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

When fermentation research requires the comparison of many strains or conditions, the major bottleneck is a technical one. Microplate approaches are not able to produce representative fermentative performance due to their inability to truly operate anaerobically, whilst more traditional methods do not facilitate sample density sufficient to assess enough candidates to be considered even medium throughput. Two robotic platforms have been developed that address these technological shortfalls. Both are built on commercially available liquid handling platforms fitted with custom labware. Results are presented detailing fermentation performance as compared to current best practice, i.e., shake flasks fitted with airlocks and sideports. The ‘TeeBot’ is capable sampling from 96 or 384 fermentations in 100 mL or 30 mL volumes, respectively, with airlock sealing and minimal headspace. Sampling and downstream analysis are facilitated by automated liquid handling, use of 96-well sample plate format and temporary cryo-storage (<0 °C).

Abstract A key driver of quality in wines is the microbial population that undertakes fermentation of grape must. Winemakers can utilise both indigenous and purposefully inoculated yeasts to undertake alcoholic fermentation, imparting wines with aromas, flavours and palate structure and in many cases contributing to complexity and uniqueness. Importantly, having a toolbox of microbes helps winemakers make best use of the grapes they are presented with, and tackle fermentation difficulties with flexibility and efficiency. Each year the number of strains available commercially expands and more recently, includes strains of non-Saccharomyces, strains that have been improved using both classical and modern yeast technology and mixed cultures. Here we review what is available commercially, and what may be in the future, by exploring recent advances in fermentation relevant strain improvement technologies. We also report on the current use of microbes in the Australian wine industry, as reported by winemakers, as well as regulations around, and sentiment about the potential use of genetically modified organisms in the future. The number of commercial yeasts available and used by winemakers is ever expanding as are techniques to develop them; the authors review these and survey Australian winemakers for opinion and use.

ABSTRACT When investigating yeast gene function in relation to fermentation, many screens rely on haploid yeast derivatives. This, however, is not representative of industrial strains, which are typically diploid. One such example is the disruption of ECM33, which was associated with improved fermentation in the haploid wine yeast C911D, but remains uncharacterised in a diploid industrial strain background. We report on the homozygous disruption of ECM33 in Lalvin EC1118 using CRISPR/Cas9. EC1118 ecm33 resulted in a reduction of fermentation duration in a defined medium with limiting and sufficient nitrogen (−20% and −13%, respectively) when shaken. Increased cell size and aggregation, a phenotype previously unidentified in ecm33∆ as haploid yeast tend to aggregate, was also observed. This phenotype led to premature settling thereby the yeast behaving similarly to EC1118 in wine-like semi-static fermentations in a chemically defined medium. Further assessment in semi-static Riesling and Chardonnay fermentations inoculated based on cell number or biomass resulted in no significant difference or significantly slower fermentation duration in comparison the EC1118, nullifying the benefits of this mutation unless agitation is applied. This study draws attention to phenotypes being condition-dependent, highlighting the need to characterise and verify fermentation efficiency mutations in industrial yeast. Gene-editing was used to disrupt the ECM33 gene in a wine yeast strain, confirming the enhanced fermentation performance of this mutation, previously only shown in a haploid, laboratory strain.

The widespread existence of bacteriophage has been of great interest to the biological research community and ongoing investigations continue to explore their diversity and role. They have also attracted attention and in-depth research in connection to fermented food processing, in particular from the dairy and wine industries. Bacteriophage, mostly oenophage, may in fact be a ‘double edged sword’ for winemakers: whilst they have been implicated as a causal agent of difficulties with malolactic fermentation (although not proven), they are also beginning to be considered as alternatives to using sulphur dioxide to prevent wine spoilage. Investigation and characterisation of oenophage of Oenococcus oeni, the main species used in winemaking, are still limited compared to lactococcal bacteriophage of Lactococcus lactis and Lactiplantibacillus plantarum (formally Lactobacillus plantarum), the drivers of most fermented dairy products. Interestingly, these strains are also being used or considered for use in winemaking. In this review, the genetic diversity and life cycle of phage, together with the debate on the consequent impact of phage predation in wine, and potential control strategies are discussed.Key points• Bacteriophage detected in wine are diverse.• Many lysogenic bacteriophage are found in wine bacteria.• Phage impact on winemaking can depend on the stage of the winemaking process.• Bacteriophage as potential antimicrobial agents against spoilage organisms.

The rotten-egg odour of hydrogen sulfide (H2S) produced by the yeast Saccharomyces cerevisiae has attracted considerable research interest due to its huge impact on the sensory quality of fermented foods and beverages. To date, the yeast genetic mechanisms of H2S liberation during wine fermentation are well understood and yeast strains producing low levels of H2S have been developed. Studies have also revealed that H2S is not just a by-product in the biosynthesis of the sulfur-containing amino acids, but indeed a vital molecule involved in detoxification, population signalling and extending cellular life span. Moreover, polysulfides have recently emerged as key players in signalling and the sensory quality of wine because their degradation leads to the release of H2S. This review will focus on the recent findings on the production of H2S and polysulfides in S. cerevisiae and summarise their potential roles in yeast survival and winemaking. Recent advances in techniques for the detection of H2S and polysulfides offer an exciting opportunity to uncover the novel genes and pathways involved in their formation from different sulfur sources. This knowledge will not only provide further insights into yeast sulfur metabolism, but could potentially improve the sensory quality of wine.

Abstract In winemaking, slow or stuck alcoholic fermentation can impact processing efficiency and wine quality. Residual fructose in the later stages of fermentation can leave the wine ‘out of specification’ unless removed, which requires reinoculation or use of a more fructophilic yeast. As such, robust, fermentation efficient strains are still highly desirable to reduce this risk. We report on a combined EMS mutagenesis and Directed Evolution (DE) approach as a ‘proof of concept’ to improve fructose utilization and decrease fermentation duration. One evolved isolate, Tee 9, was evaluated against the parent, AWRI 796 in defined medium (CDGJM) and Semillon juice. Interestingly, Tee 9 exhibited improved fermentation in CDGJM at several nitrogen contents, but not in juice. Genomic comparison between AWRI 796 and Tee 9 identified 371 mutations, but no chromosomal copy number variation. A total of 95 noncoding and 276 coding mutations were identified in 297 genes (180 of which encode proteins with one or more substitutions). Whilst introduction of two of these, Gid7 (E726K) or Fba1 (G135S), into AWRI 796 did not lead to the fermentation improvement seen in Tee 9, similar allelic swaps with the other mutations are needed to understand Tee 9’s adaption to CDGJM. Furthermore, the 378 isolates, potentially mutagenized but with the same genetic background, are likely a useful resource for future phenotyping and genome-wide association studies. Chemical mutagenesis and directed evolution as an approach to improve fructose utilization during wine fermentation.

An early burst of hydrogen sulfide (H2S) produced by Saccharomyces cerevisiae during fermentation could increase varietal thiols and therefore enhance desirable tropical aromas in varieties such as Sauvignon Blanc. Here we attempted to identify genes affecting H2S formation from cysteine by screening yeast deletion libraries via a colony colour assay on media resembling grape juice. Both Δlst4 and Δlst7 formed lighter coloured colonies and produced significantly less H2S than the wild type on high concentrations of cysteine, likely because they are unable to take up cysteine efficiently. We then examined the nine known cysteine permeases and found that deletion of AGP1, GNP1 and MUP1 led to reduced production of H2S from cysteine. We further showed that deleting genes involved in the SPS-sensing pathway such as STP1 and DAL81 also reduced H2S from cysteine. Together, this study indirectly confirms that Agp1p, Gnp1p and Mup1p are the major cysteine permeases and that they are regulated by the SPS-sensing and target of rapamycin pathways under the grape juice-like, cysteine-supplemented, fermentation conditions. The findings highlight that cysteine transportation could be a limiting factor for yeast to generate H2S from cysteine, and therefore selecting wine yeasts without defects in cysteine uptake could maximise thiol production potential.

The undesirable rotten-egg odour of hydrogen sulfide (H S) produced by yeast shortly after yeast inoculation of grape musts might be an important source of desirable varietal thiols, which contribute to tropical aromas in varieties such as Sauvign-on Blanc. In this study, we observed that Saccharomyces cerevisiae strains produce an early burst of H S from cysteine. Both Δmet2 and Δmet17 strains produce a larger burst, likely because they are unable to utilise the H S in the sulfate assimilation pathway. For the first time, we show that TUM1 is partly responsible for the early production of H S from cysteine. Overex-pressing TUM1 elevated production of H S, whilst its deletion yields only half of the H S. We further confirmed that yeast convert cysteine to H S by analysing growth of mutants lacking components of the transsulfuration pathway. High concent-rations of cysteine overcame this growth block, but required TUM1 Collectively, the data indicate that S. cerevisiae does not convert cysteine to sulfate or sulfite, but rather to sulfide via a novel pathway that requires the action of Tum1p. The findi-ngs of this study may allow the improvement of commercial yeasts through the manipulation of sulfur metabolism that are better suited towards production of fruit-driven styles.

A deficiency of nitrogenous nutrients in grape juice can cause stuck and sluggish alcoholic fermentation, which has long been a problem in winemaking. Nitrogen requirements vary between wine yeast strains, and the ability of yeast to assimilate nitrogen depends on the nature and concentration of nitrogen present in the medium. In this study, a wine yeast gene deletion collection (1844 deletants in the haploid AWRI1631 background) was screened to identify genes whose deletion resulted in a reduction in the time taken to utilise all sugars when grown in a chemically defined grape juice medium supplemented with limited nitrogen (75 mg L-1 as a free amino acid mixture). Through micro-scale and laboratory-scale fermentations, 15 deletants were identified that completed fermentation in a shorter time than the wildtype (c.a. 15%-59% time reduction). This group of genes was annotated to biological processes including protein modification, transport, metabolism and ubiquitination (UBC13, MMS2, UBP7, UBI4, BRO1, TPK2, EAR1, MRP17, MFA2 and MVB12), signalling (MFA2) and amino acid metabolism (AAT2). Deletion of MFA2, encoding mating factor-a, resulted in a 55% decrease in fermentation duration. Mfa2Δ was chosen for further investigation to understand how this gene deletion conferred fermentation efficiency in limited nitrogen conditions.