Yeasts are predominant in the ancient and complex process of winemaking. In spontaneous fermentations, there is a progressive growth pattern of indigenous yeasts, with the final stages invariably being dominated by the alcohol-tolerant strains of Saccharomyces cerevisiae. This species is universally known as the ‘wine yeast’ and is widely preferred for initiating wine fermentations. The primary role of wine yeast is to catalyze the rapid, complete and efficient conversion of grape sugars to ethanol, carbon dioxide and other minor, but important, metabolites without the development of off-flavours. However, due to the demanding nature of modern winemaking practices and sophisticated wine markets, there is an ever-growing quest for specialized wine yeast strains possessing a wide range of optimized, improved or novel oenological properties. This review highlights the wealth of untapped indigenous yeasts with oenological potential, the complexity of wine yeasts’ genetic features and the genetic techniques often used in strain development. The current status of genetically improved wine yeasts and potential targets for further strain development are outlined. In light of the limited knowledge of industrial wine yeasts’ complex genomes and the daunting challenges to comply with strict statutory regulations and consumer demands regarding the future use of genetically modified strains, this review cautions against unrealistic expectations over the short term. However, the staggering potential advantages of improved wine yeasts to both the winemaker and consumer in the third millennium are pointed out. Copyright # 2000 John Wiley & Sons, Ltd.

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Tailoring wine yeast for the new millennium: novel approaches to the ancient art of winemaking

Wine is a highly complex mixture of compounds which largely define its appearance, aroma, flavour and mouth-feel properties. The compounds responsible for those attributes have been derived in turn from three major sources, viz. grapes, microbes and, when used, wood (most commonly, oak). The grape-derived compounds provide varietal distinction in addition to giving wine its basic structure. Thus, the floral monoterpenes largely define Muscat-related wines and the fruity volatile thiols define Sauvignon-related wines; the grape acids and tannins, together with alcohol, contribute the palate and mouth-feel properties. Yeast fermentation of sugars not only produces ethanol and carbon dioxide but a range of minor but sensorially important volatile metabolites which gives wine its vinous character. These volatile metabolites, which comprise esters, higher alcohols, carbonyls, volatile fatty acids and sulfur compounds, are derived from sugar and amino acid metabolism. The malolactic fermentation, when needed, not only provides deacidification, but can enhance the flavour profile.

The aroma and flavour profile of wine is the result of an almost infinite number of variations in production, whether in the vineyard or the winery. In addition to the obvious, such as the grapes selected, the winemaker employs a variety of techniques and tools to produce wines with specific flavour profiles. One of these tools is the choice of microorganism to conduct fermentation. During alcoholic fermentation, the wine yeast Saccharomyces cerevisiae brings forth the major changes between grape must and wine: modifying aroma, flavour, mouth-feel, colour and chemical complexity. The wine bacterium Oenococcus oeni adds its contribution to wines that undergo malolactic fermentation. Thus flavour-active yeasts and bacterial strains can produce desirable sensory results by helping to extract compounds from the solids in grape must, by modifying grape-derived molecules and by producing flavour-active metabolites. This article reviews some of the most important flavour compounds found in wine, and their microbiological origin.

Yeast and bacterial modulation of wine aroma and flavour

The winemaking process includes two main steps: lactic acid bacteria are responsible for the malolactic fermentation which follows the alcoholic fermentation by yeasts. Both types of microorganisms are present on grapes and on cellar equipment. Yeasts are better adapted to growth in grape must than lactic acid bacteria, so the alcoholic fermentation starts quickly. In must, up to ten lactic acid bacteria species can be identified. They belong to the Lactobacillus, Pediococcus, Leuconostoc and Oenococcus genera. Throughout alcoholic fermentation, a natural selection occurs and finally the dominant species is O. oeni, due to interactions between yeasts and bacteria and between bacteria themselves. After bacterial growth, when the population is over 106CFU/ml, malolactic transformation is the obvious change in wine composition. However, many other substrates can be metabolized. Some like remaining sugars and citric acid are always assimilated by lactic acid bacteria, thus providing them with energy and carbon. Other substrates such as some amino acids may be used following pathways restricted to strains carrying the adequate enzymes. Some strains can also produce exopolysaccharides. All these transformations greatly influence the sensory and hygienic quality of wine. Malic acid transformation is encouraged because it induces deacidification. Diacetyl produced from citric acid is also helpful to some extent. Sensory analyses show that many other reactions change the aromas and make malolactic fermentation beneficial, but they are as yet unknown. On the contrary, an excess of acetic acid, the synthesis of glucane, biogenic amines and precursors of ethylcarbamate are undesirable. Fortunately, lactic acid bacteria normally multiply in dry wines; moreover some of these activities are not widespread. Moreover, the most striking trait of wine lactic acid bacteria is their capacity to adapt to a hostile environment. The mechanisms for this are not yet completely elucidated. Molecular biology has provided some explanations for the behaviour and the metabolism of bacteria in wine. New tools are now available to detect the presence of desirable and undesirable strains. Even if much remains unknown, winemakers and oenologists can nowadays better control the process. By acting upon the diverse microflora and grape musts, they are more able to produce healthy and pleasant wines.

Lactic acid bacteria in the quality improvement and depreciation of wine

Immobilization technologies and support materials suitable in alcohol beverages production: a review

Comprehensive knowledge regarding Saccharomyces cerevisiae has accumulated over time, and today S. cerevisiae serves as a widley used biotechnological production organism as well as a eukaryotic model system. The high transformation efficiency, in addition to the availability of the complete yeast genome sequence, has facilitated genetic manipulation of this microorganism, and new approaches are constantly being taken to metabolicially engineer this organism in order to suit specific needs. In this paper, strategies and concepts for metabolic engineering are discussed and several examples based upon selected studies involving S. cerevisiae are reviewed. The many different studies of metabolic engineering using this organism illustrate all the categories of this multidisciplinary field: extension of substrate range, improvements of producitivity and yield, elimination of byproduct formation, improvement of process performance, improvements of cellular properties, and extension of product range including heterologous protein production.

Metabolic Engineering of Saccharomyces cerevisiae

Deacidification of grape musts is crucial for the production of well-balanced wines, especially in colder regions of the world. The major acids in wine are tartaric and malic acid. Saccharomyces cerevisiae cannot degrade malic acid efficiently due to the lack of a malate transporter and the low substrate affinity of its malic enzyme. We have introduced efficient pathways for malate degradation in S. cerevisiae by cloning and expressing the Schizosaccharomyces pombe malate permease (mae1) gene with either the S. pombe malic enzyme (mae2) or Lactococcus lactis malolactic (mleS) gene in this yeast. Under aerobic conditions, the recombinant strain expressing the mae1 and mae2 genes efficiently degraded 8 g/L of malate in a glycerol-ethanol medium within 7 days. The recombinant malolactic strain of S. cerevisiae (mae1 and mleS genes) fermented 4.5 g/L of malate in a synthetic grape must within 4 days.

Engineering pathways for malate degradation in Saccharomyces cerevisiae

Malate enters Saccharomyces cerevisiae by simple diffusion. Due to the lack of a malate transporter and the low affinity of the S. cerevisiae malic enzyme, this yeast is unable to degrade malate efficiently. We have constructed a malolactic yeast strain by co-expressing the malate permease gene ( mae1 ) of the fission yeast Schizosaccharomyces pombe and the Lactococcus lactis malolactic gene ( mle S) in S. cerevisiae . The recombinant strain of S. cerevisiae transported malate and actively metabolized malate to lactate within three days in Cabernet Sauvignon and Shiraz grape musts at 20°C. The malolactic fermentation in Chardonnay grape must was completed within seven days at 15°C. The efficient degradation of malate in grape musts is important to wineries and the availability of malolactic yeasts will allow the early application of cellar operations for storage and aging of wine.

Malolactic Fermentation in Grape Musts by a Genetically Engineered Strain of Saccharomyces cerevisiae

Sequence analysis of a 4.6-kb HindIII fragment containing the malic enzyme gene (mae2) of Schizosaccharomyces pombe, revealed the presence of an open reading frame of 1695 nucleotides, coding for a 565 amino acid polypeptide. The mae2 gene is expressed constitutively and encodes a single mRNA transcript of 2.0 kb. The mae2 gene was mapped on chromosome III by chromoblotting. The coding region and inferred amino acid sequence showed significant homology with 12 malic enzyme genes and proteins from widely different origins. Eight highly homologous regions were found in these malic enzymes, suggesting that they contain functionally conserved amino acid sequences that are indispensable for activity of malic enzymes. Two of these regions have previously been reported to be NAD- and NADP-binding sites.

M. Viljoen

Papers

Engineering pathways for malate degradation in Saccharomyces cerevisiae

Malolactic Fermentation in Grape Musts by a Genetically Engineered Strain of Saccharomyces cerevisiae

Molecular analysis of the malic enzyme gene (mae2) of Schizosaccharomyces pombe

Mutational analysis of malate pathways in Schizosaccharomyces pombe

Transcriptional regulation of the Schizosaccharomyces pombe malic enzyme gene, mae2.