this assembly and annotation provides a resource for further investigation of genomic organization in this species, and functional characterization of genes that may confer important phenotypic traits. Refereed/Peer-reviewed

The past three decades have seen a global wine glut. So far, well-intended but wasteful and expensive market-intervention has failed to drag the wine industry out of a chronic annual oversupply of roughly 15%. Can yeast research succeed where these approaches have failed by providing a means of improving wine quality, thereby making wine more appealing to consumers? To molecular biologists Saccharomyces cerevisiae is as intriguing as it is tractable. A simple unicellular eukaryote, it is an ideal model organism, enabling scientists to shed new light on some of the biggest scientific challenges such as the biology of cancer and aging. It is amenable to almost any modification that modern biology can throw at a cell, making it an ideal host for genetic manipulation, whether by the application of traditional or modern genetic techniques. To the winemaker, this yeast is integral to crafting wonderful, complex wines from simple, sugar-rich grape juice. Thus any improvements that we can make to wine, yeast fermentation performance or the sensory properties it imparts to wine will benefit winemakers and consumers. With this in mind, the application of frontier technologies, particularly the burgeoning fields of systems and synthetic biology, have much to offer in their pursuit of "novel" yeast strains to produce high quality wine. This paper discusses the nexus between yeast research and winemaking. It also addresses how winemakers and scientists face up to the challenges of consumer perceptions and opinions regarding the intervention of science and technology; the greater this intervention, the stronger the criticism that wine is no longer “natural.” How can wine researchers respond to the growing number of wine commentators and consumers who feel that scientific endeavors favor wine quantity over quality and “technical sophistication, fermentation reliability and product consistency” over “artisanal variation”? This paper seeks to present yeast research in a new light and a new context, and it raises important questions about the direction of yeast research, its contribution to science and the future of winemaking. Refereed/Peer-reviewed

The vast majority of wine fermentations are performed principally by Saccharomyces cerevisiae. However, there are a growing number of instances in which other species of Saccharomyces play a predominant role. Interestingly, the presence of these other yeast species generally occurs via the formation of interspecific hybrids that contain genomic contributions from both S. cerevisiae and non-S. cerevisiae species. However, despite the large number of wine strains that are characterized at the genomic level, there remains limited information regarding the detailed genomic structure of hybrids used in winemaking. To address this, we describe the genome sequence of the thiol-releasing commercial wine yeast hybrid VIN7. VIN7 is shown to be an almost complete allotriploid interspecific hybrid that is comprised of a heterozygous diploid complement of S. cerevisiae chromosomes and a haploid Saccharomyces kudriavzevii genomic contribution. Both parental strains appear to be of European origin, with the S. cerevisiae parent being closely related to, but distinct from, the commercial wine yeasts QA23 and EC1118. In addition, several instances of chromosomal rearrangement between S. cerevisiae and S. kudriavzevii sequences were observed that may mark the early stages of hybrid genome consolidation. Refereed/Peer-reviewed

The development of new wine yeast strains with improved characteristics is critical in the highly competitive wine market, which faces the demand of ever-changing consumer preferences. Although new strains can be constructed using recombinant DNA technologies, consumer concerns about genetically modified (GM) organisms strongly limit their use in food and beverage production. We have applied a non-GM approach, adaptive evolution with sulfite at alkaline pH as a selective agent, to create a stable yeast strain with enhanced glycerol production; a desirable characteristic for wine palate. A mutant isolated using this approach produced 41% more glycerol than the parental strain it was derived from, and had enhanced sulfite tolerance. Backcrossing to produce heterozygous diploids revealed that the high-glycerol phenotype is recessive, while tolerance to sulfite was partially dominant, and these traits, at least in part, segregated from each other. This work demonstrates the potential of adaptive evolution for development of novel non-GM yeast strains, and highlights the complexity of adaptive responses to sulfite selection.

Human intervention has subjected the yeast Saccharomyces cerevisiae to multiple rounds of independent domestication and thousands of generations of artificial selection. As a result, this species comprises a genetically diverse collection of natural isolates as well as domesticated strains that are used in specific industrial applications. However the scope of genetic diversity that was captured during the domesticated evolution of the industrial representatives of this important organism remains to be determined. To begin to address this, we have produced whole-genome assemblies of six commercial strains of S. cerevisiae (four wine and two brewing strains). These represent the first genome assemblies produced from S. cerevisiae strains in their industrially-used forms and the first high-quality assemblies for S. cerevisiae strains used in brewing. By comparing these sequences to six existing high-coverage S. cerevisiae genome assemblies, clear signatures were found that defined each industrial class of yeast. This genetic variation was comprised of both single nucleotide polymorphisms and large-scale insertions and deletions, with the latter often being associated with ORF heterogeneity between strains. This included the discovery of more than twenty probable genes that had not been identified previously in the S. cerevisiae genome. Comparison of this large number of S. cerevisiae strains also enabled the characterization of a cluster of five ORFs that have integrated into the genomes of the wine and bioethanol strains on multiple occasions and at diverse genomic locations via what appears to involve the resolution of a circular DNA intermediate. This work suggests that, despite the scrutiny that has been directed at the yeast genome, there remains a significant reservoir of ORFs and novel modes of genetic transmission that may have significant phenotypic impact in this important model and industrial species., Author Summary The yeast S. cerevisiae has been associated with human activity for thousands of years in industries such as baking, brewing, and winemaking. During this time, humans have effectively domesticated this microorganism, with different industries selecting for specific desirable phenotypic traits. This has resulted in the species S. cerevisiae comprising a genetically diverse collection of individual strains that are often suited to very specific roles (e.g. wine strains produce wine but not beer and vice versa). In order to understand the genetic differences that underpin these diverse industrial characteristics, we have sequenced the genomes of six industrial strains of S. cerevisiae that comprise four strains used in commercial wine production and two strains used in beer brewing. By comparing these genome sequences to existing S. cerevisiae genome sequences from laboratory, pathogenic, bioethanol, and “natural” isolates, we were able to identify numerous genetic differences among these strains including the presence of novel open reading frames and genomic rearrangements, which may provide the basis for the phenotypic differences observed among these strains.

Increasingly, winemakers are looking for ways to introduce aroma and flavour diversity to their wines as a means of improving style and increasing product differentiation. While currently available commercial yeast strains produce consistently sound fermentations, there are indications that sensory complexity and improved palate structure are obtained when other species of yeast are active during fermentation. In this study, we explore a strategy to increase the impact of non-Saccharomyces cerevisiae inputs without the risks associated with spontaneous fermentations, through generating interspecific hybrids between a S. cerevisiae wine strain and a second species. For our experiments, we used rare mating to produce hybrids between S. cerevisiae and other closely related yeast of the Saccharomyces sensu stricto complex. These hybrid yeast strains display desirable properties of both parents and produce wines with concentrations of aromatic fermentation products that are different to what is found in wine made using the commercial wine yeast parent. Our results demonstrate, for the first time, that the introduction of genetic material from a non-S. cerevisiae parent into a wine yeast background can impact favourably on the wine flavour and aroma profile of a commercial S. cerevisiae wine yeast. Refereed/Peer-reviewed

High-throughput methodologies to screen large numbers of microorganisms necessitate the use of small-scale culture vessels. In this context, an increasing number of researchers are turning to microtiter plate (MTP) formats to conduct experiments. MTPs are now widely used as a culturing vessel for phenotypic screening of aerobic laboratory cultures, and their suitability has been assessed for a range of applications. The work presented here extends these previous studies by assessing the metabolic footprint of MTP fermentation. A comparison of Chardonnay grape juice fermentation in MTPs with fermentations performed in air-locked (self-induced anaerobic) and cotton-plugged (aerobic) flasks was made. Maximum growth rates and biomass accumulation of yeast cultures grown in MTPs were indistinguishable from self-induced anaerobic flask cultures. Metabolic profiles measured differed depending on the metabolite. While glycerol and acetate accumulation mirrored that of self-induced anaerobic cultures, ethanol accumulation in MTP ferments was limited by the increased propensity of this volatile metabolite for evaporation in microlitre-scale culture format. The data illustrates that microplate cultures can be used as a replacement for self-induced anaerobic flasks in some instances and provide a useful and economical platform for the screening of industrial strains and culture media.

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.

Saccharomyces spp. are widely used for ethanol production; however, fermentation productivity is negatively affected by the impact of ethanol accumulation on yeast metabolic rate and viability. This study used microarray and statistical two-way ANOVA analysis to compare and evaluate gene expression profiles of two previously generated ethanol-tolerant mutants, CM1 and SM1, with their parent, Saccharomyces cerevisiae W303-1A, in the presence and absence of ethanol stress. Although sharing the same parentage, the mutants were created differently: SM1 by adaptive evolution involving long-term exposure to ethanol stress and CM1 using chemical mutagenesis followed by adaptive evolution-based screening. Compared to the parent, differences in the expression levels of genes associated with a number of gene ontology categories in the mutants suggest that their improved ethanol stress response is a consequence of increased mitochondrial and NADH oxidation activities, stimulating glycolysis and other energy-yielding pathways. This leads to increased activity of energy-demanding processes associated with the production of proteins and plasma membrane components, which are necessary for acclimation to ethanol stress. It is suggested that a key function of the ethanol stress response is restoration of the NAD(+)/NADH redox balance, which increases glyceraldehyde-3-phosphate dehydrogenase activity, and higher glycolytic flux in the ethanol-stressed cell. Both mutants achieved this by a constitutive increase in carbon flux in the glycerol pathway as a means of increasing NADH oxidation.

In the second article of the ‘Food and Science' series, Paul Chambers and Isak Pretorius explain the central role of yeast in wine making and how biotechnology can contribute to improving the quality of wine.

Saccharomyces spp. are widely used for ethanologenic fermentations, however yeast metabolic rate and viability decrease as ethanol accumulates during fermentation, compromising ethanol yield. Improving ethanol tolerance in yeast should, therefore, reduce the impact of ethanol toxicity on fermentation performance. The purpose of the current work was to generate and characterise ethanol-tolerant yeast mutants by subjecting mutagenised and non-mutagenised populations of Saccharomyces cerevisiae W303-1A to adaptive evolution using ethanol stress as a selection pressure. Mutants CM1 (chemically mutagenised) and SM1 (spontaneous) had increased acclimation and growth rates when cultivated in sub-lethal ethanol concentrations, and their survivability in lethal ethanol concentrations was considerably improved compared with the parent strain. The mutants utilised glucose at a higher rate than the parent in the presence of ethanol and an initial glucose concentration of 20 g l(-1). At a glucose concentration of 100 g l(-1), SM1 had the highest glucose utilisation rate in the presence or absence of ethanol. The mutants produced substantially more glycerol than the parent and, although acetate was only detectable in ethanol-stressed cultures, both mutants produced more acetate than the parent. It is suggested that the increased ethanol tolerance of the mutants is due to their elevated glycerol production rates and the potential of this to increase the ratio of oxidised and reduced forms of nicotinamide adenine dinucleotide (NAD(+)/NADH) in an ethanol-compromised cell, stimulating glycolytic activity.

Over a period of several millennia, Saccharomyces cerevisiae yeasts have been selected for their fermentative capacities, robustness and the sensorial attributes that they impart to wine. This has led to the generation of large collections of S. cerevisiae strains that can be inoculated into grape juice to deliver reliable fermentation performance as well as a diversity of predetermined flavors and wine styles. In addition, S. cerevisiae is one of the most intensively studied eukaryotes, providing a platform for cutting-edge research into fundamental biological systems. By combining these two discrete areas, fundamental knowledge can be leveraged by industry to improve both production efficiencies and wine quality, while fundamental research can benefit from the focused research directions and financial support of industry.

Although a range of methods are available for determining protein concentration, many scientists encounter problems when quantifying proteins in the laboratory. The most commonly used methods for determining protein concentration in a modern biochemistry laboratory would probably be the Lowry and/or the Bradford protein assays. Other techniques, including direct spectrophotometric analysis and densitometry of stained protein gels, are applied, but perhaps to a lesser extent. However, the reliability of all of the above techniques is questionable and dependent to some extent on the protein to be assayed. In this paper we describe problems we encountered when using some of the foregoing techniques to quantify the concentration of poly(adenosine diphosphate-ribose) polymerase-1 (PARP-1), a nuclear enzyme found in most eukaryotes. We also describe how, by using a fluorescence-based assay and amino acid analysis, we overcame the problems we encountered.

Microbial species respond to changes in their environmental conditions by producing a range of new proteins which assist their survival and adaptation to new conditions. Normally, sudden changes in temperature (heat shock), exposure to potentially toxic substances (ethanol, hydrogen peroxide, heavy metal ions) or the onset of starvation cause the production of stress-related proteins. The most thoroughly studied stress proteins include the heat shock proteins (HSP), where the mostly highly conserved responses across genera and shock conditions is the production of the HSP60 (GroEL) and HSP70 (DnaK) group of proteins [1]. The major HSPs synthesised by Escherichia coli (HtpG, GroEL and DnaK) were designated as molecular chaperones because of their role in protein folding for both newly synthesised proteins and proteins partially denatured by stressing conditions [1], where elevated activity of these proteins is essential for cell recovery [2]. Although the heat-shock response of E. coli occurs even for mild, non-lethal temperature upshifts, the intensity of the response depends on the severity of the temperature shift: a 2-fold increase in GroEL and DnaK may occur with a shift from 30°C to 42°C, whereas a 10-fold increase is caused by a shift to 46°C [3]. Other environmental hazards also cause similar stress responses in cells, including cold shock [4], nutrient starvation [5] and exposure to pollutants such as heavy metal ions and organic compounds [6–8]. Blom et al. [6] reported that E. coli synthesises up to 39 new proteins after exposure to a range of different pollutants, where there was some commonality in the types of proteins produced in response to the different chemicals.

Interspecific hybrids are commonplace in agriculture and horticulture; bread wheat and grapefruit are but two examples. The benefits derived from interspecific hybridisation include the potential of generating advantageous transgressive phenotypes. This paper describes the generation of a new breed of wine yeast by interspecific hybridisation between a commercial Saccharomyces cerevisiae wine yeast strain and Saccharomyces mikatae, a species hitherto not associated with industrial fermentation environs. While commercially available wine yeast strains provide consistent and reliable fermentations, wines produced using single inocula are thought to lack the sensory complexity and rounded palate structure obtained from spontaneous fermentations. In contrast, interspecific yeast hybrids have the potential to deliver increased complexity to wine sensory properties and alternative wine styles through the formation of novel, and wider ranging, yeast volatile fermentation metabolite profiles, whilst maintaining the robustness of the wine yeast parent. Screening of newly generated hybrids from a cross between a S. cerevisiae wine yeast and S. mikatae (closely-related but ecologically distant members of the Saccharomyces sensu stricto clade), has identified progeny with robust fermentation properties and winemaking potential. Chemical analysis showed that, relative to the S. cerevisiae wine yeast parent, hybrids produced wines with different concentrations of volatile metabolites that are known to contribute to wine flavour and aroma, including flavour compounds associated with non-Saccharomyces species. The new S. cerevisiae x S. mikatae hybrids have the potential to produce complex wines akin to products of spontaneous fermentation while giving winemakers the safeguard of an inoculated ferment.

Oenococcus kitaharae is only the second member of the genus Oenococcus to be identified and is the closest relative of the industrially important wine bacterium Oenococcus oeni. To provide insight into this new species, the genome of the type strain of O. kitaharae, DSM 17330, was sequenced. Comparison of the sequenced genomes of both species show that the genome of O. kitaharae DSM 17330 contains many genes with predicted functions in cellular defence (bacteriocins, antimicrobials, restriction-modification systems and a CRISPR locus) which are lacking in O. oeni. The two genomes also appear to differentially encode several metabolic pathways associated with amino acid biosynthesis and carbohydrate utilization and which have direct phenotypic consequences. This would indicate that the two species have evolved different survival techniques to suit their particular environmental niches. O. oeni has adapted to survive in the harsh, but predictable, environment of wine that provides very few competitive species. However O. kitaharae appears to have adapted to a growth environment in which biological competition provides a significant selective pressure by accumulating biological defence molecules, such as bacteriocins and restriction-modification systems, throughout its genome.

Background Oenococcus oeni, a member of the lactic acid bacteria, is one of a limited number of microorganisms that not only survive, but actively proliferate in wine. It is also unusual as, unlike the majority of bacteria present in wine, it is beneficial to wine quality rather than causing spoilage. These benefits are realised primarily through catalysing malolactic fermentation, but also through imparting other positive sensory properties. However, many of these industrially-important secondary attributes have been shown to be strain-dependent and their genetic basis it yet to be determined. Results In order to investigate the scale and scope of genetic variation in O. oeni, we have performed whole-genome sequencing on eleven strains of this bacterium, bringing the total number of strains for which genome sequences are available to fourteen. While any single strain of O. oeni was shown to contain around 1800 protein-coding genes, in-depth comparative annotation based on genomic synteny and protein orthology identified over 2800 orthologous open reading frames that comprise the pan genome of this species, and less than 1200 genes that make up the conserved genomic core present in all of the strains. The expansion of the pan genome relative to the coding potential of individual strains was shown to be due to the varied presence and location of multiple distinct bacteriophage sequences and also in various metabolic functions with potential impacts on the industrial performance of this species, including cell wall exopolysaccharide biosynthesis, sugar transport and utilisation and amino acid biosynthesis. Conclusions By providing a large cohort of sequenced strains, this study provides a broad insight into the genetic variation present within O. oeni. This data is vital to understanding and harnessing the phenotypic variation present in this economically-important species.

Technology sets limits on what can be achieved in research. The advent of genetic engineering accompanied by the development of monoclonal antibody technology in the 1970s heralded the birth of modern ?molecular biology?. This revolutionised the way we approach research in the biological sciences by allowing access to cellular structures and processes that were in the realm of science fiction a decade earlier. The invention of the PCR in the 1980s built on this, making cloning easier and a great deal more rapid; with PCR we no longer required a host and vector to amplify DNA and isolate targeted DNA sequences.

One of the key challenges for industrial yeast strain development is to obtain a thorough understanding of the biology of yeast and to apply this knowledge to develop novel strains with improved features. The detailed study of individual biological components and the use of metabolic engineering have benefited the development of industrial strains enormously; however, such approaches have failed to describe yeast behaviour in the detail required to reveal the complex interactions operating within such biological systems. How can we accurately describe the biological processes and the interactions that occur during fermentation or cell growth?

Human interferon alpha 1 (HuIFN alpha 1) is known to protect bovine as well as human cells against viral infection. Hence, we investigated the specificity and tissue distribution of receptors for HuIFN alpha 1 on various cells. [35S]HuIFN alpha 1 bound specifically to homogenates of bovine tissues and particularly to bovine liver, but there was also specific binding to spleen, kidney, brain, adrenal gland, lung, thymus, skeletal muscle, heart, mammary gland and testis. There was no difference in the degree of binding of HuIFN alpha to foetal or adult liver. Competitive binding experiments showed that bovine interferon alpha C (BoIFN alpha C) competed with HuIFN alpha 1 for binding to a bovine liver plasma membrane preparation, indicating that these two IFNs bind to the same receptor. An 35S-labelled IFN alpha 1-receptor complex was isolated from bovine liver extracts by SDS/polyacrylamide gel electrophoresis, and shown to have a molecular weight of 153 kDa. Isolation of the bovine IFN alpha receptor would be a feasible approach to the characterization of the HuIFN alpha receptor.

It may come as a surprise to learn that there are over 200 commercial strains of Saccharomyces cerevisiae available for winemakers to work their magic on grape juice. Why so many? Surely one or two reliable workhorse strains should suffice; after all, don?t they just make ethanol from sugar? The answer to this is an emphatic no; the more we look at the role(s) of yeast in winemaking, the more we are learning about their influences on appearance, aroma, flavour, mouthfeel and final ethanol concentration. And different yeast are more or less robust and efficient in converting the hostile environment of grape juice into wine. Indeed, not all wine yeasts are equal.