Modifying and Improving Yeast to Increase Wine Processing Efficiency


Project Number
IWBT 05-03

Project title
Modifying and improving yeast to increase wine processing efficiency

Project leader
Bauer, F F

Institution
University of Stellenbosch. Faculty of AgriSciences. Institute for Wine Biotechnology

Team members
Arensburg, J
Louw, C
Bester, M J
Govender, P
Franken, C J
Van Rensburg, P

Project description
The program focuses on two major aspects of the improvement of wine yeast strains: (i) The secretion of enzymes that facilitate certain processing steps and (ii) the control of flocculation. (i) Saccharomyces cerevisiae does not degrade glucans and xylans efficiently. Commercial preparations of these enzymes are utilised during winemaking with the aim of improving the clarification and processing of wine, releasing varietal aromas from precursor compounds and to increase colour intensity. Some of the gene coding for these enzymes has been successfully expressed in laboratory strains of Saccharomyces cerevisiae. In this study we integrated different combinations of glucanase and xylanase gene cassettes into the genome of the wine yeast strain, VIN13, and compared the ability of these recombinant strains to utilise these carbon sources and their influence on the clarification and processing steps during the winemaking process. Four new plasmid constructs have been completed and transformed into the yeast VIN13. Analysis of the transformants indicates that the respective enzymes are secreted and form clear halos on defined media. Vinifications using these modified yeasts have commenced.

To analyse the control of flocculation, two projects are currently under way: In a first project, the pathways that regulate cellular differentiation in response to nutrient availability are investigated. The understanding of these pathways and of the underlying regulatory processes will lead to new strategies and approaches in future strain development programmes. In this process, we have cloned several genes that are involved in the regulation of gene expression in response to nutrient limitation. In some strains, the response to nutrient limitation leads to flocculation, whereas in others, the response typically involves cell elongation, the formation of pseudohyphae and the invasion of agar-containing medium. FLO11, encoding a cell surface protein, is critical for these processes. We have identified numerous genes that are critical for these processes. Previous work showed that Mss11p regulates FLO11 and performs a central regulatory role in relation to other regulatory factors and pathways. Currently, we further characterise Mss11p function by assessing the ability of Mss11p to induce invasive growth and flocculation, as well as its effect on cell shape. Furthermore, we identify additional phenotypes associated with different Mss11p expression levels. In particular, we show that differential expression of MSS11 affects growth in media containing fermentable or non-fermentable carbon sources. We also show that a deletion in MSS11 leads to the suppression of a severe growth defect on non-fermentable carbon sources of yeast from the genetic background carrying the hyperactive RAS2val19 allele. Our biochemical data indicate that Mss11p is a 97 kDa nuclear protein maintained in the cell at low levels.

In a second project, we have fused stationary phase specific promoters to dominant flocculation genes, in order to assess the possibility to design yeast strains with a flocculation behaviour that is optimal for wine fermentations. We have constructed numerous combinations of promoters and dominant flocculation genes, and assessed the consequences of these constructs on yeast flocculation. The data shows that it is possible to regulate flocculation as desired.

Presentation(s)
Bester, M C, Franken, C J and Bauer, F F. 2003. Regulation of Ca2+-dependant flocculation by the Saccharomyces cerevisiae transcriptional regulator Mss11p. Paper presented at the 21st International Conference on Yeast Genetics and Molecular Biology. 7-12 July 2003, Göteborg, Sweden

Bester, M C, Franken, C J, Van Dyk, D, Gagiano, M, Pretorius, I S and Bauer, F F. 2004. Regulation of Ca2+ dependant flocculation by the Saccharomyces cerevisiae transcriptional regulator Mss11p. Paper presented at the 13th South African Society for Microbiology Conference. 9-12 April 2004, Stellenbosch, South Africa.

Louw, C, Le Grange, D, Pretorius, I S and Van Rensburg, P. 2004. Challenging traditional winemaking practices with yeast biotechnological innovations. Paper presented at the 13th South African Society for Microbiology Conference. 9-12 April 2004, Stellenbosch, South Africa.

Louw, C, Le Grange, D, Pretorius, I S and Van Rensburg, P. 2004. Improving wine processing with polysaccharide-degrading wine yeast strains. Paper presented at the 19th International Symposium Food Micro 2004. 12-16 September, Portoroz, Slovenia.

Louw, C, Le Grange, D, Pretorius, I S and Van Rensburg, P. 2004. The development of polysaccharide-degrading wine yeast strains. Paper presented at 12th Australian Wine Industry Technical Conference. 24-29 July, Melbourne, Victoria.

Louw, C, Le Grange, D, Pretorius, I S and Van Rensburg, P. 2005. The development of polysaccharide-degrading wine yeast strains. IUMS 2005 – Microbes in a Changing World, San Francisco, United States

Bester, M C, Cordero Otero, R R, Pretorius, I S and Bauer, F F. 2005. The regulatory role of Mss11p in Ca2+-dependent flocculation and haploid invasive growth in Saccharomyces cerevisiae. Paper presented at the 22nd International Conference of Yeast Genetics and Molecular Biology. 7-12 August, Bratislava, Slovakia.

Louw, C T, Le Grange, D, Pretorius, I S and Van Rensburg, P. 2005. The development of polysaccharide-degrading wine yeast strains. Paper presented at the IUMS 2005 – Microbes in a Changing World. San Francisco, United States.

Divol, B T and van Rensburg, P. 2005. Pectinolytic activity and wine yeast? The 3rd International Congress of the South African Society for Enology and Viticulture. 7-8 November, Cape Town, South Africa.

Bester, M C and Bauer, F F. 2006. FLO gene regulation in the yeast Saccharomyces cerevisiae. Paper presented at the 14th Biennial Congress of the South African Society for Microbiology. 9-12 April, Pretoria, South Africa
14th Biennial Congress of the South African Society for Microbiology, South Africa

Bauer, F F, Næs, T, Esbensen, K H, Young, P R, Du Toit, M and Vivier, M A. 2007. Functional Wine-omics. Paper presented at the 13th Australian Wine Industry Technical Conference. 28 July – 2 August, Adelaide, Australia.

Article
Gagiano, M, Bauer, F F, Pretorius, I S. 2002. The sensing and signaling of nutritional status and the relationship to filamentous growth in the yeast Saccharomyces cerevisiae, FEMS Yeast Research, v. 2 (4) (p. 433-470)

Gagiano, M, Bester, M, Van Dyk, D, Franken, C J, Bauer, F F, Pretorius, I S. 2003. Mss11p is a transcription factor regulating pseudohyphal differentiation, invasive growth and starch metabolism in Saccharomyces cerevisiae in response to nutrient availability, Molecular Microbiology, Mnth Jan v. 47 (1) (p. 119-134)

Bayly, F C, Douglas, L M, Pretorius, I S, Bauer, F F, Dranginis, A M. 2005. Characteristics of FLO11-dependent flocculation in Saccharomyces cerevisiae, FEMS Yeast Research, v. 5 (p. 1151-1156)

Bester, M C, Pretorius, I S, Bauer, F F. 2006. The regulation of Saccharomyces cerevisiae FLO gene expression and Ca2+-dependent flocculation by Flo8p and Mss11p, Current Genetics, v. 49 (6) (p. 375-383)

Louw, C, La Grange, D, Pretorius, I S, Van Rensburg, P. 2006. The effect of polysaccharide-degrading wine yeast transformants on the efficiency of wine processing and wine flavour, Journal of Biotechnology, v. 125 (p. 447-461)

Louw, C T, La Grange, D, Pretorius, I S. 2006. The effect of polysaccharide degrading wine yeast transformants on the efficiency of wine processing and wine flavour, Journal of Biotechnology, v. 125 (p. 447-461)

Divol, B, Van Rensburg, P. 2007. PGU1 gene natural deletion is responsible for the absence of endo-polygalacturonase activity in some wine strains of Saccharomyces cerevisiae, FEMS Yeast Research, Mnth Dec v. 7 (8) (p. 1328-1339)

Van Rensburg, P, Strauss, M L A, Lambrechts, M G, Cordero Otero, R R, Pretorius, I S. 2007. The heterologous expression of polysaccharidase-encoding genes with enological relevance in Saccharomyces cerevisiae, Journal of Applied Microbiology, v. 103 (6) (p. 2248-2257)

Govender, P, Pretorius, I S, Bauer, F F. 2008. Controlled expression of the dominant flocculation genes FLO1, FLO5 and FLO11 in Saccharomyces cerevisiae, Applied and Environmental Microbiology, Mnth Oct v. 74 (19) (p. 6041-6052)

Paper
Louw, C, La Grange, D, Pretorius, I S, Van Rensburg, P. 2005. The development of polysaccharide degrading wine yeast strains, IN: Blair, R (ed). Proceedings of the 12th Australian Wine Industry Technical Conference. 24-29 July 2004, Melbourne, Australia. (p. 294) Australian Wine Research Institute, Adelaide, Australia.

Bauer, F F, Naes, T, Esbensen, K H, Young, P R, Du Toit, M, Vivier, M A. 2007. Functional wine-omics, IN: Blair, R and Williams, P J (eds). 2008. Proceedings of the 13th Australian Wine Industry Technical Conference. 28 July-2 August 2007, Adelaide, Australia. (p. 173-183) Australian Wine Research Institute, Adelaide, Australia.

Thesis
Louw, C T. 2005. The development polysaccharide degrading wine yeast strains. MSc. University of Stellenbosch, Stellenbosch.

FinalReport.pdf

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