Industry and academia-a perfect match.

My career developed very differently from those of most academic researchers. After school, I worked for 6 years in industries that employed yeast to manufacture ethanol and beer. At university, I was trained as a microbiologist with very little training in molecular biology. I retrained in 1987 in molecular yeast genetics and focused on genetic engineering of industrial yeasts to minimize the production of spoilage compounds in wine and ethyl carbamate, a carcinogen, in wine. The malolactic yeast ML01 and the urea-degrading yeast were the first genetically enhanced yeasts that obtained US FDA approval for commercial applications. Apart from applied research, I was fascinated by classic molecular yeast genetic studies using sophisticated techniques such as transcriptomics, proteomics, and metabolomics. Doing research at the University of British Columbia was stimulating and exciting, we established a core microarray and metabolomics facility that was used by many scientists at UBC and hospitals in Vancouver. I also established a state-of-the-art Wine Library that was used to study aging of wines produced in British Columbia. Finally, I have been fortunate to know and collaborate with leading yeast scientists who motivated me.

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.

Enhancing the natural folate level in wine using bioengineering and stabilization strategies.

Folate deficiency is linked to many diseases, some of which may have higher probability in individuals with alcohol-induced alterations in one-carbon metabolism. Our study shows that folate content in commercial wine is not related to white or red varieties, but associated with the yeast that is used to produce the wine. The stability of folate in these wines, once opened for consumption, did not correlate with total phenolic or sulfite content. In addition, we employed yeast bioengineering to fortify wine with folate. We confirmed by overexpression that FOL2 was the key gene encoding the rate-limiting step of folate biosynthesis in wine yeast. In this study, we also show that overexpression of other folate biosynthesis genes, including ABZ1, ABZ2, DFR1, FOL1 and FOL3, had no effect on folate levels in wine. Ensuring stability of the increased natural folate in all wines was achieved by the addition of ascorbate.

Functional characterization of individual- and mixed-Burgundian Saccharomyces cerevisiae isolates for fermentation of Pinot noir.

Pinot noir has traditionally been fermented by native flora of multiple yeasts producing a complex combination of aromas and flavors. With the use of industrial dry yeasts, winemakers gained enological reliability and consistency in their wines, but lost diversity and complexity. This research evaluated the use of co-culturing yeasts to fulfill this dual role. Fermentations of Burgundian Saccharomyces cerevisiae isolates and their mixtures were evaluated for their enological characteristics and production of volatile compounds, at 22 °C and 27 °C. The novel isolates were genetically unique and enologically equivalent to the industrial strains. Analysis of variance and principal component analysis of 25 headspace volatiles revealed differences among the yeasts and between the fermentation temperatures. Wines from the mixed-Burgundian isolates were most similar to one another and could be differentiated from the industrial strains at both 22 °C and 27 °C. Mixed-Burgundian wines at both temperatures had higher concentrations of ethyl esters and acetate esters, compared to the industrial strains which had higher concentrations of higher alcohols at 27 °C and higher concentration of other ethyl esters at 22 °C. Given the unique profiles of the co-cultured wines, this research offers winemakers a strategy for producing wines with unique and more complex characters without the risk of spontaneous fermentations.

Functional analyses of NSF1 in wine yeast using interconnected correlation clustering and molecular analyses.

Analyzing time-course expression data captured in microarray datasets is a complex undertaking as the vast and complex data space is represented by a relatively low number of samples as compared to thousands of available genes. Here, we developed the Interdependent Correlation Clustering (ICC) method to analyze relationships that exist among genes conditioned on the expression of a specific target gene in microarray data. Based on Correlation Clustering, the ICC method analyzes a large set of correlation values related to gene expression profiles extracted from given microarray datasets. ICC can be applied to any microarray dataset and any target gene. We applied this method to microarray data generated from wine fermentations and selected NSF1, which encodes a C2H2 zinc finger-type transcription factor, as the target gene. The validity of the method was verified by accurate identifications of the previously known functional roles of NSF1. In addition, we identified and verified potential new functions for this gene; specifically, NSF1 is a negative regulator for the expression of sulfur metabolism genes, the nuclear localization of Nsf1 protein (Nsf1p) is controlled in a sulfur-dependent manner, and the transcription of NSF1 is regulated by Met4p, an important transcriptional activator of sulfur metabolism genes. The inter-disciplinary approach adopted here highlighted the accuracy and relevancy of the ICC method in mining for novel gene functions using complex microarray datasets with a limited number of samples.

Functional improvement of Saccharomyces cerevisiae to reduce volatile acidity in wine.

Control of volatile acidity (VA) is a major issue for wine quality. In this study, we investigated the production of VA by a deletion mutant of the fermentation stress response gene AAF1 in the budding yeast Saccharomyces cerevisiae. Fermentations were carried out in commercial Chardonnay grape must to mimic industrial wine-making conditions. We demonstrated that a wine yeast strain deleted for AAF1 reduced acetic acid levels in wine by up to 39.2% without increasing the acetaldehyde levels, revealing a potential for industrial application. Deletion of the cytosolic aldehyde dehydrogenase gene ALD6 also reduced acetic acid levels dramatically, but increased the acetaldehyde levels by 41.4%, which is not desired by the wine industry. By comparison, ALD4 and the AAF1 paralog RSF2 had no effects on acetic acid production in wine. Deletion of AAF1 was detrimental to the growth of ald6Δ and ald4Δald6Δ mutants, but had no effect on acetic acid production. Overexpression of AAF1 dramatically increased acetic acid levels in wine in an Ald6p-dependent manner, indicating that Aaf1p regulates acetic acid production mainly via Ald6p. Overexpression of AAF1 in an ald4Δald6Δ strain produced significantly more acetic acid in wine than the ald4Δald6Δ mutant, suggesting that Aaf1p may also regulate acetic acid synthesis independently of Ald4p and Ald6p.

The fermentation stress response protein Aaf1p/Yml081Wp regulates acetate production in Saccharomyces cerevisiae.

The production of acetic acid during wine fermentation is a critical issue for wineries since the sensory quality of a wine can be affected by the amount of acetic acid it contains. We found that the C2H2-type zinc-finger transcription factor YML081Wp regulated the mRNA levels of ALD4 and ALD6, which encode a cytosolic acetaldehyde dehydrogenase (ACDH) and a mitochondrial ACDH, respectively. These enzymes produce acetate from acetaldehyde as part of the pyruvate dehydrogenase bypass. This regulation was also reflected in the protein levels of Ald4p and Ald6p, as well as total ACDH activity. In the absence of ALD6, YML081W had no effect on acetic acid levels, suggesting that this transcription factor's effects are mediated primarily through this gene. lacZ reporter assays revealed that Yml081wp stimulates ALD6 transcription, in large part from a GAGGGG element 590 base pairs upstream of the translation start site. The non-annotated ORF YML081W therefore encodes a transcription factor that regulates acetate production in Saccharomyces cerevisiae. We propose AAF1 as a gene name for the YML081W ORF.

The Saccharomyces cerevisiae fermentation stress response protein Igd1p/Yfr017p regulates glycogen levels by inhibiting the glycogen debranching enzyme.

Wine fermentation imposes a number of stresses on Saccharomyces cerevisiae, and wine yeasts respond to this harsh environment by altering their transcriptional profile (Marks et al., 2008). We have labeled this change in gene expression patterns the fermentation stress response (FSR). An important component of the FSR is the increased expression of 62 genes for which no function has been identified for their protein products. We hypothesize that a function for these proteins may only be revealed late in grape must fermentation, when the yeast cells are facing conditions much more extreme than those normally encountered in laboratory media. We used affinity copurification to identify interaction partners for the FSR protein Yfr017p, and found that it interacts specifically with the glycogen debranching enzyme (Gdb1p). The expression of both of these proteins is strongly induced during wine fermentation. Therefore, we investigated the role of Yfr017p in glycogen metabolism by constructing wine yeast strains that lack this protein. These YFR017C null cells displayed a significant reduction in their ability to accumulate glycogen during aerobic growth and fermentation. Moreover, Yfr017p inhibits Gdb1p activity in vitro. These results suggest that Yfr017p functions as an inhibitor of Gdb1p, enhancing the ability of yeast cells to store glucose as glycogen. Therefore, we propose IGD1 (for inhibitor of glycogen debranching) as a gene name for the YFR017C ORF.

Effects of nitrogen catabolite repression and di-ammonium phosphate addition during wine fermentation by a commercial strain of S. cerevisiae.

Two deletion mutants expected to be defective in nitrogen catabolite repression (NCR) were constructed in a commercial wine yeast background M2: a ure2 mutant and a dal80 gzf3 double mutant. Wild-type and both mutant strains were fermented in Sauvignon Blanc grape juice with and without addition of di-ammonium phosphate (DAP). The dal80 gzf3 double mutant exhibited a long fermentative lag phase, which was offset by DAP addition (corresponding to 300 mg/L of N). Neither the NCR mutations nor DAP addition affected the content of volatile thiols in the final wine. Microarray analyses of transcripts in the wild-type and dal80 gzf3 double-mutant strains were performed after 2% and 70% sugars were fermented. Of 80 genes previously identified as NCR-regulated, only 13 were upregulated during fermentation of the dal80 gzf3 double-mutant strain in grape juice. Following DAP addition, 34 of the known NCR genes were downregulated, including 17 that were downregulated even in the NCR mutant strain. The results demonstrate an unexpected complexity of the NCR response that may reflect differences between strains of yeast or differences in gene regulation during alcoholic fermentation compared with standard aerobic growth.

Functional analyses of PAU genes in Saccharomyces cerevisiae.

PAU genes constitute the largest gene family in Saccharomyces cerevisiae, with 24 members mostly located in the subtelomeric regions of chromosomes. Little information is available about PAU genes, other than expression data for some members. In this study, we systematically compared the sequences of all 24 members, examined the expression of PAU3, PAU5, DAN2, PAU17 and PAU20 in response to stresses, and investigated the stability of all Pau proteins. The chromosomal localization, synteny and sequence analyses revealed that PAU genes could have been amplified by segmental and retroposition duplication through mechanisms of chromosomal end translocation and Ty-associated recombination. The coding sequences diverged through nucleotide substitution and insertion/deletion of one to four codons, thus causing changes in amino acids, truncation or extension of Pau proteins. Pairwise comparison of non-coding regions revealed little homology in flanking sequences of some members. All 24 PAU promoters contain a TATA box, and 22 PAU promoters contain at least one copy of the anaerobic response element and the aerobic repression motif. Differential expression was observed among PAU3, PAU5, PAU17, PAU20 and DAN2 in response to stress, with PAU5 having the highest capacity to be induced by anaerobic conditions, low temperature and wine fermentations. Furthermore, Pau proteins with 124 aa were less stable than those with 120 or 122 aa. Our results indicate that duplicated PAU genes have been evolving, and the individual Pau proteins might possess specific roles for the adaptation of S. cerevisiae to certain environmental stresses.

Stress-induced production, processing and stability of a seripauperin protein, Pau5p, in Saccharomyces cerevisiae.

PAU genes comprise the largest multiple gene family in Saccharomyces cerevisiae with 24 members whose sequence homology ranges from 82% to 100%. Although transcriptional regulation for some of the PAU genes has been reported, none of the Pau proteins has been characterized. We constructed yeast strains encoding a C-terminal tandem affinity purification-tagged Pau5 in the PAU5 locus to study Pau5 production and properties in vivo. Pau5 is highly induced by low temperature, low oxygen and wine fermentation conditions. It is unstable in cells grown under laboratory conditions and is temporarily stabilized by low oxygen, osmotic and ethanol stresses. Pau5 degradation is accompanied by an unknown modification with a gradual increase in molecular mass by 3 kDa. Furthermore, Pau5 is O-mannosylated mainly by Pmt1; mannosylation enhances stability of the protein. The mannosylated Pau5 is soluble whereas the nonmannosylated proform Pau5 is an integral membrane protein. Our findings suggest that the intracellular concentration of Pau5 is regulated by wine making stress both at transcriptional and posttranslational levels; Pau5 might play a role in adaptation of yeast cells during alcoholic fermentations.

Dynamics of the yeast transcriptome during wine fermentation reveals a novel fermentation stress response.

In this study, genome-wide expression analyses were used to study the response of Saccharomyces cerevisiae to stress throughout a 15-day wine fermentation. Forty per cent of the yeast genome significantly changed expression levels to mediate long-term adaptation to fermenting grape must. Among the genes that changed expression levels, a group of 223 genes was identified, which was designated as fermentation stress response (FSR) genes that were dramatically induced at various points during fermentation. FSR genes sustain high levels of induction up to the final time point and exhibited changes in expression levels ranging from four- to 80-fold. The FSR is novel; 62% of the genes involved have not been implicated in global stress responses and 28% of the FSR genes have no functional annotation. Genes involved in respiratory metabolism and gluconeogenesis were expressed during fermentation despite the presence of high concentrations of glucose. Ethanol, rather than nutrient depletion, seems to be responsible for entry of yeast cells into the stationary phase.

Metabolic engineering of malolactic wine yeast.

Malolactic fermentation is essential for the deacidification of high acid grape must. We have constructed a genetically stable industrial strain of Saccharomyces cerevisiae by integrating a linear cassette containing the Schizosaccharomyces pombe malate permease gene (mae1) and the Oenococcus oeni malolactic gene (mleA) under control of the S. cerevisiae PGK1 promoter and terminator sequences into the URA3 locus of an industrial wine yeast. The malolactic yeast strain, ML01, fully decarboxylated 5.5 g/l of malate in Chardonnay grape must during the alcoholic fermentation. Analysis of the phenotype, genotype, transcriptome, and proteome revealed that the ML01 yeast is substantially equivalent to the parental industrial wine yeast. The ML01 yeast enjoys 'Generally Regarded As Safe' status from the FDA and is the first genetically enhanced yeast that has been commercialized. Its application will prevent the formation of noxious biogenic amines produced by lactic acid bacteria in wine.

Expression of the HXT13, HXT15 and HXT17 genes in Saccharomyces cerevisiae and stabilization of the HXT1 gene transcript by sugar-induced osmotic stress.

Saccharomyces cerevisiae contains a family of 17 hexose transporter (HXT) genes; only nine have assigned functions, some of which are still poorly defined. Despite extensive efforts to characterize the hexose transporters, the expression of HXT6 and HXT8-17 remains an enigma. In nature, S. cerevisiae finds itself under extreme nutritional conditions including sugars in excess of 40% (w/v), depletion of nutrients and extremes of both temperature and pH. Using HXT promoter-lacZ fusions, we have identified novel conditions under which the HXT17 gene is expressed; HXT17 promoter activity is up-regulated in media containing raffinose and galactose at pH 7.7 versus pH 4.7. We demonstrated that HXT5, HXT13 and, to a lesser extent, HXT15 were all induced in the presence of non-fermentable carbon sources. HXT1 encodes a low-affinity transporter and in short-term osmotic shock experiments, HXT1 promoter activity was reduced when cells were exposed to media containing 40% glucose. However, we found that the HXT1 mRNA transcript was stabilized under conditions of osmotic stress. Furthermore, the stabilization of HXT1 mRNA does not appear to be gene specific because 30 min after transcriptional arrest there is a fourfold more mRNA in osmotically stressed versus non-stressed yeast cells. A large portion of S. cerevisiae mRNA molecules may, therefore, have a decreased rate of turnover during exposure to osmotic stress indicating that post-transcriptional regulation plays an important role in the adaptation of S. cerevisiae to osmotic stress.

Genome-wide expression analyses: Metabolic adaptation of Saccharomyces cerevisiae to high sugar stress.

The transcriptional response of laboratory strains of Saccharomyces cerevisiae to salt or sorbitol stress has been well studied. These studies have yielded valuable data on how the yeast adapts to these stress conditions. However, S. cerevisiae is a saccharophilic fungus and in its natural environment this yeast encounters high concentrations of sugars. For the production of dessert wines, the sugar concentration may be as high as 50% (w/v). The metabolic pathways in S. cerevisiae under these fermentation conditions have not been studied and the transcriptional response of this yeast to sugar stress has not been investigated. High-density DNA microarrays showed that the transcription of 589 genes in an industrial strain of S. cerevisiae were affected more than two-fold in grape juice containing 40% (w/v) sugars (equimolar amounts of glucose and fructose). High sugar stress up-regulated the glycolytic and pentose phosphate pathway genes. The PDC6 gene, previously thought to encode a minor isozyme of pyruvate decarboxylase, was highly induced under these conditions. Gene expression profiles indicate that the oxidative and non-oxidative branches of the pentose phosphate pathway were up-regulated and might be used to shunt more glucose-6-phosphate and fructose-6-phosphate, respectively, from the glycolytic pathway into the pentose phosphate pathway. Structural genes involved in the formation of acetic acid from acetaldehyde, and succinic acid from glutamate, were also up-regulated. Genes involved in de novo biosynthesis of purines, pyrimidines, histidine and lysine were down-regulated by sugar stress.

Transcriptional profiling of wine yeast in fermenting grape juice: regulatory effect of diammonium phosphate.

The nitrogen composition of grape musts affects fermentation kinetics and production of aroma and spoilage compounds in wine. It is common practice in wineries to supplement grape musts with diammonium phosphate (DAP) to prevent nitrogen-related fermentation problems. Laboratory strains of Saccharomyces cerevisiae preferentially use rich nitrogen sources, such as ammonia, over poor nitrogen sources. We used global gene expression analysis to monitor the effect of DAP addition on gene expression patterns in wine yeast in fermenting Riesling grape must. The expression of 350 genes in the commercial wine yeast strain VIN13 was affected; 185 genes were down-regulated and 165 genes were up-regulated in response to DAP. Genes that were down-regulated encode small molecule transporters and nitrogen catabolic enzymes, including those linked to the production of urea, a precursor of ethyl carbamate in wine. Genes involved in amino acid metabolism, assimilation of sulfate, de novo purine biosynthesis, tetrahydrofolate one-carbon metabolism, and protein synthesis were up-regulated. The expression level of 86 orphan genes was also affected by DAP.