Thesis (PhD (Microbiology))--University of Stellenbosch, 2009. / ENGLISH ABSTRACT: Saccharomyces cerevisiae is the most commonly used organism in many fermentation-based
industries including baking and the production of single cell proteins, biofuel and alcoholic
beverages. In the wine industry, a consumer driven demand for new and improved products has
focussed yeast research on developing strains with new qualities. Tremendous progress in the
understanding of yeast genetics has promoted the development of yeast biotechnology and
subsequently of genetically modified (GM) wine yeast strains. The potential benefits of such
GM wine yeast are numerous, benefitting both wine makers and consumers. However, the
safety considerations require intense evaluation before launching such strains into commercial
production. Such assessments consider the possibility of the transfer of newly engineered DNA
from the originally modified host to an unrelated organism. This process of horizontal gene
transfer (HGT) creates a potential hazard in the use of such organisms. Although HGT has
been extensively studied within the prokaryotic domain, there is an urgent need for similar
studies on their eukaryotic counterparts. This study was therefore undertaken to help improve
our understanding of this issue by investigating HGT in a model eukaryotic organism through a
step-by-step approach. In a first step, this study attempted to determine whether large DNA
fragments are released from fermenting wine yeast strains and, in a second step, to assess the
stability of released DNA within such a fermenting background. The third step investigated in
this study was to establish whether “free floating” DNA within this fermenting environment could
be accepted and functionally expressed by the fermenting yeast cultures. Finally, whole
plasmid transfer was also investigated as a unified event. Biofilms were also incorporated into
this study as they constitute a possibly conducive environment for the observation of such HGT
events.
The results obtained during this study help to answer most of the above questions. Firstly,
during an investigation into the possible release of large DNA fragments (>500 bp) from a GM
commercial wine yeast strain (Parental strain: Vin13), no DNA could be detected within the
fermenting background, suggesting that such DNA fragments were not released in large
numbers. Secondly, the study revealed remarkable stability of free “floating DNA” under these
fermentation conditions, identifying intact DNA of up to ~1kb in fermenting media for up to 62
days after it had been added. Thirdly, the data demonstrate the uptake and functional
expression of spiked DNA by fermenting Vin13 cultures in grape must. Here, another
interesting discovery was made, since it appears that the fermenting natural grape must favours
DNA uptake when compared to synthetic must, suggesting the presence of carrier molecules.
Additionally, we found that spiked plasmid DNA was not maintained as a circular unit, but that
only the antibiotic resistance marker was maintained through genomic integration. Identification
of the sites of integration showed the sites varied from one HGT event to the next, indicating
that integration occurred through a process known as illegitimate recombination. Finally, we
provide evidence for the direct transfer of whole plasmids between Vin13 strains.
The overall outcome of this study is that HGT does indeed occur under the conditions
investigated. To our knowledge, this is the first report of direct horizontal DNA transfer between
organisms of the same species in eukaryotes. Furthermore, while the occurences of such
events appears low in number, it cannot be assumed that HGT will not occur more frequently
within an industrial scenario, making industrial scale studies similar to this one paramount
before drawing further conclusions. / NO AFRIKAANS SUMMARY AVAILABLE
| Identifer | oai:union.ndltd.org:netd.ac.za/oai:union.ndltd.org:sun/oai:scholar.sun.ac.za:10019.1/1216 |
| Date | 12 1900 |
| Creators | Barnard, Desire |
| Contributors | Bauer, Florian F., Wolfaardt, Gideon M., University of Stellenbosch. Faculty of Science. Dept. of Microbiology. |
| Source Sets | South African National ETD Portal |
| Language | English |
| Detected Language | English |
| Type | Thesis |
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