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Biotechnology Journal

3

Yeast molecular biology and biotechnology

The IWBT has a long history of research projects on improving yeast strains for specific applications using molecular genetic and breeding technology [17]. Yeast projects are focused on establishing a better fundamental understanding of yeast cellular and molecular biology, and to apply this knowledge to generate new wine yeast strains.Topics of inves- tigation include gene regulation and signal trans- duction networks, the molecular nature of cell wall- related phenotypes, metabolic regulation and the secretion of enzymes (see Fig. 2 for examples of genes isolated and studied in the yeast pro- gramme).

These fundamental projects feed into the biotechnology program, which uses traditional methodologies of breeding, selection, directed evo- lution and molecular modification. Applied proj- ects in the past focused on engineering yeasts with specific enzymes useful in the degradation of di- verse polysaccharides [18–21], studying the prop- erties of yeast adhesion phenotypes [22–28] that are of interest during fermentation processes in yeast during winemaking [29], the genetics of car- bohydrate source utilisation (e.g. starch, cellulose) [30–33], the metabolic engineering of aroma pro- duction pathways [34, 35], carnitine production [36, 37] and risk assessment related to the use of genet- ically modified wine yeast strains [38].

The IWBT has combined traditional technolo- gies with directed evolution to generate new strains of industrial relevance. Directed evolution refers to the application of specific selection pressures over many generations of yeast growth usually in a con- tinuous fermentation chemostat system. In such a situation strains that evolve advantageous adapta- tions to the selection pressure out-compete less well adapted strains. Through this approach the IWBT has been able to generate yeast strains with specific properties such as improved nitrogen effi- ciency or fructose utilisation. The success of the yeast breeding programme is evident in the num- ber of valuable strains produced by the IWBT for industrial applications. An example being the suc- cessfulVIN13 Saccharomyces cerevisiae strain mar- keted by Anchor Yeast and used in wine fermenta- tions both in South Africa and abroad.

Although traditional breeding methods provide useful strains of industrial relevance, this particu- lar approach is limited in two major ways. Firstly, such traditional methods can only improve pre-ex- isting characteristics present in the yeast, not gen- erate new traits that may be desirable. Secondly, ex- perience has shown that even traits within yeast can be improved up to a specific point while further

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© 2008 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

Biotechnol. J. 2008, 3, 1355–1367

improvement is only possible through the integra- tion of additional genetic material. A further disad- vantage of these technologies is their inherent ran- domness, which makes outcomes unpredictable. It is also difficult to breed strains for traits that do not have an easily selectable character as is in the case of strains with specific aroma production capabili- ties. To overcome these limitations, techniques of molecular biology and genetic engineering are em- ployed.

The specific aims of the current yeast strain de- velopment programme integrates systems biology- based approaches, and also continues to pursue strategies based on traditional breeding or genetic modification technology. The programme includes many of the areas that have been identified as be- ing of commercial relevance for the yeast and wine industries. The programme can be subdivided into five major research themes, these being: (i) pro- ducing yeast with greater fermentation efficiency, (ii) improving wine processing and filtration, (iii) developing yeast that improve the aroma and flavour of wine, (iv) increasing the wholesomeness of wine, and (v) improving wine preservation. Each of these programmes support the main thrust of the yeast biotechnology programme, which is to generate and assess new yeast strains of potential value to the South African wine industry.

A focus of the current program is the generation of yeast that would yield lower levels of ethanol. South Africa is a warm climate region with the re- sult that harvested grapes produce on average higher levels of sugar than cool climate wine re- gions. This results in wine with higher-than-aver- age alcohol levels due to the greater amount of fer- mentable sugar in the must. Export markets are more favourable to moderate alcohol levels in wine and thus the ability to control alcohol production is necessary. To this end the IWBT developed a strat- egy in which the enzyme glucose oxidase is engi- neered into yeast to convert the excess sugar (glu- cose) present during fermentation into glucono- lactone and so prevent excessive alcohol produc- tion [39]. In addition, during the fermentation of must, numerous partially soluble complex plant polysaccharides are extracted into the wine medi- um. These polysaccharides are viscous and tend to block filters as well as impede processing steps during winemaking.The general type of plant poly- saccharides present can be divided into cellulose, hemicellulose and pectin polymers.A genetic engi- neering solution to these problems consisted of en- gineering polysaccharide-degrading enzymes such as polygalacturonases, xylanases and cellulases into yeast using molecular secretion systems [19–21, 40].Thus, yeast are able to ferment the must

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