42nd annual conference on yeasts - 46th Annual Conference on Yeasts

ISSN 1336-4839

42ND ANNUAL CONFERENCE ON YEASTS PROGRAMME

ABSTRACTS

19 - 22 May, 2015 Smolenice, Slovakia

42nd ANNUAL CONFERENCE ON YEAST

PROGRAM AND ABSTRACTS

May 19-22 2015 SAS Congress Centre, Smolenice, Slovakia

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42nd Annual Conference on Yeasts

Organized by: Yeast Commission, Institute of Chemistry, Slovak Academy of Sciences, Bratislava Institute of Animal Biochemistry and Genetics, Slovak Academy of Sciences, Ivanka pri Dunaji Faculty of Chemical and Food Technology, Slovak University of Technology, Bratislava Czechoslovak Society for Microbiology, Bratislava

ORGANIZING COMMITTEE I. Hapala, E. Breierová, M. Čertík, J. Molnárová, J. Guthová

PROGRAM COMMITTEE I. Márová, M. Čertík, A. Pichová, I. Hapala, V. Raclavský, E. Paulovičová, H. Sychrová,J. Nosek, Vadkertiová R., Sipický Mathias Edited by: Ivan Hapala, Emília Breierová Reviewed by: Milan Čertík

ISSN: 1336-4839 2

Sponsors Major sponsors

. . . riešenie pre Vaše laboratórium

Sponsors

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Contents

Sponsors ................................................................................... 3 Scientific Programme ................................................................. 6 Poster Highlights session......................................................... 10 Poster session ......................................................................... 11 Abstracts ................................................................................. 14 Index........................................................................................ 83

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Scientific Programme Tuesday, May 19, 2015 Departure of bus to Smolenice (from Bratislava, 13:00 Trnavske myto, parking lot Istropolis via Bus Terminal Mlynske Nivy). 11:00 – 16:30 14:00 - 16:30 16:30 - 17:00 17:00 – 17:30 19:00

Registration Coffee, tea & snacks Opening ceremony R. Vadkertiová (Slovakia): Life with yeasts: Centennary of the birth of Dr. Anna Kocková-Kratochvílová. Welcome reception with folk music and degustation of mead (honey wine) (company Včelco Ltd., Slovakia) and wines from Small Carpathian region (company Fedor Malik & syn, Slovakia)

Wednesday, May 20, 2015 8:00 – 9:00 Breakfast Invited Lecture to the memory of Dr. A. KockováKratochvílová: J. P. Sampaio (Portugal): Taxonomy and diversity: 9:00 – 9:45 Saccharomyces in the XXI century. 9:45 – 10:40 Medical Mycology: (chairs E. Paulovičová, Slovakia, and V. Raclavský, Czech Republic) Invited lecture: 9:45-10:20 T. Boekhout, X.Z. Liu, Q.M. Wang, S. Capella, B. Theelen, M. Groenewald, S. Taj-Aldeen, M. Kostrzewa, T. Gabaldon, F.Y. Bai, A. Kolecká (Netherlands): Yeast biodiversity: towards a new taxonomy, impact for the clinician and identification by MALDI-TOF MS. 10:20 – 10:40 K. Rajkowska, A. Kunicka-Styczyńska, M. Pęczek (Poland) Hydrophobicity of clinical Candida albicans strains at the presence of thyme, clove and tea tree oils. 10:40 – 11:00 Coffee Break

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11:00 – 12:55 11:00 – 11:35 11:35 – 12:10 12:10 – 12:40 12:40 – 12:55 13:00 14:00 – 17:20

14:00 – 14:35 14:35 – 14:55 14:55 – 15:15

15:15 – 15:45 15:45 – 16:20 16:20 – 16:40 16:40 – 17:00

17:00 – 17:15

Ecology and Taxonomy of Yeasts: (chairs: R. Vadkertiová, Slovakia, and M. Sipiczki, Hungary) Invited lecture: N. Čadež (Slovenia): Alternative approaches to delineate novel Ascomycetous yeast species. Invited lecture: G. Péter, D. Dlauchy (Hungary): Methanol-assimilating yeasts, systematics and occurrence. M. Sipiczki (Hungary): Genome chimerisation in Saccharomyces: taxonomic consequences. J. Molnárová, R. Vadkertiová (Slovakia): Yeasts associated with the phyllosphere of fruit trees Lunch Molecular and Cell Biology of the Yeast I (chairs A. Pichová, Czech Republic, and I. Hapala, Slovakia) Invited lecture: P. Ludovico (Portugal): The yeast chronological aging model: proteotoxic stress and age-related dysfunctions J. Mierzejewska, K. Izdebska, M. Bakun, K. Chreptowicz, M. Dadlez (Poland):Searching for proteins interacting with Saccharomyces cerevisiae pyruvate kinase - Pyk1 O. Zimmermanová, V.Llopis-Torregrosa, J. Zemančíková, M. Dušková, H. Sychrová: pHluorin-based measurements of intracellular pH in conventional and non-conventional yeast species. Coffee Break Invited lecture: L. Pryszcz (Spain/Poland): Hybrid nature of pathogenic fungi. D. Wilkinson, V. Stovicek, L.Vachova, Z. Palkova (Czech Republic): Gene copy number and colony morphology in Saccharomyces cerevisiae K. Papoušková M. Andršová, H. Sychrová (Czech Republic): Lack of the putative ion channel Ist2 alters alkalimetal-cation accumulation in Saccharomyces cerevisiae cells Poster Highlights (Short 5-min. talks of young scientists selected from submitted abstracts) 1. D. Lajdová (Slovakia): Systematic screening of a library of Schizosaccharomyces pombe deletion strains for aberrations in mitochondrial morphology 2. K. Chreptowicz (Poland): Maf1 – an effector of RNA polymerase III, influences carbohydrate and lipid metabolism in yeast

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3. P. Nevečeřalová (Czech Republic): Functional study of

17:30 – 19:00

putative K+ transporters Kch1 and Kch2 in Saccharomyces cerevisiae 4. Sec P., Holic R. (Slovakia): High accumulation of cisvaccenic acid reduces the toxicity of unsaturated fatty acid in Saccharomyces cerevisiae strain deficient in storage lipid synthesis Presentation and degustation of wines from Small Carpathian region (company Vino Kmeťo, Slovakia) Dinner

19:00 Thursday, May 21, 2015 8:00 – 9:00 Breakfast Yeast Biotechnology: 9:00 – 12:35 (chairs I. Márová, Czech Republic, M. Čertík, Slovakia) Invited lecture: C. A. Abbas (USA): Biotechnological Developments of 9:00 – 9:45 Yeasts for Biofuels and Biobased Chemicals 9:45 – 10:05 Bucek A., Matoušková P., Svatoš A., Pichová I., (Czech Republic): Functional expression of fatty acid desaturases in Saccharomyces cerevisiae: a tool for engineering of membrane desaturases with new specificities 10:05 – 10:25 S. Landolfo, R. Cutzu, A. Tanca, M. Abbondio, M.F. Addis, M. Deligios, S. Uzzau, G. Mulas, M. Budroni, I. Mannazzu (Italy): Further insights into Rhodotorula carotenogenesis through the application of genomic and proteomic approaches. 10:25 – 10:40 O. Samek, A. Haronikova, K. Hurbanova, S. Bernatova, J. Kanka, J. Jezek, Z. Pilat, V. Kryzyanek, P. Zemanek, I. Marova (Czech Republic): SEM and Raman spectroscopy applied to yeast biomass analysis for the application in the field of biofuels and food industry. 10:40 – 11:00 Coffee Break Invited lecture: D. Porro, P. Branduardi (Italy): Molecular and process 11:00 – 11:40 design for chemicals and proteins production in yeasts 11:40 – 12:00 T. Czabany, K. Schmoelzer, C. Luley-Goedl, D. Ribitsch, S. Zitzenbacher, H. Schwab, B. Nidetzky (Austria): Expression of human glycosyltransferases in yeast 12:00 – 12:15 J. Tkáčová, T. Klempová, M. Čertík (Slovak Republic): Kinetic analysis of carotenoid pigments accumulation in red yeasts.

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12:15 – 12:35

13:00 14:00 - 16:00 16:00 – 16:45 17:30 – 18:30 19:00 – 22:00 Friday, May 22, 2015 8:00 – 9:00 9:30 – 10:15 10:15 – 10:35 10:35 - 11:50

10:35 – 11:10 11:10 - 11:30 11:30 – 11:50

12:00 12:30 13:30

Poster Highlights (Short 5-min. talks of young scientists selected from submitted abstracts) 1. S. Landolfo (Italy): Isolation of TpBGL2 gene of Tetrapisispora phaffii in view of the heterologous expression of a killer toxin of enological interest 2. P. Patelski, (Poland): C.utilis and P.stipits cultivation in sugar beet pulp hydrolysates 3. V. Hlavacek (Czech Republic): Utilization of grape pomace by S. cerevisiae CCY 21-4-47 4. A. Haronikova (Czech Republic): Using modern techniques: fluorescence microscopy and flow cytometry to molecular characterization of red yeasts Lunch Poster Session Coffee Break Presentation and degustation of wines from Small Carpathian region (company Vinko Klimko, Slovakia) Conference dinner with live music

Breakfast Community resources: A. Rieger (BioTek Instruments, USA): Automated Digital Microscopy for Cellular Research Coffee Break Molecular and Cell Biology of the Yeast II (chairs A. Pichová, Czech Republic, and I. Hapala, Slovakia) Invited lecture: J. Nosek, Ľ. Tomáška (Slovakia): Molecular diversity of yeast mitochondrial genomes M. Valachovič, M. Garaiová, I. Hapala (Slovakia) Squalene toxicit in yeast strain devoid of lipid droplets R. Sepšiová, K. Procházková, I. Nečasová, S. Willcox, C. Hofr, J.D. Griffith, J. Nosek, Ľ. Tomáška (Slovakia/Czech Republic/USA): DNA-binding properties of Taz1 and Teb1 proteins of the yeast Schizosaccharomyces pombe Closing ceremony Lunch Departure of bus to Bratislava

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Poster Highlights session

1. Lajdová D. (Slovakia): Systematic screening of a library of Schizosaccharomyces pombe deletion strains for aberrations in mitochondrial morphology 2. Chreptowicz K. (Poland): Maf1 – an effector of RNA polymerase III, influences carbohydrate and lipid metabolism in yeast 3. Nevečeřalová P. (Czech Republic): Functional study of putative K+ transporters Kch1 and Kch2 in Saccharomyces cerevisiae 4. Seč P., Holič R. (Slovakia): High accumulation of cis-vaccenic acid reduces the toxicity of unsaturated fatty acid in Saccharomyces cerevisiae strain deficient in storage lipid synthesis 5. Landolfo S. (Italy): Isolation of TpBGL2 gene of Tetrapisispora phaffii in view of the heterologous expression of a killer toxin of enological interest 6. Patelski, P. (Poland): C.utilis and P.stipits cultivation in sugar beet pulp hydrolysates 7. Hlaváček V. (Czech Republic): Utilization of grape pomace by S. cerevisiae CCY 21-4-47 8. Haroniková A. (Czech Republic): Using modern techniques: fluorescence microscopy and flow cytometry to molecular characterization of red yeasts.

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9. Poster session 1. Bacigálová, K., Petrýdesová, J., Slovák, M., Kučera, J., Sulo, P., Lopandic, K. Taxonomy of Taphrina species in Slovakia 2. Belicová L. and Pichová A. Earlier onset of mitophagy contributes to the prolonged chronological lifespan of ras2Δ yeast strain. 3. Balcerek M., Pielech-Przybylska K., Patelski P. and Dziekońska U. The Yeast used in fermentation of plum mashes and chemical composition of the obtained distillates 4. Balcerek M., Berłowska J. Pielech-Przybylska K., Patelski P. Dziekońska U. Kręgiel D., Dudkiewicz M. and Kalinowska H. Yeast recirculation in the process of ethanolic fermentation of sugar beet pulp hydrolysates 5. Breierová E., Guthová J., Sasinková V., Klempová T., Čertík M. The various distinguishing propertities of Metschnikowia pulcherrima 6. Csáky Z., Zambojová V., Hapala I. Accumulation of squalene in the yeast Saccharomyces cerevisiae with reduced squalene monooxygenase activity 7. Drozdíková E., Bardelčíková A., Geršiová A., Farkaš V., Obernauerová M. Cell wall composition of kluyveromyces lactis mutant deficient in anionic phospholipid synthesis 8. Dziekońska U., Berłowska J., Patelski P., Balcerek M., PielechPrzybylska K. Effect of lignin degradation products on ethanol fermentation by saccharomyces cerevisiae 9. Dudkiewicz M., Berlowska J., Kregiel D., Dziekońska U., Pielech– Przybylska K., Balcerek M. The influence of ultrasounds on release of saccharides during yeast autolysis. 10. Hrušková-Heidingsfeldová O., Mynářová L., Šťovíček V., Vrkoslav V., Palková Z., Pichová I. Transcription profiling of Candida albicans interacting with Pseudomonas aeruginosa 11. Stratilová B., Pavlatovská B., Omelková J., Vadkertiová R. , Stratilová E. Lactose induced galactosidases of genus Cryptococcus.

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12.Stratilová B., Ledvina V., Hamříková D., Jurnečková A.,

Molnárová J., Omelková J., Vadkertiová R., Stratilová E. Optimization of sample preparation for biotyping of Cryptococcus yeasts

13. Gajdoš P., Meravá Z., and Čertík M. In vivo study of Dga1p and Dga2p enzymes controlling triacylglycerol accumulation in Yarrowia lipolytica. 14. Goffa E., Valachovič M. Effective method for composition-specific steryl ester analysis in yeast. 15. Vránová D., Dlapalová K. Isolation and identification of yeasts from interspecific variety Hibernal on the territory of the Moravian wine region 16. Hároniková A., Turková L., Márová I., Production of pigments and carotenoid-rich biomass by red yeasts cultivated on waste substrates from winemaking industry. 17. Kováčová K.,Farkaš V. Properties and inhibition of transglycosylases of the yeast cell wall. 18. Kunicka-Styczyńska A., Rajkowska K., Maroszyńska M. Adhesion abilities of clinical Candida spp. strains to polystyrene 19. Juríková K., Tomáška Ľ., Gabaldón T. De novo gene emergence in yeasts after whole genome duplication. 20. Šimoničová L., Dudeková H., Ferenc J., Procházková K., Neboháčová M., Dušinský R., Nosek, J. and Tomáška Ľ. Saccharomyces cerevisiae as a model to study the role of mammalian telomerase in mitochondria 21. Lipták B., Knezl V., Breierová E., Frimmel K. , Križák J., Okruhlicová Ľ. Effects of yeasts biomass producing carotenoids on rat heart function after endotoxin administration 22. Mikolaskova B., Cipakova I., Chudej T., Sabova L., Anrather D., Ruchman D. , Aronica L. Cipak L. Nrl1 – a new player in the maintenance of genome stability in the Schizosaccharomyces pombe 23. Svrbická A., Balážfyová Z., Tóth Hervay N., Gbelská Y. Transcription factors Yap1p and Pdr1p in the control of multidrug resistance in Kluyveromyces lactis .

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24. Vaněk M, Márová I., Mravec F., Hároniková A. and Vacková H. Application of advanced fluorescence techniques for analysis of red yeasts 25. Rapta, M., Márová I., Hároniková A., Shapaval, V. Use of highthroughput microcultivation and ftir spectroscopy for characterization of metabolic activity of carotenogenic yeasts 26. Patelski P., Balcerek M., Pielech-Przybylska K., Dziekonska U., Berlowska J. Influence of the native yeast on the fermentation of plum mashes.. 27. Pielech-Przybylska K., Berłowska J., Balcerek M., Patelski P., Dziekońska U., Księżopolska M., Dudkiewicz M., Kalinowska H. Ethanol fermentation of sugar beet pulp hydrolysates by Kluyveromyces marxianus and Saccharomyces cerevisiae. 28. Pielech-Przybylska K., Balcerek M., Kotas M., Patelski P., Dziekońska U. Ethanol fermentation of cereal distillery mashes by mono- and mixed cultures of yeast. 29. Raclavský V., Novotný R., Zápalka M., Kopřiva F., Jakubec P., Kolek V. Burkholderia cepacia selective agar can be useful for recovery of Exophiala dermatitidis from sputum samples of cystic fibrosis patients.. 30. Schüngel M., Vadkertiová R., the MIRRI consortium MIRRI – The Microbial Resource Research Infrastructure. 31. Sulo P., Szabóová D., Jatzová K., Bielik P., Šoltýs K., Szemeš T. Cytonuclear compatibility defects in S. cerevisiae xenomitochondrial cybrids are not associated with the alteration in mtDNA sequence 32. Sulo P., Szabóová D., Zelezníková Ž., Bielik P., Šoltýs K., Szemeš T. A complete sequence of mitochondrial genomes from all Saccharomyces reveals species specific gene rearrangement. 33. Sulo P., Szabóová D., Kafková L., Poláková S., Zelezníková Ž.,Šoltýs K., Szemeš T. Yeast mitochondrial genomes with variable number of tRNA genes resulting from duplication deletion model

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Abstracts

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THE 100TH ANNIVERSARY OF THE BIRTH OF DR. ANNA KOCKOVÁ-KRATOCHVÍLOVÁ Vadkertiová R. Institute of Chemistry, Slovak Academy of Sciences, Dúbravská cesta 9, 845 38 Bratislava, Slovakia Dr. Anna Kocková-Kratochvílová, one of the most oustanding personalities of the yeast research in Czech Republic and Slovakia, was born in Tuzla, Bosnia and Herzegovina, in March 2, 1915. Dr. Kocková-Kratochvílová graduated in plant physiology from the Faculty of Natural Sciences, Charles University, Prague, in 1938. She defended her PhD thesis “The significance of fermentation types” in 1962. She received her Dr.Sc. degree in 1968. Dr. Kocková-Kratochvílová started to work in the field of microbiology at the Laboratory of Vitamin and Hormone Chemistry in Prague in 1942. She developed ways of identifying pathogenic yeasts and built up the collection of bacteria, fungi and yeasts. Dr. Kocková-Kratochvílová set up the microbiological laboratories and the collection of yeasts in the Braník brewery and thereafter, in the Research Institute of Brewing and Malting in Prague. When the activity of the Institute terminated, Dr. Kocková-Kratochvílová moved together with the collection to the Department of Microbiology and Biochemistry, Faculty of Chemistry, Slovak Technical University, Bratislava, Slovakia. Finally, in 1963 Dr. KockováKratochvílová transferred the collection to the Institute of Chemistry Slovak Academy of Sciences, where the collection has been located until now, under the name Culture Collection of Yeasts (former Czechoslovak Collection of Yeasts). Dr. Kocková-Kratochvílová’s research work was mainly dedicated to the morphotypization and phenotypization of both industrially and medically important yeasts, and to the numerical taxonomy, biochemical properties, and ecology of yeasts, which were documented in more than 300 research articles, 9 books, and several book chapters. She also discovered some new species belonging to the genus Candida. Dr. Kocková-Kratochvílova’s contribution to yeast research was recognised by the Japanese researchers when the species Rhodosporidium kratochvilovae and the genus Kockovaella were named after her. Dr. Kocková-Kratochvílová also made contact from both Czechoslovakian and abroad researchers and she organized the first International Meeting on Yeasts, in Smolenice in 1964. The meeting was succesful and therefore, two years latter, 140 participants from 27 countries took part in the meeting in Bratislava. At this meeting, the International Commission on Yeast (ICY) was estabilished and Dr. Kocková-Kratochvílová became its first chairperson. From this year, she organized the Annual Conferences on Yeasts and the International Symposia on Yeasts in Smolenice Castle on a regular basis. The Annual conferences still take place there to this day.

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TAXONOMY AND DIVERSITY: SACCHAROMYCES IN THE XXI CENTURY José Paulo Sampaio UCIBIO, Faculdade de Ciências e Tecnologia, Universidade Nova de Lisboa, Portugal

Yeast taxonomy has witnessed profound changes in the last decades. Molecular methods and DNA sequence-based identifications have gradually replaced the phenotypic recognition of species and more and more sophisticated molecular phylogenies have become the irreplaceable backbone for classification proposals. Today, every single yeast species known to science has a DNA barcode sequence publicly available. The most recent revolution concerns the abolition of the artificial dichotomy between sexual and asexual stages. Moreover, ecological surveys do not need to rely strictly on yeast cultivation approaches and as a consequence some yeasts are known only from sequence data. Saccharomyces, the most emblematic, well-known and industrially important yeast will be revisited to illustrate several of the transformations that the study of taxonomy and diversity has seen. Powerful techniques like whole genome sequencing have allowed an unprecedented capacity to analyse local and global genomic changes, thus allowing a better understanding of the evolution of species and of population dynamics and natural history. Nevertheless, and similarly with many other fields in science, the new findings have elicited a myriad of new questions and revealed the fragmentary nature of our present knowledge of yeast biology.

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L3 YEAST BIODIVERSITY: TOWARDS A NEW TAXONOMY, IMPACT FOR THE CLINICIAN AND IDENTIFICATION BY MALDI-TOF MS

Teun Boekhout1,2, Xin-Zhan Liu2, Qi-Ming Wang2, Salvador Capella1,3, Bart Theelen1,Marizeth Groenewald1, Saad Taj-Aldeen4, Markus Kostrzewa5, Toni Gabaldon3,Feng-Yan Bai1,2 Anna Kolecka1 1

CBS Fungal Biodiversity Centre, Utrecht, The Netherlands; 2State Key Laboratory of Mycology, Institute of Microbiology, Chinese Academy of Sciences, Beijing, China; 3Centro Regulación Genómica, Barcelona, Spain; 4Hamad Medical Corporation, Doha, Qatar; 5Bruker Daltonics GmbH, Bremen, Germany Email: [email protected] Our knowledge of yeast biodiversity is rapidly advancing. In the 5th edition of The Yeasts, a Taxonomic Study (2011, eds. C.P. Kurtzman, J.W. Fell & T. Boekhout) ca. 1500 species are described, but presently the number of species described is over 2000. The doubling time of species discovery was about 10 years, but likely will be (much) shorter in the future. Large parts of the biota on earth remain to be sampled with respect to yeast biodiversity. Many of the currently used genera are highly polyphyletic and, consequently, the taxonomy of yeasts needs to be completely revised. The following developments contribute to this new taxonomy: 1. Improved phylogenetic signal in multigene-based or phylogenomics-based phylogenies; 2. The introduction of the one species-one name concept; and 3. Ongoing species discovery that add species to new or sparingly sampled lineages. Attempts in order to make the classification of the yeasts more natural, i.e. in line with phylogenetic principles, are ongoing by comparative multigene sequencing and whole genome sequencing of described yeast species. The changing taxonomy of yeasts will have major consequences for the user, such as biotechnologists and clinicians [just to name two]. Fortunately, techniques such as MALDI-TOF MS allow rapid identification of many yeasts.

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HYDROPHOBICITY OF CLINICAL CANDIDA ALBICANS STRAINS AT THE PRESENCE OF THYME, CLOVE AND TEA TREE OILS

Rajkowska K.1, Kunicka-Styczyńska A.1 and Pęczek M.1 1

Lodz University of Technology, Faculty of Biotechnology and Food Sciences, Institute of Fermentation Technology and Microbiology, Wolczanska 171/173, 90-924 Lodz, Poland Members of Candida species cause significant health problems, inducing various types of superficial and deep-seated mycoses in humans. Due to the increased resistance and cross-resistance to antibiotics of C. albicans isolates, there has been a growing interest in natural products derived from medicinal plants as antifungal agents. Essential oils have already found a considerable range of applications because of their antifungal activity, low toxicity and no side effects. The aim of the research was to determine the effectiveness of chosen essential oils in reduction of hydrophobic properties of three Candida albicans clinical strains, isolated from patients with diagnosed candidiasis. Collection strain C. albicans ATCC 10231 was used for comparison. Cell surface hydrophobicity (CSH) was determined by the microbial adhesion to hydrocarbon test (MATH) to p-xylene in PBS buffer, according to Noumi et al. (2011). The results were presented as hydrophobicity index IH, i.e. the percentage of cells in the xylene layer (adhered cells). Within the study, thyme (Thymus vulgaris L.), clove (Syzygium aromaticum Merill & Perry) and tea tree (Melaleuca alternifolia Cheel) oils were used. Essential oils (EOs) were applied in their MIC concentrations (0.031-2% v/v) specific for the particular strain and oil, and used solely and in mixtures (1:1, 2:1 and 1:2 v/v). In order to determine the interactions of essential oils in mixtures, FIC factors and FIC indexes were calculated, using the following formulas: FICA= IH of mixture of oils/IH of oil A; FICB= IH of mixture of oils/IH of oil B; FICi = FICA+FICB. CSH values of strains tested were high, from 65.3% to 79.1%. EOs used solely or in combinations affected especially hydrophobic properties of collection and one clinical isolates. In these cases statistically significant decrease of hydrophobicity index (up to 1/2) was observed at the presence of tea tree and thyme oils 1:1, 1:2, 2:1, tea tree and clove oils 1:1, thyme and clove oils 1:2, 2:1 v/v. For the other two strains hydrophobicity was reduced statistically significantly only in the presence of tea tree and thyme oils (1:1), and thyme and clove oils (2:1). In general, essential oils used in mixtures showed stronger effect on hydrophobic properties of yeast tested than applicated solely. FIC indexes show additive or indifferent effect of oils in combinations, depending on the strains and the oil type. The presented results show that combinations of tea tree, thyme and clove oils are the potential effective agents decreasing the hydrophobic properties of C. albicans strains, thereby contributing to a limitation of adhesion processes. Noumi E., Snoussi M., Kammoun D., Bakhrouf A., Eulogio V. (2011) Comparison of the adhesion ability of Candida albicans strains to biotic and abiotic surfaces. Afr. J. Biotechnol. 10(6): 977-985.

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L5 ALTERNATIVE APPROACHES TO DELINEATE NOVEL ASCOMYCETOUS YEAST SPECIES

Neža Čadež Biotechnical faculty, University of Ljubljana, Jamnikarjeva 101, 1000 Ljubljana, Slovenia Species is the only non-arbitrary, real taxonomic category. However, there is no uniform definition of the species and probably there is no concept which remained consistently controversial as the concept of the species. Beside theoretical, evolutionary species concept, operational species concept for yeasts is based on the species divergence inferred from sequences for the large nuclear ribosomal RNA gene (D1/D2 domains). The generality of this operational species definition is limited by non-constant rate of in between species divergence. For example, for the species of Hanseniaspora the discriminating capacity of the ribosomal gene is mostly too low or varies between species. Therefore, in order to avoid the subjectivity of determining the limits of species we relied on the concordance of more than one gene genealogy. With progressive concatenation of independently evolving protein-coding genes, the number of phylogenetically informative sites increased and with it the accuracy of resolved relationships. Both protein-coding genes showed better capacities to resolve at the strain level and between the closely related species of Hanseniaspora compared to the ribosomal gene regions. However, the protein-coding genes as phylogenetic markers have some drawbacks, as for example the lack of universal primers, presence of paralogues genes within the genome or heterozygosity of the strains. For example, the three closely related species of the genus Ogataea showed up to 3.5% high sequence divergence in protein-coding gene for elongation transcription factor (EF-1α) due the ambiguous called nucleotides when a direct PCR sequencing approach was applied. Furthermore, by cloning of the PCR product of EF-1α gene, up to six variants of EF-1 α genes were recovered from individual strains. By analysis of the sequence variance between the three closely related species of Ogataea will be discussed. Acknowledgments The work was founded by Slovenian Research Agency (P4-0116 and MRIC-UL ZIM, IP-0510).

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METHANOL-ASSIMILATING YEASTS – SYSTEMATICS AND OCCURRENCE Péter G. and Dlauchy D. National Collection of Agricultural and Industrial Microorganisms, Faculty of Food Science, Corvinus University of Budapest, Somlói út 14-16. H-1118 Budapest, Hungary The first report on methanol utilization by yeasts was published more than forty years ago. Since then the number of methanol-assimilating yeast species has steadily increased and some species have become biotechnologically important. The application of DNA sequence-based yeast identification proved to be an excellent means for exploring the biodiversity of yeasts, including the methylotrophic ones. As a result, the number of described methanol-assimilating yeast species has increased dramatically and their current number is exceeding 80. The vast majority of the methanol-assimilating yeast species are currently assigned to the genera Komagataella, Kuraishia, Ogataea and Candida. During our investigations, following an enrichment procedure, methanolassimilating yeast strains were regularly isolated from tree exudate and rotten wood samples. The most frequently recovered species was Komagataella pastoris. It was also demonstrated that methanol-assimilating yeasts can repeatedly be isolated from the phyllosphere, a habitat usually predominated by basidiomycetous yeasts. Among the yeasts isolated primarily from the abovenoted, plant-related substrates several proved to be undescribed species distributed among the above-mentioned genera. Nineteen novel methylotrophic yeast species were described, and some additional ones are awaiting for description. The majority of the known methanol-assimilating yeast species are members of the Ogataea clade, comprising the species of the genus Ogataea and the phylogenetically related Candida species. Based on our findings the diagnosis of the genus Ogataea was emended twice to allow the inclusion of the species unable to assimilate nitrate, as a sole nitrogen-source and the species forming allantoid ascospores. Some correlations among the occurrence of the yeast species and the plant species accommodating them were documented as well.

Acknowledgments This study was partly supported by the State Secretariat for Higher Education, Hungarian Ministry of Human Resources and the Doctoral School of Food Sciences, Corvinus University of Budapest.

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GENOME CHIMERISATION IN SACCHAROMYCES: TAXONOMIC CONSEQUENCES

Sipiczki M. Department of Genetics and Applied Microbiology, University of Debrecen, Debrecen, Hungary The genus Saccharomyces harbours natural species isolated by postzygotic sterility barrier and so-called hybrid species. Whereas the natural species are constituted by interbreeding strains of similar genome structures, the hybrid species are heterogeneous groups of (mostly beer) strains of mosaic (chimerical) genomes composed of incomplete parental genomes in diverse proportions. Over the past decade, many wine strains have been described whose genomes also had mosaic structures. The mosaic genomes of those wine yeasts usually consisted of a (nearly) complete genome of a natural species and genome fragments from a different natural species. Both types of mosaic genomes are supposed to have evolved from (alloploid) hybrids of natural species by postzygotic genome chimerisation: reorganisation and reduction (segregation) of the partner genomes. However, being allodiploid, the interspecies hybrids are sterile, unable to accomplish meiosis and thus unable to segregate their genomes. We found that the sterility barrier can be overcome by tetraploidisation of the interspecies hybrid and subsequent elimination of chromosomes carrying the MAT loci of one or the other partner species. The loss of MAT heterozygosity restores fertility and allows genome reduction and rearrangement leading to segregants of chimerical genomes. The chimerical/mosaic descendants cannot be assigned to any species of the genus because they show mixed patterns of taxonomic traits, their genomes are not isolated (protected) by sterility barriers and their gene pools are polyphyletic.

21

L8 YEASTS INHABITING THE PHYLLOSPHERE OF FRUIT TREES

Jana Molnárová, Renáta Vadkertiová Institute of Chemistry, Slovak Academy of Sciences, 84538 Bratislava, Slovakia Yeasts have been considered to be important colonists of the phyllosphere that have profound effects on plant health and impact on ecosystem and agricultural functions. The diversity and density of yeasts on plant material are linked to various factors such as geographical locality, climatic conditions, season, fungicide application, cultivar, fruit developmental stage, etc. This study focused on the diversity of yeasts and yeast-like organisms associated with fruits, leaves, and blossoms of fruit trees (apple, apricot, peach, pear, plum, cherry) in 3 localities in southwest Slovakia. A total of 31 yeast and yeast-like species were isolated from fruits, leaves and blossoms. The highest yeast diversity was found with fruit samples. Aureobasidium pullulans, Candida tropicalis, Cryptococcus flavescens, Metschnikowia pulcherrima, Saccharomyces cerevisiae and Wickerhamomyces anomalus were found to be associated with all plant organs. A. pullulans, Cr. flavescens and M. pulcherrima formed major part of isolates associated with leaves, whereas fruit samples were mainly occupied with Hanseniaspora guilliermondii, H. uvarum, Metschnikowia pulcherrima, Pichia kluyveri and P. membranifaciens and Saccharomyces cerevisiae. Only A. pullulans and M. pulcherrima were isolated frequently from blossom samples. Some of the species were isolated only rarely. Pseudozyma prolifica was associated with the leaves of apricot, cherry and peach trees, Yarrowia lipolytica with leaves of cherry trees, Candida oleophila, P. manshurica and Z. bailii with the fruits and Starmerella bombicola with the blossoms of pear trees. The interactions of isolated yeasts with three fungal strains: Clonostachys rosea, Fusarium acuminatum and Penicillium brevicompactum, isolated from rotten apples, were also studied. The yeast strains exhibited antagonistic activities, grew together with the fungal strains without any activity against each other, or were suppresed by fungi. The highest spectrum of inhibition activity was found with the black yeast Aureobasidium pullulans. The inhibition activity of the species examined was found to be both strain and medium specific. This work was supported by a grant VEGA No. 2/0023/14 from the Slovak Grant Agency and Ministry of Education.

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THE YEAST CHRONOLOGICAL AGING MODEL: PROTEOTOXIC STRESS AND AGE-RELATED DYSFUNCTIONS Paula Ludovico Life and Health Sciences Research Institute (ICVS), School of Health Sciences, University of Minho, Braga, Portugal and ICVS/3B’s – PT Government Associate Laboratory, Braga/Guimarães, Portugal Yeast provides a simple and powerful model for cellular aging research. A combination of genetics, cell biology, and biochemistry has unraveled the complexities of numerous cellular processes conserved in evolution and governing, at least to some extent, the rate of aging. Several common denominators of aging found in different models, as yeast, include genomic instability, loss of proteostasis, deregulated nutrient sensing and mitochondrial dysfunction. One of the major challenges is now to understand their interconnection and relative contributions to age phenotypes. Yeast chronological aging model has been used to get new insights on these questions and results will be presented regarding the emergent phenomenon of hormesis promoted by hydrogen peroxide. We have shown that elevated levels of the reactive oxygen species (ROS), hydrogen peroxide, are generated during caloric restriction, which extends life span and retards age-related phenotypes in a variety of species, reducing the accumulation of superoxide anions. These findings established a role for hormesis effects of hydrogen peroxide in promoting longevity that has now been observed in several model systems (1,2). The beneficial role of ROS in lifespan extension challenges the free radical theory but is consistent with the essential role of these molecules in cell signaling. The study of another aging hallmark, loss of proteostasis, was performed in yeast aged cells expressing the Parkinson’s disease related protein, alpha-synuclein. Alpha-synuclein is a naturally prone to misfold protein in high concentrations impacting on cell clearance routes and protein quality control systems such as the ubiquitin-proteasome system and autophagy. The results to be presented will show that the increased autophagy and mitophagy observed in yeast cells expressing alpha-synuclein accelerates chronological aging (3). The regulation of autophagy and it’s crosstalk with genomic instability will be discussed. Overall, the data to be presented gives new insights on cellular mechanisms of aging. “All the models are wrong but some are useful” and this is the case of the yeast chronological aging model. (1) Mesquita A, Weinberger M, Silva A, Sampaio-Marques B, Almeida B, Leão C, Costa V, Rodrigues F, Burhans WC, Ludovico P. Caloric restriction or catalase inactivation extends yeast chronological lifespan by inducing H2O2 and superoxide dismutase activity. Proc Natl Acad Sci U S A. 2010 Aug 24;107(34):15123-8. (2) Ludovico P, Burhans WC. Reactive oxygen species, ageing and the hormesis police. FEMS Yeast Res. 2013 Aug 21. doi: 10.1111/1567-1364.12070. (3) Sampaio-Marques B, Felgueiras C, Silva A, Rodrigues M, Tenreiro S, Franssens V, Reichert AS, Outeiro TF, Winderickx J, Ludovico P. SNCA (α-synuclein)-induced toxicity in yeast cells is dependent on sirtuin 2 (Sir2)-mediated mitophagy. Autophagy. 2012 Oct;8(10):1494-509.

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L10

SEARCHING FOR PROTEINS INTERACTING WITH SACCHAROMYCES CEREVISIAE PYRUVATE KINASE - PYK1

Mierzejewska J.1, Izdebska K.1, Bakun M.2, Chreptowicz K. 1, Dadlez M.2 1

Warsaw University of Technology, Faculty of Chemistry, Warsaw Poland Institute of Biochemistry and Biophysics Polish Academy of Science, Warsaw Poland

2

Most tumor cells rely on the glycolytic pathway for their energy conversion processes and they display “aerobic glycolysis” – the phenomenon of decreased respiration rate and increased production of lactate in the presence of glucose, despite the availability of oxygen [1, 2]. The aerobic glycolysis in cancer cells is mediated by a key molecule, pyruvate kinase muscle isozyme 2 (PKM2), which catalyzes the last energy-generating step in glycolysis. Saccharomyces cerevisiae is a valuable model for studying mechanisms controlling basic processes in eukaryotic cells, including the glucose-induced repression of oxidative metabolism. Similar to tumor cells, yeasts prefer glucose as a carbon source regardless of the presence of oxygen, or other non-fermentable carbon sources [3, 4]. Pyruvate kinase - Pyk1, encoded by CDC19 gene in S. cerevisiae is an ortholog of mammalian PKM2, and displays an important role in switching between fermentative and oxidative metabolisms [5]. To identify the unknown proteins interacting with Pyk1, we utilized a pulldown assay combined with mass spectrometry analysis. CDC19 gene was introduced into an expression vector to create a translational fusion with histidinex6 tag sequence. This produced Pyk1-6xHis in E. coli, which was further purified by affinity chromatography. The recombinant protein Pyk1-6xHis, used as a bait, was then mixed with total protein extracts from S. cerevisiae MB159-4D, the source of the prey proteins. Further, the bait-prey complexes were co-purified on Ni-agarose resin, and were analyzed by LTQ-Orbitrap Velos mass spectrometer (Thermo Scientific) coupled with a nanoAcquity (Waters Corporation) UPLC system. In the current study, we identified 47 proteins which potentially interacts with Pyk1 including four proteins – Acs2, Ptk2, Gis2 and Pyk2, which were previously indicated to interfere with Pyk1. Most of the prey proteins identified were implicated in the glucose metabolism (glycolysis or gluconeogenesis). However, we found out that some proteins were engaged in the acetyl-CoA production, and also for its utilization of various processes, like histone acetylation. We also identified several factors involved in transcription and translation. Hence, it is more likely that Pyk1 except the enzymatic activity, may play an important role in the regulation of other intracellular processes, necessary for the proliferation of cells. Acknowledgments This work was supported by the Foundation for Polish Science (Parent-Bridge Programme/2012-6/8), and by the Warsaw University of Technology, Faculty of Chemistry.

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pHLUORIN-BASED MEASUREMENTS OF INTRACELLULAR pH IN CONVENTIONAL AND NON-CONVENTIONAL YEAST SPECIES Zimmermannová O., Llopis-Torregrosa V., Zemančíková J., Dušková M. and Sychrová H. Department of Membrane Transport, Institute of Physiology CAS, Prague, Czech Republic, [email protected] Tight regulation of intracellular ion concentrations and H+ is crucial for all living cells. There is a growing body of evidence that the activities of various transporters are coregulated and that the maintenance of intracellular pH results from a complex interaction of different proteins (transporters of H+, K+, Ca2+). To estimate the intracellular pH in living cells, we have been progressively improving the method of intracellular pH measurements in various yeast species using the pH-sensitive form of green fluorescent protein - pHluorin (Miesenbock at al. (1998) Nature 394:192-51). A set of plasmids with various promoters and auxotrophic markers for the expression of pHluorin or enabling the integration of the pHluorin sequence into the genome of various yeast species was constructed and the method of intracellular pH measurements was optimized for the estimation of intracellular pH in Saccharomyces sp., Zygosaccharomyces rouxii or Candida glabrata wild-type and mutant cells. The intracellular pH measurements in combination with measurements of the fluxes/contents of other cations (K+) proved a tight connection between potassium and proton transporters in yeasts and provided new insights into the cation homeostasis in lower eukaryotes.

Acknowledgments This work was supported by the Grant Agency of the Czech Republic (projects P302/12/1151, 15-03708S) and by ImResFun project (PITN-GA-2013-606786).

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HYBRID NATURE OF PATHOGENIC FUNGI Leszek Pryszcz Bioinformatics and Genomics Programme, CRG, Barcelona, Spain

Invasive fungal diseases pose a serious and growing health problem, and are a major cause of death worldwide. Interestingly, described human pathogens belong to several distant clades and they always have relatives that are not virulent to human, indicating that the ability to infect humans has emerged several times independently. Candida species constitute the most prevalent group of fungal pathogens. From their phylogenetic relationships it is clear that virulence to humans has emerged in this clade several, independent times. The Candida parapsilosis complex is particularly suitable to investigate the emergence of virulence, with three closely-related species of varying degree of pathogenicity and of growing relevance: C. parapsilosis, C. orthopsilosis and C. metapsilosis. We sequenced fifteen strains from this clade, sampled from clinical and environmental sources. We reported for the first time the genomic evidence for the existence of recombination, mating and hybridization in this clade, previously considered asexual. We propose the independent emergence of clinical isolates from environmental lineages and a possible role of hybridization in the acquisition of relevant traits for pathogenesis. Finally, we have compared the genomes of several Candida species to gain insight into the emergence of virulence in this clade. We have found expansions in gene families known to be involved in virulence, like adhesins, membrane transporters and extracellular enzymes, as well as expansions in gene families not implicated in virulence so far. Altogether, our findings provide the grounds for numerous hypotheses about the emergence of virulence in Candida spp. and for their future experimental testing.

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GENE COPY NUMBER AND COLONY MORPHOLOGY IN SACCHAROMYCES CEREVISIAE Derek Wilkinson, Vratislav Stovicek, Libuse Vakova, Zdena Palkova Charles University in Prague, Departmentof Genetics and Microbiology, Viničná 5, Praha 2, Czech Reublic When wild yeast are domesticated by growth under favourable conditions they may switch from a complex, structured colony morphology to a simple, smooth one. Domesticated strains, grown under less favourable conditions, may revert to a structured morphology, giving rise to feral sub-clones. In this way, the wild Saccharomyces cerevisiae strain, BR-F gave rise to the domesticated strain, BRS and the latter gave rise to the feral strain, BR-RF. Transcriptomic analysis revealed that clusters of genes in specific regions of a few chromosomes, including the left arm of chromosome XV were upregulated in the feral strain, BRRF compared with both the wild strain, BR-F and the domesticated strain, BR-S. Real time q-PCR was used to show that the copy numbers of three representative genes, YOL110W, YOL060C and YOL038W, in the left arm of chromosome XV, were higher in the feral strain, BR-RF than in the wild strain, BR-F or the domesticated strain, BR-S, relative to three reference genes, YOR194C, YOR250C and YOR288C, in the non-upregulated right arm of chromosome XV. This increased copy number of genes in particular regions of a specific chromosomes suggests the occurrence of stress-induced duplications of large areas of the genome during the reversion from domesticated to feral morphology. However, these rearrangements in BR-RF may be related to stress tolerance rather than to the structured colony phenotype.

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LACK OF THE PUTATIVE ION CHANNEL IST2 ALTERS ALKALI-METALCATION ACCUMULATION IN SACCHAROMYCES CEREVISIAE CELLS Papoušková K., Andršová M., Sychrová H. Department of Membrane Transport, Institute of Physiology of the Czech Academy of Sciences, Videnska 1083, 14220 Prague 4, Czech Republic The maintenance of ion homeostasis is a crucial task in all types of cells, including the model yeast Saccharomyces cerevisiae. The putative ion channel Ist2, which belongs to the protein family comprising mammalian anoctamin (TMEM16) ion channels, resides in the membranes of cortical endoplasmic reticulum and was shown to be one of the tethering proteins forming contacts between the plasma and ER membranes. Originally, the deletion of IST2 gene was found to increase sodium tolerance of S. cerevisiae cells, but the role of Ist2 in the maintenance of yeast alkali-metal-cation homeostasis is still unclear. In this work, we prepared a BY4741-derived series of deletion mutants lacking either IST2 gene alone or in combination with genes encoding characterized alkali-metal-cation exporters or importers. Physiological tests of these strains revealed that IST2 deletion modulates the growth of cells not only in the presence of toxic sodium and lithium ions, but also under conditions when potassium, the main intracellular cation, is limiting. Surprisingly, the lack of Ist2 results in an increased sodium accumulation in cells when they are grown in the presence of high NaCl amounts. Therefore, the presence of Ist2 is important for the maintenance of proper alkali-metal-cation homeostasis in yeast cells. As it was shown that Ist2 is involved in the trafficking and regulating the activity of some plasma membrane transporters, we tested if the function of sodium efflux systems might be affected in ist2Δ cells. Acknowledgments This work was supported by the Kontakt II LH14297 project from MSMT.

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BIOTECHNOLOGICAL DEVELOPMENTS OF YEASTS FOR BIOFUELS AND BIOBASED CHEMICALS Charles A. Abbas, Ph.D. Director Yeast & Renewables Research, Archer Daniels Midland Research Decatur, IL 62526 USA The increased demand for biofuels and bio-based chemicals has fueled a worldwide resurgence in interest in their production from renewable feedstocks. The increased focus on the use of renewable feedstocks, such as lignocellulosics, is due in part to recent advances in pretreatment and enzyme hydrolysis technology. With the increased search for alternatives to fossil fuels, renewable feedstocks have also received governmental mandates and subsidies that aim to create incentives for their commercial production. Second generation biofuels include, in addition to cellulosic ethanol, long-chain alcohols, terpenoid hydrocarbons, and diesel-length alkanes. Advances in technology have also led the chemicals industry to leverage new feedstocks that provide a cost advantage while delivering a sustainable chemical product. Today, the vast majority of industrial chemicals are produced from fossil fuels, but with the volatility of petroleum-based feedstocks, there is increased demand for more sustainable plant derived feedstocks. The development of new yeast strains of Saccharomyces cerevisiae by genetic engineering to produce biofuels and chemicals have been the focus of research efforts. This coincides with parallel efforts that use other genera of yeasts that can utilize a broader range of feedstocks. Among the chemicals of primary interest are the C3 and C4 chemicals that represent fundamental building blocks of key segments of the industrial chemical industry. The annual sales of these chemicals is estimated at more than $10 billion. These chemicals are on the list of top ten chemicals developed by the U.S. Department of Energy (DOE) and are widely used in many consumer products. The production of these chemicals by fermentation has been already demonstrated using yeasts at bench- and pilotscale and at demonstration-scale facilities.

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FUNCTIONAL EXPRESSION OF FATTY ACID DESATURASES IN SACCHAROMYCES CEREVISIAE: A TOOL FOR ENGINEERING OF MEMBRANE DESATURASES WITH NEW SPECIFICITIES Aleš Bucek, Petra Matoušková, Aleš Svatoš and Iva Pichová Institute of Organic Chemistry and Biochemistry, Academy of Sciences of the Czech Republic, v.v.i. Flemingovo nám. 2, Prague, Czech Republic

Membrane-bound fatty acid desaturases (FADs) play a prominent role in the biosynthesis of unsaturated fatty acids (FA). The large variety of FA unsaturation patterns is enabled by the high diversity of substrate, regio-, and stereospecificities of FADs but the lack of membrane desaturase crystal structures has made it difficult to elucidate the mechanism and control of these specificities. We identified novel insect desaturases that share high amino acid sequence identity. The functional expression of these FADs in S. cerevisiae and analysis of FA products by GC-MS revealed their different regio- and stereo-specificities. Construction of a number of FAD chimeras and their characterization enabled to identify the structural motif determining the desaturase specificities. The results also showed a flexibility of desaturases in accommodation of different FAs. Our findings represent a step toward understanding the mechanism of desaturation of fatty acids by membrane-bound desaturases and toward engineering of membrane desaturases with new specificities.

This work has been supported by project LO 1302 from Ministry of Education.

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L17 FURTHER INSIGHTS INTO RHODOTORULA CAROTENOGENESIS THROUGH THE APPLICATION OF GENOMIC AND PROTEOMIC APPROACHES 1 1 Sara Landolfo , Raffaela Cutzu , Alessandro Tanca2, Marcello Abbondio2, Maria Filippa Addis2, Massimo Deligios3, Sergio Uzzau2, Giuliana Mulas1, Marilena Budroni 1, Ilaria Mannazzu1 1 Dipartimento di AGRARIA, Università degli Studi di Sassari, Viale Italia 39, Sassari, Italy; 2Porto Conte Ricerche, S.P. 55 Porto Conte/Capo Caccia Km 8.400, Loc. Tramariglio, Alghero, Italy; 3Dipartimento di Scienze Biomediche, Università degli Studi di Sassari, Viale San Pietro, Sassari Italy Considering that the global market of carotenoids grows yearly of 2.3%, and that it is estimated to reach 1.4 billion dollars in 2018 (BCC Research 2011), there is great interest in the implementation of processes for the efficient production of these pigments. Carotenoids may be easily chemically synthesized. However, due to a general concern on the safety of artificial synthetic pigments, the exploitation of carotenoid producing microorganisms is an appealing alternative to the their chemical production. At present, besides Phaffia rhodozyma and Haematococcus pluvialis, already utilised for the production of astaxanthin, and Blakeslea trispora employed for the production of β-carotene (Dufossé 2006), also other microbial species present interesting potential in carotenoids production. Among these, the red yeasts ascribed to the genera Rhodosporidium, Rhodotorula, Sporobolomyces and Sporidiobolus have been described and utilized for the bioconversion of cheap substrates into added value carotenoids (Buzzini et al., 2010). Indeed, in spite of their biotechnological potential, these yeasts are poorly characterized at the genetic, genomic and proteomic level. However, things are rapidly changing. Molecular methods for gene delivery, gene targeting and random insertional mutagenesis of red yeasts have been lately proposed and the whole genome sequences of some of these yeasts have been recently released thus increasing the amount of information that can be obtained for these species.

Here, with the aim of gathering further information on the carotenogenic pathway in red yeasts, we undertook a study on Rhodotorula mucilaginosa, a highly heterogeneous species that includes promising strains for the commercial production of these pigments. In particular, we applied different molecular approaches, covering whole genome sequencing and proteomic analyses to the characterization of the wild strain C2.5t1, selected in our laboratory on the basis of its ability to produce carotenoids on low cost substrates. Project granted to IM by Regione Autonoma della Sardegna (Legge Regionale 7- 2007 Annualità 2010). References BCC Research, http://www.bccresearch.com/report/carotenoids-global-market-fod025d.html. Buzzini et al., (2010) In: Flickinger MC (ed) Encyclopedia of Industrial Biotechnology: Bioprocess, Bioseparation, and Cell Technology, Vol. 1, Wiley & Sons, New York, pp 469-481.

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SEM and Raman spectroscopy applied to yeast biomass analysis for the application in the field of biofuels and food industry O. Samek1, A. Haroniková2, K. Hrubanová1, I. Márová2, S. Bernatová1, P. Zemánek1, V. Krzyžánek1 1.

Institute of Scientific instruments of the ASCR, v.v.i., Brno, Czech Republic. Centre for Material Research, Brno University of Technology, Brno, Czech Republic. 2.

A biomass of red yeast strains has been studied due to their potential applications in the field of biofuel generation and food industry. In order to utilize biomass for efficient industrial production, the optimal cultivation parameters have to be determined which in turn lead to high production of desired substances such as oil and carotenoids in the selected cell line. Main aim of our investigations was to study how different cultivation conditions influence production of oil and carotenoids. In order to obtain as much information as possible a combination of scanning electron microscopy (SEM), and Raman spectroscopy was used. In addition we used HPLC analysis to quantify the concentration of carotenoids in selected samples. Thus, all matrix changes within the studied cells introduced by stress response mechanisms can be visualized and chemically characterized.

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L19

MOLECULAR AND PROCESS DESIGN FOR CHEMICAL AND PROTEIN PRODUCTIONS IN YEASTS

Danilo Porro and Paola Branduardi Department of Biotechnology and Bioscience, University of Milano Bicocca, Piazza della Scienza 2, Milan, Italy. Natural and engineered yeast cell factories are today extensively used for commercial productions. Based upon their innate metabolic abilities, yeasts are being employed since several decades for large-scale production of different natural compounds. In this respect, there are more than 600 yeast factories in operation in the world. Further, with the advent of recombinant DNA technology it has become possible to introduce traits for the production of desired non-natural compounds: heterologous proteins and metabolites. While the scientific and technological platforms leading to the production of a heterologous protein seems under consolidation, with the exception of membrane proteins, (i.e., being E. coli and S. cerevisiae the two microbial workhorses for the commercialization of recombinant proteins), the production of heterologous and endogenous metabolites by metabolically engineered cell factories always and strongly suffers from extensive regulation of cellular metabolism, which easily evolves in order to ensure the cellular homeostasis. This cellular homeostasis often leads to a low accumulation of the desired product. While on the market we can find about 600 recombinant pharma-proteins, the amount of recombinant metabolites is undoubtedly much lower. The main research efforts in our laboratory are related to the design of robust and stable production strains and bioprocesses to match the stress conditions occurring during industrial fermentation for both metabolite and protein productions. Here we will present how this can be reached both by manipulating specific targets involved in the pathway of interest or by modulating “hub elements” responsible for a general cellular reorganization. Finally, an innovative non-invasive strategy to monitor the accumulation of the “final product” will be presented.

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EXPRESSION OF HUMAN GLYCOSYLTRANSFERASE IN YEAST Tibor Czabany1*, Katharina Schmoelzer2, Christiane Luley-Goedl2, Doris Ribitsch2, Sabine Zitzenbacher2, Helmut Schwab3 and Bernd Nidetzky1 1) Institute of Biotechnology and Biochemical Engineering, Graz University of Technology, Graz Austria 2) Austrian Centre of Industrial Biotechnology, Graz, Austria 3) Institute of Molecular Biotechnology, Graz University of Technology, Graz, Austria * email: [email protected]

Glycosyltransferases are important enzymes of applied biocatalysis. Their application ranges from synthesis of oligosaccharides to in vitro modification of therapeutical

glycoproteins.

The

in

vitro

modification

often

requires

glycosyltransferases from mammalian sources. However, their production platforms are either very costly (mammalian cell cultures) or inefficient (bacteria, yeasts). We present a complex approach from gene design to fermentation optimization, leading towards production of an active human sialyltransferase in Pichia pastoris. The truncation of the sialyltransferase, and the introduction of Nand C-terminal tags leads to stabilization of the protein[1]. Further, optimization of the media composition and other fermentation parameters leads to higher yields of human sialyltransferase.

[1] Ribitsch, D. et al. Microb. Cell Factories 13, 138 (2014).

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KINETIC ANALYSIS OF CAROTENOID PIGMENTS ACCUMULATION IN RED YEASTS Tkáčová J., Klempová T., Čertík M. Faculty of Chemical and Food Technology, Slovak University of Technology, 81237 Bratislava, Slovakia Several species of red yeasts, as Rhodotorula sp., Rhodosporidium sp., Sporobolomyces sp. and Xanthophylomyces sp. (Phaffia sp.) are applied for commercial synthesis of various natural carotenoids. Species of genus Rhodotorula (Rh. glutinis, Rh. minuta, Rh. mucilaginosa and Rh. graminis) are known as producers of carotenoid pigments such as β-carotene, γ-carotene, torulene and torularhodin. Our preliminary experiments showed that carotenoid pigments are accumulated in yeast cells in stationary growth phase and their accumulation was stimulated by C-source exhausting from cultivation media. Therefore the goal of the present work was to deep study of kinetic parameters of yeast carotenoid biosynthesis. Because carbon and nitrogen ratio in cultivation media is one of the most important physiological factors affecting the microbial formation of lipids and pigments, different C/N ratios (20:1, 50:1, 70:1, 100:1) were applied to study their impact on carotenoid biosynthesis and profile. Oleaginous yeast Rh. glutinis is able to form carotenoids in considerable quantities when grown under low C/N ratio. It was found that total pigment accumulation in the cells was rapidly triggered by consumption of glucose when the yeast was cultivated at C/N 20:1. Especially content of torularhodin, the ending metabolite of carotenoid biosynthetic pathway, was three times higher compare to cultivation on media where glucose was not utilized completely. Similar increase in pigment formation was obtained with C/N 50:1 after glucose exhaustion. It should be noted that utilization of intracellular fatty acids (stored especially in triacylglycerols) into acetyl-CoA via the β-oxidation due to glucose deficiency in media was accompanied by enhanced carotenoid accumulation in the cells. It could be postulated that acetyl-CoA formed by the β-oxidation served as a substrate for pigment biosynthesis in mevalonate pathway. Thus, these kinetic analyses confirmed that carbon starvation is an essential factor of pigment overproduction in red yeasts. To conclude, although C/N ratio 20:1 was found as an optimal for carotenoid accumulation in cells (2.4 mg/g cells), the maximal carotenoids yield of 18.0 mg/L was achieved at C/N 50:1. Acknowledgments The work was supported by grants VEGA 1/0574/15, APVV-0662-11, bilateral APVV project SK-CZ-2013-0167 and by the project "University Science Park of STU Bratislava“ ITMS 26240220084.

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AUTOMATED DIGITAL MICROSCOPY FOR CELLULAR RESEARCH Dr. Andreas Rieger (BioTek Instruments, Inc., Winooski, Vermont, USA) A novel cellular analysis instrument – Cytation3/5 Imaging reader - comprised of a digital microscope and a PMT-based microplate reader will be described. The broad range of microscopy-based applications will be demonstrated using bright field and contrast enhancing modes such as color staining, fluorescence and phase contrast. Automated image processing and analysis capabilities will be described that allow for cell sub-population analysis such as cell counting by phenotype expression. In addition, quantitative cell population-based microplate assays using the PMT-based optics will be shown. By using PMT-based detection with the patented Hybrid Technology™ - a combination of two alternate paths for light within the same analytical instrument - light can be directed through a filterbased or a monochromator-based optical system for wavelength selection with variable bandwidths. Hence this three-optical-path system provides optimal performance and flexibility for a wide range of applications that can include Imaging, Fluorescence Intensity, UV-Vis Absorbance, Luminescence, TRF, TRFRET, FP and Alpha Assay detection modes. The instrument is particularly suited for live cell kinetic analysis as the read chamber is environmentally controlled for CO2/O2 and temperature; while injectors provide dispensing of reagents directly into the microplate in the read chamber allowing the measurement of rapid reaction kinetics.

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L23 MOLECULAR DIVERSITY OF YEAST MITOCHONDRIAL GENOMES

Nosek J. and Tomáška Ľ. Comenius University in Bratislava, Faculty of Natural Sciences, Departments of Genetics and Biochemistry, Mlynská dolina, Ilkovičova 6, Bratislava, Slovak Republic

A mitochondrial genome is typically portrayed as a circular chromosome coding for a small set of highly conserved proteins. However, comparative analyses revealed that mitochondrial genomes exhibit a number of non-canonical features and may vary dramatically in the size, base composition, genetic organization as well as in the molecular form. For example, our studies of yeast mitochondrial genomes uncovered a plethora of molecular architectures including circular, linear with different terminal structures (i.e. tandem repeat arrays, single-stranded hairpin loops, covalently bound terminal proteins) as well as linear and circular multipartite forms [1-6]. These results illustrate that mitochondria represent ‘testtubes’ for evolutionary tinkering with components of their genetic machinery that often results in emergence of peculiar novelties. As the most recent example, we discovered several families of mobile genetic elements, which are inserted in the mitochondrial coding sequences. These elements are present in matured mRNA, but are ignored by the translational machinery in a process termed programmed translational bypassing. The bypassing elements are reminiscent of guanine and cytosine rich sequence motifs termed GC clusters, which are spread across the entire subphylum Saccharomycotina. Our results indicate that they contribute to molecular diversity of mitochondrial genes and proteins [7,8].

Acknowledgments This work was supported in part by the Slovak grant agencies APVV (0035-11, 0123-10) and VEGA (1/0311/12, 1/0333/15). References 1. Nosek, J., Tomáška, Ľ., Fukuhara, H., Suyama, Y., Kováč, L. (1998). Trends Genet. 14: 183188. 2. Nosek, J., Tomáška, Ľ. (2003). Curr. Genet. 44: 73-84. 3. Kosa, P., Valach, M., Tomáška, Ľ., Wolfe, K.H, Nosek, J. (2006). Nucleic Acids Res. 34: 24722481. 4. Tomáška, Ľ., Nosek, J., Kramara, J., Griffith, J.D. (2009). Nat. Struct. Mol. Biol. 16: 1010-1015. 5. Fričová, D., Valach, M., Farkas, Z., Pfeiffer, I., Kucsera, J., Tomáška, Ľ., Nosek, J. (2010). Microbiology 156: 2153-2163. 6. Valach, M., Farkas, Z., Fričová, D., Kováč, J., Brejová, B., Vinař, T., Pfeiffer, I., Kucsera, J., Tomáška, L., Lang, B.F., Nosek, J. (2011). Nucleic Acids Res. 39: 4202-4219. 7. Lang, B.F., Jakúbková, M., Hegedűsová, E., Daoud, R., Forget, L., Brejová, B., Vinař, T., Kosa, P., Fričová, D., Neboháčová, M., Griač, P., Tomáška, Ľ., Burger, G., Nosek, J. (2014). Proc. Natl. Acad. Sci. U.S.A. 111: 5926-5931. 8. Nosek, J., Tomáška, Ľ., Burger, G., Lang, B.F. (2015). Trends Genet. 31: 187-194.

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SQUALENE TOXICITY IN YEAST STRAIN DEVOID OF LIPID DROPLETS Martin Valachovic, Martina Garaiova and Ivan Hapala Institute of Animal Biochemistry and Genetics, SAS, Ivanka pri Dunaji 90028, Slovakia. Squalene (SQ) is produced in the yeast Saccharomyces cerevisiae in the isoprenoid pathway as the first precursor dedicated to ergosterol biosynthesis. The level of squalene varies significantly dependent on the culture phase, growth conditions or restrictions in the ergosterol biosynthesis. In the wild type cells growing under normal conditions SQ can be nearly undetectable while in low oxygen environment it can accumulate up to almost 100 μg per 109 cells and up to several hundreds of μg in cells treated with the squalene epoxidase inhibitor - terbinafine. High accumulation of squalene may be one of the factors involved in the antifungal effect of terbinafine. We hypothesize that lipid droplets (LD) are significantly involved in buffering these SQ fluctuations. Cells lacking LD (e.g. quadruple mutant with defective acyltransferases responsible for synthesis of triglycerides and steryl esters) are hypersensitive to terbinafine which indicates that squalene may be toxic if it cannot be stored in LD. To study the importance of lipid droplets in coping with high levels of SQ we treated mutant strain lacking LD with terbinafine. While wild-type cells accumulate up to 500 μg of SQ per 109 cells without significant inhibition of growth, mutant lacking LD is unable to accumulate such high SQ levels and its growth is arrested already at squalene concentration around 100 μg per 109 cells. Moreover, terbinafinetreated cells lacking lipid droplets lose viability, which indicates that squalene has strong cytotoxic effect on yeast cells without neutral lipid storage organelles – lipid droplets.

This work was supported by the Slovak Grant Agency VEGA (grant No. 2/0185/14) and SRDA (grant No. APVV-0785-11).

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L25

DNA-BINDING PROPERTIES OF TAZ1 AND TEB1 PROTEINS OF THE YEAST SCHIZOSACCHAROMYCES POMBE Sepšiová, R. 1, Procházková, K. 1, Nečasová, I. 2, Willcox, S. 3, Hofr, C. 2, Griffith, J.D. 3, Nosek, J. 1, Tomáška Ľ. 1 1 Comenius University in Bratislava, Faculty of Natural Sciences, Departments of Genetics and Biochemistry, Bratislava, Slovak Republic 2 Central European Institute of Technology, Masaryk University, Brno, Czech Republic 3 Lineberger Cancer Center, University of North Carolina, Chapel Hill, USA Telomeres are specialized nucleo-protein structures protecting the ends of linear chromosomes against degradation and inappropriate DNA repair. The telomeres of eukaryotic chromosomes are composed of tandem repeat arrays and a single-stranded overhang. The double-stranded regions are bound by a special class of Myb-domain containing proteins. Although the overall organization of nuclear telomeres in ascomycetous yeasts is similar to other eukaryotes, individual yeast species exhibit remarkably high variability not only in the telomeric sequence but also in the range of telomere-binding proteins. For example, in Saccharomyces cerevisiae, the double-stranded telomeric region is bound by Rap1p, while in Yarrowia lipolytica and Schizosaccharomyces pombe this function is fulfilled by Tay1p and Taz1p, respectively. This underlines the fact that telomeres in distinct phylogenetic lineages underwent dramatic evolutionary transitions. Our long-term goal is to understand how and why these transitions occurred. S. pombe is especially valuable model to address these questions as it contains all three proteins mentioned above. Whereas Taz1p is the principal telomeric DNA-binding protein, Rap1p lost its DNA-binding activity and Teb1p (the orthologue of Tay1p) retained its ability to bind telomeric-like sequences within the promoter regions in internal chromosomal sequences, yet there is no evidence that it has any function at telomeres. This raises a question, if Teb1p functions only as a transcription factor and lost its ability to bind telomeres in S. pombe or, alternatively, it can bind the telomeres only under special circumstances. Here we address this problem by a combination of biochemical and genetic methods. Our results indicate that compared with Taz1p, Teb1p exhibits about 3-fold lower affinity toward S. pombe telomeric repeats, while retaining a high affinity to mammalian-type of telomeric DNA. Electron-microscopic analysis also emphasized differences in the mode of binding of Taz1p and Teb1p toward different model substrates. Finally, while measurements of telomere lengths in cells carrying various combinations of mutations on taz1+ and teb1+ genes do not indicate that Teb1p binds to telomeres in the absence of Taz1p, teb1- cells exhibit subtle changes in distribution of telomeric restriction fragments. These results go in line with the scenario that although Teb1p retained its potential to function at telomeres, its telomeric functions were largely replaced by Taz1p, still leaving open the possibility that Teb1p has a role in telomere maintenance under special circumstances. Acknowledgments This work was supported by the grants from the National Institutes of Health (2R01ES01377306A1) and the Slovak grant agencies APVV (0035-11) and VEGA (1/0311/12, 1/0333/15).

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SYSTEMATIC SCREENING OF A LIBRARY OF SCHIZOSACCHAROMYCES POMBE DELETION STRAINS FOR ABERRATIONS IN MITOCHONDRIAL MORPHOLOGY

Lajdová, D., Tomáška, Ľ. Department of Genetics, Faculty of Natural Sciences, Comenius University, Bratislava 842 15, Slovakia Morphology of mitochondria is closely bound to their function. This is evident from numerous studies of cells from all phylogenetic branches of Eukaryota. Mitochondria in a dividing cell usually form an interconnected reticulum of tubules undergoing dynamic fission and fusion events. The main regulators of mitochondrial dynamics are various dynamin-related GTPases (DRPs) that use energy released by GTP hydrolysis to either mechanically severe a tubule, or to bring outer (and consequently inner) mitochondrial membranes of neighboring mitochondria to close proximity, enabling them to fuse. Other proteins involved in mitochondrial morphogenesis include mitochondrial ATP-synthase subunits, components of import complexes, mitochondrial DNA maintaining factors, and proteins acting as adaptors between mitochondria and other cell compartments and structures (e. g. endoplasmic reticulum or cytoskeleton) [1]. Schizosaccharomyces pombe is a strictly aerobic yeast, whose regulation of mitochondrial morphology and distribution resembles that of higher eukaryotes. While many of the most important proteins have been identified, the complete mechanism of mitochondrial morphogenesis is far from being elucidated. In order to identify new players, we are screening the BIONEER library of haploid S. pombe strains with deletions of genes coding non-essential proteins [2]. Inner mitochondrial membranes are stained with a cationic dye DiOC6 staining membranes with high ΔΨ, and mitochondrial morphology is evaluated by fluorescence microscopy. To avoid bias, strains are classified into distinct phenotypical categories without prior knowledge about their identity. After screening 20% of the library (651 out of 3308 strains in total), 5% of strains repeatedly showed aberrant mitochondrial morphology. The candidate genes were subjected to a preliminary Gene ontology bioinformatics analysis with BiNGO software [3], that revealed presence of a class of proteins engaged in microtubule dynamics. This category is particularly interesting, as fission yeast mitochondria, similarly to their mammalian counterparts, are transported along microtubules [4], while Saccharomyces cerevisiae uses actin cables [5]. We hope to identify novel proteins mediating the contact between mitochondria and microtubules, as the means of their physical association is still unclear. References [1] Friedman, J. R., Nunnari, J., Nature, 505, 335-343 (2014) [2] http://www.bioneer.co.kr [3] Maere, S., Heymans, K., Kuiper, M., Bioinformatics, 21, 3448-3449 (2005) [4] Yaffe, M. P., et al., PNAS, 93, 11664-11668 (1996) [5] Simon, V.R., Karmon, S. L., Pon, L. A., Cell Motility and the Cytoskeleton, 37, 199-210 (1997) Acknowledgements This work was supported by the Slovak grant agencies APVV (APVV-0035-11), VEGA (1/0311/12) and Comenius University grant (UK/527/2014).

40

PH2

MAF1 – AN EFFECTOR OF RNA POLYMERASE III, INFLUENCES CARBOHYDRATE AND LIPID METABOLISM IN YEAST

Chreptowicz K. and Mierzejewska J. Faculty of Chemistry, Warsaw University of Technology, Warsaw Poland

RNA polymerase (Pol) III is the largest eukaryotic RNA polymerase, which synthesizes small RNA, such as 5S rRNA, tRNA or snRNA. The regulation of Pol III transcription is mostly related to the regulation of cell growth and the cell cycle. Under stress conditions, protein Maf1 is responsible for repression of Pol III transcriptional activity. To date, Maf1p is the first and still the only known negative regulator of Pol III activity in yeast. In the current study we have observed that changes in Pol III transcriptional activity have significant influence on yeast metabolism. Obtained results indicate that yeast maf1Δ mutant exhibits increased pyruvate kinase activity – a key enzyme of glycolysis. Further anaerobic processes, shown by alcoholic fermentation, are also intensified. Moreover, fructose-1,6-bisphophate aldolase activity is also enhanced in a medium containing non-fermentable carbon source at increased temperature. Since previous reports indicate that enhanced pyruvate kinase activity causes an apparent block of switch from fermentative to oxidative metabolism in yeast cells [1], our observations could explain why maf1Δ strain is defective in respiration. Despite the changes in carbohydrate metabolism, we also revealed that lipid metabolism is affected by Maf1p. We observed that lack of Maf1p causes accumulation of lipid droplets in yeast cells. Interestingly, the similar effect in tumor cells deprived in expression of MAF1 was recently reported [2]. Hence, Maf1p serves as a central node in yeast cells to indirectly regulate genes expression as well as biosynthetic and metabolic processes. Acknowledgments This work was supported by the Foundation for Polish Science (Parent-Bridge Program/2012-6/8), and by the Warsaw University of Technology, Faculty of Chemistry. [1] Grűning N.M. et al., (2011) Cell Metab. 14(3), 415-427 [2] Palian B.M. et al., (2014) Plos One Genetics, 10(12), e1004789. doi:10.1371/journal.pgen.1004789.

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FUNCTIONAL STUDY OF PUTATIVE K+ TRANSPORTERS KCH1 AND KCH2 IN SACCHAROMYCES CEREVISIAE Nevečeřalová P., Sychrová H. and Zimmermannová O. Department of Membrane Transport, Institute of Physiology CAS, Prague, Czech Republic, [email protected] A fundamental attribute of any living cell is the maintenance of intracellular ion homeostasis. Saccharomyces cerevisae is an excellent model for the study of transport systems in eukaryotic cells and new findings concerning not only cation homeostasis + obtained in yeasts can be widely applied in mammalian cells. Two putative low-affinity K 2+ transporters Kch1 and Kch2, homologous to animal voltage-gated Ca channels, have recently been discovered in S. cerevisiae [1]. Both proteins are necessary components of 2+ the HACS (High-Affinity Ca influx System), which is activated by mating pheromones and is essential for cell survival. During the response to mating pheromones, the expression of Kch1 and Kch2 is strongly induced [1]. Also, the ER stressors (e.g. DTT or tunicamycin) were found to influence the expression and post-translational modification of Kch1 [2]. To find out the role of Kch1 and Kch2 in the maintenance of alkali-metal-cation homeostasis, strains with deletions of KCH1 and/or KCH2 genes in BY4741 background and its derivatives lacking main potassium transporters (Trk1, Trk2) or alkali-metal-cations exporters (Tok1, Nha1 and Ena1-5) were constructed. Strains with kch1 and/or kch2 + deletions were tested for their ability to grow on solid media with limited K concentrations (15 µM – 100 mM KCl) and for their tolerance to high extracellular concentrations of alkalimetal-cation salts (KCl, NaCl, LiCl) or cationic drugs. In comparison with the wild-type + cells, the growth of strains with the kch1 deletion was improved upon the K limited conditions. To complement the phenotype of kch1 or kch2 deletions, multicopy plasmids with KCH1 and KCH2 genes (without or with GFP tag at the 3´ end) were constructed. The Kch1-GFP protein was predominantly localised in the plasma membrane of buds and in the vacuole of mother cells, while the Kch2-GFP protein was not detected in cells upon normal growth conditions. A putative role of Kch1 in the establishment of plasma membrane potential will be discussed. Acknowledgments This work was supported by the project Kontakt II LH14297 from MSMT (Czech Republic). 1. Stefan, C.P., et al., Activation of an essential calcium signaling pathway in Saccharomyces cerevisiae by Kch1 and Kch2, putative low-affinity potassium transporters. Eukaryot Cell, 2013. 12(2): p. 204-14. 2. Stefan, C.P. and K.W. Cunningham, Kch1 family proteins mediate essential responses to endoplasmic reticulum stresses in the yeasts Saccharomyces cerevisiae and Candida albicans. J Biol Chem, 2013. 288(48): p. 34861-70.

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PH4

HIGH ACCUMULATION OF CIS-VACCENIC ACID REDUCES THE TOXICITY OF UNSATURATED FATTY ACID IN SACCHAROMYCES CEREVISIAE STRAIN DEFICIENT IN STORAGE LIPID synthesis Sec P., Holic R. Institute of Animal Biochemistry and Genetics, SAS, Ivanka pri Dunaji, Slovakia The quadruple mutant (QM: dga1Ä, lro1Ä, are1Ä, are2Ä) deficient in enzymes responsible for storage lipid synthesis (triacylglycerols and sterol esters) shows high sensitivity to exogenous unsaturated fatty acids. Since mechanism of free fatty acids detoxification is in their incorporation into storage lipids, QM must employ alternative mechanism to cope with the accumulated free fatty acids. In our study we have found that cis-vaccenic acid accumulates during the cultivation time in the QM cells but not in the corresponding wild type strain. This accumulation was accompanied with reduction in palmitoleic acid (16:1n-7) content in the QM cells that is consistent with the proposed formation of cisvaccenic acid by elongation mechanism of palmitoleic acid. Fatty acid analysis of individual lipid classes from the QM strain revealed that cis-vaccenic acid was highly enriched in the free fatty acid pool. Furthermore, production of cis-vaccenic acid was arrested if mechanism of free fatty acid release to growth medium was activated. We also showed that exogenous cis-vaccenic acid did not affect the viability of the QM strain at concentrations toxic for palmitoleic or oleic acids. Moreover, addition of cis-vaccenic acid to the growth medium provided partial protection against the lipotoxic effects of exogenous oleic acid. Transformation of palmitoleic acid to cis-vaccenic acid is thus a rescue mechanism which enables S. cerevisiae cells to survive in the absence of triacylglycerol synthesis as the major mechanism for unsaturated fatty acid detoxification. Acknowledgment: This work was supported by the Slovak Research and Development Agency under the contract No. APVV-0785-11 and VEGA agency grant No. 2/0180/12.

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PH5

ISOLATION OF TPBGL2 GENE OF TETRAPISISPORA PHAFFII

IN VIEW OF THE HETEROLOGOUS EXPRESSION OF A KILLER TOXIN OF ENOLOGICAL INTEREST

Sara Landolfo1, Lucia Oro2, Francesca Comitini2, Marilena Budroni1, Severino Zara1, Maurizio Ciani2, Ilaria Mannazzu1 1

Dipartimento di Agraria, Università degli Studi di Sassari, Viale Italia 39, Sassari, Italy; 2Dipartimento di Scienze della Vita e dell’Ambiente, Via Brecce Bianche, Università Politecnica delle Marche, Ancona, Italy The yeast Tetrapisispora phaffii (previously known as Kluyveromyces phaffii) produces a glycoprotein of about 33 kDa (Kpkt) that shows β-1,3 glucanase and killer activities. This protein kills wine spoilage yeasts ascribed to the genera Kloeckera/Hanseniaspora and Zygosaccharomyces through the induction of ultrastructural modifications on the cell wall (Comitini et al., 2009). Moreover, it maintains its killer activity for at least 14 days under winemaking conditions. Therefore, Kpkt has interesting potential as a bioactive compound that can be used during the prefermentative stages of alcoholic fermentation (Comitini e Ciani 2010). Here, the isolation of the gene coding for Kpkt was carried out in view of the heterologous expression of the toxin. On this purpose, by using degenerate primers designed on the NH2-terminal sequence of Kpkt and on conserved regions of homologous proteins in S. cerevisiae and C. albicans, a portion of the gene was PCR amplified. Inverse PCR (IPCR) allowed the isolation of the complete sequence of the gene, that was named TpBGL2. TpBGL2 was knocked out by replacing its open reading frame with the cassette loxP-kanMX-loxP (Wach, 1996) and the phenotypic characterization of the mutants confirmed the involvement of TpBGL2 in both killer and β-1,3 glucanase activities of T. phaffii (Oro et al., 2014). Finally TpBGL2 was cloned in pPIC9 plasmid vector, under the control of AOX1 promoter, for the heterologous expression of Kpkt in Pichia pastoris. Project granted by Fondazione U925.2014/AI.808.MGB. PI I. Mannazzu

Banco

di

Sardegna

Prot.

References Comitini et al., (2009). Microbial Cell Factories 8:55; Comitini and Ciani (2010) Letters in Applied Microbiology 50: 50; Oro et al., (2013) FEMS YR 14: 464; Wach (1996) Yeast 12:259.

44

PH6 C.UTILIS AND P.STIPITS CULTIVATION IN SUGAR BEET PULP HYDROLYSATES

Patelski P.1, Berlowska J.1, Pielech-Przybylska K.1, Balcerek M.1, Dziekonska U.1, Irzyniec Z 1. 1 Faculty of Biotechnology and Food Sciences, Technical University of Lodz, Wolczanska 171/173, 90-924 Lodz, Poland e-mail: [email protected] Due to the increasing demand for SCP, it is necessary to look for cheap substrates for the biomass obtaining. Sugar beet pulp is a residue after sucrose extraction. In Poland, annually, about 11x109 kg of sugar beet is consumed for white sugar manufacturing, and approximately 5x108 kg (as DM) of sugar beet pulp is obtained as a by-product of this technology. Currently, sugar beet pulp is used mainly for the production of animal feed , excessive amounts are recycled to the fields as fertilizers. Possibility of C.utilis and P.stipitis cultivation in sugar beet pulp hydrolysates was evaluated in this study. Hydrolysates were obtained by enzymatic hydrolysis with use of cellulolytic preparations, and thermo-chemical pretreatment. After enriching with mineral salts and correction of pH to 5±0,2 medium was inoculated with yeast cultures obtained from pure cultures collections: Candida utilis ATCC9950 and Pichia stipitis LOCK 0047. Shaken cultures were kept in 1 dm3 flasks, with 150 cm3 filling with substrate, and inoculum quantities of dry matter 1 g dm -3. Biosynthesis of SCP yeast was carried out for 48 h at 30 ± 1 °C. Fed-batch cultivations were kept in a 3.5 dm3 working-volume benchtop bioreactor equipped with pH (4.8 ± 0.2), temperature (30 ±1°C), aeration (1vvm) and foam control systems. The inoculum was set to 20 g DM (dry matter, as 100 cm3 yeast cream); initial filling was 400 cm3 of water. During 12 h of cultivation, the “net biomass” (biomass without seed yeast) content increased from 0 g DM to 50,6 g DM for P. stipitis, and to 48.6 g DM for C. utilis in the fermentor. Crude protein content in the dry matter of C. utilis biomass was 51.2 %, and 48.6% for P. stipitis. Results of these trials demonstrate the possibility of utilisation of hydrolysed sugar beet pulp for obtaining C. utilis and P. stipitis biomass with high protein content.

Acknowledgments This work was supported by Polish National Centre for Research and Development under grant PBS1/B8/3/2012.

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PH7

UTILIZATION OF GRAPE POMACE BY S. CEREVISIAE CCY 21-4-47 Hlaváček V.1,2, Výmolová M.1, Márová I.1,2 1

Department of Food Chemistry and Biotechnology, Faculty of Chemistry, Brno University of Technology, Purkyňova 118, 612 00 Brno, Czech Republic 2 Materials Research Centre, Faculty of Chemistry, Brno University of Technology, Purkyňova 118, 612 00 Brno, Czech Republic Nowadays, utilization of food industry waste is becoming studied more widely. It’s a source of cheap, efficient and available substrate for microbial conversion and production. In the world 71% of the grape production refers to winemaking industry. Pomace is a winery byproduct consisting of skins, seeds, and stems remaining after enological fermentation and represents around 20% of grapes by weight. It is currently used as a crop fertilizer although with limited success because of its inhibitory effect on plant seed germination due to the high content of polyphenolics and flavonoids. The pomace obtained from local wine growers contains a relatively high oil content, 9.8% (w/w). This oil can be easily extracted, what leads to a higher viability of the fermenting yeasts. This is caused due to lowering the concentration of inhibitory effect of polyphenolics dissolved in the oil. The extracted pomace contains 19.7% of reducing sugars and 17.2% of cellulose, which can be also hydrolyzed to fermentable sugars. Those can be then metabolized to produce ethanol by Saccharomyces cerevisiae CCY 21-4-47. Hydrolyses have been carried out in three variations for comparison. Chemical hydrolysis performed in water bath, chemical hydrolysis enhanced by microwaves and enzymatic hydrolysis. The chemical hydrolyses were followed by fermentation, when the enzymatic hydrolysis run simultaneously with fermentation (SSF process). As the best resulted a 48 h fermentation after chemical hydrolysis in water bath. The SSF fermentation process has resulted with the same ethanol yield, but after more than 68 hours. All samples have been measured by HPLC-RI method. In conclusion grape pomace as a wine making byproduct can be a promising substrate for microbiological production. After extracting the residual oil, which can have also a further usage, the pomace has been hydrolyzed and fermented by S. cerevisiae CCY 21-4-47. This fermentation resulted in maximum ethanol yield of 31.2 g/l after 48 h of fermentation. This refers to 82 % of the theoretical ethanol yield, calculated from the available reducing sugars. Acknowledgements This work was supported by “Materials Research Centre – Sustainability and Development” project Nr. LO1212 of the Ministry of Education, Youth and Sports

46

PH8

USING MODERN TECHNIQUES: FLUORESCENCE MICROSCOPY AND FLOW CYTOMETRY TO MOLECULAR CHARACTERIZATION OF RED YEASTS

Hároniková A.1, Vacková H.2, Márová I.1,2, Samek O.3, Kryzyanek V.3 1 Material Research Centre, Brno University of Technology, Purkynova 118, 612 00, Brno, Czech Republic 2, Department of Food Chemistry and Biotechnology, Brno University of Technology, Faculty of Chemistry, Purkyňova 118 Brno, Czech Republic 3 Institute of Scientific instruments of the ASCR, v.v.i., Královopolská 147, 612 64 Brno, Czech Republic All types of cells are characterized by the ability to respond to changes in their environment. When carotenogenic yeasts cells are exposed to stress conditions they begin to accumulate even more pigments, lipids and change the morphological structure. A biomass of red yeast strains were studied due to their potential applications in food, feed and farmaceutical industry using modern techniques: flow cytometry and fluorescence microscopy. The flow cytometry can be used for determination the viability of microorganisms in combined with spectroscopy which is very important factor to study cells response on applied stress. In this study we have observed different morphology of cells of Rhodotorula and Cystofilobasidium yeast strains cultivated in two different media (control glucose medium and medium with high C/N ratio lead to an increased lipid production). We combined information about morphology of yeasts, cell viability and ability to accumulate selected metabolites during growth phase. Furthermore, the research was focused on resistence of cell wall and cell stress responses by selected stress factors - using high temperature and freezing. The yeast cells were stain by PI and Nile red. Various resistance of cell wall and specific spherical structures in yeast cells were observed, depending on yeast strain used, culture medium and stage of growth phase. These results can be applied in selected industrial sectors. However, systematic studies are still required to investigate yeast cell morphology in more details. Keywords: red yeasts, cell morphology, viability Acknowledgements: This work was supported by “Materials Research Centre – Sustainability and Development” project Nr. LO1212 of the Ministry of Education, Youth and Sports

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P1

TAXONOMY OF TAPHRINA SPECIES IN SLOVAKIA 1

Bacigálová, K., 1Petrýdesová, J., 1Slovák, M., 1Kučera, J., 2Sulo, P., and 3 Lopandic, K. 1

Institute of Botany, Slovak Academy of Sciences, Dúbravská cesta 9, 845 23 Bratislava, Slovakia; 2 Comenius University, Faculty of Natural Sciences, Department of Biochemistry, Mlynská dolina, Bratislava, 842 15 Slovakia; 3 Institute of Biotechnology, University of Natural Resources and Life Sciences, Muthgasse 18, 1190 Vienna, Austria. The genus Taphrina (Ascomycota) is remarkable group of more than 90th obligate parasites that are pathogenic of flowering plants (mainly to economicaly important fruit trees and wood species) but also to some ferns. All species of the genus Taphrina are dimorphic organisms. Whereas the filamentous state – teleomorph is strictly phytoparasitic, the yeasts state – anamorph is saprobic and can be cultivated on artificial media. The species of the genus Taphrina differentiate mainly in infections symptoms on host plants, geographical distribution, type and site of infection and morphology and the sexual stage in infected tissues. Differentiation from conventional yeasts can be accomplished by a unique combination of physiological and biochemical characteristics displayed by Taphrina yeast phase: a negative Diazonium blue B reaction (DBB); positive results in tests for the presence of urease (U) and extracellular amyloid compounds (EAS) and cell-wall carbohydrate composition. By the methods of anatomical-morphological characteristics to occurrence of 22 Taphrina species in Slovakia from 29 known species from the area of others European countries have been found. The phenotypic characteristics of some species did not show the unequivocal taxonomic identification in variability of fungi. For example determination of species of Taphrina padi and Taphrina pruni showed equivocal conclusions in European context, based on their symptomatic, anatomical-morphological and ecological similarities. Next studies of sequencing of nuclear ribosomal DNA and fragmentation analysis (RAPD fingerprinting) methods succesfully was solved the taxonomic status of these species and some other species parasitizing on Alnus sp. or Populus sp. in Slovakia. Our recent studies summarizing actual knowledge of the Taphrina species variability in global context indikate the necessity to do the taxonomic analysis of the Taphrina species from our area, because some of these species were characterizied only by methods of conventional taxonomy – Taphrina on herbaceous host plant of Rosaceae and some others. By completing molecular studies (sequencing of coding and non- coding segments of nDNA and mtDNA) mentioned Taphrina species in our territory we will to clarify their taxonomic position.

48

P2

EARLIER ONSET OF MITOPHAGY CONTRIBUTES TO THE PROLONGED CHRONOLOGICAL LIFESPAN OF RAS2Δ YEAST STRAIN Belicová L. and Pichová A. Institute of Microbiology, Academy of Sciences of the Czech Republic, Prague, Czech Republic Ras/adenylate cyclase (AC)/PKA pathway is one of the major pathways regulating chronological aging in various model organisms. Deletion of RAS2 gene in yeast Saccharomyces cerevisiae attenuates this pathway and results in prolonged chronological lifespan of a ras2Δ strain. Mitochondria play an important role in cell aging and their quality control involves autophagic removal of mitochondria – mitophagy. We aimed at investigating whether and how mitochondria and their quality control process – mitophagy – contribute to the prolonged lifespan of the ras2Δstrain. Viability, oxygen consumption, reactive oxygen species (ROS) production, and mitochondrial network morphology were assessed in wild-type and ras2Δ strain. To evaluate the importance of mitophagy, a key mitophagy gene ATG11 was deleted in respective strains (thus creating atg11Δ and double knock-out ras2Δ atg11Δ). The results showed that the extended lifespan of the ras2Δ strain is most probably enhanced by earlier removal of damaged mitochondria by mitophagy and by reducing their respiratory metabolism. In our study, a disruption of mitophagy resulted in higher ROS production and loss of viability in the ras2Δ strain. Inability to perform mitophagy did not influence the wild-type strain in any of the studied parameters. The strain atg11Δ therefore compensates the ATG11 deletion by alternative processes. Our results point to a possible interconnection between mitophagy and Ras/AC/PKA signaling pathway. Understanding the interplay of mitophagy and Ras pathway might help to elucidate how cells ensure the mitochondria quality control in the aging process.

49

P3

THE YEAST USED IN FERMENTATION OF PLUM MASHES AND CHEMICAL COMPOSITION OF THE OBTAINED DISTILLATES Balcerek M1., Pielech-Przybylska K.1, Patelski P.1 and Dziekońska U. 1 1 Department of Spirit and Yeast Technology, Institute of Fermentation Technology and Microbiology, Lodz University of Technology, Lodz 90-924, Poland In Eastern and Central Europe, plum brandy (slivovitz) is the popular spirit prepared from fresh Węgierka plums (Prunus domestica). Slivovitz can be made using indigenous yeasts found on plums, or with cultured yeasts. Its originality depends on a unique aroma profile of plum fruits, as well as yeast used for fermentation or diverse microbiota that are present during spontaneous fermentation. The aim of this study was an evaluation of the effect of yeast used for fementation of plum mashes on the chemical composition as well as taste and flavour of the obtained distillates.The raw materials for production of plum distillates were plums var. Węgierka Zwykła purchased from Polish fruit processing factories. Fermentations were carried out: (1) by using dry wine yeast Saccharomyces bayanus (Fermentis, Division of S.I. Lesaffre, France) (0.3 g/L), (2) spontaneously, or (3) by addition of raisins (1.5 g/L) treating it as the source of microorganisms. Activit preparation (Institut Oenologique de Champagne, France) was added as a nutrient for yeast. Fermentation process was conducted at 19÷20°C. Final distillates were analyzed by using Agillent 6890N gas chromatograph (USA), equipped with a flame–ionization detector (FID), a split/splitless injector and a capillary column HP-Innowax (60 m x 32 mm x 0.5 μm). Sensory assessment of obtained plum distillates was performed using the Buxbaum model of positive ranking (Tešević and others 2005). A chemical composition of the obtained distillates was diversified and dependent on the fermentation method. The addition of raisins to plum mashes resulted in an increase in the concentration of ethyl acetate and isoamyl acetate in the distillates in comparison with the spirits obtained after fermentation with S. bayanus yeast. The distillates obtained from spontaneously fermented plum mashes were characterized by a higher content of ethyl hexanoate and ethyl octanoate. The amyl alcohols/1-propanol ratio is used as an index to distinguish spontaneously fermented samples from those produced by monoculture.The ratio of these alcohols for the plum distillates obtained after fermentation with presence of microflora living at plum fruit and with raisins, was 0.6 and 0.9, respectively. The fermentation with S. bayanus yeast resulted in an increase of this ratio to 1.2. Acknowledgments The research was financed by a grant from NCBiR (Polish National Centre for Research and Development) implemented within the framework of the Programme of Applied Research PBS2/B8/9/2013 "Modern technologies of spirits production taking into account their accelerated maturation and improving of the physico-chemical stability ".

50

P4

YEAST RECIRCULATION IN THE PROCESS OF ETHANOLIC FERMENTATION OF SUGAR BEET PULP HYDROLYSATES Balcerek M1., Berłowska J.1 Pielech-Przybylska K.1, Patelski P.1 Dziekońska U.1 Kręgiel D.1, Dudkiewicz M.1 and Kalinowska H.2 1 Institute of Fermentation Technology and Microbiology, 2Institute of Technical Biochemistry, Lodz University of Technology, Wolczanska 171/173, Lodz 90-924, Poland Sugar beet pulp (SBP) is the residue of sugar beet processing and is a promising feedstock for fuel ethanol production. This raw material consists mainly of polysaccharides such as cellulose, hemicellulose and pectin which constitute up to 80% of dry matter. Converting SBP into fuel ethanol by chemical and/or enzymatic hydrolysis and next through biochemical pathways is an attractive option utilization of this by-product because these processes are under aqueous conditions.The yeast Saccharomyces cerevisiae which still remains the prime species that have been exploited for ethanol biosynthesis, produces ethanol by fermentation of hexose sugars but is unable to ferment pentose sugars. The aim of this study was to investigate the bioethanol production of sugar beet pulp hydrolysate and the effect of the type of yeast, as well as its recirculation on ethanol yield. Sugar beet pulp was obtained from the sugar factory in Dobrzelin (Poland). Hydrolysis was carried out by using enzymatic preparations such as Viscozyme and Ultraflo Max (Novozymes, Denmark). Fermentations were carried out by using Ethanol-Red yeast strain (S. cerevisiae), (Fermentis–Lesaffre, France) as well as Pichia stipitis and Pichia angusta, purchased from The National Collection of Yeast Cultures (United Kingdom). Worts were supplemented with (NH4)2HPO4 as a nutrient for yeast and fermented with Ethanol Red yeast strain (2 g d.m./L) and Pichia stipitis/angusta (2 g d.m./L), which were applied sequentially or simultaneously. Fermentation was conducted at 28–30°C and at 43–45°C. Before an inoculation with the second yeast strain, the biomass of yeast used to initiate the fermentation process was separated and then used (after disinfection in sulfuric acid solution) for a fermentation of the next portion of sugar beet pulp hydrolysate. The obtained results show that the sequential application of yeast S. cerevisiae and P. stipitis, both in the first fermentation, and after recirculation (temperature 28-30°C), resulted in an increase of fermentation efficiency as compared to using a mixed culture of S. cerevisiae and P. angusta. The separation of yeast biomass used for initiation of fermentation before inoculation with the second yeast strain, had a positive effect on a fermentation performance at the temperature of 43-45°C. The worts fermented by using recirculated biomass of yeast, were characterized by the relatively high intakes of sugars in relation to the ethanol yields.

Acknowledgments The research was financed by a grant from NCBiR implemented within the framework of the Programme of Applied Research - PBS1/B8/3/2012 "Biomass sugar beet pulp as a new raw material for the production of fermentation substrates"

51

P5

THE VARIOUS DISTINGUISHING PROPERTITIES OF METSCHNIKOWIA PULCHERRIMA

Breierová E.1, Guthová J.1, Sasinková V.1, Klempová T.2, and Čertík M.2 1 Institute of Chemistry, Slovak Academy of Sciences, 84538 Bratislava, Slovakia, and 2 Faculty of Chemical and Food Technology, Slovak University of Technology, Radlinského 9, 812 37 Bratislava, Slovakia

The ecological distribution of Metschnikowia pulcherrima is broad and includes flowers, fruits and other plants. The strains of this species have strong biocontrol activity against various microorganisms. The yeast can persist on winery equipment leading to subsequent inoculations if the equipment is not properly sanitized. Different strains have been used as a highly effective biocontrol agent against several fungal species such as Penicillium expansum and Botrytis cinerea. Antagonistic effects of various M. pulcherrima strains on different species of Candida, Aspergillus, E. coli, Proteus vulgaris, Trichosporon mucoides, and on Trichoderma spp., were also reported (1). M. pulcherrima produces a secondary metabolite pulcherrimin and exopolymers it to the growth medium. Pulcherrimin forms a chelate complex and immobilizes the iron ions in the growth medium. Hence, it seems that M. pulcherrima strains exert their antagonistic effects on the other microorganisms by the depletion of iron in the growth medium. A distinctive feature of this type of yeast is accumulation of lipids in cells (visualized by Nile blue A). Oleic acid (C18:1-9c – 49%) and linoleic acid (C18:2-9c,12c – 26 %) are dominant fatty acids in total intracellular lipids. Elementary analysis also revealed that this yeast species produced into extracellular medium compounds included consisting of nitrogen (7.1%), carbon (26.2%), hydrogen (4.5%) and sulphur(2.6 %). Acknowledgment: This work was supported by the Slovak Research and Development Agency under the contract No. APVV-0662-11 and VEGA agency grant No. 2/0023/14. (1).Oro L., Ciani M., Comitini F. Antimicrobial activitz of Metschnikowia pulcherrima on wine yeasts. J Appl Microbiol 116, 1209-1217, 2014.

52

P6

ACCUMULATION OF SQUALENE IN THE YEAST SACCHAROMYCES CEREVISIAE WITH REDUCED SQUALENE MONOOXYGENASE ACTIVITY Zsófia Csáky, Veronika Zambojová, Ivan Hapala Institute of Animal Biochemistry and Genetics, Slovak Academy of Sciences, Moyzesova 61, 900 28 Ivanka pri Dunaji, Slovakia Squalene is a polyunsaturated hydrocarbon which plays a key role in sterol biosynthesis in eukaryotes. Due to its beneficial effects as antioxidant, immune stimulator and skin hydrating substance, it has considerable potential in nutraceutical, cosmetic and pharmaceutical industries. The richest natural source of squalene is the oil fromdeep-sea sharks, but it can be found also in some vegetable oils e.g. olive oil, palm oil, wheat-germ oil and amaranth oil. Since the natural sources of squalene are limited, alternative options of squalene production are explored. Yeast Saccharomyces cerevisie represents one of promising producents of this valuable substance. Under normal growth conditions, squalene is rapidly converted to ergosterol and therefore does not accumulate in yeast cells. Through genetic or/and physiological manipulations of sterol biosynthetic pathway the amount of squalene in cells can be increased. The aim of our study was to examine the accumulation of squalene in the yeast Saccharomyces cerevisiae with reduced activity of squalene monooxygenase, which catalyzes the epoxidation of squalene into 2, 3oxidosqualene. We analyzed two mutants in the ERG1 gene encoding this enzyme – one ts

isolated as terbinafine-hypersensitive mutant [1], the other as supressor of the erg26 mutation [2]. Both mutants showed significant accumulation of squalene in standard

growth conditions. To explore the potential of these mutants for squalene production we estimated the dependence of cellular squalene levels on the growth phase (late o

o

o

exponential/early stationary/late stationary) and growth temperature (22 C/28 C/35 C). Both mutants showed highly specific response both to the growth phase and to growth temperature indicating that optimization of cultivation conditions will be necessary for each individual mutant.

This work was supported by the Slovak Research and Development Agency under the contract No. APVV-0785-11 and VEGA grant No. 2/0058/11. [1] Ruckenstuhl C, et al.Antimicrob Agents Chemother. 2007 Jan;51(1):275-84 [2] Germann M, et al., J Biol Chem. 2005 Oct 28;280(43):35904-13

53

P7

CELL WALL COMPOSITION OF KLUYVEROMYCES LACTIS MUTANT DEFICIENT IN ANIONIC PHOSPHOLIPID SYNTHESIS

Drozdíková E. 1, Bardelčíková A.1, Geršiová A.1, Farkaš V.2, Obernauerová M.1 1

Depertment of Microbiology and Virology, Faculty of Natural Sciences, Comenius University in Bratislava, Slovakia; 2 Institut of Chemistry, Center for Glycomic, Slovak Academy of Sciences, Bratislava, Slovakia. Kluyveromyces lactis is a strictly aerobic petite negative yeast for which the loss of mtDNA or anionic phospholipids is lethal. Previous work has shown that

the

anionic

phospholipids

cardiolipin

(CL)

and

its

precursor

phosphatidylglycerol (PG) are not only essential for the mitochondrial biogenesis and cell´s energy metabolism but also for some other non-mitochondrial functions, e.g. for cell wall biogenesis. The cell wall is a complex structure which affects the integrity of the organism during the growth and cell division. A connection between the absence of anionic phospholipids and the cell wall biogenesis was reported by Lussier et al., (1997) who analysed Scpgs1 mutant (deficient in CL and PG synthesis) and showed its altered sensitivity to cell wall-perturbing agents. The aim of this work was to analyze the impact of the absence of anionic phospholipids on the cell wall composition of K. lactis pgs1 mutant, carrying the suppressoric atp2.1 mutation. Our results showed that Klpgs1atp2.1 mutant in comparison to the wild type strain was hypersensitive to calcofluor white (chitin binding fluorescent dye), but resistant to the lytic action of zymolyase (cell wall degrading β-1,3-glucanase). Analyses of cell wall composition in K. lactis strain deficient in anionic phospholipids

has shown increased content of cell wall

chitine, while major changes in levels of β-glucan were not observed. Acknowledgement: This work was supported by the project VEGA 1/0077/14.

54

P8 EFFECT OF LIGNIN DEGRADATION PRODUCTS ON ETHANOL FERMENTATION BY SACCHAROMYCES CEREVISIAE Dziekońska U., Berłowska J., Patelski P., Balcerek M., Pielech-Przybylska K. Institute of Fermentation Technology and Microbiology, Lodz University of Technology, Wolczanska 171/173, 90-924 Lodz, Poland

Second generation bioethanol production technology is currently in the area of interest of many research centers in the world. Biofuels can be produced from non-food organic lignocellulosic materials. The typical conversion process involves pretreatment of lignocelluloses with mild sulphuric acid to increase the susceptibility of the cellulose to enzymatic hydrolysis. During pretreatment of lignocellulosic substrates, various toxic compounds are formed that can inhibit fermentation of the released sugars to ethanol. The pretreatment usually degrades the hemicellulose leading to the formation of products such as pentose and hexose sugars, sugar acids, aliphatic acids and furan aldehydes. After hydrolysis of lignocellulose polysaccharides, lignin remains as a solid residue, although a minor part is degraded to phenolics and other aromatic compounds. Apart from furans formed by sugar degradation, phenol monomers from lignin degradation are important co-factors in hydrolysate inhibition, and inhibitory effects of these aromatic compounds on different ethanol producing microorganisms is reviewed. Aim of the study was to examine the inhibiting effects of model compounds generated during acid hydrolysis of lignin (vanillin and 4-hydroxybenzaldehyde) on ethanol fermentation of glucose by commercial dried distillers yeasts Saccaromyces cerevisiae Theromsacc Dry. Fermentations were carried out in 250 ml flasks with cotton wool plugs containing 100 ml of YPG medium. Flasks were sterilized at 121°C for 20 minutes and then inoculated with S. cerevisiae yeast (0,5 g/L). Solutions of inhibitors (vanillin at 1; 2; 3 g/L and 4hydroxybenzaldehyde at 0,5; 1; 2 g/L) were sterilized by filtration and added to the fermenters before inoculation. The dynamics of fermentation processes and it’s yield were evaluated during 120 h of process conducted at 30°C. Obtained result showed that there was no complete inhibition of the fermentation in the tested range of vanillin concentrations. In the control test the turbulent phase began at approx. 14 h of fermentation, while in the sample with the highest dose of this compound (3 g/L) the beginning of this phase was observed at approx. 40 h. In the case of the 4-hydroxybenzaldehyde it has been shown that the addition of this compound causes a significant slowdown of the process. The higher the concentration of the compound in the medium, the greater the delay of the adaptive phase was observed, but none of tested doses does not completely inhibit the fermentation. For samples with the highest content of 4hydroxybenzaldehyde adaptive phase lasted for approx. 60 h. Acknowledgments The research was financed by a grant from NCBiR implemented within the framework of the Programme of Applied Research - PBS1/B8/3/2012 "Biomass sugar beet pulp as a new raw material for the production of fermentation substrates".

55

P9

THE INFLUENCE OF ULTRASOUNDS ON RELEASE OF SACCHARIDES DURING YEAST AUTOLYSIS. Dudkiewicz M., Berlowska J., Kregiel D., Dziekońska U., Pielech–Przybylska K., Balcerek M. Institute of Fermentation Technology and Microbiology, Lodz University of Technology, Wolczanska 171/173, 90-924 Lodz, Poland The term ‘autolysis’ means ‘self-degradation’ and refers to the breakdown of a cell by its own enzymes following the death of the cell. The process of yeast autolysis begins naturally following the death of the cell, with the disintegration of the membranous systems of yeast cells. Mechanical disintegration such as ultrasounds cause profound changes in regulatory and biosynthetical cellular processes therefore can be used as inductors of yeast cell lysis. In our study we examined the effect of ultrasound on the process of yeast autolysis. The concentrations of glucose, mannose, mannan and β-glucan present in supernatants (after the process) were taken as evaluation cryterion of autolysis process modifications. The conventional distillery yeasts (Saccharomyces cerevisiae Ethanol Red) and unconventional strains Scheffersomyces stipitis (NCYC 1541) and Kluyveromyces marxianus (LOCK0026) were used as post-fermentation mixed cultures. Autolysis was conducted at 55°C for 48 hours. Ultrasounds (40kHz, 180 minutes, 55oC) were used directly before or after 48 hours incubation. After the process, the yeast suspensions were centrifuged. The supernatants and the reaction mixture (yeast suspension) were analyzed. Analysis of glucans requires prior partial acid hydrolysis to remove gel-forming properties and covalent links to other polysaccharides (e.g. chitin) or proteins. 1,3:1,6-β-D-Glucan, 1,3-β-Dglucans and α-glucans were extensively hydrolysed by 1.3 N HCl at 100°C for 2 h. Hydrolysis to D-glucose was completed by incubation with a mixture of highly purified exo-1,3- β-glucanase and β-glucosidase. The assay was conducted using a β-Glucan (Yeast & Mushroom) Assay Kit (Megazyme). Mannans were hydrolysed in 2 M hydrochloric acid at 100°C for 2 h. After this time, a volume of 2 M sodium hydroxide equal to the amount of acid used was added. The amounts of mannose and glucose were determined using a D-Mannose/D-Fructose/DGlucose Assay Kit (Megazyme). Ultrasounds have a positive stimulatory effect on the release of intracellular substances of yeast only in the case where they are used after the conducted autolysis process, and not directly before it. The usage of a 180-minute incubation of a yeast cells suspension in an ultrasonic bath in the process of autolysis, supported the release and extraction of the polysaccharide compounds associated with the cell wall structures. Acknowledgments The research was financed by a grant from NCBiR implemented within the framework of the Programme of Applied Research - PBS1/B8/3/2012 "Biomass sugar beet pulp as a new raw material for the production of fermentation substrates".

56

P10 TRANSCRIPTION PROFILING OF CANDIDA ALBICANS INTERACTING WITH PSEUDOMONAS AERUGINOSA Olga Hrušková-Heidingsfeldová1, Lenka Mynářová1, Vratislav Šťovíček2, Vladimír Vrkoslav1, Zdena Palková2 and Iva Pichová1 1

Institute of Organic Chemistry and Biochemistry, Academy of Sciences of the Czech Republic, Prague 6, 166 10; 2Department of Genetics and Microbiology, Faculty of Science, Charles University in Prague, Viničná 5, Prague 2, 128 44. The yeast Candida albicans and Gram-negative bacterium Pseudomonas aeruginosa were found to form mixed communities in respiratory tract of patients suffering from cystic fibrosis or ventilator-associated pneumonia. Interactions between C. albicans and P. aeruginosa (Ca-Pa) can be either antagonistic or synergistic, depending on overall environmental conditions. While yeast form of C. albicans is resistant to P. aeruginosa and retains full viability in mixed cultures, the bacterium readily adheres to C. albicans filaments and kills them. Surface structures such as cell wall glycans that differ between yeast and hyphal form of C. albicans play an important role in resistance or susceptibility of C. albicans to P. aeruginosa2. Ca-Pa interactions are controlled also by secreted factors. Some of them are known to be constituents of quorum sensing (QS) systems of either of the species. In our study, C. albicans and P. aeruginosa were co-cultivated in planktonic settings and C. albicans transcription profile was analyzed using DNA arrays. Transcriptional changes in C. albicans were rather moderate. Transcription of approximately 700 C. albicans genes appears to be affected by presence of P. aeruginosa. This set includes genes involved in transport of nutrients, protein glycosylation, mating pathway; genes known to be regulated by pH or iron; multidrug transporters. Interestingly, presence of P. aeruginosa caused downregulation of ARO2 and ARO7 genes that are involved in shikimate pathway. Shikimate is a precursor of aromatic amino acids that can be converted to QS molecules. Analysis of the cultivation media revealed decreased concentration of phenetyl alcohol in Ca-Pa co-cultures. Phenetyl alcohol is known to inhibit hyphal growth. The decrease of phenetyl alcohol concentration may render the Candida cells more prone to form hyphae and to become a target of P. aeruginosa killing. Acknowledgments This work was supported by grants GA14-23022S from the Czech Science Foundation and by project LO1302 (NPU I, LO) from Ministry of Education of the Czech Republic , and RVO 61388963 project.

57

P11

LACTOSE INDUCED GALACTOSIDASES OF GENUS CRYPTOCOCCUS Stratilová B.1,2, Pavlatovská B.3, Omelková J.3, Vadkertiová R.1 and Stratilová E.1 1

Institute of Chemistry, Slovak Academy of Sciences, Bratislava, Slovakia Faculty of Natural Sciences, Comenius University in Bratislava, Bratislava, Slovakia 3 Faculty of Chemistry, Technical University of Brno, Brno, Czech Republic 2

This work focuses on induction of galactosidases by yeasts of genus Cryptococcus growing on lactose medium and relations between this production and growth of yeasts. Each of the 18 tested yeast strains, including species Cr. carnescens (CCY 17-3-13), Cr. flavescens (CCY 17-3-6, CCY 17-3-15, CCY 17-3-29, CCY 17-3-31, CCY 17-3-33, CCY 17-3-38), Cr. flavus (CCY 17-3-5), Cr. laurentii (CCY 17-3-2, CCY 173-9, CCY 17-3-17, CCY 17-3-24), Cr. magnus (CCY 17-4-39, CCY 17-4-40), Cr. saitoi (CCY 17-3-18), Cr. victoriae (CCY 17-3-26) and Bulleromyces albus (CCY17-3-35, CCY 17-3-37) from the Culture Collection of Yeasts, Institute of Chemistry of SAS, was cultured for 96 hours in a liquid medium with lactose. During cultivation, the quantity of cells was determined as well as enzyme activities of α- and β-galactosidase in the medium and on the cell surface. Lactose medium was shown not to be suitable culture medium for all Cryptococcus strains, because some of them grew on it very slowly (CCY 17-3-29, CCY 17-3-5, CCY 17-3-26, CCY 17-3-35), or showed a long adaptation phase (CCY 17-3-2, CCY 17-3-6). A comparison of the growth and surface β-galactosidase production curves showed that the link between lactose medium and production of this enzyme does not exist in generally. The results did not confirm neither the expected impact of this enzyme on the growth of strains nor the anticipated induction of β-galactosidase by lactose. For instance, the fastest growth on lactose showed the strain Cr. carnescens CCY 17-3-13 which exhibited a very low activity of this enzyme on its surface. Relatively increased production of this enzyme was observed on the surfaces of the type strain Cr. laurentii CCY 17-3-2, strain Cr. flavescens CCY 17-3-31 and Cr. flavus CCY 17-3-5. The production of α-galactosidase by capsular yeasts was strain-dependent with the exception of the members of Cr. flavescens species. Because of this, the expected general influence of this enzyme on the re-building of the protective cryptococcal capsule [Mészároszová et al. 2012] can be excluded. Only Cr. flavescens strains can be generally considered as producers of lactose-inducible surface α-galactosidase. In other cases, the production of this enzyme matters on single strain and not on species, as can be seen in the case of type strain Cr. laurentii and the other Cr. laurentii strains. Similar induction by lactose medium was also observed with non-cryptococcus yeasts and fungi and therefore the link with the capsules can be also excluded. Acknowledgments This contribution is the result of the project implementation: Centre of Excellence for Glycomics, ITMS 26240120031, supported by the Research & Development Operational Programme funded by the ERDF. The financial support from VEGA grants Nos. 2/0020/12 and 2/0023/14 is greatly appreciated, too.

58

P12

OPTIMIZATION OF SAMPLE PREPARATION FOR BIOTYPING OF CRYPTOCOCCUS YEASTS

Stratilová B.1,2, Ledvina V.3, Hamříková D.3, Jurnečková A.3, Molnárová J.1, Omelková J.3, Vadkertiová R.1, Stratilová E.1 1

Institute of Chemistry, Slovak Academy of Sciences, Bratislava, Slovakia Faculty of Natural Sciences, Comenius University in Bratislava, Bratislava, Slovakia 3 Faculty of Chemistry, Technical University of Brno, Brno, Czech Republic 2

The species Cryptococcus laurentii was assumed to be heterogeneous for a long time. The application of molecular biology and bioinformatic methods led to dividing of yeast strains to two distinct phylogenetic groups, the first one containing species Cr. laurentii, Cr. flavescens and Cr. aureus, the second one at least Cr. carnescens, Cr. peneaus and Cr. victoriae. The locution „Cryptococcus laurentii group“ was introduced [Takashima et al., 2003]. This work was firstly focused on 28 strains of the species Cr. laurentii, Cr. flavescens and Cr. victoriae from the Culture Collection of Yeasts, Institute of Chemistry of SAS, which were identified by the sequence analysis of the D1/D2 domains of 26rRNA gene and/or by the identity of mass spectra of strains growing on malt agar (biotyping). Biotyping is a method successfully used in identification of prokaryotic and ever-increasing number of eukaryotic microorganism in clinical diagnostic laboratories. It is based on mass spectrometry measurement of so called “molecular fingerprint” given mostly by ribosomal proteins which is species (in some cases even variety) dependent. The production of ribosomal proteins is independent on such factors as cultivation medium or the period of the cultivation. The yeasts of the genus Cryptococcus are specific because of the production of huge amounts of extracellular polysaccharides and the polysaccharide capsule. Unlike ribosomal proteins, the production of these saccharides is dependent on cultivation medium or the culture age. Althoug the cells growing on malt agar (maintenance medium of CCY) gave spectra with satisfactory quality, YPD, Sabouraud and potato agar were tested as cultivation media. Their effect on spectra quality and the possibilities to influence it using modified methods of sample preparation, application and measurement are studied. From the best set of spectra MSP dendrogram was created and compared with phylogram created from the sequences of the D1/D2 domains of 26rRNA gene using Clustalw2.Further experiments were based on the procedures applied on the strains from the Cryptococcus laurentii group. Eighteen Cryptococcus, but non-laurentii group strains were examined but the resulting effect was not uniform. The best spectra were achieved, when the yeasts were cultivated four days on YPD medium at 20 ºC but the only general rule for all strains was to use matrices visualizing preferentially proteins (mixture of sinapic and ferulic acid) instead of peptides (α-cyano-4-hydroxycinnamic acid) as was recommended. The spectra, although poorer, show more exactly typical proteins because of effective elimination of saccharide peaks with lover m/z. However, a manual checking of spectra quality is required before their normalization. Acknowledgments This contribution is the result of the project implementation: Centre of Excellence for Glycomics, ITMS 26240120031, supported by the Research & Development Operational Programme funded by the ERDF. The financial support from VEGA grants Nos. 2/0020/12 and 2/0023/14 is greatly appreciated, too. The study states of Bc. Vojtěch Ledvina and Bc. Dominika Hamříková in the Institute of Chemistry of SAS was financed by the project” BiochemNet - Vytvoření sítě pro podporu spolupráce biomedicínských pracovišť a zvýšení uplatnitelnosti absolventů biochemických oborů v praxi” (reg. No. CZ.1.07/2.4.00/31.0133). Project was realized within “Operační program vzdělávání pro konkurenceschopnost”, funded by the ESF and the state budget of Czech Republic.

59

P13

IN VIVO STUDY OF DGA1P AND DGA2P ENZYMES CONTROLLING TRIACYLGLYCEROL ACCUMULATION IN YARROWIA LIPOLYTICA Gajdoš P., Meravá Z., and Čertík M. Faculty of Chemical and Food Technology, Slovak University of Technology, Bratislava, Slovak Republic Yarrowia lipolytica is an oleaginous yeast with unique lipid metabolism. It is adapted to grow in environments rich in various hydrophobic substances e. g. n-alkanes, oils, and fats. Cells of Y. lipolytica produce lipases and emulsifiers enabling transport of such hydrophobic substances across the plasma membrane. Once the hydrophobic molecules are in the cell, they can be degraded in βoxidation pathway to provide energy for the cell. Another possibility is conversion of these molecules into storage lipids, which is known as ex novo lipid accumulation. The major storage lipid species in Y. lipolytica’s cells are triacylglycerols (TAG). TAG in yeast cells are stored in organelles called lipid bodies. Synthesis of TAG in yeasts is mediated predominantly by Dga1p, which belongs to and acyl-CoA:diacylglycerol acyltransferase 2 (DGAT2) family. However, it has been recently shown that Y. lipolytica harbors also an acylCoA:diacylglycerol acyltransferase 1 belonging to the DGAT1 family, represented by Dga2p. Thus, Y. lipolytica is the only known microorganism possessing two DGAT enzymes up to date. Overexpression of DGA1 or DGA2 gene completely restored TAG synthesis in Q4 strain, a quadruple deletion mutant unable to synthesize TAG. Strains overexpressing either DGA1 or DGA2 accumulated considerable amounts of lipids. However, strain overexpressing DGA1 showed higher lipid accumulation under certain conditions. Fatty acid composition of lipids in DGA1 and DGA2 expressing strains was different. Strain overexpressing DGA1 gene had a tendency to form stearic acid (C18:0) enriched TAG. This feature was clearly visible also in the presence of extracellular fatty acids.

Acknowledgments The work was supported by grants VEGA 1/0574/15, APVV-0662-11, bilateral APVV project SK-CZ-2013-0167 and by the project "University Science Park of STU Bratislava“ ITMS 26240220084. The yeast strains were kindly provided by Dr. Jean-Marc Nicaud (France).

60

P14 EFFECTIVE METHOD FOR COMPOSITION-SPECIFIC STERYL ESTER ANALYSIS IN YEAST

Goffa E., Valachovič M. Institute of Animal Biochemistry and Genetics, Slovak Academy of Sciences, 90028 Ivanka pri Dunaji, Slovakia Fatty acid esters of ergosterol and its biosynthetic precursors are exclusively stored in lipid droplets. Under the condition of sterol deficiency steryl esters are hydrolyzed and freed to be used as building blocks for membrane formation. Esterification of sterols can also be an effective mechanism for yeast cell to get rid of some toxic ergosterol precursors when the biosynthetic pathway is blocked by antifungals, e.g. azoles or by mutations in the erg genes. To find out which of the ergosterol precursors in erg mutants are preferably used in membranes and which are stored in lipid droplets we established an effective method to separate free sterols from steryl esters with subsequent hydrolysis of steryl esters. In the first step total lipids are extracted (Blight Dyer, 1959). The whole lipid extract is separated into several fractions using Chromabond NH2 column (Macherey-Nagel). The steryl ester fraction is split in two parts and analyzed separately for sterol and fatty acid content. Critical step in the sterol analysis part is the solubilization of highly non-polar steryl esters which are only poorly soluble in the hydrolysis mixture (60% KOH, 50% methanol) and they remain adhered to the surface of the tube. To avoid this we introduced solubilization of steryl esters in 3% Triton-X100 for 2 hours at 50°C prior to actual hydrolysis. Hexane extracted sterols are then analysed by liquid chromatography. To analyze the fatty acid content of the steryl sters they are dissolved in hexane and hydrolyzed by methanolic HCl for 6 hours at 60°C. After this procedure the methylated fatty acids are subjected to gas chromatography analysis. We optimized an efficient method for steryl esters purification and their quantitative analysis. Important part of our approach is analysis of both sterol and fatty acid content of steryl esters. This method can be used to study metabolism of lipid particles not only in yeast but in other organisms as well. Acknowledgments This work was supported by the Slovak grant agencies VEGA under contract No. 2/0185/14 and APVV under contract No. 0785-11. References: E. G. Bligh, W. J. Dyer (1959) A rapid method of total lipid extraction and purification. Canadian Journal of Biochemistry and Physiology, 37(8): 911917, 10.1139/o59-099

61

P15

ISOLATION AND IDENTIFICATION OF YEASTS FROM INTERSPECIFIC VARIETY

HIBERNAL ON THE TERRITORY OF THE MORAVIAN WINE

REGION

Dana Vránová and Kristýna Dlapalová Brno University of Technology, Brno , Czech republic [email protected] This work was elaborated in cooperation with winery Stephan Maňák from Žádovice. This Moravian region has a tradition in winemaking and the wine is produced by a spontaneous fermentation, so it could represent a reservoir of wine natural yeasts with important oenological features. The aim of this study was to isolate and identify the yeast community of Hibernal interspecific wine grapes growing on Žádovice region in order to create our Saccharomyces cerevisiae bank for potential industrial applications. PCR-RFLP was applied for the identification at the species level. The grapes were sterile collected in the autumn 2014. The unstable meteorological conditions throughout the growing season caused that we managed to isolate only nine species of yeast, and among them Saccharomyces cerevisiae, too. Quality of this yeast was already tested in the winery in sterile grape must. Oenological properties of yeast will be further tested.

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PRODUCTION OF PIGMENTS AND CAROTENOID-RICH BIOMASS BY RED YEASTS CULTIVATED ON WASTE SUBSTRATES FROM WINEMAKING INDUSTRY

Hároniková A.1, Turková L.2, Márová I.1,2 1Brno University of Technology, Centre for Materials Research, Faculty of Chemistry, Purkyňova 118 Brno, Czech Republic 2 Brno University of Technology, Department of Food Chemistry and Biotechnology, Faculty of Chemistry, Purkyňova 118 Brno, Czech Republic

Carotenoids are naturally occurring pigments of plants also produced in many bacteria, and fungi. The area of their application concerns mainly food industry; however, they are used in chemical, pharmaceutical, and cosmetics industry as well. One possibility is study of potential of red yeasts that are able to convert various substrates into carotenoid pigments. When carotenogenic yeasts cells are exposed to stress conditions they begin to accumulate even more pigments. In presented comparative study production properties of four red yeast strains (Cystofilobasidium capitatum, Rhodotorula glutinis, Sporobolomyces roseus and Sporobolomyce sshibatanus) were tested. Production of carotenoids and biomass using waste substrates from winemaking (grape peelings and stemps) as nutrient sources were studied. The substrates were also subjected to chemical and enzymatic hydrolysis with complex enzyme mixtures isolated from selected fungi in our laboratory. Pigments were analyzed by RP-HPLC/PDA, enzyme activities were measured spectrophotometrically. All tested strains were able to utilize using substrates and produced biomass and pigments. The smallest production of biomass and carotenoids was observed in media with unhydrolysed waste substrates. The highest biomass production was reached in media with hydrolysed substrates. Yeast strains from genus Sporobolomyces produced significant amounts of pigments on hydrolysed grape peeling and stamps. In some cases 5 – 12x higher production than in control glucose medium was obtained. In conclusion, grapes waste substrates can be used as medium component, which can induce production of pigments or carotenoid-rich biomass. Keywords: carotenoids, waste substrates, red yeasts Acknowledgements: This work was supported by “Materials Research Centre – Sustainability and Development” project Nr. LO1212 of the Ministry of Education, Youth and Sports

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PROPERTIES AND INHIBITION OF TRANSGLYCOSYLASES OF THE YEAST CELL WALL Kristína Kováčová 1 and Vladimír Farkaš1 1 Institute of Chemistry, Slovak Academy of Sciences, 84538 Bratislava, Slovakia The yeast cell wall has many essential functions. It is composed of an inner and an outer layer. The outer layer is formed by mannoproteins, whereas the inner layer consists of polysaccharides β-1,3-glucan, β-1,6-glucan and chitin. These individual polysaccharide polymers are mutually cross-linked into large supramolecular complexes by mechanism of transglycosylation catalysed by enzymes called transglycosylases. Transglycosylases of the yeast cell wall are not very known and they have been relatively little studied and only few of them have been characterized on the basis of their transglycosylating activities. Our attention was focused on pH-regulated β-1,3glucanosyltransferases Phr1p and Phr2p (GH 72) from Candida albicans and chitin:β1,3/β-1,6-glucan specific transglycosylases Crh1p and Crh2p (GH 16) of Saccharomyces cerevisiae. These transglycosylases belong to yeast cell wall remodeling enzymes. Explanation of properties of fungal transglycosylases operating on β-1,3-glucan and chitin and their inhibition may lead to development of new antimycotics, because β-1,3-glucan and chitin are widely distributed in fungi but absent in mammalian cells. The transglycosylation activities of Crh1p and Crh2p, Phr1p and Phr2p were determined by a fluorescent in vitro assay and confirmed or disproved by size-exclusion chromatography. Subsequently, the products of the transglycosylation reactions catalysed by these enzymes were identified by MALDI-TOF analyses as hybrid molecules composed of the portions of the donor molecule attached to non-reducing end of the acceptor molecule (1,2). Several potential inhibitors of yeast cell wall transglycosylases were tested by the fluorescent dot-blot method (1). As the most effective showed the compound termed ramihyfin A (3).

References 1

Mazáň, M., Blanco, N., Kováčová, K., Firáková, Z., Řehulka, P., Farkaš, V., Arroyo, J.: A novel fluorescent assay and catalytic properties of Crh1 and Crh2 yeast cell wall transglycosylases. Biochem J. 2013; 455(3), p. 307-318. 2 Kováčová, K., Degani, G., Stratilová, E., Farkaš, V., Popolo, L.: Catalytic properties of Phr family members of cell wall glucan remodeling enzymes. FEMS Yeast Research, 2015, vol. 15, p. 1-15. 3 Baráth, Z., Baráthová, H., Betina, V., Nemec, P.: Ramihyphins – Antifungal and morfogenic antibiotics from Fusarium sp. S-435. Folia Microbiol., 1974, vol. 19, p. 507511.

Acknowledgments This work was supported by the scientific grant agency of The Ministry of Education, Science, Research and Sport of the Slovak Republic VEGA 2/0020/12.

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ADHESION ABILITIES OF CLINICAL CANDIDA SPP. STRAINS TO POLYSTYRENE Kunicka-Styczyńska A.1, Rajkowska K.1 and Maroszyńska M.1,2 1 Lodz University of Technology, Faculty of Biotechnology and Food Sciences, Institute of Fermentation Technology and Microbiology, Wolczanska 171/173, 90924 Lodz, Poland, 2 Biotechnology Laboratory, Lodz Regional Park of Science and Technology Ltd., Dubois 114/116, 93-465 Lodz, Poland The ability of Candida spp. to adhere to biotic and abiotic surfaces is regarded as prerequisite for the permanent colonization of a site exposed and for the formation of biofilm. Candida is recognized as the most frequent etiological agent of systemic and invasive infections in humans. However the most common pathogen is Candida albicans, the other Candida species are also isolated from clinical samples. The aim of this study was to assess adhesion abilities of 26 Candida clinical strains: 24 C. albicans, one C. glabrata, one C. lusitaniae, isolated from patients with diagnosed candidiasis. C. albicans ATCC 10231 was used for comparison. The cell adherence ability was estimated to polystyrene 96 well flat bottom plates, according to Raut et al. (2010). Cell density was adjusted to 107 cells/ml in PBS buffer. The number of adhering cells was counted by a hemocytometer after 90 minutes incubation at 37°C with gentle shaking. The results were expressed as the adherence index (the percentage of yeast cells adhering). In general, adherence abilities of clinical Candida spp. to polystyrene varied over a wide range, from 7% to 89%. Eight of the isolates expressed adherence indexes of less than 20%. Most of the isolates (46%) and the standard strain were characterized by high adherence capacity of more than 60%. This group consisted of ten Candida albicans strains, and C. glabrata and C. lusitaniae clinical isolates as well. About 62% of the strains tested showing wide range of adherence abilities, from 7% to 89%, were isolated from faeces. Similar differentiation of adherence indexes was found for strains isolated from throat (1370%) and from urogenital tract (8-70%). The study showed that adhesion to polystyrene varies over a broad range in clinical Candida spp. isolates, independent of their clinical source. Raut J., Rathod V., Karuppayil S. M. (2010) Cell surface hydrophobicity and adhesion: a study on fifty clinical isolates of Candida albicans. Jpn. J. Med. Mycol. 51: 131-136.

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P19

DE NOVO GENE EMERGENCE IN YEASTS AFTER WHOLE GENOME DUPLICATION

JuríkováK.1, Tomáška Ľ.1 and Gabaldón T.2 1Department of Genetics, Comenius University, Mlynská dolina, Ilkovičova 6, 842 01 Bratislava, Slovakia, and 2Bioinformatics and Genomics Programme, Centre for Genomic Regulation, Dr. Aiguader 88, 080 03 Barcelona, Spain De novo genes originate in evolution more often than previously thought and some were shown to play a role in vital cell processes. In Saccharomyces cerevisiae, genes from scratch were proposed to emerge and disappear in a continuous manner, typical for the evolutionary continuum existing between genes and pseudogenes. However, the factors contributing to the emergence of a de novo gene are not precisely characterized. In our work, we attempt to study de novo genes emergence in several yeast species. We developed a bioinformatic pipeline to find transcribed de novo genes corresponding to a non-coding homologous sequence in sister species. Our pipeline is designed with the aim to discern de novo originated genes from rapidly diverging genes that were shown to be present in yeast, especially in the species after a whole genome duplication (WGD) event. Post-WGD yeasts may represent an intriguing model to study mechanisms of de novo gene emergence. In these yeasts, WGD events were followed by a phase of gene loss, where many duplicated genes disappeared, presumably to compensate for elevated gene dose after the WGD. The loci freed by such loss may represent a vacant niche in the tightly packed yeast genomes and may thus represent a suitable environment for a de novo gene emergence. Such event may be further facilitated by the presence of an open-state chromatin or residual regulation sequences. By the use of our pipeline, we attempted to search for such cases of de novo genes originated on these free niches that originated from the residual non-coding sequence. Several interesting cases in post-WGD species show that the specific properties of post-WGD yeast genomes may indeed allow for the emergence of de novo genes on the remnants of the ceased, evolutionary more ancient genes. Acknowledgments We would like to thank all members of KovLab and Comparative Genomics Group that contributed by ideas and support. This work was supported by grants from the Slovak grant agencies APVV (0123-10, 0035-11), VEGA (1/0405/11, 1/0311/12), and the Comenius University (UK/29/2015).

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SACCHAROMYCES CEREVISIAE AS A MODEL TO STUDY THE ROLE OF MAMMALIAN TELOMERASE IN MITOCHONDRIA Šimoničová L., Dudeková H., Ferenc J., Procházková K., Neboháčová M., Dušinský R., Nosek, J. and Tomáška Ľ. Comenius University in Bratislava, Faculty of Natural Sciences, Departments of Genetics and Biochemistry, Mlynská dolina, Bratislava, Slovak Republic Telomerase is a ribonucleoprotein complex involved in the maintenance of nuclear chromosomal ends (telomeres) and genome integrity of eukaryotic cells. The core components of this enzymatic complex are represented by the telomerase RNA subunit and catalytic subunit (TERT) with reverse transcriptase activity. Somatic cells are characterized by the lack of telomerase activity resulting in progressive telomere shortening. Once the length of telomeres reaches the critical size, cells stop to divide and enter the state of cellular senescence. In contrast, cells that reactivate the telomerase are able to proliferate indefinitely and can transform into immortalized cell lines. The telomerase activity is considered as a marker of carcinogenesis and the core subunits of telomerase are suitable targets for anticancer therapies. Except from its conventional role in telomere homeostasis, telomerase exhibits various extratelomeric functions that may increase the resistance of cancer cells to various chemotherapeutics. Interestingly, under conditions of oxidative stress human catalytic subunit hTERT translocates from nucleus to mitochondria where it participates in cellular responses to oxidative stress. Despite numerous findings regarding the effects of mitochondrial hTERT, the data are often inconsistent and the details of underlying molecular mechanisms remain elusive. To address the role of telomerase in mitochondria, we developed a heterologous system, which allows investigating the effects of mammalian TERT on cellular responses to oxidative stress. We ectopically expressed both mouse and human TERT in mitochondria of baker’s yeast Saccharomyces cerevisiae. Furthermore, we demonstrated that telomeres of yeast cells expressing mammalian TERT are stably maintained. By analyzing the ability of S. cerevisiae cells to retain functional mitochondrial genome and tolerate oxidative stress we demonstrate, that in agreement with observations in mammalian cells, TERT targeted exclusively to mitochondria improves the organellar functions. Importantly, we provide evidence that this protective effect might represent the universal feature of reverse transcriptases located in mitochondria. In summary, we showed that the yeast system represents an elegant experimental model to elucidate extratelomeric functions of mammalian telomerase.

Acknowledgments This work was supported by the National Institutes of Health Grant 2R01ES013773-06A1, the Slovak grant agencies APVV (0035-11 and 0123-10) and VEGA (1/0311/12), and the Comenius University grant (UK/409/2014).

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P21 EFFECTS OF YEASTS BIOMASS PRODUCING CAROTENOIDS ON RAT HEART FUNCTION AFTER ENDOTOXIN ADMINISTRATION

Lipták B.1, Knezl V.1, Breierová E.2, Frimmel K. 3, Križák J. 3, Okruhlicová Ľ. 3 1

Institute of Experimental Pharmacology and Toxicology, Slovak Academy of Sciences, 2 Dúbravská cesta 9, 841 04 Bratislava, Slovakia, Institute of Chemistry, Slovak Academy 3 of Sciences, Dúbravská cesta 9, 845 38 Bratislava,Slovakia, and Institute for Heart Research, Slovak Academy of Sciences, Dúbravská cesta 9, 840 05 Bratislava, Slovakia Major civilization diseases that are related to cardiovascular system are accompanied with inflammation. Inflammatory process is known to change function of vascular endothelium which plays a crucial role in atherosclerotic plaque development and related disturbances in heart function. In addition to pharmacotherapy the natural substances have been recommended to reduce incidence and severity of atherosclerotic heart diseases. Therefore, the aim of our study was to examine if acetylcholine (Ach)modulated heart function may be affected by endotoxin and carotenoids produced by yeast strain Rhodontorula characterized with antioxidative properties. Ach is an important neurotransmitter of the parasympathicus nerve system, opposite to the sympathicus nerve system, slowing the heart functions. The experiment was performed on male Wistar rats. The inflammation was induced with a single dose of endotoxin (LPS E. coli, 1 mg/kg). Rats were treated with yeast biomass (10 mg/kg/day) for 10 days after LPS application. At the end of experiments we isolated the heart of the animals according to Langendorff with constant perfusion pressure. Functional parameters of the heart were evaluated in controls (C), LPS, yeast (Y), and LPS+Y groups. After the stabilization period Ach (10 min, -7 -1 3x10 mol/l ) was added to the perfusion, following 10 min perfusion without Ach. We measured heart frequency (HR), coronary flow (CF) and heart contraction (LVP). In the next experiment we examined ischemia/reperfusion (30 min/30 min)-induced arrhythmias. Selected enzymes of energetic and metabolic processes (AlPh, DAP IV, GlP, ATPases, 5NC) were also studied in the heart tissue using in situ histochemistry. Significant reduction in LVP was recorded only in LPS group in comparison to other groups. Ach significantly decreased the HR, CF and LVP in comparison with values without Ach in all experimental groups. Induced inflammation attenuated the heart propensity to arrhythmia, significantly increasing the ventricular tachycardia (VT) in comparison to the control group. The ventricular fibrillation and VT were decreased when yeast biomass administered (LPS+Y), shifting the ventricular premature contractions, which is a less dangerous arrhythmia. Yeast biomass partially improved endotoxin-related reduction of enzyme activities. The yeast carotenoids had protective effect on the heart function against the depressant effect of LPS on LVP and decreased the ischemia/reperfusion-induced severe arrhythmias. Acknowledgments Supported with VEGA 2/0065/13.

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P22 NRL1 – A NEW PLAYER IN THE MAINTENANCE OF GENOME STABILITY IN THE SCHIZOSACCHAROMYCES POMBE

Mikolaskova B.1, Cipakova I.1, Chudej T.1, Sabova L.1, Anrather D.2, Ruchman D. 2, Aronica L.2 and Cipak L.1 1

Cancer Research Institute, Slovak Academy of Sciences, Vlarska 7, 83391 Bratislava, Slovakia, and 2 Department of Chromosome Biology, Max F. Perutz Laboratories, University of Vienna, Dr. Bohr-Gasse 9, 1030 Vienna, Austria Genomic instability is defined as a process prone to genomic changes or an increased propensity for genomic alterations. Splicing of annotated introns regulates both qualitative and quantitative aspects of gene expression by producing different functional and non-functional isoforms of a transcript, respectively. Additionally, metazoan genes contain large numbers of nonannotated introns, referred as to cryptic introns, which display weaker splice sites and are spliced at lower levels. The weaker nature of cryptic intron splice sites is likely due their sequence features, which favour the re-annealing of the nascent mRNA to the DNA template forming the so-called R-loops, thus preventing the primary steps of spliceosome assembly. Aberrant cryptic intron splicing is associated with many human diseases including cancer. Here we provide new functional insight on the role of Nrl1 in splicing and its implications on genome stability. We show that Nrl1 co-purified with a complex closely resembling the endogenous S. pombe U2·U5·U6 spliceosome complex. A distinctive feature of the Nrl1-associated complex was its increased enrichment in the spliceosome disassembly factors Brr2, Prp43, Ntr1 and Ntr2, which suppress disease-associated cryptic intron splicing in human. Furthermore, we propose that Nrl1 is essential to prevent the accumulation of genome-threatening R-loops, thus safeguarding genome integrity. Future studies of Nrl1 physical interaction network and its regulation by post-translational modifications might reveal in more details the role of this protein in maintenance of genome stability. Since Nrl1 is conserved in higher eukaryotes including humans, this study paves the way to a deeper understanding of the mechanism and functional significance of regulation of metazoan splicing. Acknowledgments This work was supported by the Slovak Research and Development Agency under the contract no. APVV-0111-12 and by VEGA grant 2/0014/14.

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TRANSCRIPTION FACTORS YAP1P AND PDR1P IN THE CONTROL OF MULTIDRUG RESISTANCE IN KLUYVEROMYCES LACTIS Svrbická A., Balážfyová Z., Tóth Hervay N., Gbelská Y. Department of Microbiology and Virology, Faculty of Natural Sciences, Comenius University in Bratislava, 842 15 Bratislava, Slovak Republic Multidrug resistance (MDR), known as pleiotropic drug resistance (PDR) is a ubiquitous cellular defence mechanism to deal with potential toxicity from environmental small molecular toxins or bioactives. The rapid induction of MDR in the model yeast Saccharomyces cerevisiae is controled by two networks of genes, YAP and PDR. In these networks two types of transcription factors are involved: with the structural motif of leucine zipper (bZIP) or zinc finger motifs Zn2Cys6, respectively. Many duplicated or multiplicated genes encoding transcriptional regulators in S. cerevisiae are present in fewer copies in Kluyveromyces lactis. Yap1p is a key determinant of oxidative stress response. It regulates expression of genes encoding proteins that maintain a favourable redox balance in the cell and proteins conferring metal and drug resistance. The main determinants of MDR in S. cerevisiae include the transcription factors Pdr1p and Pdr3p that control expression of ABC, MFS transporters and several other genes. KlPdr1p is the only homologue of ScPdr1p and ScPdr3p in K. lactis. In silico analysis of KlPDR1 gene promoter revealed the presence of one PDRE element and two YRE elements. In vitro binding of KlYap1p to the KlPDR1 promoter showed Hodúrová et al. (2011). In this work, we studied the mutual functional interaction between KlPdr1p and KlYap1p in more detail. We compared the sensitivity of standard strains and its isogenic deletion mutants (Klpdr1Δ, Klyap1Δ, Klpdr1Δyap1Δ) to antifungal azoles. Our results showed differences in strain´s susceptibilities to the cytotoxic drugs used. The influence of the transcription factors on the expression of the main ABC transporter gene KlPDR5 in K. lactis was also analysed. The activity of the reporter gene (promoter of KlPDR5 gene fused in frame with bacterial β-D-glucuronidase; PKlPDR5- gusA) was assessed together with the analysis of KlPDR5 gene expression in the strains studied using qPCR. Our results indicate that KlPdr1p influences the expression of KlPDR5 gene. Based on the results obtained, we propose that the transcription factor KlYap1p binds to the YRE elements present in the KlPDR1 gene promoter and controls the KlPDR5 gene expression. Further clarification of the relations between the main transcriptional regulators KlYap1p and KlPdr1p could help to understand the origin and the control of the multidrug resistance phenomenon in K. lactis yeast.

Acknowledgments This work was supported by grants from the Slovak Grant Agency of Science (VEGA 1/0077/14 and 2/0111/15), the Slovak Research and Developmental Agency (APVV-0282-10) and Comenius University (UK 246/2014). The contribution is also supported by the Research and Development Operational Programme funded by the ERDF (ITMS 26240220086). HODUROVA Z, TOTH HERVAY N, BALAZFYOVA Z, et al. 2011. Interplay among regulators of multidrug resistance in Kluyveromyces lactis. Gen Physiol Biophys 30:S1–92.

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APPLICATION OF ADVANCED FLUORESCENCE TECHNIQUES FOR ANALYSIS OF RED YEASTS

Vaněk M, Márová I., Mravec F., Hároniková A. and Vacková H. Materials Research Centre,Faculty of Chemistry, Brno University of Technology, 612 00 Brno, Czech Republic Carotenogenic yeasts are widely used in biotechnology as well as in model studies of yeast physiology, morphology and metabolic activity in various conditions. Molecular studies of red yeast strains are of increased interest due to their applications in food, feed and pharmaceutical industry. In this study advanced fluorescence techniques were applied to determine formation, distribution and localization of lipids and pigments in yeast cells. Two yeast strains - Rhodotorula glutinis CCY 20-2-26 and Cystofilobasidium capitatum CCY 10-1-2 were cultivated in simple glucose medium under permanent lighting and stirring. Cells of late exponential phase (35-38 hours of cultivation in production medium) were used for analysis by fluorescence microscopy, flow cytometry (Apogee) and fluorescence correlation spectroscopy (FCS; MicroTime 200 system; PicoQuant GmbH). FCS is based on monitoring fluctuations of the fluorescence intensity originating from species diffusing through a very small observation volume. Along with higher sensitivity the availability of FCS instrumentation with laser con-focal scanning microscopes (LCSM) multiplied its utility for biology. Auto-fluorescence can be specifically emitted by particular molecule in complex system (e.g. proteins, polymers, metal-complexes, etc.), thus, it is possible to study the behavior of individual molecules and molecular dynamics in living cells. Using these techniques auto-fluorescence of yeast cells was evaluated. Fluorescence dyes were used too, staining of cell nuclei was done by propidium iodine, while lipids were stained by Nile red. Yeas cells exhibited weak auto-fluorescence of carotenoids in fluorescence microscopy as well as in flow cytometry. Using FC technique, monitoring of viability and rapid determination of total yeast biomass was performed. Using LCSM (excitation, emission) sub-cellular structures were observed (mainly mitochondria, nuclei and pigments). Using combination of carotenoid auto-fluorescence and Nile red staining localization of pigments in yeast cells was determined in plasma membrane and lipid bodies. The results could contribute to better understanding of intracellular metabolic processes and, thus, to better control and effectiveness of biotechnological process. However, further study is still required to investigate red yeast cell physiology and intracellular organization in more details. Acknowledgments This contribution is the result of the project implementation: “Materials Research Centre – Sustainability and Development” project Nr. LO1212 of the Ministry of Education, Youth and Sports g/l30

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USE OF HIGH-THROUGHPUT MICROCULTIVATION AND FTIR SPECTROSCOPY FOR CHARACTERIZATION OF METABOLIC ACTIVITY OF CAROTENOGENIC YEASTS

Rapta, M. 1,, Márová I.1, Hároniková A.1 and Shapaval, V.2 1

Materials Research Centre, Faculty of Chemistry, Brno University of Technology, 612 00 Brno, Czech Republic, and 2 NNofima Mat AS, Ctr Biospect & Data Modeling, Osloveien 1, N-1430 As, Norway Characterization of metabolic activity of yeasts in biotechnological applications is an important issue both for routine analysis and trouble-shooting incidences. Present microbial techniques for yeasts characterization suffer from a low throughput and are time consuming. In this study we present a results of use of high-throughput microcultivation and spectral characterization of metabolic activity of some strains of red yeasts by Fourier transform infrared spectroscopy. For the study 4 species of in total three different yeast genera (Rhodotorula, Sporobolomyces, Cystofilobasidium) were analyzed by FTIR spectroscopy. The cultivation was performed simple glucose or glycerol liquid medium in a Bioscreen C system, allowing high-throughput cultivation of 200 samples at the same time. Each sample was cultivated in triplicates and differences were evaluated by PCA. Biomass was subsequently investigated by high-throughput Fourier transform infrared spectroscopy. Three spectral regions, fatty acids + lipid (3200-2800 cm(-1), 1300-1000 cm(-1)), proteinlipid (1800-1200 cm(-1) and "finger print" (900-700 cm(-1)) were evaluated for reproducibility and discrimination ability. Selected samples were then cultivated in liquid medium with particular C/N ratio and results of microcultivatin were verified. Biomass was determined gravimetrically, pigment production was measured by RP/HPLC and lipid production was measured enzymatically. The results show that all spectral regions evaluated can be used as spectroscopic biomarkers for evaluation of biomass and lipid production by FTIR. The influence of different glucose concentration and C/N ratios on lipid production by FTIR spectroscopy was investigated. In glucose medium (glucose concentration 30 g/l) an optimal C/N ratio 70 – 90 was observed after four days of growth. In glycerol medium lower lipid production was observed in all strains. This study presents a novel concept for high-throughput cultivation of yeasts for FTIR spectroscopy that enables characterization of metabolic activity of hundreds of strains per day.

. Acknowledgments This contribution is the result of the project implementation: “Materials Research Centre – Sustainability and Development” project Nr. LO1212 of the Ministry of Education, Youth and Sports.

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P26 INFLUENCE OF THE NATIVE YEAST ON THE FERMENTATION OF PLUM MASHES

Patelski P.1, Balcerek M.1, Pielech-Przybylska K.1, Dziekonska U.1, Berlowska J.1 1

Faculty of Biotechnology and Food Sciences, Technical University of Lodz, Wolczanska 171/173, 90-924 Lodz, Poland e-mail: [email protected] Fruit distillates may be obtained with use of the pure cultures of selected yeast strains, but in some cases also indigenous microflora of the raw material used for mash preparation may be responsible for course of the fermentation. Yeast cells present on the fruit peels represent wide spectrum of fermentative properties and various capabilities to create chemical compounds responsible for sensory features of fruit distillates. Aim of this work was to evaluate the influence of native yeast of plum fruits and raisins on the fermentation of plum mashes prepared with use “wegierka zwykla” plums ( Prunus domestica L.) Sterile and not-sterile plum mashes were inoculated with microflora isolated from plums and raisins surface. Fermentations with use of pure wine strain S.bayanus were carried out as a reference. Fermentation dynamics was evaluated gravimetrically (as a carbon dioxide released) during fermentation. Ethanol and selected volatile compounds concentration were evaluated by GC in the fermented mash and in the distillates. Ethanol yield was calculated. Mashes inoculated with yeast isolated from plum fruits and raisins were distinguished by the fastest fermentation. GC analysis showed that distillates prepared from mash fermented with native yeast contain even 15,2 g of the methanol per 1dm3 of the ethanol in the distillate. These distillates were also distinguished by elevated concentration of the volatile fermentation byproducts. Acknowledgements The research was financed by a grant from NCBiR (Polish National Centre for Research and Development) implemented within the framework of the Programme of Applied Research - PBS2/B8/9/2013 "Modern technologies of spirits production taking into account their accelerated maturation and improving of the physico-chemical stability"

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P27 ETHANOL FERMENTATION OF SUGAR BEET PULP HYDROLYSATES BY KLUYVEROMYCES MARXIANUS AND SACCHAROMYCES CEREVISIAE

Pielech-Przybylska K.1, Berłowska J.1, Balcerek M.1, Patelski P.1, Dziekońska U.1, Księżopolska M.-1, Dudkiewicz M. 1 and Kalinowska H.2 1

Institute of Fermentation Technology and Microbiology, 2Institute of Technical Biochemistry, Lodz University of Technology, Wolczanska 171/173, 90-924 Lodz, Poland The sugar beet pulp is a waste product of the sugar industry, which could be an attractive raw material for ethanol and yeast production. Due to the presence in the dry mass of the pulp, except sucrose and simple sugars, polysaccharides such as cellulose and hemicelluloses, it is appropriate to carry out its pre-hydrolysis. Also yeast strains selection is important to carry out fermentation of pentoses and hexoses obtained after hydrolysis of the mentioned polysaccharides. It should be regarded as justifiable use of mixed cultures of yeast with the ability to ferment hexoses and pentoses. It will allow better consumption of sugars and higher ethanol yield. The aim of the study was to examine fermentation efficiency of the sugar beet pulp hydrolysates obtained from the fresh beet pulp by enzymatic treatment with Viscozyme and UltrafloMax preparations (Novozymes, Denmark). Ozonation as a pretreatment method was also used in some trials. Fermentations were carried out with use of mono- and mixed cultures of yeast (each strain in dosage of 2 g d.m./L, applied simultaneously at the start of fermentation or sequentially) in thermostatic room in 3234°C, by 72 hours. Kluyveromyces marxianus (NCYC, United Kingdom) and Saccharomyces cerevisiae (Ethanol Red, Lesaffre, France) yeast strains were reported to ferment sucrose, glucose, galactose and mannose, as well as to assimilate rhamnose, arabinose and xylose. The obtained results showed that the preatreatment of sugar beet pulp hydrolysates by ozonation influenced positively on the fermentation dynamics and ethanol yield, in comparison with trials without ozone treatment. Particularly advantageous of ozonation was proved for samples of beet pulp hydrolysate which were fermented by the K. marxianus yeast strain. Higher oxygen concentration in the medium caused an increase in the efficiency of fermentation (calculated to the total sugars before fermentation) by 76% (K. marxianus dm 2 g d.m./L), by 85% (S. cerevisiae 2 g d.m./L K marxianus 2 g d.m./L, applied simultaneously) and by 98% (S. cerevisiae 2 g d.m./L, K. marxianus 2 g d.m./L, applied sequentially, with an interval of 24 h), in comparison with the appropriate control samples without ozonation. Acknowledgments The research was financed by a grant from NCBiR implemented within the framework of the Programme of Applied Research - PBS1/B8/3/2012 "Biomass sugar beet pulp as a new raw material for the production of fermentation substrates".

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P28 ETHANOL FERMENTATION OF CEREAL DISTILLERY MASHES BY MONOAND MIXED CULTURES OF YEAST Pielech-Przybylska K.1, Balcerek M.1, Kotas M.1, Patelski P.1 and Dziekońska U.1 Institute of Fermentation Technology and Microbiology, Lodz University of Technology, Wolczanska 171/173, 90-924 Lodz, Poland The aim of this study was to evaluate the dynamics and efficiency of fermentation of cereal mashes by distillery yeast strains used separately and as mixed cultures, as well as their impact on the qualitative and quantitative composition of volatile by-products in distillates obtained. The fermentation process was carried out using dry distillery yeast strains (Saccharomyces cerevisiae): As-4, D2, I-7-43 (Madra, Poland) and Ethanol Red (Lesaffre, France). The distillery sweet mashes were prepared by two methods of starch liberation: thermal-pressure and non-pressure. The fermentations were carried out at 30°C by 72 hours. During the process samples of mashes were collected every 24 hours and analyzed for extract (dry matter), sugars and ethanol content, as well as pH value was measured. The obtained results indicate that the mashes prepared with both of methods of starch liberation, fermented by I-7-43 yeast strain and mixed cultures of strains As-4 and Ethanol Red, were characterized by the highest ethanol yield 72 and 82% of the theoretical yield, respectively. The use of mixed cultures of yeast for the fermentation of mashes obtained by thermal-pressure method, resulted in lower (by 15%) ethanol yield, in comparison with trials fermented by monocultures of investigated yeast strains. Distillates obtained through distillation of mashes after fermentation were subjected to gas chromatography analysis. Concentration of acetaldehyde in the distillates varied between 0.055 and 0.465 g/L alcohol 100% v/v for thermal-pressure method and between 0.037 and 0.130 g/L alcohol 100% v/v for non-pressure method. The lowest acetaldehyde concentrations were observed in spirits produced from mashes obtained by non-pressure method and fermented by monocultures of yeast I-7-43 and Ethanol Red, and mixed cultures of yeast (As-4 and Ethanol Red; I-7-43 and D-2). The presence of aldehydes in agricultural distillates is a result of high temperature of steaming. It stimulates formation of alcohol dehydrogenase inhibitors that slow down reduction of acetaldehyde to ethanol. The distillates obtained from rye mashes fermented by mono – and mixed cultures of D-2 and I-7-43 yeast strains were characterized by the lower content of higher alcohols, in comparison with the other spirit samples. Acknowledgments The research was financed by a grant from NCBiR (Polish National Centre for Research and Development) implemented within the framework of the Programme of Applied Research - PBS2/B8/9/2013 "Modern technologies of spirits production taking into account their accelerated maturation and improving of the physico-chemical stability."

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P29

BURKHOLDERIA CEPACIA SELECTIVE AGAR CAN BE USEFUL FOR RECOVERY OF EXOPHIALA DERMATITIDIS FROM SPUTUM SAMPLES OF CYSTIC FIBROSIS PATIENTS Raclavský V1, Novotný R.1, Zápalka M.2, Kopřiva F.2, Jakubec P.3, Kolek V.3 1 Department of Microbiology, Faculty of Medicine & Dentistry, Palacký University, Hněvotínská 3, 779 00 Olomouc, Czech Republic 2 Department of Pediatrics and Department of 3Respiratory Medicine, University Hospital Olomouc, Czech Republic The black-pigmented fungus Exophiala dermatitidis is recovered from 5-20% of sputum samples of patients suffering cystic fibrosis, but not from other patient groups. Cystic fibrosis is a genetic disease that results in defective clearance of highly viscous mucus in respiratory pathways, resulting in chronic colonisation by pathogenic bacteria with recurrent infectious exacerbations. Whereas in some bacterial species e.g. Staphylococcus aureus, Pseudomonas aerugunisa, Burkholderia cepacia, and in the fungus Aspergillus fumigatus their pathogenic role is well established, in others it remains unclear, whether their presence results in worsened lung function and prognosis or not. E. dermatitidis is traditionally viewed as having low virulence and thus is considered to be a harmless inhabitant of lower respiratory airways. However, recent serological analyses revealed significantly higher levels of IgG antibodies to E. dermatitidis cell wall fragments in the E. dermatitidis culture-positive patients in a scandinavian CF center. These patients had also increased markers of inflammation and some of them who did not respond to antibacterial treatment (4/17) underwent prolonged antifungal treatment. In one case this treatment had to be terminated because of intolerance, in another case because of lack of clinical effect and in 2 cases the clinical status was improved. Therefore, E. dermatitidis should be viewed as an emerging opportunistic pathogen in the CF setting. Unfortunately, most routine laboratories do not recover this fungus effficiently, because of the need of extended cultivation periods (up to 35 days) on mycological media. In our cohort of CF patients, we apply 10-days cultivation on SGA plates and Burkholderia cepacia selective agar (BCSA) plates. In a 1-year period, we have recovered Exophiala dermatitidis from sputum samples of 6 patients (14 % of patients). Surprisingly, all the isolates, except for one morhological variant, were observed both on SGA and BCSA plates. Therefore, we performed a dilution test with our isolates to compare the sensitivity of both media in detection of E. dermatitidis growth. No significant differences were observed, indicating that BCSA is not inferior to SGA in detection of E. dermatitidis. This may facilitate its recovery in routine settings, because BCSA incubation is recommended to be extended for at least 5 days, therefore, further extension should not interfere with typical routine settings too much. Acknowledgments This work was supported by IGA MH CR (NT/13560), by RVO (61989592), by IGA LF UP (LF_2013_012), and by Ministry of Education, Youth and Sports (LO

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P30

MIRRI – THE MICROBIAL RESOURCE RESEARCH INFRASTRUCTURE Manuela Schüngel1, Renáta Vadkertiová2, the MIRRI consortium 1

Leibniz Institute DSMZ-German Collection of Microorganisms and Cell Cultures, Inhoffenstraße 7 B, 38124 Braunschweig, Germany 2 Culture Collection of Yeasts, Institute of Chemistry SAS, Dúbravská cesta 9, 842 38 Bratislava, Slovakia The vision of MIRRI is to be a unique pan-European high-performance platform adding value to known and yet unknown microbial biodiversity and exploiting unknown sources and knowledge to discover and disclose for the bio-economy and bioscience. MIRRI will generate solutions to societal challenges by stimulating interaction between academia and bio-industry. Placed on the European Strategy Forum on Infrastructure (ESFRI) roadmap in 2010, MIRRI started its preparatory phase in November 2012. During this initial phase a number of strategies for the implementation of MIRRI will be developed. These will, among others, include definition of a governance structure, operational practices as well as modes for sustainable funding of partner resource centres (for a detailed description of our work please visit the website www.mirri.org). MIRRI aims to provide a collaborative work environment inspiring excellence and to drive collaboration across borders and disciplines. Within concerted efforts, the consortium partners coherently and jointly work together with the objective to alleviate the current fragmentation of bioresource holdings and information, and eliminate duplication and redundancy at the national and pan-European level. We bring together research, education and industry to promote global responsibility towards biodiversity and to construct an innovative Europe. This project has received funding from the European Union’s Seventh Framework Programme for research, technological development and demonstration under grant agreement no 312251.

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P31

CYTONUCLEAR COMPATIBILITY DEFECTS IN S. CEREVISIAE XENOMITOCHONDRIAL CYBRIDS ARE NOT ASSOCIATED WITH THE ALTERATION IN MTDNA SEQUENCE Sulo P.1, Szabóová D.1, Jatzová K.1, Bielik P.1, Šoltýs K.2, Szemeš T.2 1

Department of Biochemistry, 2Department of Molecular Biology, Comenius University, Faculty of Natural Sciences, Mlynská Dolina, Bratislava, 842 15, Slovakia Nucleo-mitochondrial interactions, particularly those determining the primary divergence of biological species, can be studied by means of xenomitochondrial cybrids, which are cells where the original mitochondria (mitochondrial DNA, mtDNA) are substituted by their counterparts from related species. S. cerevisiae cybrids prepared by the mating of the ρ0 strain with impaired karyogamy and germinating spores from other Saccharomyces species exhibit different rates of re-established oxidative phosphorylation. Some of them are metabolically and genetically similar to cybrids containing mtDNA from different S. cerevisiae. However most of them require a period of adaptation to establish efficient oxidative phosphorylation resulting from inefficient intron cox1I3β splicing that is associated with the reduced cytochrome aa3 content. The limited circumstances are S. cerevisiae cybrids with S. bayanus mtDNA unable to respire and grow on ethanol-glycerol. To verify that transferred mtDNA did not undergo to any mutations linked to adaptation (or transplacement) process we sequenced mtDNA from cybrids by Illumina Miseq and compared them to their origin from parental “noncerevisiae” species. Comparison of mtDNAs did not reveal any significant changes in DNA sequence. Negligible differences can be assigned to sequencing errors or clonal (strain) dependent single nucleotide polymorphism.

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P32

A COMPLETE SEQUENCE OF MITOCHONDRIAL GENOMES FROM ALL SACCHAROMYCES REVEALS SPECIES SPECIFIC GENE REARRANGEMENT. Sulo P.1, Szabóová D.1, Zelezníková Ž.1, Bielik P.1, Šoltýs K.2, Szemeš T.2 1

Department of Biochemistry, 2Department of Molecular Biology, Comenius University, Faculty of Natural Sciences, Mlynská Dolina, Bratislava, 842 15, Slovakia Saccharomyces are excellent model to study mitochondrial genome evolution. Currently, the genus consists of the species S. cerevisiae, S. cariocanus, S. paradoxus, S. mikatae, S. kudriavzevii, S. arboricolus, S. bayanus var. bayanus, S. bayanus var. uvarum, and S. pastorianus. But, the last three species are alloploids of S. cerevisiae and the newly described species S. eubayanus and S. cariocanus is considered to be more a S. paradoxus variant as this species is reproductively isolated by four translocations but not by sequence. Despite large numbers of nuclear genome sequences now available for different isolates, completed mitochondrial genome sequences from Saccharomyces are limited to five. The problem is associated with the short DNA reads (approx. 100 nt) generated by the most popular next-generation sequencing platform Ilumina Miseq. Due to the AT-rich and redundant intergenic regions, this system can be routinely used for sequencing mtDNA different yeast but not Saccharomyces. We solved Ilumina Miseq 100- 250 nt reads assembly problems and obtained whole mitochondrial genomes sequences from all Saccharomyces species. The sequence comparison revealed identical gene content but species specific alterations in gene order, except S. cerevisiae and S. cariocanus. The translocations involve only three gene clusters trnfM-rnpB-trnP; trnW-rns and trnS1-rps3. Our data emphasize the fact that important evolution events such as is divergence of species are in Saccharomyces accompanied by the alternation of gene order in mitochondrial genome.

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P33 YEAST MITOCHONDRIAL GENOMES WITH VARIABLE NUMBER OF TRNA GENES RESULTING FROM DUPLICATION DELETION MODEL Sulo P.1, Szabóová D.1, Kafková L., Poláková S.1, Zelezníková Ž.1, Šoltýs K.2, Szemeš T.2 1

Department of Biochemistry, 2Department of Molecular Biology, Comenius University, Faculty of Natural Sciences, Mlynská Dolina, Bratislava, 842 15, Slovakia and 3 Institute of Animal Biochemistry and Genetics, Slovak Academy of Sciences, Ivanka pri Dunaji, 900 28, Slovakia Saccharomyces yeasts offer a great opportunity to study the evolutionary processes and “speciation” experimentally. The process is called “hybrid speciation”, and it uses two closely related species to create a third, “unique” species. In some interspecific hybrids significant mtDNAs recombination initiated by intron transposition accompanies the speciation process. Rarely transposition of mitochondrial ORF1 (coding for free standing homing endonuclease) generates S. paradoxus/S. cerevisiae hybrid with recombined mtDNA and two copies of the genes for cox3, rnpB, rns and six tRNAs. This molecule allows to examine “the duplication gene loss model” that is preferably used to explain why the important evolution events such as divergence of species are in Saccharomyces accompanied by the alternation of gene order in mitochondrial genome. We sequenced original mtDNA and mtDNA in several clones of meiotic progeny (F2 generation) with altered restriction patterns by Illumina Miseq. Sequencing revealed the different extend of elimination in duplicated regions. The most common variant results from the multiple recombination events among ACTAATG repeats and three distant intergenic GC clusters. All redundant genes were deleted and the middle intergenic segment inverted. Less common F2 generation variants produced a large number of petites. Therefore grande colonies (with wild type mtDNA) were obtained and studied after subcloning or cytoduction. In these variants complete mtDNA sequencing revealed only partial deletion of duplicated segment, remaining one odd tRNA gene (trnF) or entire odd tRNA cluster (F,T1,V). This is the first experimental evidence elucidating the variability in the number and locations of tRNA genes in yeast mtDNAs.

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Index

83

Abbas Ch. A. Abbondio M. Addis M. F. Andršová M. Anrather D. Aronica L. Bacigálová K. Bai F-Y Bakun M. Balážfyová Z. Balcerek M. Bardelčíková A. Belicová L. Berlowska J Bernatova S. Bielik P. Boekhout T. Branduardi P Breierová E. Bucek A. Budroni M. Capella S Ciani M. Cipak L. Cipakova I. Comitini F. Csáky Z. Cutzu R. Czabany T. Čadež N. Čertík M. Dadlez M. Deligios M. Dlapalová K. Dlauchy D. Drozdíková E.

Dudeková H. Dudkiewicz M. Dušinský R. Dušková M.

29 31 31 28 69 69 48 17 24 70 45, 50, 51, 55, 56, 73, 74, 75, 54

Dziekońska U. Farkaš V. Ferenc J. Frimmel K. Gabaldon T. Gajdoš P. Garaiova M. Gbelská Y. Geršiová A. Goffa E. Griffith J.D. Groenewald M. Guthová J. Hamříková D. Hapala I.

49 45, 51, 55, 56, 73, 74 32 78, 79 17 33 52, 68 30 31, 44, 68 17 44, 68 69 69 44, 68 53 31 34 19 35, 52, 60 24 31 62 21 54

Haronikova A. Hlaváček V. Hofr C. Holic R. HruškováHeidingsfeldová O. Hurbanova K. Chreptowicz K. Chudej T. Irzyniec Z. Izdebska K. Jakubec P Jatzová K. Juríková K. Jurnečková A. Kafková L. Kalinowska H.

84

67 51, 55,56, 74 67 25 45, 50, 51, 55, 56, 73, 74, 75, 54, 64 67 68 17, 66 60 38 70 54 61 39 17 52 59 38, 53 32, 47, 63, 71, 72 46, 61 39 43 57 32 24, 41, 64 70 75 24 76 78 66 59 80 51, 74

Klempová T. Knezl V. Kolecka A. Kolek V. Kopřiva F. Kostrzewa M. Kotas M.

35, 52 68 17 76 76 17 75

Kováčová K. Kręgiel D. Križák J. Kryzyanek V. Krzyžanek V. Księżopolska M. Kučera J. Kunicka-Styczyńska A. Lajdová, D. Landolfo S. Ledvina V. Lipták B. Liu X-Z Llopis-Torregrosa V. Lopandic K Ludovico P. Luley-Goedl Ch. Mannazzu I. Maroszyńska M.

64 51, 55, 56 68 47 32 74 48 18, 65 40, 67 31, 44, 68 59 68 17 25 48 23 34 31, 44, 68 65 32, 46, 47, 61, 63, 71,72 30 60 24, 41, 64 69 22, 59 71 31 57 67 39

Marova I. Matoušková P. Meravá Z. Mierzejewska J. Mikolaskova B. Molnárová J. Mravec F. Mulas G. Mynářová L. Neboháčová M. Nečasová I

Nevečeřalová P. Nidetzky B Nosek J. Novotný R. Obernauerová M. Okruhlicová Ľ. Omelková J. Oro L. Palkova Z. Papoušková K.

Pichová A. Pichová I. Poláková S. Porro D. Procházková K.

42, 71 34 37,39, 67 76 54 69 58, 59 44,68 27,57 28 45, 50, 51, 73, 74, 75 58 18 20 48 45, 50,51, 55, 56, 73, 74, 75 49 30, 5 80 33 67

Pryszcz L. Raclavský V. Rajkowska K. Rapta M. Ribitsch D. Rieger A. Ruchman D. Sabova L. Samek O. Sampaio J.P. Sasinková V. Sec P. Sepšiová R. Shapaval V Schmoelzer K. Schüngel M.

26 76 18, 65 72 34 36 69 69 32, 47, 16 52 43 39 72 34 77

Patelski P. Pavlatovská B. Pęczek M. Péter G. Petrýdesová J. Pielech-Przybylska K.

85

Schwab H. Sipiczki M Slovák M. Stovicek V. Stratilová B. Stratilová E. Sulo P. Svatoš A. Svrbická A. Sychrová H. Szabóová D. Szemeš T. Šimoničová L. Šoltýs K. Šťovíček V. Taj-Aldeen S. Tanca A. Theelen B Tkáčová J. Tomáška Ľ. Tóth Hervay N. Turková L.

34 21 48 27,57 58, 59 58, 59 48, 78, 79, 80 30 70 25, 28, 42, 71 78, 79, 80 78, 79, 80 67 78, 79, 80 87 17 31 17 35 37, 39, 40,66, 67, 68, 70 63

Uzzau S. Vacková H. Vadkertiová R. Vakova L. Valachovic M. Vaněk M. Vránová D. Vrkoslav V. Výmolová M. Wang Q-M Wilkinson D. Willcox S. Zambojová V. Zápalka M. Zara S. Zelezníková Ž. Zemančíková J. Zemanek P Zimmermannová O. Zitzenbacher S.

86

31 47, 71 15,22, 58, 59, 77 27 38, 61 71 62 57 46, 61 17 27 39 53 76 44, 68 79, 80 25 32 25, 42,71 34

87

88

89

90

91

92

93

94

95

96