Browsing by Author "Van Staden, J."

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    Genetic engineering of an industrial strain of saccharomyces cerevisiae for l-malic acid degradation via an efficient malo-ethanolic pathway
    (South African Society of Enology & Viticulture, 2004) Volschenk, H.; Bloom, M.; Van Staden, J.; Husnik, J.; Van Vuuren, H. J. J.
    The optimal ratio of L-malic and L-tartaric acid in relation to other wine components is one of the most important aspects that ultimately determine wine quality during winemaking. Winemakers routinely rely on the judicious use of malolactic fermentation (MLF) after alcoholic fermentation to deacidify and stabilise their wines. However, due to the unreliability of the process and unsuitable sensory modifications in some grape cultivars, especially for fruity-floral wines, MLF is often regarded as problematic and undesirable. Alternative methods for reducing the amounts of L-malic acid in wine will contribute to improving the production of quality wines in the future, especially in coolclimate regions. Most wine yeast strains of Saccharomyces are unable to effectively degrade L-malic acid, whereas the fission yeast Schizosaccharomyces pombe efficiently degrades high concentrations of L-malic acid by means of malo-ethanolic fermentation. However, strains of S. pombe are not suitable for vinification due to the production of undesirable off-flavours. Previously, the 5. pombe malate permease (mael) and malic enzyme (mae2) genes were successfully expressed under the 3-phosphoglycerate kinase (PGK1) regulatory elements in 5. cerevisiae, resulting in a recombinant laboratory strain of S. cerevisiae with an efficient malo-ethanolic pathway. Stable integration of the S. pombe malo-ethanolic pathway genes has now been obtained through the construction of a unique integration strategy in a commercial wine yeast strain. Co-transformation of the linear integration cassette containing the mael and mae2 genes and PGK1 regulatory elements and a multi-copy plasmid containing the phleomycin-resistance marker into a commercial Saccharomyces cerevisiae strain resulted in the successful transformation and integration of the malo-ethanolic genes. The recombinant 5. cerevisiae strain was successfully cured of phleomycin-resistance plasmid DNA in order to obtain malo-ethanolic yeast containing only yeast-derived DNA. The integrated malo-ethanolic genes were stable in 5. cerevisiae and during synthetic and grape must fermentation, L-malic acid was completely fermented to ethanol without any negative effect on fermentation kinetics and wine quality.
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    Malic acid distribution and degradation in grape must during skin contact : the influence of recombinant malo-ethanolic wine yeast strains
    (South African Society for Enology and Viticulture, 2005) Van Staden, J.; Volschenk, H.; Van Vuuren, H. J. J.; Viljoen-Bloom, M.
    Wine acidity plays an important role in determining wine quality and ensuring physiochemical and microbiological stability. In high-acid wines, the L-malic acid concentration is usually reduced through bacterial malolactic fermentation, while acidulation in low-acidity wines is usually done during final blending of the wine before bottling. This study showed that skin contact did not influence the relative concentration of L-malic acid in the pulp and juice fractions from Colombard, Ruby Cabernet and Cabernet Sauvignon grape musts, with 32%-44% of the L-malic acid present in the pulp fraction. Four recombinant malo-ethanolic (ME) Saccharomyces wine yeast strains containing the malic enzyme (mae2) and malate transporter (mael) genes of Schizasaccharomyces pombe, effectively degraded the L-malic acid in both the juice and pulp fractions of all three cultivars, with a complete degradation of malic acid in the juice fraction within 2 days.
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    Transformation of potato (cv. Late Harvest) with the potato leafroll virus coat protein gene, and molecular analysis of transgenic lines
    (Academy of Science for South Africa, 1998) Murray, S. L.; Burger, J. T.; Oelofse, D.; Cress, W. A.; Van Staden, J.; Berger, D. K.
    Potato leafroll virus (PLRV) is one of the most destructive potato viruses in South Africa. In order to establish resistance against PLRV in the potato cultivar Late Harvest, the coat protein (CP) gene of a South African isolate of the virus was isolated, cloned into the plant transformation vector pBI121 and inserted into potatoes using Agrobacterium tumefaciens-mediated transformation. Six plantlets, which appeared to be phenotypically normal, were regenerated from leaf disks under kanamycin selection. These lines were analysed for stable transgene insertion and expression. The presence of the PLRV CP, uidA (GUS) and nptII (kanamycin resistance) genes were shown using PCR! Southern blot analysis verified that the PLRV CP gene had been inserted into the genome of the transgenic potato lines. Coat protein could not be detected, but RNA dot blots demonstrated PLRV CP gene expression at the mRNA level. Expression of the uidA gene was investigated using a fluorometric assay, and it was observed that lines containing the PLRV CP gene in the antisense orientation exhibited GUS activity.