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Background and Aims. Shiraz (Syrah) is a dark-skinned cultivar of the wine grape Vitis vinifera that forms the basis of some of the world’s most iconic wines. Worldwide, Shiraz is the fourth most planted grapevine cultivar; however, it represents the most planted cultivar in Australia. Given the importance of Shiraz to worldwide wine production, this study aimed to produce a reference genome for this cultivar while investigating the unique genetic variants and ancestral origins of this iconic variety. Methods and Results. Long-read ONT data were selected to produce a highly contiguous genome assembly for Shiraz. Phylogenetic reconstruction using high-quality genome assemblies for wine grape cultivars provided further support of a kinship between Shiraz and Pinot Noir. Harnessing long-read data, transposable element insertions potentially affecting gene function were characterized in Shiraz and assessed relative to other cultivars. This revealed a heterogenous landscape of transposon insertion points across cultivars and uncovered a specific combination of allelic variants at the VviTPS24 terpene synthase locus. Conclusions. This establishment of a Shiraz genome provides a detailed view of the genetics that underpin this cultivar, including the discovery of a specific combination of VviTPS24 variants, which when combined with appropriate environmental triggers may allow Shiraz to produce high levels of rotundone, the aroma compound responsible for the distinctive peppery characteristics of this cultivar. Significance of the Study. The availability of a reference genome for Shiraz expands the pool of genomes available for wine grapes while providing a foundation resource for whole-genome studies involving this iconic cultivar, including intracultivar variant identification and transcriptomic studies using a matching reference genome.

Shiraz (Syrah) is a dark-skinned cultivar of the wine grapeVitis viniferathat forms the basis of some of the world’s most iconic wines. Worldwide, Shiraz is the fourth most planted grapevine cultivar, however it represents the most planted cultivar in Australia. Given the importance of Shiraz to worldwide wine production, a long-read based reference genome assembly for this cultivar was produced and used to investigate the unique genetic diversity and ancestral origins of this iconic variety. Phylogenetic reconstruction using high-quality genome assemblies for wine grape cultivars provided further support of a kinship between Shiraz and Pinot Noir. Harnessing long-read data, transposable element insertions potentially affecting gene function were characterized in Shiraz and assessed relative to other cultivars. This revealed a heterogenous landscape of transposon insertion points across cultivars and uncovered a specific combination of allelic variants at theTPS24terpene synthase locus, which when combined with appropriate environmental triggers, may allow Shiraz to produce high levels of rotundone, the aroma compound responsible for the distinctive peppery characteristics of this cultivar.

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.

The ability to genetically manipulate microorganisms has been essential for understanding their biology and metabolism. Targeted genome editing relies on highly efficient homologous recombination, and while this is readily observed in the yeast Saccharomyces cerevisiae, most non-conventional yeast species do not display this trait and remain recalcitrant to targeted editing methods. CRISPR-based editing can bypass the requirement for high levels of native homologous recombination, enabling targeted modification to be more broadly implemented. While genetic transformation has been reported previously in Brettanomyces bruxellensis, a yeast with broad biotechnological potential and responsible for significant economic losses during the production of fermented beverages, targeted editing approaches have not been reported. Here, we describe the use of an expression-free CRISPR-Cas9 system, in combination with gene transformation cassettes tailored for B. bruxellensis, to provide the means for targeted gene deletion in this species. Deletion efficiency was shown to be dependent on homologous flanking DNA length, with higher targeting efficiencies observed with cassettes containing longer flanking regions. In a diploid strain, it was not possible to delete multiple alleles in one step, with heterozygous deletants only obtained when using DNA cassettes with long flanking regions. However, stepwise transformations (using two different marker genes) were successfully used to delete both wild-type alleles. Thus, the approach reported here will be crucial to understand the complex physiology of B. bruxellensis. Key points • The use of CRISPR-Cas9 enables targeted gene deletion in Brettanomyces bruxellensis. • Homozygous diploid deletions are possible with step-wise transformations. • Deletion of SSU1 confirmed the role of this gene in sulphite tolerance.

Glycosides are sugar conjugates of aroma compounds that are found in many fruits and vegetables, and while glycosides are non-volatile, they can release flavor during eating, through enzyme hydrolysis from oral microbiota. Recently, a range of sensory phenotypes for glucoside perception have been observed, reflecting interindividual variation in response to precursors of floral and smoky flavors, geranyl glucoside and guaiacyl glucoside. To understand this variation and investigate the role of oral microbiota on in vitro hydrolysis of glucosides in saliva, metagenomic screening was conducted using individuals representing the range of sensory phenotypes for geranyl and guaiacyl glucosides. In parallel, sensory retronasal detection thresholds for geranyl glucoside, guaiacyl glucoside, and the volatile odorants geraniol and guaiacol were determined. Oral microbial communities correlated with hydrolysis of glucosides in saliva, but the relationship did not extend to sensory phenotypes. Overall, the retronasal detection threshold of the volatile odorants studied was the main factor determining sensory phenotype. Refereed/Peer-reviewed

Eutypa dieback of grapevine is an important disease caused by the generalist Ascomycete fungus Eutypa lata. Despite the relevance of this species to the global wine industry, its genomic diversity remains unknown, with only a single publicly available genome assembly. Whole-genome sequencing and comparative genomics was performed on forty Australian E. lata isolates to understand the genome evolution, adaptation, population size and structure of these isolates. Phylogenetic and linkage disequilibrium decay analyses provided evidence of extensive gene flow through sexual recombination between isolates obtained from different geographic locations and hosts. Investigation of the genetic diversity of these isolates suggested rapid population expansion, likely as a consequence of the recent growth of the Australian wine industry. Genomic regions affected by selective sweeps were shown to be enriched for genes associated with secondary metabolite clusters and included genes encoding proteins with a role in nutrient acquisition, degradation of host cell wall and metal and drug resistance, suggesting recent adaptation to both abiotic factors and potentially host genotypes. Genome synteny analysis using long-read genome assemblies showed significant intraspecific genomic plasticity with extensive chromosomal rearrangements impacting the secondary metabolite production potential of this species. Finally, k-mer based GWAS analysis identified a potential locus associated with mycelia recovery in canes of Vitis vinifera that will require further investigations.

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.

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.

The Synthetic Yeast Genome Project (Sc2.0) represents the first foray into eukaryotic genome engineering and a framework for designing and building the next generation of industrial microbes. However, the laboratory strain S288c used lacks many of the genes that provide phenotypic diversity to industrial and environmental isolates. To address this shortcoming, we have designed and constructed a neo-chromosome that contains many of these diverse pan-genomic elements and which is compatible with the Sc2.0 design and test framework. The presence of this neo-chromosome provides phenotypic plasticity to the Sc2.0 parent strain, including expanding the range of utilizable carbon sources. We also demonstrate that the induction of programmable structural variation (SCRaMbLE) provides genetic diversity on which further adaptive gains could be selected. The presence of this neo-chromosome within the Sc2.0 backbone may therefore provide the means to adapt synthetic strains to a wider variety of environments, a process which will be vital to transitioning Sc2.0 from the laboratory into industrial applications.

The use of non-Saccharomyces yeast species generally involves sequential or co-inoculation of a Saccharomyces cerevisiae strain to complete fermentation. While most studies have focused on characterising the impact that S. cerevisiae has on the growth and metabolic activity of these non-Saccharomyces species, microbial interactions work reciprocally. Antagonism or competition of non-Saccharomyces species against S. cerevisiae has been shown to impact subsequent fermentation performance. To date, it remains unclear whether these negative interactions are strain specific. Hence, characterisation of strain-specific responses to co-inoculation would enable the identification of specific S. cerevisiae strain/non-Saccharomyces combinations that minimise the negative impacts of sequential fermentation on fermentation performance. The competitive fitness response of 93 S. cerevisiae strains to several non-Saccharomyces species was simultaneously investigated using a barcoded library to address this knowledge gap. Strain-specific fitness differences were observed across non-Saccharomyces treatments. Results obtained from experiments using selected S. cerevisiae strains sequentially inoculated after Metschnikowia pulcherrima and Torulaspora delbrueckii were consistent with the competitive barcoded library observations. The results presented in this study indicate that strain selection will influence fermentation performance when using non-Saccharomyces species, therefore, appropriate strain/yeast combinations are required to optimise fermentation.

Brettanomyces bruxellensis is considered one of the most problematic microbes associated with wine production. Sulfur dioxide is commonly used to inhibit the growth of B. bruxellensis and limit the potential wine spoilage. Brettanomyces bruxellensis wine isolates can grow at higher concentrations of this preservative than isolates from other sources. Thus, it has been suggested that the use of sulfite may have selected for B. bruxellensis strains better adapted to survive in the winemaking environment. We utilized laboratory adaptive evolution to determine the potential for this to occur. Three B. bruxellensis strains, representative of known genetic variation within the species, were subjected to increasing sublethal sulfur dioxide concentrations. Individual clones isolated from evolved populations displayed enhanced sulfite tolerance, ranging from 1.6 to 2.5 times higher than the corresponding parental strains. Whole-genome sequencing of sulfite-tolerant clones derived from two of the parental strains revealed structural variations affecting 270 genes. The region containing the sulfite efflux pump encoding gene, SSU1, showed clear copy number variants in all sequenced clones. Regardless of parental strain genetic background, SSU1 copy number changes were reproducibly associated with one SSU1 haplotype. This work clearly demonstrates adaptive evolution of B. bruxellensis when exposed to sublethal sulfites and suggests that, similar to Saccharomyces cerevisiae wine yeast, the mechanism responsible involves the gene SSU1.

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.

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 β-malic acid) and enzymes with pectinase, β - 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.

An industrial wastewater treatment plant (WWTP) in Australia has long suffered from bulking problems associated with the proliferation of Thiothrix spp. The WWTP consists of a covered anaerobic lagoon (CAL) followed by a sequencing batch reactor (SBR). The CAL functions as both an anaerobic digester and surge lagoon for the irregular flow of wastewater generated from the production of seasonal products. Chemical analysis of the raw influent showed it was composed of a mixture of organic acids, phenols and alcohols. The CAL effluent was characterised by high acetic acid and phenolic concentrations. An attempt was made to manipulate the SBR microbial community to improve settling by direct feeding small volumes of raw influent into the SBR. After raw feeding, the plant ceased bulking as the settled sludge volume reduced from 930 to 200 mL L−1. 16S rRNA gene profiling and biovolumes of SBR samples revealed major changes in the microbial community. The Thiothrix spp. population decreased from 36.8% to 0.2%, and Zoogloea spp. dominated all samples after raw feeding. Therefore, direct feeding is proposed as a control method for industrial plants with surge/anaerobic lagoons in order to manage the bulking problems caused by Thiothrix spp. in downstream SBRs.

Torulaspora delbrueckii and Saccharomyces cerevisiae are yeast species found concurrently in wine. In order to commence fermentation, they adapt to the initial harsh environment, maintaining cellular homeostasis and promoting metabolism. These actions involve an intricate regulation of stress tolerance, growth and metabolic genes. Their phenotypes are influenced by the fermentation environment and physiological state of the cell, but such gene-environment interactions are poorly understood. This study aimed to compare the cell physiology of the two species, through genome-wide analysis of gene expression, coupling Oxford Nanopore MinION and Illumina Hiseq sequencing platforms. The early transcriptional responses to stress, nutrients and cell-to-cell communication were analysed. Particular attention was given to the fundamental gene modulations, leading to an understanding of the physiological changes needed to maintain cellular homeostasis, exit the quiescent state and establish dominance in the fermentation. Our findings suggest the existence of species-specific adaptation strategies in response to growth in a high sugar synthetic grape juice medium.

Filamentous cluster III Defluviicoccus (DF3) are known to proliferate and cause bulking issues in industrial wastewater treatment plants. Members of the genus Defluviicoccus are also known to exhibit the glycogen accumulating organism (GAO) phenotype, which is suggested to be detrimental to enhanced biological phosphorus removal (EBPR). Despite the reported negative impact members of the DF3 have on activated sludge wastewater treatment systems, limited research has focused on understanding the physiological traits that allow them to compete in these environments. In this study, a near complete genome of an abundant filamentous DF3 named 'Candidatus Defluviicoccus seviourii' was obtained from a full-scale sequencing batch reactor (SBR) treating winery wastewater. Annotation of the 'Ca. D. seviourii' genome revealed interesting metabolic features that help to understand the abundance of this microorganism in industrial wastewater treatment plants. Their potential for the storage of polyhydroxyalkanoates (PHA) is suggested to favour these organisms with the intermittent availability of carbon in these systems. An ability to fix nitrogen and take up urea may provide them with an additional advantage with the characteristically high carbon to nitrogen content of industrial waste. The genome and preliminary findings of this study provide a foundation for further research into these biotechnologically relevant organisms.

Enhanced biological phosphorus removal (EBPR) involves the cycling of biomass through carbon-rich (feast) and carbon-deficient (famine) conditions, promoting the activity of polyphosphate accumulating organisms (PAOs). However, several alternate metabolic strategies, without polyphosphate storage, are possessed by other organisms, which can compete with the PAO for carbon at the potential expense of EBPR efficiency. The most studied are the glycogen accumulating organisms (GAOs), which utilize aerobically stored glycogen to energize anaerobic substrate uptake and storage. In full-scale systems the Micropruina spp. are among the most abundant of the proposed GAO, yet little is known about their ecophysiology. In the current study, genomic and metabolomic studies were performed on Micropruina glycogenica str. Lg2 T and compared to the in situ physiology of members of the genus in EBPR plants using state-of-the-art single cell techniques. The Micropruina spp. were observed to take up carbon, including sugars and amino acids, under anaerobic conditions, which were partly fermented to lactic acid, acetate, propionate, and ethanol, and partly stored as glycogen for potential aerobic use. Fermentation was not directly demonstrated for the abundant members of the genus in situ, but was strongly supported by the confirmation of anaerobic uptake of carbon and glycogen storage in the absence of detectable polyhydroxyalkanoates or polyphosphate reserves. This physiology is markedly different from the classical GAO model. The amount of carbon stored by fermentative organisms has potentially important implications for phosphorus removal - as they compete for substrates with the Tetrasphaera PAO and stored carbon is not made available to the "Candidatus Accumulibacter" PAO under anaerobic conditions. This study shows that the current models of the competition between PAO and GAO are too simplistic and may need to be revised to take into account the impact of potential carbon storage by fermentative organisms.

Here, we report the first sequenced genome of an indigenous Australian wine isolate of Torulaspora delbrueckii using the Oxford Nanopore MinION and Illumina HiSeq sequencing platforms. The genome size is 9.4 Mb and contains 4,831 genes.

Enhanced biological phosphorus removal (EBPR) involves the cycling of biomass through carbon-rich (feast) and carbon-deficient (famine) conditions, promoting the activity of polyphosphate accumulating organisms (PAOs). However, several alternate metabolic strategies, without polyphosphate storage, are possessed by other organisms, which can compete with the PAO for carbon at the potential expense of EBPR efficiency. The most studied are the glycogen accumulating organisms (GAOs), which utilize aerobically stored glycogen to energize anaerobic substrate uptake and storage. In full-scale systems the Micropruina spp. are among the most abundant of the proposed GAO, yet little is known about their ecophysiology. In the current study, genomic and metabolomic studies were performed on Micropruina glycogenica str. Lg2T and compared to the in situ physiology of members of the genus in EBPR plants using state-of-the-art single cell techniques. The Micropruina spp. were observed to take up carbon, including sugars and amino acids, under anaerobic conditions, which were partly fermented to lactic acid, acetate, propionate, and ethanol, and partly stored as glycogen for potential aerobic use. Fermentation was not directly demonstrated for the abundant members of the genus in situ, but was strongly supported by the confirmation of anaerobic uptake of carbon and glycogen storage in the absence of detectable polyhydroxyalkanoates or polyphosphate reserves. This physiology is markedly different from the classical GAO model. The amount of carbon stored by fermentative organisms has potentially important implications for phosphorus removal – as they compete for substrates with the Tetrasphaera PAO and stored carbon is not made available to the “Candidatus Accumulibacter” PAO under anaerobic conditions. This study shows that the current models of the competition between PAO and GAO are too simplistic and may need to be revised to take into account the impact of potential carbon storage by fermentative organisms.

Defluviicoccus vanus -related glycogen accumulating organisms (GAO) regularly proliferate in industrial wastewater treatment plants handling high carbon but nitrogen deficient wastes. When GAO dominate, they are associated with poor performance, characterised by slow settling biomass and turbid effluents. Although their ecophysiology has been studied thoroughly in domestic waste treatment plants, little attention has been paid to them in aerobic industrial systems. In this study, the effect of nitrogen addition on GAO carbon metabolism was investigated during an 8 h cycle. Activated sludge dominated by GAO from a winery wastewater sequencing batch reactor was incubated under different carbon to nitrogen (COD:N) ratios (100:1, 60:1 and 20:1) using 13 C — acetate and 15 N — urea. GAO cell assimilation was quantified using FISH-NanoSIMS. The activated sludge community was assessed by 16S rRNA gene profiling, DNA and storage polymer production. Carbon and nitrogen quantification at the cellular level by NanoSIMS revealed that low (COD:N of 100:1) or null nitrogen concentrations enhanced GAO carbon uptake. COD:N ratios of 60:1 and 20:1 reduced GAO carbon uptake and promoted whole microbial community DNA production. Nitrogen dosing at COD:N ratios of 60:1 or higher was demonstrated as feasible strategy for controlling the excessive GAO growth in high COD waste treatment plants.

Red grape musts from overripe grapes are characterised by high pH and sugar concentration. Corrections with organic acids are commonly used to secure the alcoholic fermentation and improve the organoleptic characteristics of the wine. In this study we test an alternative biological acidification method using the ability of Lactobacillus plantarum to produce high concentrations of lactic acid. The time course of sugars, organic acids and pH were measured. Available sugars were consumed by L. plantarum producing up to 8.3 g L(-1) of lactic acid. Lactic acid changed the pH from 3.9 to 3.4 after 14 days post-inoculation without yielding a relevant concentration of acetic acid (0.34 g L(-1)).