Transformation of Wine Yeasts with the Saccharomyces Pombe Malate Permease and L. Lactic Malolactic Genes


Project Number
MV 01

Project title
Transformation of wine yeasts with the Saccharomyces pombe malate permease and L. lactic malolactic genes

Project leader
Bloom, M

Institution
University of Stellenbosch. Department of Microbiology

Team members
Volschenk, H
Van Staden, J

Project description
The production of well balanced wines requires the reduction of excess acidity, especially in the colder regions of the world. Strains of Saccharomyces cerevisiae metabolise only small and insignificant amounts of l-malic acid and therefore most red wines and some white wines are subjected to the process of bacterial malolactic fermentation (MLF) during or soon after alcoholic fermentation. Lactic acid bacteria, primarily strains of Oenococcus oeni, are added to the wine and this results in the decarboxylation of l-malic acid to l-lactic acid and carbon dioxide. Malolactic fermentation reduces the levels of titratable acidity, increases the pH of the wine and leads to enhanced microbial stability of the wine. However, a major disadvantage of malolactic fermentation is the unpredictability of its occurrence and control during vinification.

The lack of a malic acid transporter and the low substrate affinity of the malic enzyme renders the wine yeast Saccharomyces cerevisiae unable to degrade malic acid efficiently. In contrast, the fission yeast Schizosaccharomyces pombe is able to effectively degrade up to 20 g/l malic acid due to the active transport of malate and a malic enzyme with a very high substrate affinity. We were able to co-express the malate transporter gene (mae1) and the malic enzyme gene (mae2) from Schizosaccharomyces pombe in a laboratory strain of Saccharomyces. cerevisiae. The recombinant strain could degrade up to 8 g/l l malic acid in synthetic and 6.75 g/l l malic acid in Chardonnay grape must and produced higher levels of ethanol during the fermentation. Co-expression of the mae1 gene and the Oenococcus oeni malolactic gene (mleA) in Saccharomyces cerevisiae resulted in a malolactic yeast strain that degraded 8 g/l malic acid in less than three days in synthetic grape must.

The mae1-mae2 expression cassette was subsequently transformed and integrated into several wine yeast strains of Saccharomyces cerevisiae and Saccharomyces bayanus. The recombinant malo-ethanolic yeasts were evaluated for their ability to successfully remove malic acid during alcoholic fermentation in synthetic and natural grape must, as well as for their sensory quality. The four different malo-ethanolic yeast strains and their corresponding parental strains (negative controls) were inoculated in Cabernet Sauvignon and Chardonnay grape must. All further treatments of the red and white fermentations were according to standard procedures for small-scale winemaking. All the recombinant yeast strains tested were able to degrade the malic acid in the Chardonnay must within 70 hours, while the malic acid in Cabernet Sauvignon was removed within 31 hours. Organoleptic evaluation of the Chardonnay wines indicated that, although undesired off-flavours were not detected, the complete absence of malic acid in the wine resulted in an acid imbalance in the wine.

Presentation(s)
Van Vuuren, H J J, Grobler, J, Viljoen, M, Volschenk, H and Bauer, F F. 1994. Genetic construction of wine yeast strains for the utilisation of malate in grape must. Paper presented at the 20th National Congress of the South African Society for Enology and Viticulture. 10-11 November, Cape Town, South Africa.

Volschenk, H, Viljoen, M, Grobler, J, Bauer, F F, Subden R E and Van Vuuren, H J J. 1995. Genetic engineering of a pathway for malate degradation in Saccharomyces cerevisiae. Presentation at the Postgraduate Biotechnology Day. University of the Western Cape, Bellville, South Africa.

Van Vuuren, H J J, Viljoen, M, Grobler, J, Volschenk, H, Bauer F and Subden, R E. 1995. Engineering a pathway for malate degradation in Saccharomyces cerevisiae. Paper presented at the NCP TECHKNOW Conference, Scottburgh, South Africa.

Van Vuuren, H J J, Viljoen, M, Grobler, J, Volschenk, H, Bauer, F F and Subden, R E. 1995. Genetic analysis of the Schizo-saccharomyces pombe malate permease (mae1) and malic enzyme (mae2) genes and their expression in Saccharomyces cerevisiae. Paper presented at the International Enology Symposium. Bordeaux, France.

Van Vuuren, H J J, Viljoen, M, Grobler, J, Volschenk, H, Bauer, F F and Subden, R E. 1995. Genetic analysis of the Schizosaccharomyces pombe malate permease (mae1) and malic enzyme (mae2) genes and their expression in Saccharomyces cerevisiae. Paper presented 1st International Congress of the South African Society for Enology and Viticulture. 8-10 November, Cape Town, South Africa.

Bauer, F F, Van Vuuren, H J J, Viljoen, M, Grobler, J, Volschenk, H and Subden, R E. 1995. Engineering a pathway for malate degradation in Saccharomyces cerevisiae. Paper presented at the All African Conference on Biotechnology, Pretoria, South Africa.

Van Vuuren, H J J, Viljoen, M, Grobler, J, Volschenk, H, Bauer, F F, Subden, R E, Denayrolles, M and Lonvaud, A. 1996. Malolactic fermentation in grape must by a genetically engineered strain of Saccharomyces cerevisiae. Paper presented at the 4th International Symposium of the Cool Climate Enology and Viticulture. 16-20 July, New York, United States.

Van Vuuren, H J J, Viljoen, M, Grobler, J, Volschenk, H, Bauer, F F, Subden R E, Denayrolles M and Lonvaud A. 1996. Malolactic fermentation in grape must by a genetically engineered strain of Saccharomyces cerevisiae. Paper presented at the 9th International Specialised Symposium on Yeast. Sydney, Australia.

Volschenk, H, Viljoen, M, Grobler, J, Bauer, F F, Subden, R E, Denayrolles, M, Lonvaud, A and Van Vuuren, H J J. 1996. Engineering pathways for malate degradation in Saccharomyces cerevisiae. Paper presented at the 9th Conference of the South African Society for Microbiology. Pretoria, South Africa.

Volschenk, H, Viljoen, M, Grobler, J, Bauer, F F, Subden, R E, Denayrolles, M, Lonvaud, A and Van Vuuren, H J. 1997. Malolactic fermentation in grape must by a genetically engineered strain of Saccharomyces cerevisiae. Paper presented at the 2nd Conference on Biotechnology and Development in Southern Africa. Grahamstown, South Africa.

Volschenk, H, Viljoen, M, Grobler, J, Bauer, F, Subden, R E, Denayrolles, M, Lonvaud, A and Van Vuuren, H J J. 1997 Development of malolactic and malo-alcoholic strains of Saccharomyces cerevisiae. Paper presented at the 21st National Congress of South African Society for Enology and Viticulture. 15-17 November, Cape Town, South Africa.

Volschenk, H, Viljoen, M, Grobler, J, Bauer, F F, Subden, R E, Denayrolles, M, Lonvaud, A and Van Vuuren, H J J. 1997. Malolactic fermentation in grape must by a genetically engineered strain of Saccharomyces cerevisiae. Paper presented at the 18th International Conference on Yeast Genetics and Molecular Biology. Stellenbosch, South Africa.

Viljoen, M, Volschenk, H and Van Vuuren, H J J. 1997. Malo-alcoholic and malolactic yeast strains: A dream or reality? Paper presented at the 22nd National Congress of the South African Society for Enology and Viticulture. 12-13 November, Stellenbosch, South Africa.

Bauer, F F, Van Vuuren, H J J, Viljoen, M, Grobler, J, Volschenk, H and Subden, R E. 1998. Engineering pathways for malate degradation in wine yeast. Paper presented at the 18th Great Lakes Mycology Workshop. St Catherines, Canada.

Volschenk, H, Viljoen, M, Grobler, J, Lonvaud-Funel, A, Denayrolles, M, Subden, R E and Van Vuuren, H J J. 1998. Genetic construction of malo-ethanolic and malolactic strains of S. cerevisiae. Paper presented at the 10th Australian Wine Industry Technical Conference. 2-5 August 1998, Sydney, Australia .

Volschenk, H, Viljoen, M, Husnik, J and Van Vuuren, H J J. 1998. Malate degradation by recombinant strains of Saccharomyces cerevisiae. Paper presented at the 23rd National Congress of the South African Society for Enology and Viticulture. 12-13 November, Stellenbosch, South Africa.

Volschenk, H, Viljoen, M and Van Vuuren, H J J. 1999. Construction of recombinant strains of Saccharomyces cerevisiae for malo-ethanolic and malolactic fermentation. Paper presented at the 29th International Conference on Yeast Genetics and Molecular Biology. Rimini, Italy.

Viljoen-Bloom, M, Volschenk, H, Husnik, J and Van Vuuren, H J J. 2000. Wine fermentation by malo-ethanolic strains of Saccharomyces cerevisiae. Paper presented at the 10th International Symposium on Yeast. Arnhem, The Netherlands.

Volschenk, H, Viljoen-Bloom, M and Van Vuuren, H J J. 2000. Wine fermentation by malo-ethanolic strains of Saccharomyces cerevisiae. Paper presented at the 2nd International Congress of the South African Society for Enology and Viticulture. 8-10 November, Cape Town, South Africa.

Volschenk, H, Viljoen, M, Husnik, J and Van Vuuren, H J J. 2000. Malate degradation by recombinant strains of Saccharomyces cerevisiae. Paper presented at BIOY2K Combined Millennium Meeting. 23-28 January 2000, Grahamstown, South Africa.

Article
Volschenk, H, Viljoen, M, Grobler, J, Bauer, F F, Lonvaud-Funel, A, Denayrolles, M, Subden, R E, Van Vuuren, H J J. 1997. Malolactic fermentation of grape musts by a genetically engineered strain of Saccharomyces cerevisiae, American Journal of Enology and Viticulture, v. 48 (2) (p. 193-197)

Volschenk, H, Viljoen, M, Grobler, J, Petzold, B, Bauer, F F, Subden, R E, Young, R, A, Lonvaud-Funel, A, Denayrolles, M, Van Vuuren, H J J. 1997. Engineering pathways for malate degradation in Saccharomyces cerevisiae, Nature Biotechnology, v. 15 (3) (p. 253-257)

Saayman, M, Van Vuuren, H J J, Van Zyl, W H, Viljoen-Bloom, M. 2000. Differential uptake of fumarate by C. utilis and S. pombe, Applied Microbiology and Biotechnology, v. 54 (6) (p. 792-798)

Redzepovic, S, Orlic, S, Kozina, B, Majdak, A, Volschenk, H, Viljoen-Bloom, M. 2003. Differential malic acid degradation by selected strains of Saccharomyces during alcoholic fermentation, International Journal of Food Microbiology, v. 83 (1) (p. 49-61)

Volschenk, H, Van Vuuren, H J J, Viljoen-Bloom, M. 2003. Malo-ethanolic fermentation in Saccharomyces and Schizosaccharomyces – a review, Current Genetics, v. 294 (p. 379-391)

Volschenk, H, Viljoen-Bloom, M, Van Staden, J, Husnik, J, Van Vuuren, H J J. 2004. Genetic engineering of an industrial strain of Saccharomyces bayanus for malic acid degradation via an efficient malo-ethanolic pathway, South African Journal of Enology and Viticulture, v. 25 (2) (p. 63-73)

Van Staden, J, Volschenk, H, Van Vuuren, H J J, Viljoen-Bloom, M. 2005. Malic acid distribution and degradation in grape must during skin contact: The influence of recombinant malo-ethanolic wine yeast strains, South African Journal of Enology and Viticulture, v. 26 (1) (p. 16-20)
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