Marius G. Lambrechts

Screening for the production of extracellular hydrolytic enzymes by non-Saccharomyces wine yeasts

Aims: The objective of this study was to investigate what types of enzymes are being produced by non‐Saccharomyces yeasts isolated from grapes in South Africa vineyards and clarified grape juice. These enzyme profiles could pave the way for attributing specific effects in wine to some of these enzymes produced by so‐called wild yeasts associated with grape must.

Metabolic engineering of Saccharomyces cerevisiae for the synthesis of the wine-related antioxidant resveratrol

The stilbene resveratrol is a stress metabolite produced by Vitis vinifera grapevines during fungal infection, wounding or UV radiation. Resveratrol is synthesised particularly in the skins of grape berries and only trace amounts are present in the fruit flesh. Red wine contains a much higher resveratrol concentration than white wine, due to skin contact during fermentation. Apart from its antifungal characteristics, resveratrol has also been shown to have cancer chemopreventive activity and to reduce the risk of coronary heart disease. It acts as an antioxidant and anti-mutagen and has the ability to induce specific enzymes that metabolise carcinogenic substances. The objective of this pilot study was to investigate the feasibility of developing wine yeasts with the ability to produce resveratrol during fermentation in both red and white wines, thereby increasing the wholesomeness of the product. To achieve this goal, the phenylpropanoid pathway in Saccharomyces cerevisiae would have to be introduced to produce p-coumaroyl-CoA, one of the substrates required for resveratrol synthesis. The other substrate for resveratrol synthase, malonyl-CoA, is already found in yeast and is involved in de novo fatty-acid biosynthesis. We hypothesised that production of p-coumaroyl-CoA and resveratrol can be achieved by co-expressing the coenzyme-A ligase-encoding gene (4CL216) from a hybrid poplar and the grapevine resveratrol synthase gene (vst1) in laboratory strains of S. cerevisiae. This yeast has the ability to metabolise p-coumaric acid, a substance already present in grape must. This compound was therefore added to the synthetic media used for the growth of laboratory cultures. Transformants expressing both the 4CL216 and vst1 genes were obtained and tested for production of resveratrol. Following beta-glucosidase treatment of organic extracts for removal of glucose moieties that are typically bound to resveratrol, the results showed that the yeast transformants had produced the resveratrol beta-glucoside, piceid. This is the first report of the reconstruction of a biochemical pathway in a heterologous host to produce resveratrol.

The effect of increased yeast alcohol acetyltransferase and esterase activity on the flavour profiles of wine and distillates

The fruity odours of wine are largely derived from the synthesis of esters and higher alcohols during yeast fermentation. The ATF1‐ and ATF2‐encoded alcohol acetyltransferases of S. cerevisiae are responsible for the synthesis of ethyl acetate and isoamyl acetate esters, while the EHT1‐encoded ethanol hexanoyl transferase is responsible for synthesizing ethyl caproate. However, esters such as these might be degraded by the IAH1‐encoded esterase. The objectives of this study were: (a) to overexpress the genes encoding ester‐synthesizing and ester‐degrading enzymes in wine yeast; (b) to prepare Colombard table wines and base wines for distillation using these modified strains; and (c) to analyse and compare the ester concentrations and aroma profiles of these wines and distillates. The overexpression of ATF1 significantly increased the concentrations of ethyl acetate, isoamyl acetate, 2‐phenylethyl acetate and ethyl caproate, while the overexpression of ATF2 affected the concentrations of ethyl acetate and isoamyl acetate to a lesser degree. The overexpression of IAH1 resulted in a significant decrease in ethyl acetate, isoamyl acetate, hexyl acetate and 2‐phenylethyl acetate. The overexpression of EHT1 resulted in a marked increase in ethyl caproate, ethyl caprylate and ethyl caprate. The flavour profile of the wines and distillates prepared using the modified strains were also significantly altered as indicated by formal sensory analysis. This study offers prospects for the development of wine yeast starter strains with optimized ester‐producing capability that could assist winemakers in their effort to consistently produce wine and distillates such as brandy to definable flavour specifications and styles. Copyright

Msn1p/Mss10p, Mss11p and Muc1p/Flo11p are part of a signal transduction pathway downstream of Mep2p regulating invasive growth and pseudohyphal differentiation in Saccharomyces cerevisiae

In Saccharomyces cerevisiae, a network of signal transduction pathways governs the switch from yeast‐type growth to pseudohyphal and invasive growth that occurs in response to nutrient limitation. Important elements of this network have been identified, including nutrient signal receptors, GTP‐binding proteins, components of the pheromone‐dependent MAP kinase cascade and several transcription factors. However, the structural and functional mapping of these pathways is far from complete. Here, we present data regarding three genes, MSN1/MSS10, MSS11 and MUC1/FLO11which form an essential part of the signal transduction network establishing invasive growth. Both MSN1 and MSS11 are involved in the co‐regulation of starch degradation and invasive growth. Msn1p and Mss11p act downstream of Mep2p and Ras2p and regulate the transcription of both STA2 and MUC1. We show that MUC1 mediates the effect of Msn1p and Mss11p on invasive growth. In addition, our results suggest that the activity of Msn1p is independent of the invasive growth MAP kinase cascade, but that Mss11p is required for the activation of pseudohyphal and invasive growth by Ste12p. We also show that starch metabolism in S. cerevisiae is subject to regulation by components of the MAP kinase cascade.

The occurrence of malolactic fermentation in brandy base wine and its influence on brandy quality

Aims: In this study we determined the extent to which lactic acid bacteria (LAB) occurred in brandy base wines, their ability to catalyse the malolactic fermentation (MLF) and the effect of MLF on the quality of the base wine and the brandy distillate.

MSS11, A NOVEL YEAST GENE INVOLVED IN THE REGULATION OF STARCH METABOLISM

Abstract Expression of the STA1-3 glucoamylase genes, responsible for starch degradation in Saccharomyces cerevisiae, is down regulated by the presence of STA10. In order to elucidate the role of STA10 in the regulation of the glucoamylase system, a multicopy genomic library was constructed and screened for genes that enhanced growth of a STA2-STA10 S. cerevisiae strain on starch media. This screen allowed us to clone and characterize a novel activator gene of STA2 (and by extrapolation, STA1 and STA3), designated MSS11. A strain transformed with multiple copies of MSS11 exhibits increased levels of STA2 mRNA and, consequently, increased glucoamylase activity. Deletion of MSS11, located on chromosome XIII, results in media-dependent absence of glucoamylase synthesis. MSS11 has not been cloned previously and the encoded protein, Mss11p, is not homologous to any other known protein. An outstanding feature of Mss11p is that the protein contains regions of 33 asparagine residues interrupted by only three serine residues, and 35 glutamine residues interrupted by a single histidine residue. Epistasis studies showed that deletion of MSS11 abolishes the activation of STA2 caused by the over-expression of MSS10, a previously identified gene. In turn, it was found that deletion of MSS10 still allows activation of STA2 by over-expression of MSS11. Mss11p therefore appears to be positioned below Mss10p in a signal transduction pathway.

A multicopy suppressor gene, MSS10, restores STA2 expression in Saccharomyces cerevisiae strains containing the STA10 repressor gene

Abstract Transcription of the three unlinked, homologous STA1–3 glucoamylase-encoding genes, involved in starch degradation by Saccharomyces cerevisiae, was previously shown to be down-regulated by the presence of STA10, acting via three upstream repression sequence regions that were identified in the STA2 promoter. Here we report the cloning and characterization of a putative transcriptional activator gene, MSS10 (multicopy suppressor of STA10), which, when present in multiple copies, overcomes STA10 repression. Deletion of MSS10, located on chromosome XV, resulted in media-specific extinction of glucoamylase synthesis. The nucleotide sequence of MSS10 is identical to three other genes from S. cerevisiae identified as: FUP1, a gene that enhances iron-limited growth; PHD2, a gene identified for its ability to induce pseudohyphal growth in diploid cells grown on nitrogen-limited media; and MSN1, a gene encoding a transcriptional activator involved in invertase regulation.

Multiple positive and negative cis-acting elements of the STA2 gene regulate glucoamylase synthesis in Saccharomyces cerevisiae.

Expression of the glucoamylase-encoding gene (STA2) in Saccharomyces cerevisiae was previously shown to be regulated transcriptionally by both positive and negative factors. The objective of this work was to identify the cis-acting elements responsible for STA2 transcriptional activation as well as the transcriptional repressor effects of STA10 and MATa/MAT alpha. We identified two upstream activation regions (UAS). Three repressor regions responsive to STA10-mediated repression were identified, as well as two regions for down-regulation of STA2 expression. MATa/MAT alpha repression appears to effect STA2 expression either downstream from the translational start site or, indirectly, since no functional a1/alpha 2-responsive sequence was identified in the promoter region.

The heterologous expression of polysaccharidase-encoding genes with oenological relevance in Saccharomyces cerevisiae

Aims:  The main objective of this study was to develop polysaccharide‐degrading wine strains of Saccharomyces cerevisiae, which are able to improve aspects of wine processing and clarification, as well as colour extraction and stabilization during winemaking.

The influence of different winemaking techniques on the extraction of grape tannins and Anthocyanins

The aim of this study was to determine the effect of different maceration techniques on the extraction of grape tannins and anthocyanins. Two cultivars (Cabernet Sauvignon and Shiraz) were harvested in two different climatic regions (Durbanville and Simondium) at two different ripeness levels for the 2008 and 2009 harvest seasons. Five basic winemaking processes were applied, namely a normal alcoholic fermentation (C), enzyme treatment (E), cold soaking (CM), post-maceration (PM), and a combination of cold and post-maceration (CM+PM). At harvest the phenolic ripeness was determined with the Glories method, while the tannin concentration was determined with the methyl cellulose (MCP) method. The grapes in the warmer area had higher tannin levels than grapes harvested in the cooler area in both years. In the 2009 harvest season, the enzyme treatment extracted the highest levels of tannin. CM+PM showed the best results of tannin extraction with early ripeness (Cabernet Sauvignon), and CM with fuller ripeness in the warm area. CM showed the best results with both early and fuller ripeness levels in the cooler area. PM showed the best results with the early ripeness levels, and the E treatment with the fuller ripeness levels, in the warm area. CM+PM showed the best results with the early ripeness level in the cooler area, and varied results with the fuller ripeness levels. In both years, grapes from the cooler area contained more anthocyanin than those from the warmer area. At a fuller ripeness level (2009) the treatments had no effect.