Modern yeast development: finding the balance between tradition and innovation in contemporary winemaking.

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.

Fructilactobacillus cliffordii sp. nov., Fructilactobacillus hinvesii sp. nov., Fructilactobacillus myrtifloralis sp. nov., Fructilactobacillus carniphilus sp. nov. and Fructobacillus americanaquae sp. nov., five novel lactic acid bacteria isolated from insects or flowers of Kangaroo Island, South Australia.

Six strains, KI11_D11T, KI4_B1, KI11_C11T, KI16_H9T, KI4_A6T and KI3_B9T, were isolated from insects and flowers on Kangaroo Island, South Australia. On the basis of 16S rRNA gene phylogeny, strains KI11_D11T, KI4_B1, KI11_C11T, KI16_H9T, KI4_A6T were found to be closely related to Fructilactobacillus ixorae Ru20-1T. Due to the lack of a whole genome sequence for this species, whole genome sequencing of Fructilactobacillus ixorae Ru20-1T was undertaken. KI3_B9T was found to be closely related to Fructobacillus tropaeoli F214-1T. Utilizing core gene phylogenetics and whole genome analyses, such as determination of AAI, ANI and dDDH, we propose that these six isolates represent five novel species with the names Fructilactobacillus cliffordii (KI11_D11T= LMG 32130T = NBRC 114988T), Fructilactobacillus hinvesii (KI11_C11T = LMG 32129T = NBRC 114987T), Fructilactobacillus myrtifloralis (KI16_H9T= LMG 32131T = NBRC 114989T) Fructilactobacillus carniphilus (KI4_A6T = LMG 32127T = NBRC 114985T) and Fructobacillus americanaquae (KI3_B9T = LMG 32124T = NBRC 114983T). Chemotaxonomic analyses detected no fructophilic characters for these strains of member of the genus Fructilactobacillus. KI3_B9T was found to be obligately fructophilic, similarly to its phylogenetic neighbours in the genus Fructobacillus. This study represents the first isolation, to our knowledge, of novel species in the family Lactobacillaceae from the Australian wild.

A special drop: Characterising yeast isolates associated with fermented beverages produced by Australia's indigenous peoples.

Way-a-linah, an alcoholic beverage produced from the fermented sap of Eucalyptus gunnii, and tuba, a fermented drink made from the syrup of Cocos nucifera fructifying bud, are two of several fermented beverages produced by Australian Aboriginal and Torres Strait people. Here we describe the characterisation of yeast isolates from samples associated with the fermentation of way-a-linah and tuba. Microbial isolates were obtained from two different geographical locations in Australia - the Central Plateau in Tasmania, and Erub Island in the Torres Strait. While Hanseniaspora species and Lachancea cidri were the most abundant species in Tasmania, Candida species were the most abundant in Erub Island. Isolates were screened for tolerance to stress conditions found during the production of fermented beverages and for enzyme activities relevant to the appearance, aroma and flavour of these beverages. Based on screening results, eight isolates were evaluated for their volatile profile during the fermentation of wort, apple juice and grape juice. Diverse volatile profiles were observed for beers, ciders and wines fermented with different isolates. These findings reveal the potential of these isolates to produce fermented beverages with unique aroma and flavour profiles and highlight the vast microbial diversity associated with fermented beverages produced by Australia's Indigenous peoples.

Characterization of polysulfides in Saccharomyces cerevisiae cells and finished wine from a cysteine-supplemented model grape medium.

Polysulfide degradation in wine can result in hydrogen sulfide (H2S) release, imparting a rotten-egg smell that is detrimental to wine quality. Although the presence of wine polysulfides has been demonstrated, their biogenesis remains unclear. This study investigated the role of Saccharomyces cerevisiae in polysulfide formation during fermentation, with and without 5 mM cysteine supplementation as an H2S source. Using an established liquid chromatography-tandem mass spectrometry method, monobromobimane derivatives of hydropolysulfides, including CysSSSH, CysSSSSH and GSSSSH, and two oxidized polysulfides, GSSG and GSSSSG, were detected in yeast cells at the end of fermentation in a grape juice-like medium. Polysulfide production by four S. cerevisiae single deletion mutants (BY4743 Δcys3, Δcys4, Δmet17 and Δtum1) showed no significant differences compared to BY4743, suggesting that uncharacterized pathways maintain cellular polysulfide homeostasis. Five mM cysteine addition increased the formation of shorter sulfur chain species, including GSS-bimane and GSSG, but did not elevate levels of longer sulfur chain species. Additionally, polysulfides with even numbers of sulfur atoms tended to predominate in cellular lysates. Oxidized polysulfides and longer chain hydropolysulfides were not detected in finished wines. This evidence suggests that these polysulfides are unstable in wine-like environments or not transported extracellularly. Collectively, our data illustrate the complexity of yeast polysulfide metabolism under fermentation conditions.

Directed evolution as an approach to increase fructose utilization in synthetic grape juice by wine yeast AWRI 796.

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.

Apilactobacillus apisilvae sp. nov., Nicolia spurrieriana gen. nov. sp. nov., Bombilactobacillus folatiphilus sp. nov. and Bombilactobacillus thymidiniphilus sp. nov., four new lactic acid bacterial isolates from stingless bees Tetragonula carbonaria and Austroplebeia australis.

Four strains, SG5_A10T, SGEP1_A5T, SG4_D2T, and SG4_A1T, were isolated from the honey or homogenate of Australian stingless bee species Tetragonula carbonaria and Austroplebeia australis. Based on 16S rRNA gene phylogeny, core gene phylogenetics, whole genome analyses such as determination of amino acid identity (AAI), cAAI of conserved genes, average nucleotide identity (ANI), and digital DNA-DNA hybridization (dDDH), chemotaxonomic analyses, and the novel isolation sources and unique geography, we propose three new species and one genus with the names Apilactobacillus apisilvae sp. nov. (SG5_A10T = LMG 32133T = NBRC 114991T), Bombilactobacillus thymidiniphilus sp. nov. (SG4_A1T = LMG 32125T = NBRC 114984T), Bombilactobacillus folatiphilus sp. nov. (SG4_D2T = LMG 32126T = NBRC 115004T) and Nicolia spurrieriana sp. nov. (SGEP1_A5T = LMG 32134T = NBRC 114992T). Three out of the four strains were found to be fructophilic, where SG5_A10T and SGEP1_A5T belong to obligately fructophilic lactic acid bacteria, and SG4_D2T representing a new type denoted here as kinetically fructophilic. This study represents the first published lactic acid bacterial species associated with the unique niche of Australian stingless bees.

Sulfate transport mutants affect hydrogen sulfide and sulfite production during alcoholic fermentation.

Hydrogen sulfide is a common wine fault, with a rotten-egg odour, which is directly related to yeast metabolism in response to nitrogen and sulfur availability. In grape juice, sulfate is the most abundant inorganic sulfur compound, which is taken up by yeast through two high-affinity sulfate transporters, Sul1p and Sul2p, and a low affinity transporter, Soa1p. Sulfate contributes to H2 S production under nitrogen limitation, by being reduced via the Sulfur Assimilation Pathway (SAP). Therefore, yeast strains with limited H2 S are highly desirable. We report on the use of toxic analogues of sulfate following ethyl methane sulfate treatment, to isolate six wine yeast mutants that produce no or reduced H2 S and SO2 during fermentation in synthetic and natural juice. Four amino acid substitutions (A99V, G380R, N588K and E856K) in Sul1p were found in all strains except D25-1 which had heterozygous alleles. Two changes were also identified in Sul2p (L268S and A470T). The Sul1p (G380R) and Sul2p (A470T) mutations were chosen for further investigation as these residues are conserved amongst SLC26 membrane proteins (including sulfate permeases). The mutations were introduced into EC1118 using Crispr cas9 technology and shown to reduce accumulation of H2 S and do not result in increased SO2 production during fermentation of model medium (chemically defined grape juice) or Riesling juice. The Sul1p (G380R) and Sul2p (A470T) mutations are newly reported as causal mutations. Our findings contribute to knowledge of the genetic basis of H2 S production as well as the potential use of these strains for winemaking and in yeast breeding programmes.

Disruption of ECM33 in diploid wine yeast EC1118: cell morphology and aggregation and their influence on fermentation performance.

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.

Exploring the diversity of bacteriophage specific to Oenococcus oeni and Lactobacillus spp and their role in wine production.

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.

QTL mapping: an innovative method for investigating the genetic determinism of yeast-bacteria interactions in wine.

The two most commonly used wine microorganisms, Saccharomyces cerevisiae yeast and Oenococcus oeni bacteria, are responsible for completion of alcoholic and malolactic fermentation (MLF), respectively. For successful co-inoculation, S. cerevisiae and O. oeni must be able to complete fermentation; however, this relies on compatibility between yeast and bacterial strains. For the first time, quantitative trait loci (QTL) analysis was used to elucidate whether S. cerevisiae genetic makeup can play a role in the ability of O. oeni to complete MLF. Assessment of 67 progeny from a hybrid S. cerevisiae strain (SBxGN), co-inoculated with a single O. oeni strain, SB3, revealed a major QTL linked to MLF completion by O. oeni. This QTL encompassed a well-known translocation, XV-t-XVI, that results in increased SSU1 expression and is functionally linked with numerous phenotypes including lag phase duration and sulphite export and production. A reciprocal hemizygosity assay was performed to elucidate the effect of the gene SSU1 in the SBxGN background. Our results revealed a strong effect of SSU1 haploinsufficiency on O. oeni's ability to complete malolactic fermentation during co-inoculation and pave the way for the implementation of QTL mapping projects for deciphering the genetic bases of microbial interactions. KEY POINTS: • For the first time, QTL analysis has been used to study yeast-bacteria interactions. • A QTL encompassing a translocation, XV-t-XVI, was linked to MLF outcomes. • S. cerevisiae SSU1 haploinsufficiency positively impacted MLF by O. oeni.

Yeast bioprospecting versus synthetic biology-which is better for innovative beverage fermentation?

Producers often utilise some of the many available yeast species and strains in the making of fermented alcoholic beverages in order to augment flavours, aromas, acids and textural properties. But still, the demand remains for more yeasts with novel phenotypes that not only impact sensory characteristics but also offer process and engineering advantages. Two strategies for finding such yeasts are (i) bioprospecting for novel strains and species and (ii) genetic modification of known yeasts. The latter enjoys the promise of the emerging field of synthetic biology, which, in principle, would enable scientists to create yeasts with the exact phenotype desired for a given fermentation. In this mini review, we compare and contrast advances in bioprospecting and in synthetic biology as they relate to alcoholic fermentation in brewing and wine making. We explore recent advances in fermentation-relevant recombinant technologies and synthetic biology including the Yeast 2.0 Consortium, use of environmental yeasts, challenges, constraints of law and consumer acceptance.

Early adaptation strategies of Saccharomyces cerevisiae and Torulaspora delbrueckii to co-inoculation in high sugar grape must-like media.

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.

Removal of Volatile Phenols From Wine Using Crosslinked Cyclodextrin Polymers.

Volatile phenols have been implicated as contributors to off-odors associated with taints from bushfire smoke and microbial spoilage. Various methods for the amelioration of off-odors have been evaluated, but to date, they have not included cyclodextrin (CD) polymers. In the current study, two CD polymers were prepared from ß- and γ-CD, using hexamethylene diisocyanate (HDI) as a crosslinking agent. Adsorption tests were performed with four volatile phenols (guaiacol, 4-methylguaiacol, 4-ethylguaiacol and 4-ethylphenol) at concentrations up to 1 mg/L. The removal of volatile phenols by CD polymers achieved equilibrium almost instantly, with isotherm tests suggesting an adsorption capacity of 20.7 µg of volatile phenol per gram of polymer. Langmuir and Freundlich models were subsequently used to fit the data. In batch adsorption tests, the CD polymers achieved 45 to 77% removal of volatile phenols. Polymer reusability was also evaluated and was found to be excellent. A comparison between volatile phenol adsorption by CDs vs. CD polymers, determined using a novel four-phase headspace solid-phase microextraction (HS-SPME) method for gas chromatography-mass spectrometry (GC-MS), suggests CD polymers offer several advantages for use by the wine industry.

The microbial challenge of winemaking: yeast-bacteria compatibility.

The diversity and complexity of wine environments present challenges for predicting success of fermentation. In particular, compatibility between yeast and lactic acid bacteria is affected by chemical and physical parameters that are strain and cultivar specific. This review focuses on the impact of compound production by microbes and physical interactions between microbes that ultimately influence how yeast and bacteria may work together during fermentation. This review also highlights the importance of understanding microbial interactions for yeast-bacteria compatibility in the wine context.

TAMMiCol: Tool for analysis of the morphology of microbial colonies.

Many microbes are studied by examining colony morphology via two-dimensional top-down images. The quantification of such images typically requires each pixel to be labelled as belonging to either the colony or background, producing a binary image. While this may be achieved manually for a single colony, this process is infeasible for large datasets containing thousands of images. The software Tool for Analysis of the Morphology of Microbial Colonies (TAMMiCol) has been developed to efficiently and automatically convert colony images to binary. TAMMiCol exploits the structure of the images to choose a thresholding tolerance and produce a binary image of the colony. The images produced are shown to compare favourably with images processed manually, while TAMMiCol is shown to outperform standard segmentation methods. Multiple images may be imported together for batch processing, while the binary data may be exported as a CSV or MATLAB MAT file for quantification, or analysed using statistics built into the software. Using the in-built statistics, it is found that images produced by TAMMiCol yield values close to those computed from binary images processed manually. Analysis of a new large dataset using TAMMiCol shows that colonies of Saccharomyces cerevisiae reach a maximum level of filamentous growth once the concentration of ammonium sulfate is reduced to 200 µM. TAMMiCol is accessed through a graphical user interface, making it easy to use for those without specialist knowledge of image processing, statistical methods or coding.

Measures to improve wine malolactic fermentation.

This review focuses on the considerable amount of research that has been directed towards the improvement of efficiency and reliability of malolactic fermentation (MLF), which is important in winemaking. From this large body of work, it is clear that reliable MLF is essential for process efficiency and prevention of spoilage in the final product. Impediments to successful MLF in wine, the impact of grape and wine ecology and how this may affect MLF outcome are discussed. Further focus is given to how MLF success may be enhanced, via alternative inoculation strategies, MLF progress sensing technologies and the use of different bacterial species. An update of how this information may be used to enhance and improve sensory outcomes through metabolite production during MLF and suggestions for future research priorities for the field are also provided.

Development and Evaluation of a HS-SPME GC-MS Method for Determining the Retention of Volatile Phenols by Cyclodextrin in Model Wine.

Volatile phenols exist in wine and can be markers for Brettanomyces and smoke taint off-odors. Cyclodextrins (CDs) are found to be capable of forming inclusion complexes with volatile phenols. Cross peaks on 2D 1H ROESY nuclear magnetic resonance (NMR) spectra demonstrated inclusion of volatile phenols in the ß-CD cavity, while difference tests confirmed this resulted in a perceptible reduction of their sensory impact. However, a conventional headspace solid phase microextraction (HS-SPME) method using an isotopically labelled normalizing standard failed to quantify the residual volatile phenols by gas chromatography-mass spectrometry (GC-MS) because of inclusion of the standard by the CDs. A new method involving an additional liquid phase was developed and validated for quantitation of volatile phenols in the presence of CDs. The retention of eight volatile phenols by α-, ß-, and γ-CD was subsequently studied.

Dissection of the molecular bases of genotype x environment interactions: a study of phenotypic plasticity of Saccharomyces cerevisiae in grape juices.

BACKGROUND: The ability of a genotype to produce different phenotypes according to its surrounding environment is known as phenotypic plasticity. Within different individuals of the same species, phenotypic plasticity can vary greatly. This contrasting response is caused by gene-by-environment interactions (GxE). Understanding GxE interactions is particularly important in agronomy, since selected breeds and varieties may have divergent phenotypes according to their growing environment. Industrial microbes such as Saccharomyces cerevisiae are also faced with a large range of fermentation conditions that affect their technological properties. Finding the molecular determinism of such variations is a critical task for better understanding the genetic bases of phenotypic plasticity and can also be helpful in order to improve breeding methods. RESULTS: In this study we implemented a QTL mapping program using two independent cross (~ 100 progeny) in order to investigate the molecular basis of yeast phenotypic response in a wine fermentation context. Thanks to whole genome sequencing approaches, both crosses were genotyped, providing saturated genetic maps of thousands of markers. Linkage analyses allowed the detection of 78 QTLs including 21 with significant interaction with the environmental conditions. Molecular dissection of a major QTL demonstrated that the sulfite pump Ssu1p has a pleiotropic effect and impacts the phenotypic plasticity of several traits. CONCLUSIONS: The detection of QTLs and their interactions with environment emphasizes the complexity of yeast industrial traits. The validation of the interaction of SSU1 allelic variants with the nature of the fermented juice increases knowledge about the impact of the sulfite pump during fermentation. All together these results pave the way for exploiting and deciphering the genetic determinism of phenotypic plasticity.

Disruption of the cell wall integrity gene ECM33 results in improved fermentation by wine yeast.

Severe oenological conditions, such as limited assimilable nitrogen and high sugar contents restrict yeast's ability to successfully complete fermentation. In the absence of a comprehensive commercially available deletion collection in a wine yeast background, a screening approach was applied to a transposon library in a wine yeast derivative to identify clones with superior fermentation performance. Five candidate genes, when disrupted by Ty insertion, were identified as enabling yeast to efficiently complete a model oenological fermentation with limited nitrogen availability. Analogous single gene disruptions were subsequently constructed in the haploid wine yeast strain C911D, and the performance of these during fermentation was analysed. Deletion of ECM33 resulted in the shortest fermentation (up to 31% reduction) in both synthetic medium and grape juice. Interestingly, no significant differences were found in nitrogen utilization, cell viability or biomass yield between ∆ecm33 and the wild type. ∆ecm33 did, however, display growth hypersensitivity to the dyes Calcofluor White and Congo Red, suggesting a link to cell wall integrity. Transcriptional profiling of ∆ecm33 during fermentation demonstrated the up-regulation of SLT2 and HOG1, encoding mitogen activated protein kinases involved in the cell wall integrity (CWI) and high osmolarity glycerol (HOG) pathways, respectively. CHS3 a major chitin synthase gene was also found to be upregulated, and the transcript abundance of key genes of central nitrogen metabolism, GLN1, GLT1, GDH1 and GDH2 in mutant ∆ecm33 were also altered. The findings highlight the complexity of the robust fermentation phenotype and provide clues for further improvement of industrial strains.

Use of a wine yeast deletion collection reveals genes that influence fermentation performance under low-nitrogen conditions.

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.