Assessing the occurrence and mechanisms of horizontal gene transfer during wine making

[摘要] ENGLISH ABSTRACT: Saccharomyces cerevisiae is the most commonly used organism in many fermentation-basedindustries including baking and the production of single cell proteins, biofuel and alcoholicbeverages. In the wine industry, a consumer driven demand for new and improved products hasfocussed yeast research on developing strains with new qualities. Tremendous progress in theunderstanding of yeast genetics has promoted the development of yeast biotechnology andsubsequently of genetically modified (GM) wine yeast strains. The potential benefits of suchGM wine yeast are numerous, benefitting both wine makers and consumers. However, thesafety considerations require intense evaluation before launching such strains into commercialproduction. Such assessments consider the possibility of the transfer of newly engineered DNAfrom the originally modified host to an unrelated organism. This process of horizontal genetransfer (HGT) creates a potential hazard in the use of such organisms. Although HGT hasbeen extensively studied within the prokaryotic domain, there is an urgent need for similarstudies on their eukaryotic counterparts. This study was therefore undertaken to help improveour understanding of this issue by investigating HGT in a model eukaryotic organism through astep-by-step approach. In a first step, this study attempted to determine whether large DNAfragments are released from fermenting wine yeast strains and, in a second step, to assess thestability of released DNA within such a fermenting background. The third step investigated inthis study was to establish whether 'free floating DNA within this fermenting environment couldbe accepted and functionally expressed by the fermenting yeast cultures. Finally, wholeplasmid transfer was also investigated as a unified event. Biofilms were also incorporated intothis study as they constitute a possibly conducive environment for the observation of such HGTevents.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 GMcommercial wine yeast strain (Parental strain: Vin13), no DNA could be detected within thefermenting background, suggesting that such DNA fragments were not released in largenumbers. Secondly, the study revealed remarkable stability of free 'floating DNA under thesefermentation conditions, identifying intact DNA of up to ~1kb in fermenting media for up to 62days after it had been added. Thirdly, the data demonstrate the uptake and functionalexpression of spiked DNA by fermenting Vin13 cultures in grape must. Here, anotherinteresting discovery was made, since it appears that the fermenting natural grape must favoursDNA 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 thatonly the antibiotic resistance marker was maintained through genomic integration. Identificationof the sites of integration showed the sites varied from one HGT event to the next, indicatingthat integration occurred through a process known as illegitimate recombination. Finally, weprovide 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 conditionsinvestigated. To our knowledge, this is the first report of direct horizontal DNA transfer betweenorganisms of the same species in eukaryotes. Furthermore, while the occurences of suchevents appears low in number, it cannot be assumed that HGT will not occur more frequentlywithin an industrial scenario, making industrial scale studies similar to this one paramountbefore drawing further conclusions.

[发布机构] Stellenbosch University