SO2 and copper tolerance exhibit an evolutionary trade-off in Saccharomyces cerevisiae.

Copper tolerance and SO2 tolerance are two well-studied phenotypic traits of Saccharomyces cerevisiae. The genetic bases of these traits are the allelic expansion at the CUP1 locus and reciprocal translocation at the SSU1 locus, respectively. Previous work identified a negative association between SO2 and copper tolerance in S. cerevisiae wine yeasts. Here we probe the relationship between SO2 and copper tolerance and show that an increase in CUP1 copy number does not always impart copper tolerance in S. cerevisiae wine yeast. Bulk-segregant QTL analysis was used to identify variance at SSU1 as a causative factor in copper sensitivity, which was verified by reciprocal hemizygosity analysis in a strain carrying 20 copies of CUP1. Transcriptional and proteomic analysis demonstrated that SSU1 over-expression did not suppress CUP1 transcription or constrain protein production and provided evidence that SSU1 over-expression induced sulfur limitation during exposure to copper. Finally, an SSU1 over-expressing strain exhibited increased sensitivity to moderately elevated copper concentrations in sulfur-limited medium, demonstrating that SSU1 over-expression burdens the sulfate assimilation pathway. Over-expression of MET 3/14/16, genes upstream of H2S production in the sulfate assimilation pathway increased the production of SO2 and H2S but did not improve copper sensitivity in an SSU1 over-expressing background. We conclude that copper and SO2 tolerance are conditional traits in S. cerevisiae and provide evidence of the metabolic basis for their mutual exclusivity. These findings suggest an evolutionary driver for the extreme amplification of CUP1 observed in some yeasts.

Analysis of Transcriptomic Response to SO2 by Oenococcus oeni Growing in Continuous Culture.

To successfully complete malolactic fermentation (MLF), Oenococcus oeni must overcome wine stress conditions of low pH, high ethanol, and the presence of SO2. Failure to complete MLF may result in detrimental effects to the quality and stability of the resulting wines. Research efforts to date have focused on elucidating the mechanisms and genetic features that confer the ability to withstand low pH and high ethanol concentrations on O. oeni; however, the responses to SO2 stress are less well defined. This study focused on characterizing the transcriptional response of O. oeni to SO2 challenge during cultivation in a continuous system at wine-like pH (3.5). This experimental design allowed the precise discrimination of transcriptional changes linked to SO2 stress from responses associated with growth stage and cultivation parameters. Differential gene expression analysis revealed major transcriptional changes following SO2 exposure and suggested that this compound primarily interacts with intracellular proteins, DNA, and the cell envelope of O. oeni. The molecular chaperone hsp20, which has a demonstrated function in the heat, ethanol, and acid stress response, was highly upregulated, confirming its additional role in the response of this species to SO2 stress. This work also reports the first nanopore-based complete genome assemblies for O. oeni. IMPORTANCE Malolactic fermentation is an indispensable step in the elaboration of most wines and is generally performed by Oenococcus oeni, a Gram-positive heterofermentative lactic acid bacterium species. While O. oeni is tolerant to many of the wine stresses, including low pH and high ethanol concentrations, it has high sensitivity to SO2, an antiseptic and antioxidant compound regularly used in winemaking. Understanding the physiological changes induced in O. oeni by SO2 stress is essential for the development of more robust starter cultures and methods for their use. This study describes the main transcriptional changes induced by SO2 stress in the wine bacterium O. oeni and provides foundational understanding on how this compound interacts with the cellular components and the induced protective mechanisms of this species.

Aromatic Higher Alcohols in Wine: Implication on Aroma and Palate Attributes during Chardonnay Aging.

The higher alcohols 2-phenylethanol, tryptophol, and tyrosol are a group of yeast-derived compounds that have been shown to affect the aroma and flavour of fermented beverages. Five variants of the industrial wine strain AWRI796, previously isolated due to their elevated production of the 'rose-like aroma' compound 2-phenylethanol, were characterised during pilot-scale fermentation of a Chardonnay juice. We show that these variants not only increase the concentration of 2-phenylethanol but also modulate the formation of the higher alcohols tryptophol, tyrosol, and methionol, as well as other volatile sulfur compounds derived from methionine, highlighting the connections between yeast nitrogen and sulfur metabolism during fermentation. We also investigate the development of these compounds during wine storage, focusing on the sulfonation of tryptophol. Finally, the sensory properties of wines produced using these strains were quantified at two time points, unravelling differences produced by biologically modulating higher alcohols and the dynamic changes in wine flavour over aging.

Novel Methods to Manipulate Autolysis in Sparkling Wine: Effects on Yeast.

Sparkling wine made by the traditional method (Méthode Traditionelle) develops a distinct and desirable flavour and aroma profile attributed to proteolytic processes during prolonged ageing on lees. Microwave, ultrasound and addition of ß-glucanase enzymes were applied to accelerate the disruption of Saccharomyces cerevisiae, and added to the tirage solution for secondary fermentation in traditional sparkling winemaking. Scanning electron microscopy and flow cytometry analyses were used to observe and describe yeast whole-cell anatomy, and cell integrity and structure via propidium iodide (PI) permeability after 6-, 12- and 18-months post-tirage. Treatments applied produced features on lees that were distinct from that of the untreated control yeast. Whilst control yeast displayed budding cells (growth features) with smooth, cavitated and flat external cell appearances; microwave treated yeast cells exhibited modifications like 'doughnut' shapes immediately after treatment (time 0). Similar 'doughnut'-shaped and 'pitted/porous' cell features were observed on progressively older lees from the control. Flow cytometry was used to discriminate yeast populations; features consistent with cell disruption were observed in the microwave, ultrasound and enzyme treatments, as evidenced by up to 4-fold increase in PI signal in the microwave treatment. Forward and side scatter signals reflected changes in size and structure of yeast cells, in all treatments applied. When flow cytometry was interpreted alongside the scanning electron microscopy images, bimodal populations of yeast cells with low and high PI intensities were revealed and distinctive 'doughnut'-shaped cell features observed in association with the microwave treatment only at tirage, that were not observed until 12 months wine ageing in older lees from the control. This work offers both a rapid approach to visualise alterations to yeast cell surfaces and a better understanding of the mechanisms of yeast lysis. Microwave, ultrasound or ß-glucanase enzymes are tools that could potentially initiate the release of yeast cell compounds into wine. Further investigation into the impact of such treatments on the flavour and aroma profiles of the wines through sensory evaluation is warranted.

Comparative genome analysis proposes three new Aureobasidium species isolated from grape juice.

Aureobasidium pullulans is the most abundant and ubiquitous species within the genus and is also considered a core component of the grape juice microflora. So far, a small number of other Aureobasidium species have been reported, that in contrast to A. pullulans, appear far more constrained to specific habitats. It is unknown whether grape juice is a reservoir of novel Aureobasidium species, overlooked in the course of conventional morphological and meta-barcoding analyses. In this study, eight isolates from grape juice taxonomically classified as Aureobasidium through ITS sequencing were subjected to whole-genome phylogenetic, synteny and nucleotide identity analyses, which revealed three isolates to likely represent newly discovered Aureobasidium species. Analyses of ITS and metagenomic sequencing datasets show that these species can be present in grape juice samples from different locations and vintages. Functional annotation revealed the Aureobasidium isolates possess the genetic potential to support growth on the surface of plants and grapes. However, the loss of several genes associated with tolerance to diverse environmental stresses suggest a more constrained ecological range than A. pullulans.

Investigating the effects of Aureobasidium pullulans on grape juice composition and fermentation.

Aureobasidium pullulans has been observed as one of the most abundant species in freshly pressed grape juice. Despite this, little is known about the consequences for the wine-making process associated with the presence and proliferation of this fungus, including its interaction with other ferment-derived microorganisms and impact on the composition of the resulting wine. In this study, the physiology of abundant A. pullulans grape juice isolates was investigated through lab scale fermentation trials, demonstrating the ability of this species to survive in grape juice while producing polysaccharides, polymers of malic acid (poly ß-malic acid) and enzymes with pectinase, ß - glucosidase and tannase activity. A possible antagonistic effect against yeast through competition for metals including Fe and Zn was also observed. Overall, the data suggests this abundant species could have important implications for wine production and quality.

Evaluation of Saccharomyces cerevisiae Wine Yeast Competitive Fitness in Enologically Relevant Environments by Barcode Sequencing.

When a wine yeast is inoculated into grape juice the potential variation in juice composition that confronts it is huge. Assessing the performance characteristics of the many commercially available wine yeasts in the many possible grape juice compositions is a daunting task. To this end we have developed a barcoded Saccharomyces cerevisiae wine yeast collection to facilitate the task of performance assessment that will contribute to a broader understanding of genotype-phenotype relations. Barcode sequencing of mixed populations is used to monitor strain abundance in different grape juices and grape juice-like environments. Choice of DNA extraction method is shown to affect strain-specific barcode count in this highly related set of S. cerevisiae strains; however, the analytical approach is shown to be robust toward strain dependent variation in DNA extraction efficiency. Of the 38 unique compositional variables assessed, resistance to copper and SO2 are found to be dominant discriminatory factors in wine yeast performance. Finally, a comparison of competitive fitness profile with performance in single inoculum fermentations reveal strain dependent correspondence of yeast performance using these two different approaches.

Purge Haplotigs: allelic contig reassignment for third-gen diploid genome assemblies.

BACKGROUND: Recent developments in third-gen long read sequencing and diploid-aware assemblers have resulted in the rapid release of numerous reference-quality assemblies for diploid genomes. However, assembly of highly heterozygous genomes is still problematic when regional heterogeneity is so high that haplotype homology is not recognised during assembly. This results in regional duplication rather than consolidation into allelic variants and can cause issues with downstream analysis, for example variant discovery, or haplotype reconstruction using the diploid assembly with unpaired allelic contigs. RESULTS: A new pipeline-Purge Haplotigs-was developed specifically for third-gen sequencing-based assemblies to automate the reassignment of allelic contigs, and to assist in the manual curation of genome assemblies. The pipeline uses a draft haplotype-fused assembly or a diploid assembly, read alignments, and repeat annotations to identify allelic variants in the primary assembly. The pipeline was tested on a simulated dataset and on four recent diploid (phased) de novo assemblies from third-generation long-read sequencing, and compared with a similar tool. After processing with Purge Haplotigs, haploid assemblies were less duplicated with minimal impact on genome completeness, and diploid assemblies had more pairings of allelic contigs. CONCLUSIONS: Purge Haplotigs improves the haploid and diploid representations of third-gen sequencing based genome assemblies by identifying and reassigning allelic contigs. The implementation is fast and scales well with large genomes, and it is less likely to over-purge repetitive or paralogous elements compared to alignment-only based methods. The software is available at https://bitbucket.org/mroachawri/purge_haplotigs under a permissive MIT licence.

Population sequencing reveals clonal diversity and ancestral inbreeding in the grapevine cultivar Chardonnay.

Chardonnay is the basis of some of the world's most iconic wines and its success is underpinned by a historic program of clonal selection. There are numerous clones of Chardonnay available that exhibit differences in key viticultural and oenological traits that have arisen from the accumulation of somatic mutations during centuries of asexual propagation. However, the genetic variation that underlies these differences remains largely unknown. To address this knowledge gap, a high-quality, diploid-phased Chardonnay genome assembly was produced from single-molecule real time sequencing, and combined with re-sequencing data from 15 different Chardonnay clones. There were 1620 markers identified that distinguish the 15 clones. These markers were reliably used for clonal identification of independently sourced genomic material, as well as in identifying a potential genetic basis for some clonal phenotypic differences. The predicted parentage of the Chardonnay haplomes was elucidated by mapping sequence data from the predicted parents of Chardonnay (Gouais blanc and Pinot noir) against the Chardonnay reference genome. This enabled the detection of instances of heterosis, with differentially-expanded gene families being inherited from the parents of Chardonnay. Most surprisingly however, the patterns of nucleotide variation present in the Chardonnay genome indicate that Pinot noir and Gouais blanc share an extremely high degree of kinship that has resulted in the Chardonnay genome displaying characteristics that are indicative of inbreeding.

Systems-based approaches enable identification of gene targets which improve the flavour profile of low-ethanol wine yeast strains.

Metabolic engineering has been vital to the development of industrial microbes such as the yeast Saccharomyces cerevisiae. However, sequential rounds of modification are often needed to achieve particular industrial design targets. Systems biology approaches can aid in identifying genetic targets for modification through providing an integrated view of cellular physiology. Recently, research into the generation of commercial yeasts that can produce reduced-ethanol wines has resulted in metabolically-engineered strains of S. cerevisiae that are less efficient at producing ethanol from sugar. However, these modifications led to the concomitant production of off-flavour by-products. A combination of transcriptomics, proteomics and metabolomics was therefore used to investigate the physiological changes occurring in an engineered low-ethanol yeast strain during alcoholic fermentation. Integration of 'omics data identified several metabolic reactions, including those related to the pyruvate node and redox homeostasis, as being significantly affected by the low-ethanol engineering methodology, and highlighted acetaldehyde and 2,4,5-trimethyl-1,3-dioxolane as the main off-flavour compounds. Gene remediation strategies were then successfully applied to decrease the formation of these by-products, while maintaining the 'low-alcohol' phenotype. The data generated from this comprehensive systems-based study will inform wine yeast strain development programmes, which, in turn, could potentially play an important role in assisting winemakers in their endeavour to produce low-alcohol wines with desirable flavour profiles.

Whole Genome Comparison Reveals High Levels of Inbreeding and Strain Redundancy Across the Spectrum of Commercial Wine Strains of Saccharomyces cerevisiae.

Humans have been consuming wines for more than 7000 yr . For most of this time, fermentations were presumably performed by strains of Saccharomyces cerevisiae that naturally found their way into the fermenting must . In contrast, most commercial wines are now produced by inoculation with pure yeast monocultures, ensuring consistent, reliable and reproducible fermentations, and there are now hundreds of these yeast starter cultures commercially available. In order to thoroughly investigate the genetic diversity that has been captured by over 50 yr of commercial wine yeast development and domestication, whole genome sequencing has been performed on 212 strains of S. cerevisiae, including 119 commercial wine and brewing starter strains, and wine isolates from across seven decades. Comparative genomic analysis indicates that, despite their large numbers, commercial strains, and wine strains in general, are extremely similar genetically, possessing all of the hallmarks of a population bottle-neck, and high levels of inbreeding. In addition, many commercial strains from multiple suppliers are nearly genetically identical, suggesting that the limits of effective genetic variation within this genetically narrow group may be approaching saturation.

At the cutting-edge of grape and wine biotechnology.

Wine is arguably the oldest biotechnological endeavor, with humans having been involved in wine production for at least 7000 years. Despite the artisan nature of its production, work by pioneering scientists such as Antoine-Laurent de Lavoisier and Louis Pasteur placed wine research in a prominent position for the application of cutting-edge biological and chemical sciences, a position it still holds to this day. Technologies such as whole-genome sequencing and systems biology are now revolutionizing winemaking by combining the ability to engineer phenotypes rationally, with a precise understanding of the genetic makeup and key phenotypic drivers of the key organisms that contribute to this age-old industry.

Impacts of variations in elemental nutrient concentration of Chardonnay musts on Saccharomyces cerevisiae fermentation kinetics and wine composition.

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.

Microvinification--how small can we go?

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.

Hpf2 glycan structure is critical for protection against protein haze formation in white wine.

Grape-derived proteins can form haze in wine. Some cell-wall glycoproteins of Saccharomyces cerevisiae are capable of reducing protein haze formation. The basis of their haze protective activity is not yet understood. One of the S. cerevisiae cell-wall proteins, Hpf2, was produced in Pichia pastoris . An altered glycan structure in the P. pastoris -produced protein was associated with decreased solubility in water and reduced capacity to mitigate haze formation compared to native Hpf2 protein from S. cerevisiae. alpha-1,2-Linked mannose in the glycan chain was shown to be required for haze protective activity using a series of S. cerevisiae deletion mutants (mnn1-Delta, mnn2-Delta, mnn4-Delta, and mnn5-Delta), defective in different aspects of glycan processing. The effect of media additives phthalate, casamino acids, and yeast nitrogen base on Hpf2 production in P. pastoris were also evaluated. Casamino acids were shown to suppress Hpf2 production in P. pastoris .

Purification of the M flax-rust resistance protein expressed in Pichia pastoris.

The M flax-rust resistance (R) gene is predicted to encode a 150-kDa protein of the Toll-interleukin-like receptor-nucleotide binding site-leucine rich repeat (TIR-NBS-LRR) class of plant disease resistance proteins and provides resistance against the Melampsora lini (flax rust) fungus carrying the AvrM avirulence gene. The extremely low level of this class of R proteins found in plant tissue has precluded their biochemical and structural analysis, and the study of these proteins has been largely restricted to genetic analyses and in vivo investigations. Here we report the production and purification of the M protein in the methalotrophic yeast, Pichia pastoris. Expression trials with five different constructs reveals optimum levels of soluble native M protein can be obtained as an N-terminally 9x His-tagged protein, in which the first 21 amino acids of the predicted wild-type protein are deleted. Expression was achieved using a high cell density fed-batch bioreactor culture at low temperature. M protein was purified to near homogeneity from whole-cell lysates using cation exchange, immobilised metal ion affinity chromatography and gel filtration with a final yield of approximately 3 mg of protein/1000 g wet weight of yeast cells lysed. The successful expression and purification of soluble M protein opens the way for biochemical and structural analysis of this class of important plant proteins.