VAAM-Jahrestagung 2012 18.–21. März in Tübingen

239YEV7-FGThe genetics of ester synthesis in Hanseniaspora uvarumduring winemakingS. Fischer 1 , E. Sieber 2 , Z. Zhang 2 , J. Heinisch 3 , C. von Wallbrunn* 11 Geisenheim Research Center, Department of Microbiology andBiochemistry, Geisenheim, Germany2 Hochschule RheinMain, Fachbereich Geisenheim, Geisenheim, Germany3 University of Osnabrück, Department of Genetics, Faculty of Biology,Osnabrück, GermanyIt is well known that the so called Non-Saccharomycetes have a stronginfluence on the chemical composition and the sensorical quality of wines.Hanseniaspora uvarum is a common yeast found in the early stages ofspontaneous wine fermentations but also sometimes in starter culturesinoculated fermentations. Depending on the phytosanitary status of thegrapes in a vineyard during ripening up to 90% of a yeast population at thebeginning of fermentation can belong to this species. In some cases,fermentations with strains of this organism show interestingly positivebouquets affected by positive esters flavours. But it is also known thatfermentations with high amounts of H. uvarum during the beginning canbe characterized by high amounts of acidic acid and the resulting ethylacetate ester, both typical off flavours in wine.The composition and pH of musts, availability of nutrients for yeastgrowth, the temperature during fermentation and viticultural andoenological methods are parameters which can influence differentpathways of the yeast metabolism involved in the production of flavoursand aromas, for example esters (Lilly et al., 2000)In S. cerevisiae, esters are produced by two alcohol acyltransferases ATF1and ATF2 and an acyl-coenzyme A: ethanol O-acyltransferase EEB1.These enzymes and genes are well characterized.The question is in which metabolic pathway(s) are esters produced by H.uvarum and how potential genes are regulated to show the formerlydescribed observations concerning the ester production in a positive ornegative way.In contrast to S. cerevisiae genomic data of H. uvarum were not availableso far. In a cooperation project with J. Heinisch, Department of Genetics,University of Osnabrück, a type strain was sequenced. Using thesesequences possible candidates of ATF and EEB genes in H. uvarum wereidentified. Derived primers were used to amplify these genes by PCR. ThePCR products were characterized by sequencing and cloned in E. coli. Onepart of the work was to reconstitute the corresponding EuroScarf knockout-mutantsand aftergrape must fermentations the ester formation byanalyzed by GC-MS. Another part of this work was to observe underwhich conditions high amounts of several ester compounds are produced.The next steps will be the development of an efficient transformationprotocol, the generation of knock-out and over expression mutants and theanalysis of the promotor sequences.1. M. Lilly, M.G. Lambrechts, I.S. Pretorius, Yeast 23 (2000), p. 641-659YEV8-FGFeel me, thrill me, kill me - when K. lactis meets S. cerevisiaeR. Schaffrath* 1,2 , C. Bär 1,2 , D. Jablonowski 1,21 Universität Kassel, Institut für Biologie, Abteilung Mikrobiologie, Kassel,Germany2 University of Leicester, Department of Genetics, Leicester, GermanyRecent studies have shown that transfer RNAs (tRNAs) are not onlyessential for decoding messenger RNAs (mRNAs) but also serve aspathorelevant targets for microbial endoribonuclease toxins (ribotoxins)from bacteria, yeast and fungi that cleave within tRNA anticodons andthereby inhibit growth of sensitive target cells. Strikingly, these tRNaseribotoxins ensure survival of their producers against other microbialcompetitors in the same ecological niche and often, their attacks on tRNAslead to cell death by way of tRNA depletion. Antifungal tRNase ribotoxinsinclude the zymocin complex from dairy yeast Kluyveromyces lactis whichkills sensitive cells of baker’s yeast Saccharomyces cerevisiae.Intriguingly, zymocin’s tRNase activity targets tRNA species that possessspecific nucleobase modifications at their anticodon wobble position andthese modifications are functionally conserved among prokaryal andeukaryal organisms. Therefore, our idea was to take the basic biology oftRNase ribotoxins and apply this to cell systems, including HeLa tumourcells, whose proliferation heavily relies on proper tRNA functions inmRNA translation and de novo protein synthesis. Our pilot findingsindicate that expression of the zymocin tRNase from K. lactis not onlykills sensitive cells of S. cerevisiae but also affects the growth and viabilityof higher eukaryal cells including plants and mammals. Hence, weconclude and propose in this session that microbial tRNase ribotoxins maybe invoked as novel anti-proliferative factors for biomedical oragrobiological intervention schemes [1].1. Support of the work to RS by funds from the alumni programme of the Alexander von Humboldtfoundation, Bonn, Germany, Department of Genetics, University of Leicester, UK, theBiotechnology and Biological Sciences Research Council, UK (grant BB/F019106/1) andUniversität Kassel, Germany, is greatly acknowledged.BIOspektrum | Tagungsband 2012