Coexpression of α-l-arabinofuranosidase and ß-glucosidase in Saccharomyces cerevisiae.

Monoterpenes are important aroma compounds in grape varieties such as Muscat, Gewürztraminer and Riesling, and are present as either odourless, glycosidically bound complexes or free aromatic monoterpenes. Commercial enzymes can be used to release the monoterpenes, but they commonly consist of crude extracts that often have unwanted and unpredictable side-effects on wine aroma. This project aims to address these problems by the expression and secretion of the Aspergillus awamoriα-l-arabinofuranosidase in combination with either the ß-glucosidases from Saccharomycopsis fibuligera or from Aspergillus kawachii in the industrial yeast Saccharomyces cerevisiae VIN13. The concentration of five monoterpenes was monitored throughout alcoholic fermentation of Gewürztraminer grapes. The recombinant yeast strains that caused an early boost in the geraniol concentration led to a reduction in the final geraniol levels due to the downregulation of the sterol biosynthetic pathway. Monoterpene concentrations were also analysed 9 and 38 days after racking and the performance of the VB2 and VAB2 recombinant strains was similar, and in many cases, better than that of a commercial enzyme used in the same experiment. The results were backed by sensorial analysis, with the panel preferring the aroma of the wines produced by the VAB2 strain.

Epigenetic regulation of PGU1 transcription in Saccharomyces cerevisiae.

The PGU1 gene of Saccharomyces cerevisiae has been shown to encode a polygalacturonase. The polygalacturonase activity in S. cerevisiae is strain specific. There are no significant differences in the PGU1 promoter regions of strains with and without polygalacturonase activity. The PGU1 gene is subtelomeric because it is located within 25 kb of the right telomere of chromosome X. Expressions of genes located in subtelomeric regions in the yeast S. cerevisiae are inhibited compared with the rest of the genome. In this study, we showed that the deletion of genes involved in telomere silencing enhances polygalacturonase activity. PGU1 transcription and polygalacturonase activity are increased when PGU1 is shifted to a different location in the genome, away from the telomere located close to this gene, and the depletion of the histone H4 leads to an increase in PGU1 transcription. We concluded that PGU1 is silenced in strains without polygalacturonase activity due to an epigenetic effect. The results of this study suggest that PGU1 is silenced by being folded into a heterochromatin-like structure at its subtelomeric position on chromosome X. Formation of this silent structure is dependent on the Isw2p chromatin remodeling complex, its histone fold motif containing subunit Dls1p and the N-terminal tail of the H4 histone.

Regulation of endo-polygalacturonase activity in Saccharomyces cerevisiae.

Pectolytic activity in Saccharomyces cerevisiae is due to the secretion of an endo-polygalacturonase encoded by the PGU1 gene. The ability to degrade polygalacturonic acid has been shown to vary between different strains. In this study, we attempted to elucidate how pectolytic activity is regulated in S. cerevisiae and to determine whether the means of regulation differ between strains. Saccharomyces cerevisiae strains from different genetic backgrounds, with varying ability to degrade pectin, were compared. Activity was found not to be regulated by sequence differences in the PGU1 gene, but by the transcription level of the gene. Expression of PGU1 was found to be determined by the transcription level of its two transcription factors TEC1 and STE12. The activation of PGU1 transcription by galactose was found to be strain specific, independent of the strain being an industrial or a domesticated one. The EUROSCARF yeast deletion library was screened for genes encoding inhibitors and activators of polygalacturonase activity. Fourteen strains were identified, in which deletion of a specific gene resulted in a recovery of polygalacturonase activity; these genes were identified as encoding inhibitors of polygalacturonase activity, and two activators were identified.

Establishing a risk-assessment process for release of genetically modified wine yeast into the environment.

The use and release of genetically modified organisms (GMOs) is an issue of intense public concern and, in the case of food and beverages, products containing GMOs or products thereof carry the risk of consumer rejection. The recent commercialization of 2 GM wine yeasts in the United States and Canada has made research and development of risk assessments for GM microorganisms a priority. The purpose of this study was to take a first step in establishing a risk-assessment process for future use and potential release of GM wine yeasts into the environment. The behaviour and spread of a GM wine yeast was monitored in saturated sand columns, saturated sand flow cells, and conventional flow cells. A widely used commercial Saccharomyces cerevisiae wine yeast, VIN13, a VIN13 transgenic strain (LKA1, which carries the LKA1 alpha-amylase gene of Lipomyces kononenkoae), a soil bacterium (Dyadobacter fermentens), and a nonwine soil-borne yeast (Cryptococcus laurentii) were compared in laboratory-scale microcosm systems designed to monitor microbial mobility behaviour, survival, and attachment to surfaces. It was found that LKA1 cells survived in saturated sand columns, but showed little mobility in the porous matrix, suggesting that the cells attached with high efficiency to sand. There was no significant difference between the mobility patterns of LKA1 and VIN13. All 3 yeasts (VIN13, LKA1, and C. laurentii) were shown to form stable biofilms; the 2 S. cerevisiae strains either had no difference in biofilm density or the LKA1 biofilm was less dense than that of VIN13. When co-inoculated with C. laurentii, LKA1 had no negative influence on the breakthrough of the Cryptococcus yeast in a sand column or on its ability to form biofilms. In addition, LKA1 did not successfully integrate into a stable mixed-biofilm community, nor did it disrupt the biofilm community. Overall, it was concluded that the LKA1 transgenic yeast had the same reproductive success as VIN13 in these 3 microcosms and had no selective advantage over the untransformed parental strain.

Characterization of selected South African young cultivar wines using FTMIR spectroscopy, gas chromatography, and multivariate data analysis.

The powerful combination of analytical chemistry and chemometrics and its application to wine analysis provide a way to gain knowledge and insight into the inherent chemical composition of wine and to objectively distinguish between wines. Extensive research programs are focused on the chemical characterization of wine to establish industry benchmarks and authentication systems. The aim of this study was to investigate the volatile composition and mid-infrared spectroscopic profiles of South African young cultivar wines with chemometrics to identify compositional trends and to distinguish between the different cultivars. Data were generated by gas chromatography and FTMIR spectroscopy and investigated by using analysis of variance (ANOVA), principal component analysis (PCA), and linear discriminant analysis (LDA). Significant differences were found in the volatile composition of the cultivar wines, with marked similarities in the composition of Pinotage wines and white wines, specifically for 2-phenylethanol, butyric acid, ethyl acetate, isoamyl acetate, isoamyl alcohol, and isobutyric acid. Of the 26 compounds that were analyzed, 14 had odor activity values of >1. The volatile composition and FTMIR spectra both contributed to the differentiation between the cultivar wines. The best discrimination model between the white wines was based on FTMIR spectra (98.3% correct classification), whereas a combination of spectra and volatile compounds (86.8% correct classification) was best to discriminate between the red wine cultivars.

PGU1 gene natural deletion is responsible for the absence of endo-polygalacturonase activity in some wine strains of Saccharomyces cerevisiae.

The PGU1 gene encodes an endo-polygalacturonase enzyme in Saccharomyces cerevisiae. The literature reports that most S. cerevisiae strains possess this gene, despite a wide range of enzyme activity levels. Nevertheless, a few wine strains lack the PGU1 gene. We investigated the PGU1 locus sequence in these strains. The results indicated that the gene had been replaced by a partial Ty mobile element, whereas the gene promoter was still at the expected location. As all the strains lacking the PGU1 gene experienced the same phenomenon, it was tempting to hypothesize a common phylogenetic origin. However, fingerprints only allowed grouping of a few of them within one cluster.

Enhancing volatile phenol concentrations in wine by expressing various phenolic acid decarboxylase genes in Saccharomyces cerevisiae.

Phenolic acids, which are generally esterified with tartaric acid, are natural constituents of grape must and wine and can be released as free acids (principally p-coumaric, caffeic, and ferulic acids) by certain cinnamoyl esterase activities during the wine-making process. Some of the microorganisms present in grape can metabolize the free phenolic acids into 4-vinyl and 4-ethyl derivatives. These volatile phenols contribute to the aroma of wine. The Saccharomyces cerevisiae phenyl acrylic acid decarboxylase gene (PAD1) is steadily transcribed, but its encoded product, Pad1p, shows low activity. In contrast, the phenolic acid decarboxylase (PADC) from Bacillus subtilis and the p-coumaric acid decarboxylase (PDC) from Lactobacillus plantarum display substrate-inducible decarboxylating activity in the presence of phenolic acids. In an attempt to develop wine yeasts with optimized decarboxylation activity on phenolic acids, the padc, pdc, and PAD1 genes were cloned under the control of S. cerevisiae's constitutive phosphoglyceratekinase I gene promoter (PGK1(P)()) and terminator (PGK1(T)()) sequences. These gene constructs were integrated into the URA3 locus of a laboratory strain of S. cerevisiae, Sigma1278b. The overexpression of the two bacterial genes, padc and pdc, in S. cerevisiae showed high enzyme activity. However, this was not the case for PAD1. The padc and pdc genes were also integrated into an industrial wine yeast strain, S. cerevisiae VIN13. As an additional control, both alleles of PAD1 were disrupted in the VIN13 strain. In microvinification trials, all of the laboratory and industrial yeast transformants carrying the padc and pdc gene constructs showed an increase in volatile phenol formation as compared to the untransformed host strains (Sigma1278b and VIN13). This study offers prospects for the development of wine yeast starter strains with optimized decarboxylation activity on phenolic acids and the improvement of wine aroma in the future.

Cloning and characterization of a second alpha-amylase gene (LKA2) from Lipomyces kononenkoae IGC4052B and its expression in Saccharomyces cerevisiae.

Lipomyces kononenkoae secretes a battery of highly effective amylases (i.e. alpha-amylase, glucoamylase, isoamylase and cyclomaltodextrin glucanotransferase activities) and is therefore considered as one of the most efficient raw starch-degrading yeasts known. Previously, we have cloned and characterized genomic and cDNA copies of the LKA1 alpha-amylase gene from L. kononenkoae IGC4052B (CBS5608T) and expressed them in Saccharomyces cerevisiae and Schizosaccharomyces pombe. Here we report on the cloning and characterization of the genomic and cDNA copies of a second alpha-amylase gene (LKA2) from the same strain of L. kononenkoae. LKA2 was cloned initially as a 1663 bp cDNA harbouring an open reading frame (ORF) of 1496 nucleotides. Sequence analysis of LKA2 revealed that this ORF encodes a protein (Lka2p) of 499 amino acids, with a predicted molecular weight of 55,307 Da. The LKA2-encoded alpha-amylase showed significant homology to several bacterial cyclomaltodextrin glucanotransferases and also to the alpha-amylases of Aspergillus nidulans, Debaryomyces occidentalis, Saccharomycopsis fibuligera and Sz. pombe. When LKA2 was expressed under the control of the phosphoglycerate kinase gene promoter (PGK1(p)) in S. cerevisiae, it was found that the genomic copy contained a 55 bp intron that impaired the production of biologically active Lka2p in the heterologous host. In contrast to the genomic copy, the expression of the cDNA construct of PGK1p-LKA2 in S. cerevisiae resulted in the production of biologically active alpha-amylase. The LKA2-encoded alpha-amylase produced by S. cerevisiae exhibited a high specificity towards substrates containing alpha-1,4 glucosidic linkages. The optimum pH of Lka2p was found to be 3.5 and the optimum temperature was 60 degrees C. Besides LKA1, LKA2 is only the second L. kononenkoae gene ever cloned and expressed in S. cerevisiae. The cloning, characterization and co-expression of these two genes encoding these highly efficient alpha-amylases form an important part of an extensive research programme aimed at the development of amylolytic strains of S. cerevisiae for the efficient bioconversion of starch into commercially important commodities.