# Evolutionnary and reverse engineering of commercially important metabolic traits in wine yeast

Axelle Cadière, Valentin Tilloy, Carole Camarasa, Frederic Bigey, Stephane

Guezenec, Brigitte Cambon, Virginie Galeote, Sylvie Dequin

► To cite this version:

Axelle Cadière, Valentin Tilloy, Carole Camarasa, Frederic Bigey, Stephane Guezenec, et al. Evolutionnary and reverse engineering of commercially important metabolic traits in wine yeast. 31. International Specialised Symposium on Yeast, 2014, Nova Gorica/Vipava, Slovenia. hal-01605611

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https://hal.science/hal-01605611

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issy31

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JP
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31st International Specialised Symposium on Yeast

Nova Gorica / Vipava

Slovenia

October 9 – 12, 2014

BOOK OF ABSTRACTS
Editorial Board: Jure Piškur, Uroš Petrovič, Marko Dolinar, Sofia Dashko

Graphical support: Andrey Polupanov, Jernej Šribar

Published by Jubi kinase, Baunebjerg Park 36, 3520 Farum, Denmark

Printed by: Garamond d.o.o., Ljubljana, Slovenia

ISSY 31 is organized under the auspices of the International Commission on Yeast (ICY).

Organizing committee of the symposium:

Jure Piškur (1960 - 2014) – Honorary Chairman and Inspirator; Uroš Petrovič – Executive Chairman,
Birgitte Munch-Petersen – Treasurer, Marko Dolinar, Jernej Šribar, Lorena Butinar, Sofia Dashko,
Melita Sternad Lemut

Scientific committee:

J. Thevelein, T. Boekhout, C. Compagno, K. Natter, D. Delneri, J. Hansen, G. Liti, L. Butinar, U. Petrovič

Co-organized by:

Lund University, Jožef Stefan Institute, Cornucopia EU ITN project, University of Nova Gorica and Jubi kinase

CIP - Kataložni zapis o publikaciji

Narodna in univerzitetna knjižnica, Ljubljana

582.282.23(082)

663.14(082)

INTERNATIONAL Specialised Symposium on Yeasts (31 ; 2014 ; Nova gorica / Vipava)

Book of abstracts / 31st International Specialised Symposium on Yeast, Nova Gorica, Vipava, Slovenia, October 9 -12,
2014 ; [editorial board Jure Piškur ... et al.]. - Farum : Jubi kinase, 2014

1. Piškur, Jure

275780352
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Dear Colleagues,

It is our great pleasure to welcome you to the 31st *International Specialised Symposium on Yeast* in Slovenia!

Through the years, ISSY has become a popular venue for the yeast researchers to meet and discuss the many aspects of their favourite study organism. This year's edition is dedicated to yeast fermentations, from the basic concepts to the applications, and from the aspects of biodiversity and evolution to genetic regulation. It is also dedicated to the new generation of yeast researchers, as all of them who have submitted an abstract have been given an opportunity to present their work to the scientific community in the form of a lecture. We hope that this format of the symposium will spark even more ideas, hypotheses and theories, and new collaborations.

Both, the venue and the topic of the conference, have been selected by late Jure Piškur, who, sadly, will only be present at this symposium in spirit. Jure felt at home in Slovenia, and in the science of yeast fermentations. The Law of Nature, to use his words, returned his physical existence into the grand metabolism of all living and non-living things on the planet, before he could enjoy the renovated Lanthieri palace in the company of his friends, colleagues and scientific competitors alike. He was deeply engaged and enthusiastic about this conference and planned it as a highlight of his life and career, and as a tribute to the numerous collaborations and friendships he had created during his productive life. Our wish for the ISSY31 is that together we will, through lectures, discussions and social events, celebrate Jure's life and work.

The ISSY31 Organizing Committee

Uroš Petrovič, Executive Chairman of the Committee
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## General information about the ISSY31 Symposium

The majority of participants will be accommodated in the Perla Hotel, the actual venue of all the plenary sessions of the symposium, as well as of the get-together on the first day of the programme and the conference dinner on Sunday evening. Perla is a casino hotel, so all the guests are granted access to the casino section on the ground level. You would need to register, however, before your first access to the premises.

Parallel sessions will be organized in Vipava, a countryside town about 35 km east of Nova Gorica. Transportation to/from Vipava will be organized by a total of 5 coaches which will depart from in front of the Perla Hotel in the direction of Vipava and from the Vipava Grammar School parking area in the direction of Nova Gorica. Please keep in mind that coaches will leave at hours listed in the programme and that there are very limited possibilities for commuting between the two towns in the evening hours.

For easier orientation in Vipava, please consult the map on page 216 of this booklet. Parallel sessions will take place at two different locations in Vipava, the Catholic Grammar School and the Lanthieri palace. The distance between the two is about 400 m. Lanthieri palace is the building where the University of Nova Gorica Centre for Wine Research is located, as well as the Centre for Biomedical Sciences and Engineering.

Light lunches on Friday and Saturday will be served in the Lanthieri palace, while coffee and tea will be served both at the grammar school and the palace.

In the case of serious problems, call emergency numbers which are:

* Mr. Boštjan Marcina: +386 31 628 477 (transportation)

* Mrs. Mija Rijavec: +386 41 766 367 (accommodation and general).
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# Symposium Programme Outline

## Thursday, October 9, Nova Gorica 9

from 12:00 Registration

18:00 – 18:10 Symposium Opening

18:10 – 19:55 Opening plenary lectures:
ICY lecture, Novozymes lecture, Pivovarna Laško lecture

from 20:15 ISSY31 Banquet

## Friday, October 10, Nova Gorica and Vipava 15

08:30 – 12:00: Plenary sessions (Nova Gorica)

Biodiversity – Basidioworld

Genetic Regulation – Metabolic Modelling

Industrial Applications – Bulk and Fine Chemicals

12:15 – 13:00 Transportation to Vipava – buses leave from in front of the Perla Hotel

13:00 – 14:00 Lunch at the Lanthieri Palace

14:00 – 19:00: Parallel sessions (Vipava)

Biodiversity & Wine

Genetic Regulation of Stress Response

Bioethanol & Bioenergy (Visby project)

Elucidating Regulation through Functional Genomics

YEASTCELL EU project session

19:15 – 20:00 Transportation to Nova Gorica – buses leave from in front of the Grammar School
08:00 – 08:45 Transportation to Vipava – buses leave from in front of the Perla Hotel

09:00 – 12:30

Parallel sessions

Beverages 1 & 2

Biodiversity for Applications

2<sup>nd</sup> Generation Biofuels

Regulation of and by Lipids

Non-conventional Yeasts in Bioproduction

12:30 – 14:00 Lunch / Vipava sightseeing & meeting local food/wine producers

14:00 – 19:00

Parallel sessions

Ecology and Genomics

*Cornucopia* EU project session

Regulation of Metabolism and Nutrient Sensing

Biofactories: Tools, Physiology & Products

Polygenic Traits

YeSVitE EU project session

19:15 – 20:00 Transportation to Nova Gorica – buses leave from in front of the Grammar School
Plenary Sessions

09:00 – 12:00 Novo Nordisk session on Yeast Evolution

12:00 – 13:30 Lunch

13:30 – 14:30 Genialis lectures

14:30 – 15:00 Announcement of the best young investigator

15:30 – 17:15 Carlsberg session – Closing plenary lectures

17:15 – 18:00 Presentation of the new ICY and ISSY meetings & Closing Ceremony

20.00 Conference Dinner
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DETAILED DAILY PROGRAMMES AND ABSTRACTS
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Thursday, October 9

Perla Hotel Auditorium

from 12:00 Registration in the Perla Hotel Lobby

18:00 Opening of the Symposium

Opening plenary lectures (chair: Teun Boekhout)

18:10 - 18:45 Wen-Hsiung Li

Evolutionary genetics and genomics of yeast carbon metabolism (ICY lecture) 11

18:45 - 19:20 Johan Thevelein

Polygenic analysis of commercially-important complex traits in yeast (Novozymes lecture) 12

19:20 - 19:55 Amparo Querol

Molecular bases of the regulation of the glycerol production in the *Saccharomyces* species and relevance for wine fermentations (Pivovarna Laško lecture) 13

from 20:15 ISSY31 Get-Together Banquet
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Wen-Hsiung Li

Biodiversity Research Center, Academia Sinica, Taipei, Taiwan

Department of Ecology and Evolution, University of Chicago, Chicago

Yeasts display highly diversified physiological characteristics. The most distinct physiological character in many yeasts is their special sugar metabolism. In particular, the baker's yeast *Sacharomyces cerevisiae* and its relatives predominantly ferment sugars into ethanol even in the presence of oxygen, which is known as Crabtree effect or aerobic fermentation. It has been postulated that this unusual metabolism provides these yeasts selective advantages in sugar-rich environments. However, it has long been a mystery as to genetically how these yeasts evolved a predominantly fermentative life style. The rapid accumulation of genomic, transcriptomic and epigenetic data in many yeast species in recent years has greatly increased our understanding of the genetic basis and molecular mechanism for the diversified sugar metabolisms among yeasts. In this talk, I shall provide a review of recent comparative genomics and evolutionary studies related to the metabolisms of glucose and galactose, whose metabolic pathways have been extensively studied in yeasts. A series of studies suggested that the evolution of aerobic fermentation involved many different factors, including increases in copy numbers of genes involved in glucose transport, glycolysis and ethanol production; sequence divergence; and transcriptional reprogramming of genes involved in mitochondrial functions through changes of *cis*-regulatory elements and promoter structures. It has also been found that the different abilities among yeasts to use galactose is strongly correlated with the presence of the galactose pathway genes in their genomes. These studies revealed that the adaptation of yeasts to specific niches has greatly shaped their genomic content and regulatory program.
POLYGENIC ANALYSIS OF COMMERCIALLY-IMPORTANT TRAITS IN YEAST

Johan M. Thevelein, María R. Foulquié-Moreno, Edgard Belo, Tom den Abt, Mekonnen Demeke, Françoise Dumortier, Annelies Goovaerts, Sylvester Holt, Yingying Li, Raquel Quintilla Mateo, Hygor Mezadri, Vaskar Mukherjee, Benjamin Offei, Dorota Radecka, Ben Souffriau, Marija Stojiljkovic, Bruna Trindade de Carvalho

Laboratory of Molecular Cell Biology, Institute of Botany and Microbiology, KU Leuven and Department of Molecular Microbiology, VIB, Kasteelpark Arenberg 31, B-3001 Leuven-Heverlee, Flanders, Belgium

Contact email: johan.thevelein@mmbio.vib-kuleuven.be

Most traits of industrial importance in yeast are polygenic traits, i.e. traits determined by multiple genes acting together. Screening of *Saccharomyces cerevisiae* strain collections has revealed a wide diversity for such traits, with certain strains often being superior for a desirable trait compared to industrial yeast strains. The genetic analysis of polygenic traits has been a major challenge for many years. We have developed pooled-segregant whole-genome sequence analysis to map all QTLs (Quantitative Trait Loci) determining a complex trait in such superior yeast traits relative to an industrial strain of interest. Reciprocal hemizygosity analysis and allele exchange are then used to identify and confirm the causative genes in the QTLs. The superior alleles found in this way are transferred into industrial yeast strains creating self-cloned, 'cisgenic' industrial yeast strains. This allows for predictable strain improvement with naturally occurring mutant alleles, minimizing the risk of side-effects on other essential traits. We have applied this technology platform to several yeast traits of prime industrial importance: ethanol tolerance of cell proliferation, maximal ethanol accumulation capacity, thermotolerance, reduced glycerol/enhanced ethanol production, acetic acid tolerance, xylose fermentation rate, osmotolerance, flavour compound production, etc. We have identified multiple causative alleles of which several had never been connected to the trait-of-interest. Different approaches have been used to improve the detection of minor loci. This work has also revealed the importance of mimicking as closely as possible the industrial conditions in phenotyping the trait of interest. For instance, we have found new genes responsible for ethanol tolerance at industrially relevant concentrations of 16-17% and we have shown that different superior alleles underlie maximal ethanol accumulation capacity compared to high ethanol tolerance of cell proliferation. Polygenic analysis of an industrial yeast strain with superior performance in bioethanol production with lignocellulose hydrolysates has revealed new genes controlling xylose fermentation capacity.
MOLECULAR BASES OF THE REGULATION OF THE GLYCEROL PRODUCTION IN
THE *Saccharomyces* SPECIES AND RELEVANCE FOR WINE FERMENTATIONS

Bruno M. Oliveira<fnref n="1" />, Eladio Barrio<fnref n="1" />,<fnref n="2" />, Roberto Pérez-Torrado<fnref n="1" /> and Amparo Querol<fnref n="1" />

Contact email: aquerol@iata.csic.es

The wine fermentation is a complex process produced as a result of the activities of a
succession of microorganisms, being *Saccharomyces* yeasts (mainly *S. cerevisiae*) the
responsible for the alcoholic fermentation. Although *S. cerevisiae* is the most frequent species
in wines, and the subject of most studies, *S. bayanus* var. *uvarum* and natural hybrids between
*Saccharomyces* species such as *S. cerevisiae* x *S. kudriavzevii* and *S. cerevisiae* x *S. bayanus*
var. *uvarum* are also involved in wine fermentations and can be preponderant in certain wine
regions.

Studies performed in our laboratory, which compared the properties of natural wine hybrids
with their parent species, showed that strains of non-conventional *Saccharomyces* species,
such as *S. bayanus* var. *uvarum* and *S. kudriavzevii*, exhibit physiological properties of
potential interest in enology because they can respond to the new demands of the wine
industry, such as their ability to ferment at low temperatures, their increased **production of glycerol**, their lower ethanol yield (1, 2, 3). By using DNA chips (4), we have also observed, a significant lower expression levels in *S. cerevisiae* compared to the rest of species for genes involved in the glycerol metabolism. These observations attracted our attention due to the potential use of these non-conventional *Saccharomyces* species as starters to solve some demands of the wine industry to improve wine quality.

The main objective of this work is to decipher the molecular mechanism responsible of the overproduction of glycerol in *S. kudriavzevii*, *S. bayanus* var. *uvarum* and natural strains of *S. cerevisiae*. To explain this observation at the molecular level we studied the expression of glycerol biosynthetic pathway genes (Oliveira et al, in preparation). We observed higher enzymatic activity of Gpd1p in *S. kudriavzevii* in response to osmotic and cold stress. Also, we determined that *S. kudriavzevii* Gpd1p enzyme presents increased catalytic properties that will contribute to increase glycerol production. (5).

References:

1. Gamero et al., (2014). J Appl Microbiol. 2013 May;114(5):1405-14.

2. González et al., (2007). Int J Food Microbiol. 2007 May 1;116(1):11-8.

3. Salvadó et al., (2011). Appl Environ Microbiol. 2011 Apr;77(7):2292-302.

4. Combina et al., (2012). Int J Food Microbiol. 2012 Jul 16;157(3):340-5.

5. Oliveira et al., (2014). PLoS One. 2014 Jan 30;9(1):e87290.

This work was supported by AGL2012-39937-C02 from Ministerio de Educación y Ciencia and the EU's Seventh Framework Programme (FP7) under grant agreement PITN-GA-2010-264717.

<footnote n="1">¹ Instituto de Agroquímica y Tecnología de los Alimentos, IATA-CSIC, Calle Agustín Escardino, 7. E-46980, Paterna (Valencia), Spain</footnote>

<footnote n="2">²Departament de Genética, Universitat de Valencia, Valencia, Spain.</footnote>
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Friday, October 10

08:30 – 12:00

Plenary sessions (Nova Gorica)

Biodiversity – Basidioworld (chair: Lorena Butinar)

08:30 - 08:55 Teun Boekhout: Basidiomycetous yeasts: prospects, diversity and taxonomy 23

08:55 - 09:10 Tomotake Morita: Genome and transcriptome analysis of a yeast
*Pseudozyma antarctica* T-34 24

09:10 - 9:20 Virginia de Garcia: Poliextremophilic yeasts from Patagonia Argentina and
Antarctic sea 25

Genetic Regulation - Metabolic Modelling (chair: Klaus Natter)

09:25 - 09:55 Steve Oliver: Making, mending, and extending the yeast metabolic model 26

09:55 - 10:20 Diethard Mattanovich: Model based engineering of *Pichia pastoris* central
metabolism enhances recombinant protein production 27

Industrial Applications – Bulk and Fine Chemicals (chair: Concetta Compagno)

10:30-11:00 Charles Abbas: Recent developments in biofuels and bio-based chemicals
production by yeasts: Opportunities, challenges and technical progress 28

11:00-11:15 Kaisa Karhumaa: Robust high-performance *S. cerevisiae* strains for
C5 fermentation in lignocellulosic ethanol production 29

11:15-11:45 Jørgen Hansen: Using baker's yeast for production of sustainable ingredients
in health, nutrition and wellness 30

11:45-12:00 Tomas Linder: Exploring “peripheral” metabolic pathways in budding yeasts 31

12:15 – 13:00 Transportation to Vipava – buses leave from in front of the Perla Hotel

13:00 – 14:00 Lunch in the Lanthieri Palace
14:00 – 16:15

Parallel sessions (Vipava)

Biodiversity & Wine (chairs: Gianluigi Cardinali and Angelica Ganga) -- see p. 17 for details

Genetic Regulation of Stress Tolerance (chairs: Xinqing Zhao and Tilen Konte) – see p. 17 for details

Bioethanol & Bioenergy – Visby project
(chairs: Volkmar Passoth and Andriy Sibirny) – see p. 18 for details

Coffee break 16:15 – 16:45

Bioethanol & Bioenergy – Visby project – continued
(chairs: Volkmar Passoth and Andriy Sibirny) – see p. 18 for details

Elucidating Regulation through Functional Genomics
(chairs: Mojca Mattiazzi Ušaj and Károly Kovács) – see p. 20 for details

YEASTCELL EU project session (chair: John Morissey) – see p. 20 for details

19:15 – 20:00 Transportation to Nova Gorica – buses leave from in front of the Grammar School
## Friday, October 10 afternoon sessions in Vipava – parallel sessions in:

Aula Magna and Aula Parva – Lanthieri Palace

Grammar School Hall (5 min walk from the Lanthieri Palace)

## Biodiversity & Wine / in Aula Magna

<table><tr><td>14:00 - 14:30</td><td>Gianluigi Cardinali: Defining the unit of yeast biodiversity</td><td>32</td></tr><tr><td>14:30 - 14:55</td><td>Angelica Ganga: Yeasts from the end of the world: Technological potential?</td><td>33</td></tr><tr><td>14:55 - 15:05</td><td>Gabriella De Lorenzis: Survey on yeast biodiversity in Georgian vineyards: a pristine environment for the selection of wine strains</td><td>34</td></tr><tr><td>15:05 - 15:15</td><td>Aspasia Nisiotou: Multi-strain indigenous yeast starters for 'wild-ferment' wine production</td><td>35</td></tr><tr><td>15:15 - 15:25</td><td>Gemma Beltran: Effect of mixed Saccharomyces and non-Saccharomyces starters on alcoholic fermentations and wines</td><td>36</td></tr><tr><td>15:25 - 15:35</td><td>Souhir Marsit: Adaptive role of horizontally transferred oligopeptide transporters in wine yeasts</td><td>37</td></tr><tr><td>15:35 - 15:45</td><td>Tanja Vojvoda: Physiological and genetic characterisation of Saccharomyces strains isolated from ice harvest of chardonnay grapes</td><td>38</td></tr><tr><td>15:45 - 16:00</td><td>Francisco Carrau: Application of Hanseniaspora vineae strains. Searching for genes to explain increased flavor complexity in wines</td><td>39</td></tr></table>

## Genetic Regulation of Stress Response / in Aula Parva

<table><tr><td>14:00 - 14:20</td><td>Xinqing Zhao: Exploration of genetic resources from yeast genome to develop robust yeast strains for biofuels production</td><td>40</td></tr><tr><td>14:20 - 14:30</td><td>Rakesh Koppram: The water insoluble solids of pretreated lignocellulosic materials increases the resistance of Saccharomyces cerevisiae to lignocellulosic inhibitors – a proteomic study of the effects</td><td>41</td></tr></table>
14:30 - 14:40 Magnus Ask: Targeting the intracellular redox state in the development of more robust *Saccharomyces cerevisiae* strains for lignocellulosic bioethanol production 42

14:40 - 14:50 Cecilia Picazo Campos: Role of antioxidant defense systems on aging in wine yeast 43

14:50 - 15:00 Chiara Nadai: Transcriptome analysis of wine yeast strains under sulphite-induced stress conditions 44

15:00 - 15:10 Estefani Garcia-Rios: Multiple global approaches for deciphering the molecular basis of low temperature adaptation in wine yeast 45

15:10 - 15:20 Noreen Suliani binti Mat Nanyan: The role of yeast transcriptional factor Msn2 in *Saccharomyces cerevisiae* 46

15:20 - 15:30 Tilen Konte: Investigation of HOG signaling pathway architecture of the halophilic basidiomycete *Wallolemia ichthyophaga* with the functional complementation of *Saccharomyces cerevisiae* mutants 47

15:30 - 15:40 Hyunah Kim: The protein o-mannosyltransferase Pmt4 is crucial for cell wall integrity and stress resistance of *Hansenula polymorpha* 48

15:40 - 15:50 Kristína Kováčová: In vitro analyses of Phr1 and Phr2 transglycosylases of the *Candida albicans* cell wall 49

15:50 - 16:00 Natalia Sybirna: Methylotrophic yeast *Hansenula polymorpha* as a novel tool for studying mechanisms underlying Parkinson's disease 50

Bioethanol & Bioenergy (Visby project) / in Grammar School Hall

14:00 - 14:30 Volkmar Passoth: Non-conventional yeasts for biofuel production 51

14:30 - 15:00 Andriy Sibirny: Construction of the improved ethanol producers from xylose in the thermotolerant yeast *Hansenula polymorpha* 52

15:00 - 15:10 Albina Bakeeva: The biocontrol yeast *Wickerhamomyces anomalus* in maize biopreservation 53
15:10 - 15:20 Ievgeniia Tiukova: *Dekkera bruxellensis* competitive non-conventional ethanol production yeast 54

15:20 - 15:30 Iryna Salii: Deletion of *ADH1* gene, encoding alcohol dehydrogenase for the improvement of glycerol production in *Saccharomyces cerevisiae* 55

15:30 - 15:40 Iwona Kata: Metabolic engineering of *Hansenula polymorpha* for improving the conversion of glycerol to ethanol 56

15:40 - 15:50 Johanna Blomqvist: Ethanol and biogas from steam exploded ISP (integrated storage and pre-treatment) sawdust inoculated with *Arxula adeninivorans* 57

15:50 - 16:00 Jule Brandenburg: Bio-lipid production from lignocelluloses material with oleaginous yeasts 58

16:00 - 16:10 Justyna Ruchala: Construction of *CAT8* gene deletion strains in the yeast *Hansenula polymorpha* 59

(coffee break)

16:45 - 16:55 Krzysztof Berezka: Insertional tagging of the *Pichia stipitis* genes involved in xylose alcoholic fermentation 60

16:55 - 17:05 Marianna Yurkiv: Construction of glutathione overproducers in methylotrophic yeast *Hansenula polymorpha* by engineering of γ-glutamylcysteine synthetase 61

17:05 - 17:15 Marta Semkiv: Metabolic engineering of yeast *Saccharomyces cerevisiae* for rerouting of carbon flux towards glycerol production 62

17:15 - 17:25 Oleksii Lyzak: The role of transcription factor Sef1 in the regulation of flavinogenesis in yeast *Candida famata* 63

17:25 - 17:35 Olena Kurylenko: Metabolic engineering of the methylotrophic yeast *Hansenula polymorpha* for construction of the high-temperature ethanol producers from xylose 64

17:35 - 17:45 Orest Hryniv: Disruption of *ATG13* gene causes the increas of ethanol production during xylose alcoholic fermentation of the thermotolerant yeast *Hansenula polymorpha* 65

17:45 - 17:55 Taras Luzhetskyi: Improvement of high temperature alcoholic fermentation of *Saccharomyces cerevisiae* 66
Elucidating Regulation through Functional Genomics / in Aula Magna

16:45 - 17:15 Mojca Mattiazzi Ušaj: Exploring the yeast endocytic pathway by combining high-throughput genetics and high-content microscopy 67

17:15 - 17:30: Elena Kuzmin: Complex genetic interaction profiles reveal evolutionary fates of duplicated genes 68

17:30 - 17:45 Zoltán Farkas: Chaperone overload is a major determinant of dosage sensitivity 69

17:45 - 18:00 Károly Kovács: General principles of transcriptomic changes following single-gene deletion in *Saccharomyces cerevisiae* 70

18:00 - 18:20 Steven Parker: RNA functional profiling by gene deletion in *S. cerevisiae* 71

18:20 - 18:40 Yu Sasano: Discovery of novel genetic interactions by chromosomal segmental deletions and its application to ethanol fermentation in *Saccharomyces cerevisiae* 72

18:40 - 19:00 Josephine Peter: Use of prototrophic deletion libraries as a tool to identify nitrogen related genes in lab and wine yeast 73

YEASTCELL EU project session / in Aula Parva

16:45 - 17:05 John Morissey: Development of *Kluyveromyces marxianus* for cell factory applications 74

17:05 - 17:25 Paola Branduardi: Engineering the yeast mRNA metabolism for fishing the desired phenotype 75

17:25 - 18:05 Elevator Pitch talks (YEASTCELL ESRs):

Anja Brickwedde: Characterization of beer-related physiological processes in *Saccharomyces cerevisiae*, *Saccharomyces eubayanus* and their interspecific hybrid, the best of both worlds 76

Ângela Carvalho: Production of specialized fatty acids in *Saccharomyces cerevisiae* 77

Matthias Eder: Deciphering the genetic and metabolic bases of yeast aroma properties 78

David Ferreira: Multi-stress resistance and lag-phase time reduction in wine fermentation 79
<table>
  <tbody>
    <tr>
      <td>Manuela Gottardi: Reprogramming of <i>Saccharomyces cerevisiae</i> shikimate pathway for the production of bulk chemicals</td>
      <td>80</td>
    </tr>
    <tr>
      <td>Nurzhan Kuanyshev: Evaluation of <i>Zygosaccharomyces bailii</i> for lactic acid tolerance</td>
      <td>81</td>
    </tr>
    <tr>
      <td>Frederico Magalhães: Elucidating mechanisms that determine differential maltotriose utilization between strains of the hybrid yeast <i>Saccharomyces pastorianus</i></td>
      <td>82</td>
    </tr>
    <tr>
      <td>Anna Malak: Gallic acid production by the two tannases Atan1p and Atan2p using the yeast <i>Arxula adeninivorans</i></td>
      <td>83</td>
    </tr>
    <tr>
      <td>Raúl A. Ortiz-Merino: Automated annotation, structural validation, and metabolic modeling of industrial yeast genomes</td>
      <td>84</td>
    </tr>
    <tr>
      <td>Javier Varela: Towards a genome scale metabolic model of <i>K. marxianus</i>: a key tool to improve its industry-relevant properties</td>
      <td>85</td>
    </tr>
    <tr>
      <td>Leonie Wenning: Synthesis of wax esters in <i>Saccharomyces cerevisiae</i> by enzymatically catalyzed esterification of long-chain fatty acids and primary long-chain fatty alcohols</td>
      <td>86</td>
    </tr>
    <tr>
      <td>18:05 - 18:25 Jean-Roch Mouret: Kinetic analysis and gas-liquid balances of the production of fermentative aromas during winemaking fermentations</td>
      <td>87</td>
    </tr>
    <tr>
      <td>18:25 - 18:35 Selected Elevator Pitch talk no. 1 - TBA</td>
      <td></td>
    </tr>
    <tr>
      <td>18:35 - 18:45 Selected Elevator Pitch talk no. 2 - TBA</td>
      <td></td>
    </tr>
    <tr>
      <td>18:45 - 18:55 Selected Elevator Pitch talk no. 3 - TBA</td>
      <td></td>
    </tr>
  </tbody>
</table>
<figure><img src="image_11.png" /></figure>
BASIDIOMYCETOUS YEASTS: PROSPECTS, DIVERSITY AND TAXONOMY

Teun Boekhout<sup>1,2</sup>, Xin-Zhan Liu<sup>2</sup>, Qi-Ming Wang<sup>2</sup>, Bart Theelen<sup>1</sup>, Marizeth Groenewald<sup>1</sup>
AND Feng-Yan Bai<sup>1,2</sup>

Contact email: t.boekhout@cbs.knaw.nl

Our knowledge on the biodiversity of basidiomycetous yeast is rapidly expanding, but the taxonomy used is still largely outdated. Here we present novel insights based on a multigene phylogeny analysis of all described basidiomycetous yeasts of the Agaricomycotina, Pucciniomycotina and Ustilaginomycotina. Their taxonomy will be revised accordingly, based on this multigene phylogeny backbone as many well supported clades were observed. We will address some specific phenotypic features that are of applied interest, such as the ability to control the growth of pest organisms, the ability to ferment, the ability to cause disease, etc. highlighting the great applied potential of these fungi.

<footnote n="1"><sup>1</sup> CBS Fungal Biodiversity Centre, Utrecht, The Netherlands</footnote>

<footnote n="2"><sup>2</sup> State Key Laboratory of Mycology, Institute of Microbiology, Chinese Academy of Sciences, Beijing, China</footnote>
Tomotake Morita¹, Hideaki Koike², Shun Sato¹, Hiroshi Habe¹, Dai Kitamoto¹

¹Research Institute for Innovation in Sustainable Chemistry, National Institute of Advanced Industrial Science and Technology (AIST), Tsukuba, Ibaraki, Japan

²Bioproduction Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), Tsukuba, Ibaraki, Japan

Contact email: morita-tomotake@aist.go.jp

*Pseudozyma antarctica* is a basidiomycetous yeast belonging to the Ustilagomycetes, a group including the smut fungus *Ustilago maydis*, and has been known as a producer of industry-relevant lipases. *P. antarctica* T-34 was isolated from phyllosphere as the producer of extracellular glycolipids, mannosylerythritol lipids (MELs). The MELs are known as multi-functional glycolipids exhibiting not only excellent properties as bio-surfactant but also unique properties for practical cosmetic ingredients. Recently, we reported the genome sequence (18Mb) of *P. antarctica* T-34, and found its oleaginous character. Among 6,543 of the total genes on the *P. antarctica* genome, 219 genes were categorized into the lipid transport and metabolism classification, while the ascomycetous yeast *Saccharomyces cerevisiae* (non-oleaginous) possessed a smaller amount of genes (140 genes) categorized into the same classification on the basis of KOG analysis. Particularly, the gene encoding an ATP/citrate lyase (ACL) related to acetyl-CoA synthesis commonly conserved in oleaginous strains was found in the *P. antarctica* genome. Moreover, the genome of *P. antarctica* (non-plant pathogen) exhibited a remarkable degree of synteny to the genome of *U. maydis* (plant pathogen), whereas the gene expression profiles of the two strains were significantly different by the DNA microarray method under the oily conditions. The expression of the gene sets relating fatty acid metabolism were markedly up-regulated under the oily conditions compared with glucose in *P. antarctica*. In addition, the MEL biosynthesis cluster was expressed at high intensity in *P. antarctica* regardless of the carbon source compared to *U. maydis*. These results indicate that *P. antarctica* shows oleaginous nature on the basis of the sequence analysis, and these genome and transcriptome analysis would facilitate developing the improved strains with customized properties for high production of the functional bio-based materials.
POLIEXTREMOPHILIC YEASTS FROM PATAGONIA ARGENTINA AND ANTARTCTIC SEA

de Garcia V.<fnref n="1" />, Martina Turk<fnref n="2" />, Polona Zalar<fnref n="2" />, Nina Gunde - Cimerman<fnref n="2" />, Giraudo M. R.<fnref n="1" />

Contact email: vdegarcia@comahue-conicet.gob.ar

Microorganisms inhabiting cold environments have the ability to tolerate freezing since they are exposed to low temperatures, dehydration, which is caused by water being bounded to ice crystals, and to survive in ultraoligotrophic conditions. Cellular adaptations to low temperatures and high salinity are generally similar and the species that inhabit these environments have specialized mechanisms for stress tolerance (osmotic and oxidative). These conditions trigger the synthesis of various metabolites that help the cell to survive. Physiological adaptations of psychrophilic and psychrotolerant microorganisms include modifications of membrane lipid composition, the accumulation of cryoprotectants like polyols (glycerol, trehalose), synthesis of the enzymes that are active at low temperatures and the production of antifreeze proteins. The presence of yeasts in sea water and sea ice of Antarctic (Bellingshausen, Weddell and Scotia seas) was studied. From 26 samples (19 of sea water and 7 of sea ice), 281 yeast strains (65 species) were isolated and identified. On selected yeast strains (psychrophilic, psychrotolerant and halophilic) cellular stress was estimated by measuring phosphoglucomutase activity at various osmotic conditions and temperature (at 5 and 15 °C, with 3 % NaCl (w/v) and without NaCl). Results showed that strains have a higher phosphoglucomutase activity at 15 °C with 3 % NaCl (w/v). We also studied the capability of yeasts to grow on media with urea as the only carbon source at 4 °C, were only basidiomycetous yeasts were able to grow. Furthermore we have searched for the presence of antifreeze protein genes in psychrophilic yeasts (Glaciozyma, Mrakia and Rhodotorula), and for all genera positive results were obtained. The results contribute to the general knowledge of the diversity, ecology, and biotechnological potential of these yeasts and will help to identify the impact of stress conditions on the major metabolic pathways of poliextremophilic yeasts from the marine environments of Patagonia and the Antarctic seas.

<footnote n="1">¹ Laboratory of Applied Microbiology and Biotechnology, Centro Regional Universitario Bariloche, Universidad Nacional del Comahue, INIBIOMA (CONICET-UNCo), Quintral 1250, San Carlos de Bariloche, Río Negro C.P. 8400, Argentina.</footnote>

<footnote n="2">² Department of Biology, Biotechnical Faculty, University of Ljubljana, Večna pot 111, SI-1000 Ljubljana, Slovenia.</footnote>
MAKING, MENDING, AND EXTENDING THE YEAST METABOLIC MODEL

Stephen G Oliver

Cambridge Systems Biology Centre & Dept. Biochemistry, University of Cambridge, Sanger
Building, 80 Tennis Court Road, Cambridge CB2 1GA, UK

Stoichiometric (or constraint-based) models of microbial metabolic networks have provided new insights into metabolism and have enormous potential as tools with which synthetic biologists can design novel strains for the production of fuels, chemicals, and pharmaceutical agents. Such models are also of use to systems biologists in understanding the complex network of interactions that are required for the functioning of a single cell.

The metabolic model of the yeast *Saccharomyces cerevisiae* is the most complete for any eukaryotic organism and is very effective at predicting which gene deletions will have a lethal phenotype. However, despite early successes, it is very much less effective at predicting genetic interactions. At a coarse-grained level, the yeast metabolic model is capable of successfully predicting the overall topology of the network of interactions between genes involved with the organism's metabolism. However, the model only predicts < 3% of all the epistatic interactions revealed from synthetic genetic array (SGA) experiments. Moreover, predictions from the metabolic models for bacteria are much more accurate than is that for yeast. While, it is clear that the content and connectivity of the yeast model can be improved, can it really be that our view of yeast metabolism is so far behind that for bacteria, or is something else missing?

In this presentation, I shall demonstrate how machine-learning (sometimes assisted by a Robot Scientist), more careful strain construction, and the use of quantitative data on the localisation of individual proteins can all be used to improve the accuracy of the yeast metabolic model. I shall also discuss the impact of such improvements on the predictive power of the model and its use in strain design.
MODEL BASED ENGINEERING OF *Pichia pastoris* CENTRAL METABOLISM ENHANCES RECOMBINANT PROTEIN PRODUCTION

Justyna Nocon<fnref n="1" />, Matthias G. Steiger<fnref n="1" />,<fnref n="2" />, Martin Pfeffer<fnref n="1" />, Seung Bum Sohn<fnref n="3" />, Tae Yong Kim<fnref n="3" />, Hannes Rußmayer<fnref n="1" />,<fnref n="2" />, Stefan Pflügl<fnref n="1" />, Christina Haberhauer-Troyer<fnref n="2" />,<fnref n="4" />, Karin Ortmayr<fnref n="4" />, Stephan Hann<fnref n="2" />,<fnref n="4" />, Gunda Köllensperger<fnref n="2" />,<fnref n="4" />, Brigitte Gasser<fnref n="1" />,<fnref n="2" />, Sang Yup Lee<fnref n="3" />, *Diethard Mattanovich*<fnref n="1" />,<fnref n="2" />

Contact email: diethard.mattanovich@boku.ac.at

Genome scale metabolic models have been successfully applied to predict genetic interventions to redirect metabolic fluxes towards desired products of the primary and secondary metabolism. Complex polymeric products like heterologous proteins equally demand redistributions of primary metabolic fluxes, their rational design however is far less obvious. Therefore cell engineering for protein overproduction has concentrated mainly to transcription, codon usage, protein folding and secretion. We have developed the first genome scale metabolic model for the yeast *Pichia pastoris* (PipMBEL1254), and integrated the synthesis of heterologous protein. The model could successfully predict the increase of protein production in oxygen limited conditions, as well as changes of central metabolic fluxes in production strains, as measured by ¹³C flux analysis. Metabolic engineering targets for enhanced protein productivity were predicted with FSEOF for gene overexpression and MOMA for gene deletion. After deleting or overexpressing the respective genes as predicted more than 50 % of the interventions led to an enhanced production of cytosolic human superoxide dismutase (hSOD), and similarly of other proteins. Beneficial mutations were mainly related to reduction of the NADP/H pool and the deletion of fermentative pathways. We demonstrate that genome scale metabolic modeling is suitable to describe metabolic changes in recombinant strains and can be successfully applied to design genetic interventions to the primary metabolism to increase recombinant protein production.

<footnote n="1">¹ Department of Biotechnology, University of Natural Resources and Life Sciences Vienna, Austria</footnote>

<footnote n="2">² Korea Advanced Institute of Science and Technology (KAIST), Daejeon, Korea</footnote>

<footnote n="3">³ Austrian Centre of Industrial Biotechnology, Vienna, Austria</footnote>

<footnote n="4">⁴ Department of Chemistry, University of Natural Resources and Life Sciences Vienna, Austria</footnote>
RECENT DEVELOPMENTS IN BIOFUELS AND BIO-BASED CHEMICALS
PRODUCTION BY YEASTS

Charles A. Abbas, Ph.D.
Director Yeast & Renewables Research
Archer Daniels Midland Research
Decatur, IL 62526 USA

In the past decade, the demand for energy has fueled a worldwide resurgence in interest in the production of biofuels and bio-based chemicals 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 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 with fossil fuel-based feedstocks; but with the volatility of petroleum-based feedstocks, there is increased demand for more sustainable products.

The development of new yeast strains of *Saccharomyces cerevisiae* by genetic engineering and the use of other genera of yeasts that can utilize a broader range of feedstocks have been the focus of research efforts. The chemicals of primary interest are the C3 and C4 chemicals that represent the fundamental building blocks of key segments of the industrial chemical industry, which today have annual sales valued at more than $10 billion. Used for a broad range of industrial and consumer products, these chemicals are on the list of top ten chemicals developed by the U.S. Department of Energy (DOE). The production of these chemicals has been already demonstrated at bench- and pilot-scale and at demonstration-scale facilities.
ROBUST HIGH-PERFORMANCE *S. cerevisiae* STRAINS FOR C5 FERMENTATION IN LIGNOCELLULOSIC ETHANOL PRODUCTION

Violeta Sanchez Nogue, Malin Sendelius, Carl Fredrik Klintner, Bärbel Hahn-Hägerdal and
**Kaisa Karhumaa**

C5 Ligno Technologies AB, P.O. box 124, SE-22100 Lund, Sweden.

Contact e-mail: kaisa.karhumaa@c5lt.se

The process consolidation of lignocellulose based ethanol production at industrial scale is now becoming a reality. Feedstocks, including agricultural crop residues, and wood and paper mill discards, represent a relatively cheap and abundant source of biomass. However, the sustainable production of ethanol requires the combination of efficient hydrolysis and successful fermentation. During biomass pretreatment and hydrolysis, several potential inhibitory compounds including weak acids, furaldehydes and phenolics are released, which reduces the microbial fermentation performance. While fermentation of lignocellulose based sugars such as glucose and xylose by recombinant yeasts have frequently been demonstrated in laboratory media, efficient fermentation in realistic process conditions is much more challenging.

The yeast *Saccharomyces cerevisiae* is currently the preferred microorganism for the fermentation step of lignocellulose ethanol production. While *S. cerevisiae* is naturally able to metabolize hexose sugars, genetic engineering is required to develop strains harboring heterologous pathways for the consumption of pentose sugars. However, lignocellulosic hydrolysates from different raw materials are very different in both sugar composition and inhibitor content. To meet the demand for strains suitable for different raw materials, we have developed a tool-box with a flexible modular system to generate collections of strains based on various *S. cerevisiae* strain backgrounds. Our xylose assimilation pathway based on a mutated xylose reductase (XR) eliminates xylitol formation and results in close to theoretical ethanol yields. C5/C6 fermentation in different lignocellulosic hydrolysates will be presented.
USING BAKER'S YEAST FOR PRODUCTION OF SUSTAINABLE INGREDIENTS IN
HEALTH, NUTRITION AND WELLNESS

Jørgen Hansen

Evolva, Copenhagen, Denmark

Contact e-mail: jorgenh@evolva.com

Most valuable small molecules are made from fossil oils, from extraction of plant or other
natural materials or obtained through specialized agriculture. The use of fossils for
manufacturing has its obvious drawbacks, but less appreciated is the fact that extraction from
natural sources may lead to exhaustion of these, and though some agricultural production
systems are perfectly sustainable, not all are. Thus, growing the plant or raising the animal
may take more land, more water or more energy than it really should. Finally, any extraction
process may require solvents or other processes which generate significant waste.

Making the compounds by fermentation instead can improve the product's sustainability greatly, avoiding deleterious use of fossils, natural source extraction or freeing land or other resources for other uses. Evolva's Genetic Chemistry technologies [1,2] allow for this. State-of-the art methodologies for establishment of heterologous biosynthesis pathways in Baker's yeast allow for fast development of fermentation-based sustainable manufacturing routes. Added benefits are higher product quality (only one product formed at a time), improved supply chain stability (no seasonal variation) and the possibility for customization (blends of single components). Production of vanillin, resveratrol and Stevia sweeteners will be discussed.

Klein, J., Heal J., Hamilton W.D.O., Boussemghoune T., Tange T.O., Delegrange F.,† Jaeschke G., Hatsch A. and Heim J. Yeast Synthetic Biology Platform Generates Novel Chemical Structures as Scaffolds for Drug Discovery. *ACS Synthetic Biology* (January 16., 2014)

Naesby N., Nielsen S.V.S., Nielsen C.A.F., Green T.1, Tange T.O., Simón E., Knechtle P., Hansson A., Schwab M.S.1, Titiz1 O., Folly C., Archila R.E., Maver M., Fiet S.v.S., Boussemghoune T., Janes M., Kumar A.S.S., Sonkar S.P., Mitra P.P., Benjamin V.A.K., Korrapati N., Suman I., Hansen E.H., Thybo T., Goldsmith N. and Sorensen A.S. Yeast artificial chromosomes employed for random assembly of biosynthetic pathways and production of diverse compounds in *Saccharomyces cerevisiae*. *Microbial Cell Factories* **8**:45 (2009)
EXPLORING “PERIPHERAL” METABOLIC PATHWAYS IN BUDDING
YEASTS

Tomas Linder

Department of Microbiology, Swedish University of Agricultural Sciences, Box 7025,
Uppsala SE-750 07, Sweden

Contact email: tomas.linder@slu.se

The budding yeasts (phylum *Ascomycota*, sub- phylum *Saccharomycotina*) play a
number of important roles in human society such as model systems for understanding
basic cellular function, opportunistic pathogens, spoilage organisms and the
production of fermented beverages to name but a few. From a metabolic perspective,
budding yeasts have a much simpler metabolic circuitry than the filamentous
ascomycetes (sub- phylum *Pezizomycotina*) or the basidiomycetes (phylum
*Basidiomycota*). This metabolic simplicity in combination with the relative ease of
genetic manipulation make the budding yeasts an ideal system to explore poorly
characterized, non-universal metabolic pathways – so-called “peripheral” metabolism
(to distinguish it from universally conserved central pathways such as glycolysis etc).
Peripheral metabolism includes both catabolic pathways (the assimilation of non-
conventional sources of carbon, nitrogen and sulfur) as well as anabolic pathways (for
example the biosynthesis of sophorolipids, exopolysaccharides and volatiles). My
research is devoted to characterizing these pathways and identifying the genes
involved as well as discovering new pathways. An increased understanding of these
pathways have a wide variety of applications including the improvement of substrate
utilization in industrial fermentations, degradation of xenobiotics as well as
production of biofuels and fine chemicals.
SPECIES DELIMITATION: DEFINING THE UNIT OF YEAST BIODIVERSITY

Claudia Colabella<fnref n="1" />, Luca Roscini<fnref n="1" />, Duong Vu<fnref n="2" />, Laura Corte <fnref n="1" />, Vincent Robert<fnref n="2" /> and Gianluigi Cardinali<fnref n="1" />

Delimiting yeast species for reliable and rapid identification is a complex operation conditioned by philosophical, biological and technical problems that one can summarize in the three following questions: i. do yeast species exist?, ii. do the delimited species have a biological meaning?, iii. how to combine tools for the best classification and identification?.

The first issue is an exquisitely ontological question transformable in the other interrogation: "do yeast species present relevant discontinuities (gaps) among themselves?". Data from the literature are sometimes conflicting, maybe due to different behaviors of the analyzed taxa.

The evidence that molecular tools often based on very short DNA sequences (as barcoding), are increasingly popular in yeast identification, poses the question on whether the species delimited with these systems have also a biological meaning. This can be translated in the question: "do barcode clusters correlate with relevant biological factors?".

Finally, the presence of an overwhelming amount of data collected by yeast biologists over the decades suggests the urgent need to efficiently use existing databases, possibly developing next generation analytical tools to facilitate data mining.

Discontinuity analyses were carried out on various taxa, predominantly those involved in wine production, using LSU and ITS markers. Results showed that discontinuities are often not obvious, especially for species with many isolates like *Saccharomyces cerevisiae*. These results suggested that yeast taxa involved in the fermentation industry were subject to correlation between DNA markers and other available data. The high variability of these analyses outcome will be presented in detail. Finally, we will present an example of a state of the art database for yeast taxonomy and some lines of development for next generation bioinformatics tools focused on this topic.

<footnote n="1">¹ Department of Pharmaceutical Sciences, University of Perugia, Via Borgo 20 Giugno, 74, Perugia - Italy</footnote>

<footnote n="2">²Centraalbureau voor Schimmelcultures - Uppsalalaan 8, 3435CT, Utrecht - The Netherlands
Contact email: gianluigi.cardinali@unipg.it</footnote>
YEASTS OF “END OF WORLD”: TECHNOLOGICAL POTENTIAL

Ma. Angélica Ganga<fnref n="1" />, Liliana Godoy<fnref n="1" />, Lourdes Carmona<fnref n="1" />, Verónica García<fnref n="1" />, Francisco Cubillos<fnref n="1" />, Claudio Martínez <fnref n="1" />,<fnref n="2" />, Gustavo Lorca<fnref n="1" />, Rubén Peña<fnref n="1" />, Elías Franco<fnref n="1" />.

Obispo Manuel Umaña 050. Estación Central. Chile.

Contact email: angelica.ganga@usach.cl

Grant: Gore-Atacama 3303248.

In the applied science, the main goal of our group has been the study of native yeasts isolated in different areas of Chile to determine its potential use in the wine industry. Our group has a collection of 1.800 yeasts isolates between *Saccharomyces* and *non-Saccharomyces* genera. During 8 years, we have researched about the potential use of *Saccharomyces* spp. species in winery industry. From this work, we have defined a native yeast named Fermicru XL (Oenobrands) to be used in young wines. This yeast is characterized to give fruity red wines, with enhanced mouth-feel and soft tannins.

In addition, we have performed a screening of different enzymatic activities xilanase, β-glycosidase, etc.) using our non-*Saccharomyces* collection. Thus, we have obtained the strain *Metchiniscowia pulcherrima* that produced β glycosidase activity. During the wine fermentation, this yeast produces high thiols concentration, which have special interest for the oenologists because providing fruity aromas to wine, especially in white wine. As in the previous case, this yeast is being marketed as Flavia (Lallemand Inc).

On the other hand, our group has selected a native yeast from a typical wine of north of our country, named Pajarete. This wine is sweet and is produced with dried grapes. This native yeast provides fruits aromas, especially banana characteristic of this type of wine. At the moment, we are selecting more yeasts from this wine and we are studying to produce the yeasts at laboratory.

<footnote n="1">¹ Departamento en Ciencia y Tecnología de los Alimentos. Universidad de Santiago de Chile.</footnote>

<footnote n="2">² Centro de Estudios en Ciencia y Tecnología de los Alimentos. Universidad de Santiago de Chile.</footnote>
SURVEY ON YEAST BIODIVERSITY IN GEORGIAN VINEYARDS: A PRISTINE
ENVIRONMENT FOR THE SELECTION OF WINE STRAINS

Gabriella De Lorenzis<fnref n="1" />, Ileana Vigentini<fnref n="1" />, Atina Cote<fnref n="2" />, Luca Roscini<fnref n="3" />, Laura Corte<fnref n="3" />, Gianluigi Cardinali<fnref n="3" />, Osvaldo Failla<fnref n="1" />, David Maghradze<fnref n="4" />, Frederick P. Roth<fnref n="2" />,<fnref n="5" />, Roberto Foschino<fnref n="1" />

Contact email: roberto.foschino@unimi.it

The wine market is changing due to ever-growing demand for improved sensory and nutritional properties of the final product. New styles of wines and innovative ways of managing fermentations have intensified interest in the search for new starter strains. Here we investigate wine yeast biodiversity in Georgia, an ancient vine growing area where the use of starter cultures has not yet spread, to select non-conventional yeast species for wine-making. Yeast isolation was carried out from grapes, musts and wines in six different Georgian regions. One hundred and seventy six isolates were identified as belonging to 14 different species: *S. cerevisiae* (36.4%), *Hanseniaspora gulliermondi* and *Metschnikowia pulcherrima* (about 20%), *Cryptococcus flavescensis* (8%), *Cryptococcus carnescens* and *Torulaspora delbrueckii* (about 3%), *Candida intermedia*, *Pichia guilliermondi* and *Pichia kluyveri* (about 2%), *Candida gotoi*, *Issatchenkia terricola*, *Cystofilobasidium infirmominiatum*, *Kluyveromyces marxianus* and *Hanseniaspora osmophila* (≤ 1%). *S. cerevisiae* isolates were further submitted to whole-genome comparison to establish phylogenetic relationships and genome structure. Some non-*Saccharomyces* yeasts were investigated biochemically (alcohol and SO₂ tolerance, acetic acid, glycerol and H₂S production). Using micro-vinification experiments, one *K. marxianus* and one *T. delbrueckii* strain are proved to be promising for wine production.

<footnote n="1">¹ Department of Food, Environmental and Nutritional Sciences and Department of Agricultural and Environmental Sciences, Università degli Studi di Milano, Milan, Italy</footnote>

<footnote n="2">² Donnelly Centre, University of Toronto and Lunenfeld-Tanenbaum Research Institute, Mt Sinai Hospital, Toronto, Canada</footnote>

<footnote n="3">³ Department of Applied Biology, Università degli Studi di Perugia, Perugia, Italy</footnote>

<footnote n="4">⁴ Institute of Horticulture, Viticulture and Oenology, Agricultural University of Georgia, Tbilisi, Georgia</footnote>

<footnote n="5">⁵ Departments of Molecular Genetics and Computer Science, University of Toronto, Toronto, Canada</footnote>
MULTI-STRAIN INDIGENOUS YEAST STARTERS FOR ‘WILD-FERMENT’ WINE
PRODUCTION

Nisiotou Aspasia, Banilas Georgios, Chorianopoulos Nikolaos, Tassou Chrysoula

Hellenic Agricultural Organisation DEMETER, Institute of Technology of Agricultural
Products and Wine, Athens, Greece
ctassou@nagref.gr

Industrial wine-making is based on commercial *Saccharomyces cerevisiae* starter cultures, which are applied to confer manageability and reproducibility. However, the use of a single starter culture, which typically overwhelms the indigenous microbiota, deprives wines from the complexity and authenticity. Yet, global competition in the wine market calls for the production of quality wines with typical character. Indigenous *S. cerevisiae* and non-*Saccharomyces* (wild) yeast species may confer added value to wines, by adding to the wine complexity and authenticity. In the above context, the scope of the current project is to combine selected native *S. cerevisiae* with wild yeasts species in the development of novel multi-species yeast starter blends to serve as starters in induced ‘wild’ fermentations. Therefore, the biodiversity of key EU viticultural regions is being thoroughly surveyed to identify yeast strains of enological importance. With respect to the yeast populations from Peza and Nemea, two key Greek viticultural regions, a relatively high species as well as strain biodiversity was observed. Taking *S. cerevisiae* populations as an example, it was shown that the percentage of biodiversity ranged between 10 – 13%, with only 7 common genotypes (< 5%) between the two zones. Furthermore, present results clearly point to spatial structure of *S. cerevisiae* populations in separate regions, albeit the within-zone distribution seems rather random. Different genotypes were then evaluated as per their technological properties, e.g. SO₂ or ethanol resistance, and selected strains were applied in mixed fermentations. The best performing strain combinations will be examined in plant-scale fermentations. Project results are expected to allow the launching of ‘wild ferment’ technology in winemaking and the production of quality *terroir*-driven wines. By these means, the project is expected to assist the companies to enhance marketing abilities towards a more competitive and sustainable wine industry.

Funded by EU FP7 Grant Agreement 315065-WILDWINE (www.wildwine.eu).
EFFECT OF MIXED SACCHAROMYCES AND NON-SACCHAROMYCES STARTERS ON ALCOHOLIC FERMENTATIONS AND WINES

Beltran, Gemma; González, Beatriz; López, Paula; Izidoro, Iara; Traver, Iris; Padilla, Beatriz; Esteve-Zarzoso, Braulio; Mas, Albert

Dept. Bioquímica i Biotecnologia, Universitat Rovira i Virgili, c/ Marcel·lí Domino n°1
Tarragona 43007, Spain

Contact email: gemma.beltran@urv.cat

Alcoholic fermentation is characterized by a succession of yeast species from grapes to final wines. In grapes, non-*Saccharomyces* yeasts are dominant, yet *Saccharomyces cerevisiae* soon takes over and dominates the fermentation process. Although non-*Saccharomyces* yeasts have often been considered as a source of microbial spoilage, there is substantial contrary evidence pointing to a positive contribution by these yeasts. The role of non-*Saccharomyces* yeasts in wine fermentation is therefore receiving increasing attention by wine microbiologists. In the present study, wine yeasts belonging to different species have been isolated from grapes and spontaneous fermentations in DOQ Priorat (Spain). After molecular yeast identification (RFLP-PCR of the ITS region and sequencing D1/D2 region of the 26S rRNA gene), isolates were typified at the strain level using different molecular techniques depending on the yeast species (analysis of inter-delta elements, PCR-based method of tandem repeat tRNA, RAPD-PCR, microsatelites, etc). *Hanseniaspora uvarum* was the dominating non-*Saccharomyces* species, followed by *Candida zemplinina*. Strains with good oenological characteristics have been then selected to be used as starters in mixed culture fermentations, performing sequential inoculation in pilot-scale fermentations. Natural grape musts were initially inoculated with either *Hanseniaspora uvarum*, *Candida zemplinina* or a mixture of them, and *S. cerevisiae* was added to the must after two days of fermentation. Fermentation kinetics development, yeast growth and evolution of the different species were followed through the process, and the main metabolites and aromatic compounds were determined at the end of the fermentation. Significant differences were observed in most of the parameters analyzed, confirming the higher complexity and diversity in flavor and aromatic compounds when yeast multi-starters are used. Thus, the development of yeast starter blends composed of indigenous, privileged strains, could ensure the production of elegant wines reflecting sense of place and compatible with *terroir*-driven approaches.
ADAPTIVE ROLE OF HORIZONTALLY TRANSFERRED OLIGOPEPTIDE
TRANSPORTERS IN WINE YEASTS

Souhir Marsit, Frédéric Bigey, Jean-Luc Legras, Viginie Galeote and Sylvie Dequin

UMR 1083 Sciences Pour l'Oenologie, INRA, 2 Place Viala, F-34060 Montpellier, France
Contact email: souhir.marsit@supagro.inra.fr

Wine yeasts have unique properties within the *Saccharomyces cerevisiae* species. However, the molecular mechanisms behind these features remain largely unknown. We previously showed that horizontal gene transfers (HGT) of three large regions (A, B and C) containing 34 genes from distant yeasts have shaped the genome of wine yeasts. These new genes potentially encode important metabolic functions in winemaking (sugar and nitrogen metabolism), suggesting a role in adaptation to the wine environment.

To assess the adaptive value of these HGT, we carried out a functional analysis of a mutant deleted for the region C (65kb). We found that this region confers a better cell viability and capacity to complete the fermentation in grape must. We showed that this phenotype is associated to a better assimilation of the oligopeptide fraction of grape must by the Fot1/2 oligopeptide transporters present in this region. We also showed that Fot-mediated peptides uptake affects the glutamate node and the NADPH/NADP+ balance, resulting in altered metabolite profile. By carry out competition assays, we demonstrated that the presence of *FOT* genes improves yeast fitness on grape must. Indeed, *FOT1/2* deletion mutants were completely outcompeted by the wild-type strain after three successive co-cultures. Finally, using population genomics data, we found a strong conservation of *FOT* genes in the genome of yeast strains possessing the region C, even when this region was incomplete. These findings demonstrate the adaptive value of *FOT* genes which improve nutrient resource utilization in a nitrogen-limited environment, and highlight the role of HGT in genome evolution and adaptation of yeasts to their ecological niche.
PHYSIOLOGICAL AND GENETIC CHARACTERISATION OF *Saccharomyces*
STRAINS ISOLATED FROM ICE HARVEST OF CHARDONNAY GRAPES

Tanja Vojvoda Zeljko¹, Lucilla Iacumin², Katarina Huić Babić³, Giuseppe Comi²,
Sandi Orlić⁴

¹ Division of Molecular Biology, Institute Ruđer Bošković, Bijenička 54, Zagreb, Croatia

² Department of Food Science, Faculty of Agraria, University of Udine, Via Sondrio 2,
Udine, Italy

³ Department of Microbiology, Faculty of Agriculture, Svetošimunska cesta 25, Croatia

⁴ Division of Materials Chemistry, Institute Ruđer Bošković, Bijenička 54, Zagreb, Croatia
Contact email: sandi.orlic@irb.hr

Specific ecological environment in the production of wine is ice harvest. Such harvest takes
place in extreme temperature conditions while the grapes are still frozen and the microflora of
those grapes has specific features like stress tolerance. Yeasts present during fermentation of
ice wine have to be adapted to stressful conditions like high sugar content and low pH. In this
study wine yeasts present during the Chardonnay ice wine must fermentation were identified
and a physiological and genetic characterization of them was performed. In total, twenty-eight
yeast strains were isolated during the fermentation and fourteen of them belonged to the
*Saccharomyces* group. Identification at the species level was performed by restriction of the
ITS region and six strains of *S. cerevisiae* as well as eight strains of *S. paradoxus* were
identified. These strains were tested in more detail regarding physiological characteristics,
which are important for fermentation of such specific substrate. Most of the isolates (79%)
showed significant osmotolerance, which could result from adaptation or genetic inheritance.
Flocculation capacity was tested as well but neither strain showed true flocculation in all
conditions. A certain degree of flocculation was obtained for two strains, but only when
grown in preculture containing calcium ions. SOY 28L showed interesting values and
therefore investigation should be continued in the manner of detection and determination of
regulation mechanisms leading to that pattern of behavior. The finding indicates that the
indigenous isolates could really be competitors of other yeasts in ice wine fermentation and in
the future used as a starter culture.
APPLICATION OF *Hanseniaspora vineae* STRAINS. SEARCHING FOR GENES TO
EXPLAIN INCREASED FLAVOR COMPLEXITY IN WINES

Facundo Giorello¹; Valentina Martin¹ Valentina Salzman², Laura Camesasca³ Laura Fariña¹,
Eduardo Boido¹, Karina Medina¹, Eduardo Dellacassa¹. Luisa Berna²; Pablo S. Aguilar²;
Carina Gaggero³; Francisco Carrau¹

¹ Sección Enología, Facultad de Química, Universidad de la República, 11800, Montevideo, Uruguay. ²Laboratorio de Biología Celular de Membranas, Institut Pasteur de Montevideo, 11400 Montevideo, Uruguay. ³Departamento de Biología Molecular, Instituto de Investigaciones Biológicas Clemente Estable (IIBCE), 11600 Montevideo, Uruguay.

Contact email: fcarrau@fq.edu.uy

Use of novel yeast strains for winemaking is increasingly regarded as a way for improving quality and to provide variation including subtle characteristic differences in fine wines. Furthermore, spontaneous fermentations are associated with greater wine body, unusual or odd aromas and flavors, creamy texture and greater complexity.

More than 800 non-*Saccharomyces* strains were isolated from the Uruguayan winemaking environment over a 5 year period. We selected 11 strains of *Hanseniaspora vineae* to study sequential fermentations strategies with and without the presence of a conventional *Saccharomyces* wine strain.

Based on winemaking parameters, key chemical and sensory analysis we were able to define specific metabolic and sensory traits for each strain. In general, the co-fermentation strategy with *H.vineae* provided significant increases in glycerol and acetyl and ethyl ester flavour compounds and relative decreases in alcohols, fatty acids, and biogenic amines which correlates with wine differences found between these alternative fermentation procedures. In order to understand the genetic base of “flavour phenotypes” we also sequenced and assembled the genomes of two *H. vineae* strains. More than 4000 highly reliable coding sequences were predicted for *H. vineae* and the presence-absence of genes belonging to several metabolic pathways were analyzed. One hundred twenty eight *S. cerevisiae* genes associated with fermentation as those participating on Glycolysis/ Gluconeogenesis, Citrate cycle, Pentose pathway, Steroid biosynthesis, Fatty acid degradation and Fatty acid biosynthesis pathways were screened. Despite the great sequence divergence observed between *H. vineae* and industrial *S. cerevisiae* genomes, 87 of those genes (68%) were found in *H. vineae*. Interestingly, increased divergences in genes related to aroma compounds metabolism are discussed. For example, gene associated with the biosynthesis of acetates esters (ATF2) was highly divergent in *H. vineae* compared to that present in *S. cerevisiae* and genes involved in the regulation of higher alcohols (ARO8 and ARO9) had more than 3 copies compared to one present in *S. cerevisiae*.
# EXPLORATION OF GENETIC RESOURCES FROM YEAST GENOME TO DEVELOP ROBUST YEAST STRAINS FOR BIOFUELS PRODUCTION

Xinqing Zhao

School of Life Science and Biotechnology, Dalian University of Technology
Linggong Road 2, Dalian, 116024, China
E-mail: xqzhao@dlut.edu.cn

*Saccharomyces cerevisiae* is widely used in brewing industry and for biofuels production and biorefinery. Industrial *S. cerevisiae* strains are exposed to various stressful conditions during bioethanol fermentation, such as toxic level of ethanol and inhibitors from cellulosic hydrolysates, high temperature and so on. Improvement of stress tolerance of yeast strains benefits economic production of fuel ethanol. In our previous work, the protective effect of zinc against ethanol toxicity and high temperature was revealed. When the effect of different concentrations of zinc ion on ethanol production of self-flocculating yeast SPSC01 in presence of high concentration of acetic acid was studied in batch fermentation, it showed that appropriate concentration of zinc ions significantly shortened the lag time of cell growth of the self-flocculating yeast. Transcriptome analysis revealed the remodeling of gene expression by zinc in presence of toxic level of acetic acid. Functional analysis of about 20 differentially expressed genes in the transcriptomic data showed that overexpression of these genes improved tolerance to toxic levels of acetic acid and ethanol. We proposed that exploration of differentially expressed genes under stressful conditions in different zinc status benefits development of robust yeast strains for biofuels production.

## Acknowledgements

This work was supported by Program for New Century Excellent Talents in University (NCET-11-0057) and National Science Foundation of China (21376043).

## References

1. Zhao XQ, Xue C, Ge XM, Wang JY, Yuan WJ, Bai FW. Impact of zinc supplementation on the improvement of ethanol tolerance and ethanol yield of self-flocculating yeast in continuous ethanol fermentation. J Biotechnol. 2009, 139: 55-60.

2. Xue C, Zhao XQ, Bai FW. Effect of the size of yeast flocs and zinc supplementation on continuous ethanol fermentation performance and metabolic flux distribution under very high gravity conditions. Biotechnol Bioeng. 2010, 105: 935-944.
THE WATER INSOLUBLE SOLIDS OF PRETREATED LIGNOCELLULOSIC
MATERIALS INCREASES THE RESISTANCE OF *Saccharomyces cerevisiae* TO
LIGNOCELLULOSIC INHIBITORS – A PROTEOMIC STUDY OF THE EFFECTS

Rakesh Koppram<sup>1,2</sup>, Valeria Mapelli<sup>2</sup>, Eva Albers<sup>2</sup> and Lisbeth Olsson<sup>2</sup>

Contact email: rakesh.koppram@boku.ac.at

The pretreatment of lignocelluloses usually results in a material of semi-solid consistency containing liquid and solid fraction. The monomeric sugars and inhibitory compounds present in the liquid fraction have been extensively characterized and their effects on *Saccharomyces cerevisiae* based fermentation are well documented. However, there is no information on the influence of water-insoluble solid (WIS) fraction on the cellular performance. Here we show that when WIS was added to the minimal medium containing synthetic inhibitors, a reduced lag phase and enhanced volumetric ethanol productivity of *S. cerevisiae* CEN.PK 113-7D were observed compared to when no solid fraction was present in the medium. To investigate the underlying molecular reasons for the effects of WIS, acetic acid was used as an inhibitor to determine the response to WIS at the proteome level in yeast cells. Comparisons were made with cells grown in the presence of acetic acid but without WIS in the medium. Altogether, 729 proteins were detected and quantified, however when applying Student's t-test with 95% confidence we found 264 proteins were over-expressed and 280 proteins were under-expressed with a fold change≥1.2 in the presence of WIS compared to absence of WIS. The cells in the presence of WIS over-expressed several proteins related to glycolysis, electron transport chain, oxidative stress response, oxygen and radical detoxification and unfolded protein response; and under-expressed most proteins related to biosynthetic pathways including amino acid, purine, isoprenoid biosynthesis, aminoacyl-tRNA synthetases and pentose phosphate pathway. Differentially expressed proteins may indicate that the likelihood of increased ATP generation in the presence of WIS was used to defend against acetic acid stress at the expense of reduced biomass formation. Although, comparative proteomics of cells with and without WIS in the acetic acid containing medium revealed numerous changes, a direct effect of WIS on cellular physiology remains to be investigated.

<footnote n="1"><sup>1</sup> Austrian Center of Industrial Biotechnology (ACIB), Vienna, Austria</footnote>

<footnote n="2"><sup>2</sup> Industrial Biotechnology, Department of Chemical and Biological Engineering, Chalmers
University of Technology, Gothenburg, Sweden</footnote>
TARGETING THE INTRACELLULAR REDOX STATE IN THE DEVELOPMENT OF
MORE ROBUST *Saccharomyces cerevisiae* STRAINS FOR LIGNOCELLULOSIC
BIOETHANOL PRODUCTION

*Magnus Ask*<fnref n="1" />,<fnref n="2" />, *Maurizio Bettiga*<fnref n="1" />, *Valeria Mapelli*<fnref n="1" />, *Varuni Duraiswamy*<fnref n="1" />, *Heidi Höck*<fnref n="1" />,<fnref n="3" />, and *Lisbeth Olsson*<fnref n="1" />

Contact e-mail: magnus.ask@acib.at

Bioethanol produced from lignocellulosic raw materials is a promising alternative to fossil
fuels and to decrease greenhouse gas emissions, but several challenges still exist. When
lignocellulosic biomass is pretreated, a number of undesired degradation products are
generated, among which the furaldehydes furfural and hydroxymethylfurfural (HMF) have
shown to impede growth and limit ethanol productivity of the yeast *Saccharomyces
cerevisiae*. In the present study, a recombinant, xylose-utilizing *S. cerevisiae* strain was
challenged with sub-lethal concentrations of furfural and HMF in anaerobic batch
cultivations. By pulsing furaldehydes in either the glucose or the xylose consumption phase,
perturbations in the intracellular NAD(P)H/NAD(P)⁺ ratios could be demonstrated. A
genome-wide study of transcription found that genes related to NADPH-requiring processes,
such as nitrogen and sulphur assimilation, were significantly induced. Moreover, the
protective metabolite and antioxidant glutathione was identified as the highest scoring
reporter metabolite in the transcriptome analysis. *S. cerevisiae* strains overproducing
glutathione were constructed and the resulting strains were evaluated in simultaneous
saccharification and fermentation (SSF) of pretreated spruce. The results from the present
study provide valuable insights of how *S. cerevisiae* responds to stress imposed by HMF and
furfural and how such information could be used to engineer more robust yeast strains.

<footnote n="1">¹ Department of Chemical and Biological Engineering, Chalmers University of Technology, Gothenburg, Sweden</footnote>

<footnote n="2">² Current affiliation: Austrian Centre of Industrial Biotechnology, Vienna, Austria</footnote>

<footnote n="3">³ Current affiliation: Lehrstuhl für Biotechnologie, RWTH Aachen University, Aachen, Germany</footnote>
ROLE OF ANTIOXIDANT DEFENSE SYSTEMS ON AGING IN WINE YEAST

Cecilia Picazo<fnref n="1" />, María Cano<fnref n="1" />, Helena Orozco<fnref n="1" />, Esther Gamero-Sandemetrio<fnref n="2" />, Emilia Matallana<fnref n="1" />, Agustín Aranda<fnref n="2" />

Contact email: cecilia.picazo@iata.csic.es

Under aerobic and anaerobic conditions, biomass production and grape juice fermentation respectively, *S. cerevisiae* is subjected to many types of stress. Oxygen is responsible for the production of toxic reactive oxygen species (ROS) and the cells have defense mechanisms against oxidative stress. In *S. cerevisiae* there is a large group of ROS detoxifying enzymes where Tsa1 is a cytosolic peroxiredoxin (PRX) that reduce peroxides with the help of thioredoxins (TRX), usually acting as an electron donor. *S. cerevisiae* has two cytosolic thioredoxins, Trx1 and Trx2, that obtain the redox power of the thiorredoxin reductase Trr1 action and finally from NADPH. Furthermore, thioredoxin are involved in a reversible posttranslational modification mechanism, whereby a glutathione (GSH) is added to a cysteine residue of a protein to prevent its irreversible oxidation. Thioredoxins have a role of deglutathionylation in stationary phase and this role may be related to the control of longevity. Tsa1 activity is regulated by sulfiredoxin Srx1. In our laboratory has shown the importance of *TRX2* gene in the biomass production. Due to this importance in the regulation of redox homeostasis and longevity, we studied the role of these proteins on chronological life span (measured as viability in stationary phase) and in protein glutathionylation in different environmental conditions. Double deletion of thioredoxins *TRX1* and *TRX2* cause an increase in sensitivity to oxidants and shortening on CLS in aerobic conditions as expected, but it increases longevity in winemaking conditions. Peroxiredoxin Tsa1 is analyzed in these different environmental conditions too. Double deletion with nutrient regulator *GCN2* leads to a very short chronological longevity in all growth media. *SRX1* mutation extends longevity during winemaking and its combination with *GCN2* deletion extends it even further. The role of mitochondria in stress tolerance and longevity is also addressed. Mitochondrion is a key factor in the tolerance to drying.

<footnote n="1">¹ Departament of biochemistry and Molecular Biology University of Valencia</footnote>

<footnote n="2">² Institute of Agrochemistry and Food Technology IATA-CSIC</footnote>
TRANSCRIPTOME ANALYSIS OF WINE YEAST STRAINS UNDER SULPHITE-INDUCED STRESS CONDITIONS

Chiara Nadai¹, Stefano Campanaro³, Alessio Giacomini¹,² and Viviana Corich¹,²

1 Department of Agronomy Food Natural Resources Animals and Environment, University of Padova, Legnaro (PD), Italy

2 Centro Interdipartimentale per la Ricerca in Viticoltura ed Enologia (CIRVE), University of Padova, Conegliano (TV), Italy

3 CRIBI Biotechnology Centre, Department of Biology, University of Padova (PD), Italy
Contact e-mail: viviana.corich@unipd.it

Sulphite is widely used in winemaking for its antimicrobial and antioxidant properties, although its toxic effect on human health is proven. For this reason strategies for reducing chemical preservatives in winemaking is strongly demanded. Wine yeasts can cope with SO2 by different systems, such as acetaldehyde production, sulphite uptake and reduction or SO2 export.

The aim of this work was to study the genes involved in sulphites response in *S. cerevisiae* and see how different strains respond to SO2 addition in the early stage of fermentation.

In this study 10 strains has been chosen among those whose genome has been sequenced: 6 commercial yeasts, EC1118, AWRI796, AWRI1631, VIN13, QA23, VL3, and 4 isolated directly from vineyard, R008, R103, P301, P283. The strain S288C was added to the analysis, as reference. Fermentation trials in synthetic must were conducted at laboratory scale to assess the main technological and quality traits and to investigate strain behaviours towards sulphite. The 4 strains showing the strongest differences in terms of response to SO2 were selected. To clarify the genetic basis of this complex enological trait we performed transcription profiling using SOLID technology. The analysis was conducted during fermentation process mimicking winemaking condition, in synthetic must supplemented with 25 mg/l of SO2 in 1l-capacity bioreactors. For all strains, fermentation rate was determined overall the process, together with sulphite and acetaldehyde production. RNA seq was performed at early exponential phase (6 g/l of CO2 produced) to investigate yeast adaptation under sulphite conditions. Gene expression analysis suggested that both specific gene activities and more general genetic pathways are involved in sulphite response.
MULTIPLE GLOBAL APPROACHES FOR DEPHICERING THE MOLECULAR BASIS
OF LOW TEMPERATURE ADAPTATION IN WINE YEAST

Estéfani García-Ríos<fnref n="1" />, María López-Malo<fnref n="1" />, <fnref n="2" />, José Manuel Guillamón<fnref n="1" />

Contact email: e.garcia.rios@iata.csic.es

Low temperature alcoholic fermentations (10-15°C) are becoming more frequent due to the winemaker's tendency to produce wines with more pronounced aromatic profile. However fermentation at low temperature presents some disadvantages: reduced growth rate, long lag phase, sluggish or stuck fermentations. The aim of this study is to phenotype a collection of 27 S. cerevisiae commercial wine strains growing within temperatures (4-45°C) in both minimal media (SD) and synthetic must (SM) and, taking into account μmax value, we selected two strains with divergent phenotype in their capacity to grow at low temperature. To confirm this differential phenotype, we design a competition between both strains during wine fermentations. As expected, at low temperature fermentation, the strain showing a good performance out-compete to the strain growing badly in cold, whereas the percentages of both strains were kept around 50% throughout the fermentation process at 28°C.

Finally we aimed to decipher the molecular basis underlying this divergent phenotype by analyzing the genomic, proteomic and transcriptional differences between both strains at low temperature. The up-regulation of genes in the good strain implicated in biosynthesis of sulfur-containing amino acids and S-Adenosilmethionine (SAM) biosynthesis suggest the implication of this pathway in cold adaptation, likely for the requirement of SAM in the biosynthesis of phospholipids (Hickman *et al.*, 2011). We also found genes implicated in glutathione redox reaction, together with the presence of thioredoxins proteins, could be indicating a bigger oxidative stress at low temperature. Moreover, we found glycolysis enzymes of the lower part of the pathway, the part that leads to ATP generation and enhances the glycolytic flux and the fermentative capacity of the strain.

<footnote n="1">¹Departamento de Biotecnología de los alimentos, Instituto de Agroquímica y Tecnología de los Alimentos (CSIC), Avda. Agustín Escardino, 7, E-46980-Paterna, Valencia, Spain</footnote>

<footnote n="2">²Biotecnologia Enològica. Departament de Bioquímica i Biotecnologia, Facultat de Enologia, Universitat Rovira i Virgili. Marcel·li Domingo s/n, 43007, Tarragona, Spain</footnote>
THE ROLE OF YEAST TRANSCRIPTIONAL FACTOR Msn2 IN
*Saccharomyces cerevisiae*

Noreen Suliani binti Mat Nanyan¹, Daisuke Watanabe¹, Keisuke Hashida¹, Yu Sasano¹
and Hiroshi Takagi¹

¹Graduate School of Biological Sciences, Nara Institute of Science and Technology,
Ikoma-shi 630-0192 Nara, Japan
Contact email: hiro@bs.naist.jp

During fermentation processes, yeast *Saccharomyces cerevisiae* cells are exposed to multiple environmental stresses, which often impair the cellular functions and limit their fermentation properties. To protect the cells from these harsh conditions, the transcriptional factor Msn2 plays a pivotal role in response to a variety of stresses in *S. cerevisiae*. When cells are exposed to such stresses, Msn2 is imported from the cytoplasm into the nucleus and binds to the stress response elements, normally found in the promoter regions, and subsequently activates the transcription of many stress-response genes. To improve the fermentation properties of industrial yeasts, we constructed the *MSN2*-overexpressing strain used for bioethanol production. The engineered strains exhibited reduced intracellular reactive oxygen species levels and showed rapid growth compared to the control strain in the presence of furfural, one of the major fermentation inhibitors generated by pretreatment of lignocellulosic biomass. Furthermore, a baker's yeast strain overexpressing *MSN2* accumulated trehalose in the cells and improved the gassing power in frozen dough compared with wild-type strain. These results suggest that overexpression of Msn2 is an effective strategy to enhance the stress tolerance of *S. cerevisiae* cells through affecting antioxidative mechanism and carbon metabolism. Intriguingly, we found that *MSN2*-overexpressing strains showed higher sensitivity towards several toxic amino acid analogues, such as azetidine-2-carboxylic acid, canavanine, and *o*-fluoro-DL-phenylalanine, than wild-type cells. Based on this result, we hypothesize that the overexpression of *MSN2* is involved in the regulation of amino acid transporters. Our preliminary data indicated that Gap1 and Gnp1 transporters were located on the plasma membrane of *MSN2*-overexpressing cells, whereas they were internalized in wild-type cells. Altogether, this study will aid in revealing a novel regulatory role of Msn2 in the uptake of amino acids, in addition to its function in stress response.
INVESTIGATION OF HOG SIGNALING PATHWAY ARCHITECTURE OF THE
HALOPHILIC BASIDIOMYCETE *Walloemia ichthyophaga* WITH THE FUNCTIONAL
COMPLEMENTATION OF *Saccharomyces cerevisiae* MUTANTS

Tilen Konte¹ and Ana Plemenitaš¹

¹Institute of Biochemistry, Faculty of Medicine, University of Ljubljana, Vrazov trg 2, 1000 Ljubljana, Slovenia

Contact email: tilen.konte@mf.uni-lj.si

Exotic and uncommon model organisms are attractive research objects. Besides being a good source of potentially interesting new discoveries, they are hard to work with, because the complexity of their manipulation largely exceeds that of the well established models like *Saccharomyces cerevisiae*. High evolutionary conservation of signaling pathways allows yeast to be used as a system for investigation of heterologously expressed orthologous signaling proteins. We used *S. cerevisiae* knockout mutants to study the architecture of *W. ichthyophaga* HOG signaling pathway, with the focus on MAPKK WiPbs2 and its interacting proteins. We expressed in the yeast *hog1Δpbs2Δ* cells two paralogous MAPKs, WiHog1A or WiHog1B, along with the WiPbs2 kinase. When grown on salt, only the cells expressing WiHog1B displayed salt tolerance similar to that of the wt yeast cells, suggesting that most probably WiHog1B, but not WiHog1A, is the primary HOG pathway kinase also in *W. ichthyophaga*. The *pbs2Δste11Δ* cells, where MAPKKK Ssk2/22 is the only activator of WiPbs2, tolerated salt like the wt yeast, but in the *pbs2Δssk2/22Δsho1Δ* cells the salt-tolerance of the wt yeast cells could not be reached with the expression of WiPbs2 and WiSho1. In this strain, we only recovered the salt-tolerance by inserting the *S. cerevisiae* SH3-binding proline-rich motif to the N-terminal part of WiPbs2 kinase. Additionally, the polymyxin B sensitivity of the WiPbs2 expressing cells suggests, that the Ssk2/22 kinase of the SLN1 branch in the HOG pathway is the primary WiPbs2 activating MAPKKK also in *W. ichthyophaga*.
THE PROTEIN O-MANNOSYLTRANSFERASE PMT4 IS CRUCIAL FOR CELL WALL INTEGRITY AND STRESS RESISTANCE OF *Hansenula polymorpha*

<u>Hyunah Kim</u><fnref n="1" />, Dong-jik Lee<fnref n="1" />, Hye Yun Moon<fnref n="1" />, Michael O. Agaphonov<fnref n="2" />, Hyun Ah Kang<fnref n="1" />

Protein O-mannosyltransferase (PMT) initiates the addition of mannose residue to serine or threonine residue of secretory proteins, which is an important post-translational modifications evolutionarily conserved from bacteria to human. PMTs are significant differences in their organization and properties, reflecting the diversity of O-mannosylation machineries. In this study we carried out functional and molecular characterization of the *PMT4* gene in the thermotolerant methylotrophic yeast *Hansenula polymorpha*. Deletion of *HpPMT4* did not affect thermotolerance but resulted in increased sensitivity to several cell wall destabilizers, and various salt stresses. Notably, the analysis of cell wall mannoproteins with lectin blotting indicated significant decrease in O-mannosylation in the *Hppmt4* mutant. Furthermore, just single deletion of *HpPMT4* generated severe glycosylation defects of the surface sensor proteins HpWsc1p and HpMid2p without alteration of the secreted glycoproteins, chitinase and HpYps1p lacking GPI anchor. Interestingly, the basal level of the phosphorylated HpMpk1p, a key kinase in the cell wall integrity pathway, was markedly reduced in the *Hppmt4* strain, although the phosphorylation of HpMpk1p was also increased to a high extent in the *Hppmt4* mutant upon treatment of cell wall disturbing agents. Altogether, our results indicate that Pmt4p plays important roles particularly in O-mannosylation of surface glycoproteins involved in signal pathways for cell wall integrity and stress resistance in *H. polymorpha*.

<footnote n="1">¹ Department of Life Science, Chung-Ang University, Seoul 156-756, Korea;</footnote>

<footnote n="2">² A.N.Bach Institute of Biochemistry of the Russian Academy of Sciences, Moscow, Russia
Contact e-mail: Hyunkang@cau.ac.kr</footnote>
*In vitro* ANALYSES OF PHR1 AND PHR2 TRANSGLYCOSYLASES OF THE *Candida albicans* CELL WALL

Kováčová K.¹, Farkaš V.¹ and Popolo, L.²

¹Institute of Chemistry, Slovak Academy of Sciences, 84538 Bratislava, Slovakia,

²Università degli Studi di Milano, Dipartimento di Bioscienze, Via Celoria 26, 20133 Milano, Italy

Contact email: kristina.kovacova@savba.sk

The yeast cell wall is an essential component that defines cell shape, preserves the cell's osmotic integrity, is involved in flocculation, sporulation, mediates cell-cell interactions and plays a crucial role in host - pathogen interaction and in virulence. The individual structural components of the polysaccharide polymers of the *Candida albicans* cell wall, β-(1,3)-glucan, β-(1,6)-glucan and chitin, are mutually cross-linked by covalent bonds into large macromolecular complexes based on β-(1,3)-glucan backbone. We are focused on transglycosylases, specifically to pH-regulated enzymes Phr1 and Phr2 from *Candida albicans*. Phr1 and Phr2 proteins belong to β-(1,3)-glucanosyltransferases (family GH72) which catalyze the linking of part of β-(1,3)-glucan to another molecule of β-(1,3)-glucan.

In this part of our research, the biochemical characterization and determination of substrate specificity of Phr1 and Phr2 transglycosylases heterologously expressed in *Pichia pastoris* were found out. A fluorescence *in vitro* assay was used for biochemical characterization of Phr1p and Phr2p. The pH optimum was 5,6 for Phr1 and 3 for Phr2. The optimum temperature was 30°C for both. The transglycosylation activities of Phr1p and Phr2p were determined by a fluorescent *in vitro* assay and size-exclusion chromatography (SEC). Laminarin was used as the donor and the oligosaccharides labeled by sulforhodamine (SR) as the artificial acceptors. The wide spectrum of (SR)-oligosaccharides (OS-SR) was tested as acceptors: laminari-OS-SR; N-acetyl-chito-OS-SR, cello-OS-SR and SR-oligosaccharides derived from xyloglucan, mixed-linkage β-(1,3/1,4)-glucan; β-(1,6)-glucan (pustulan), β-(1,4)-linked mannan and α-(1,4)-glucan (starch). Results based on relative rates of transglycosylation and elution profile of the reaction mixtures measured by SEC have shown that Phr1 and Phr2 are specific homotransglycosylases catalyzing transfer from β-(1,3)-linked laminarin to β-(1,3)-linked laminarioligosaccharides. The rate of transglycosylation mediated by Phr2 was higher than by Phr1. The products of the transglycosylation reactions were identified by MALDI-TOF mass spectrometry as the molecules composed of the acceptor and portions of the donor molecule attached to its non-reducing end.

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.
METHYLOTROPHIC YEAST *Hansenula polymorpha* AS A NOVEL TOOL FOR STUDYING MECHANISMS UNDERLYING PARKINSON’S DISEASE

Iryna O. Denega<fnref n="1" />, <fnref n="2" />, Natalia O. Sybirna<fnref n="1" />, Olena G. Stasyk<fnref n="1" />, <fnref n="2" />

Contact e-mail: deneha.ira@gmail.com

Several features of Parkinson’s disease (PD) have been recreated in yeasts (e.g. formation of protein aggregates, cellular toxicity mediated by misfolded proteins), enabling researchers to use them as a powerful artificial model for investigation of molecular mechanisms of PD. We suggest expanding a small number of model yeast species using a methylotrophic yeast *Hansenula polymorpha*. This yeast as a new model of PD has several advantages, including its extreme thermotolerance with a consequent ability to grow at temperatures up to 45 °C. This allows to study PD-related mechanisms at physiological for both human organism and this yeast temperature, 37 °C, and also the general influence of the temperature on the amyloid formation. We have created novel yeast models of PD with regulated expression of α-synuclein gene (*SNCA*) fused with the gene of green fluorescent protein. These models are based on *H. polymorpha* wild type strain NCYC495 and mutant strain *gcr1-2* defective in glucose transport and metabolism. Epidemiological studies have identified diabetes mellitus as an independent risk factor for multiple diseases of the nervous system including PD. Combination of defective glucose metabolism and α-synuclein overproduction in single strain will help us to understand relationship between Parkinson's disease and abnormal sugar metabolism. Heterologous α-syn production was detrimental for yeast cells and decreased their growth rate on alternative carbon sources. We observed that NCYC495-*SNCA* strain did not form visible α-syn aggregates but exhibited plasma membrane perforations and cytoplasm leakage which are most probably caused by α-syn oligomers. *gcr1-2-SNCA* mutant strain exhibited enhanced aggregation of fluorescently tagged α-syn. However, such accelerated amyloid formation is not a direct consequence of the impaired glucose metabolism as were also observed in cells grown on glycerol. The question of what molecular mechanisms evoked by *gcr1* mutation result in α-syn aggregation will be addressed in our prospective studies.

<footnote n="1">¹ Ivan Franko National University of Lviv, 4, Hrushevskyi St., Lviv 79005, Ukraine</footnote>

<footnote n="2">² Institute of Cell Biology NAS of Ukraine, 14–16, Drahomanov St., Lviv 79005, Ukraine</footnote>
NON-CONVENTIONAL YEASTS FOR BIOFUEL PRODUCTION

Volkmar Passoth

Swedish University of Agricultural Sciences, Department of Microbiology, Box 7025, SE-75007 Uppsala, Sweden

Contact email: volkmar.passoth@slu.se

Production of biofuels is a complex technology of handling and converting biomass. Yeasts represent a highly diverse group of organisms with an amazing metabolic potential. We have tested several non-*Saccharomyces* yeasts in biofuel production and as biopreservation and pre-treatment agents for both first generation raw materials and lignocellulose. The yeast, *Dekkera bruxellensis*, had outcompeted *Saccharomyces cerevisiae* in an industrial ethanol process, by this challenging the dogma that *S. cerevisiae* is always the most appropriate species for ethanol production. *Scheffersomyces (Pichia) stipitis* is the best known xylose-fermenting organism. Recently we discovered that the yeast, when used as biopreservative in airtight storage of moist wheat straw, inhibits moulds and at the same time improves the accessibility of polysaccharides to pre-treatment. Beyond ethanol production, the production of lipids as components of biodiesel or- as omega 3 fatty acids as feed- and food additives is a novel approach of using yeasts. Certain oleaginous species can accumulate more than 60% of their biomass as lipids, and some form substantial amounts of essential fatty acids (linoleic and linolenic acid). Apart from this, many of them can naturally assimilate sugars released from hemicellulose, which cannot be converted to ethanol by *S. cerevisiae* or *D. bruxellensis*. Utilising the metabolic diversity of non-conventional yeasts and integrating different steps of biofuel- and chemical production can push the development towards a biorefinery that is sustainable and competitive against the traditional, fossil based production systems.
CONSTRUCTION OF THE IMPROVED ETHANOL PRODUCERS FROM XYLOSE IN
THE THERMOTOLERANT YEAST Hansenula polymorpha

Olena Kurylenko<fnref n="1" />, Justyna Ruchala<fnref n="2" />, Orest Hryniv<fnref n="1" />, Oleh Stasyk<fnref n="1" />, Kostyantyn Dmytruk<fnref n="1" />, Charles Abbas<fnref n="3" />, Andriy Sibirny<fnref n="1" />,<fnref n="2" />

*Hansenula polymorpha* belongs to the best studied non-conventional yeasts and is apparently the most thermotolerant yeast known with maximal growth temperature of 50 °C. It was also shown that this yeast is capable of xylose alcoholic fermentation though ethanol yield and productivity are low. Using methods of classical selection and metabolic engineering, the strains of *H. polymorpha* have been constructed which accumulate elevated amounts of ethanol from xylose, up to 10-12 g/L. For this, overexpression of the modified gene *XYL1m* (encoding xylose reductase) and the native genes *XYL2* (xylitol dehydrogenase), *XYL3* (xylulokinase) and *PDC1* (pyruvate decarboxylase) and, additionally, the isolation of 3-bromopyruvate-resistant mutants from metabolically engineered strains have been conducted. Additionally, the gene *CAT8* involved in regulation of gluconeogenesis has been deleted and role of this defect on xylose fermentation was studied. It was unexpectedly found by us that the *pex* mutants of the yeast *Hansenula polymorpha* which do not contain peroxisomes poorly grow on and do not ferment xylose. The mutants of this yeast deleted in genes *DAS1* and *TAL2* coding for peroxisomal dihydroxyacetone synthase and transketolase, respectively, show normal growth in xylose medium though fermentation of this sugar is severely damaged. Inversely, overexpression of *DAS1* and *TAL2* resulted in activation of xylose alcoholic fermentation whereas overexpression of cytosolic genes of pentose phosphate pathway *TKT1*, *TAL1*, *RPI1* and *RPE1* had only slight effect on this process. Derepression of *DAS1* and *TAL2* in the best ethanol producing strain further increased ethanol yield and productivity. Defect in peroxisomes autophagic degradation (pexophagy) due to insertion or deletion of the gene *ATG13* activated xylose alcoholic fermentation. All mentioned genetic changes have no effect on glucose alcoholic fermentation. Further improvement of high-temperature xylose alcoholic fermentation in *H. polymorpha* is discussed.

<footnote n="1">¹ Department of Molecular Genetics and Biotechnology, Institute of Cell Biology, NAS of Ukraine, Drahomanov Street, 14/16, Lviv 79005 Ukraine</footnote>

<footnote n="2">² Department of Biotechnology and Microbiology, University of Rzeszow, Zelwerowicza 4, Rzeszow 35-601, Poland</footnote>

<footnote n="3">³ Archer Daniels Midland Company, 1001 Brush College Road, 62521 Decatur, IL, USA
Contact email: sibirny@cellbiol.lviv.ua</footnote>
THE BIOCONTROL YEAST *Wickerhamomyces anomalus* IN MAIZE
BIOPRESERVATION

Albina Bakeeva<fnref n="1" />, Su-lin L. Leong<fnref n="1" />, Aziwo T. Niba<fnref n="2" />, Matilda Olstorpe<fnref n="1" />

Contact email: albina.bakeeva@slu.se

Preservation of moist crimped grain (barley, wheat) is made possible through fermentation by lactic acid bacteria (LAB) in an airtight environment, in a process similar to silage production. This yields a hygienic product with reduced levels of potentially pathogenic bacteria and spoilage moulds. Additional of the biocontrol yeast *Wickerhamomyces anomalus* (syn. *Hansenula anomala*, formerly *Pichia anomala*) further inhibits mould growth by production of ethyl acetate; this minimizes the risk for mycotoxin production. The biocontrol strain has also been shown to reduce *Enterobacteriaceae* by an unknown mechanism (1). Biocontrol with *W. anomalus* was tested for the storage of moist harvested maize in Cameroon, where key microbiota of maize kernels had been characterized earlier (2). The white maize cultivar 'Kasai' was harvested and stored in airtight plastic barrels, with and without inoculation of *W. anomalus*. Microbes were enumerated at harvest and after 2, 5 and 8 months of storage; nutritional contents were compared before and after storage. High levels of LAB naturally present on maize (10⁸ cfu/g) were maintained throughout storage. Activity of LAB in both control and inoculated maize likely contributed to the decline in *Enterobacteriaceae* in both treatments to < 10 cfu/g after 2 months storage. The biocontrol yeast, *W. anomalus*, was not found in the inoculated treatment after 2 months, but a positive effect was seen in significantly reduced mould counts to < 100 cfu/g compared with the uninoculated maize. *W. anomalus* has previously been shown to improve protein and amino acid contents during storage of moist crimped grain. This was not observed for white maize, probably, due to the poor survival of the biocontrol yeast. Preliminary results suggested that *W. anomalus* survives better on the yellow ATP (acid-tolerant population) maize cultivar from Cameroon than on the white 'Kasai' cultivar; survival of the biocontrol yeast may be affected by cultivar.

1. Olstorpe M & Passoth V (2011): *Pichia anomala* in grain biopreservation. *Antonie van Leeuwenhoek* 99:57–62.

2. Leong SL, Niba AT, Ny S, Olstorpe M (2012): Microbial populations during maize storage in Cameroon. *Afr J Biotechnol* 11:8692-8697.

<footnote n="1">¹ Department of Microbiology, BioCenter, SLU, Box 7025, 75007 Uppsala, Sweden</footnote>

<footnote n="2">² College of Technology, University of Bamenda, Cameroon</footnote>
*Dekkera bruxellensis* COMPETITIVE NON-CONVENTIONAL ETHANOL PRODUCTION YEAST

Ievgeniia Tiukova<fnref n="1" />, Will de Baros Pita<fnref n="2" />, Marcos Antonio de Morais<fnref n="2" />,<fnref n="3" />, Volkmar Passoth<fnref n="1" />

Contact mail: Ievgeniia.Tiukova@slu.se

The outcompetition of an initial *Saccharomyces cerevisiae* inoculum by *Dekkera bruxellensis* has been reported in several ethanol production plants, while process efficiency has remained high. In most cases the co-occurrence of *D. bruxellensis* together with *Lactobacillus vini* has been observed. The outcompetition phenomenon was observed exclusively under fermentation conditions with glucose-limited continuous cultivation. Research was done to understand different aspects of *D. bruxellensis* competitiveness, recently reconsidered as essential trait for a production strain. Possible reasons for the competitive advantage of *D. bruxellensis* such as glucose uptake and metabolism, nitrate assimilation and interaction with *L. vini* were investigated. Global gene expression analysis allowed identifying genes responsible for efficient carbon metabolism of *D. bruxellensis*. Nitrate has been shown to be an important component of fermentation broth in Brazilian ethanol production plants. Ability to assimilate nitrate allows *D. bruxellensis* to utilize it as sole nitrogen source when ammonium is depleted. Regulation of genes involved in nitrate assimilation was investigated. Pseudomycelium structure and cell wall composition of *D. bruxellensis* were shown to be involved in the interaction with *L. vini*.

<footnote n="1">¹ Uppsala Biocenter, Department of Microbiology, Swedish University of Agricultural Sciences, Box 7025, 750 07 Uppsala, Sweden</footnote>

<footnote n="2">²Interdepartmental Research Group in Metabolic Engineering, Federal University of Pernambuco, Recife, PE, Brazil</footnote>

<footnote n="3">³Department of Genetics, Federal University of Pernambuco, Recife, PE, Brazil</footnote>
DELETION OF *ADH1* GENE, ENCODING ALCOHOL DEHYDROGENASE FOR THE IMPROVEMENT OF GLYCEROL PRODUCTION IN *Saccharomyces cerevisiae*

Iryna Salii¹, Marta Semkiv¹, Kostyantyn Dmytruk¹, Andriy Sibirny¹,²

Department of Molecular Genetics and Biotechnology, Institute of Cell Biology, NAS of Ukraine, Drahomanov Street, 14/16, Lviv 79005 Ukraine;
e-mail:saliirka@gmail.com

Glycerol is widely used in cosmetics, food, tobacco, pharmaceutical, leather and textile industries. Glycerol is one the dominant by-product of glucose conversion during alcoholic fermentation by *S. cerevisiae*. In addition, it is considered as a cheap raw material for microbial fermentation. That is why the construction of yeast strains-producers of glycerol became an actual objective for modern metabolic engineering.

Our strategy comprises the deletion of *ADH1* gene, encoding alcohol dehydrogenase in *S. cerevisiae* in order to redirect the glycolytic pathway to a glycerol synthesis. Glycerol is synthesized in a two reaction process, consisting of the reduction of dihydroxyacetone phosphate by a NADH-dependent glycerol-3-phosphate dehydrogenase (GPD), followed by the dephosphorylation of glycerol-3-phosphate by a glycerol-3-phosphate phosphatase (GPP).

Deletion of *ADH1* gene will eliminate the alcohol dehydrogenase reaction during glycolysis and lead to the accumulation of NADH, which will further be used in glycerol-3-phosphate dehydrogenase reaction.

The deletion cassette pUC57_ADHpr_NTC_ADHterm, including terminal and promoter part of *ADH1* gene and *natNT2* gene was constructed. Plasmid pUC57_ADH1term_NTC was introduced into *S. cerevisiae* strain BY4742. As a result, several recombinants were obtained. The correct disruption of *ADH1* gene was confirmed by analytical PCR.

The specific activity of alcohol dehydrogenase in recombinant strains was decreased in comparison with wild type strain. The ethanol productivity was also significantly decreased in recombinant strains compared with wild type strain BY4742. A glycerol production was considerably enhanced in transformants if to compare with the wild type strain. Recombinant strains consumed less amounts of glucose than the wild type strain. An ethanol production was essentially reduced in transformants in comparison with the wild type strain.
METABOLIC ENGINEERING OF *Hansenula polymorpha* FOR IMPROVING THE
CONVERSION OF GLYCEROL TO ETHANOL

Iwona Kata<fnref n="1" />, Justyna Ruchała<fnref n="1" />, Olena Kurylenko<fnref n="2" />, Kostyantyn Dmytruk<fnref n="2" />, Andriy Sibirny<fnref n="1" />,<fnref n="2" />

Email address: kataiwona@tlen.pl

Decreasing fossil fuels, drastic increase in prices, global warming and environmental pollution caused that development of alternative renewable fuels, such as biodiesel, has become attractive. The increase in biodiesel production generates an excess of crude glycerol since this compound is a significant waste by-product accumulating directly from the trans-esterification of the plant oil. Glucose fermentation is efficiently carried out by microorganisms, at the same time; there are few microorganisms that are capable of converting glycerol to ethanol. Therefore, the tools of metabolic engineering along with classical selection should be used for construction of the recombinant strains with improved ethanol production.

Thermotolerant methylotrophic yeast *Hansenula polymorpha* is able to convert glycerol as well as glucose and xylose, to ethanol. However, the yield of ethanol from glycerol by wild-type strains is too low and needs to be substantially improved before introduction of those strains for production of ethanol on the industrial scale.

The aim of this work was to improve production of ethanol from glycerol using thermotolerant methylotrophic yeast *Hansenula polymorpha*. For this, vectors for multicopy integration of the overexpressed *PDC1* and *ADH1* genes under control of strong promoters have been constructed.

According to previous data, overexpression of a pyruvate decarboxylase gene (PDC1) improved the production of ethanol from glycerol. Compared to the wild type strain, overexpression of PDC1 led to threefold increase of ethanol production. In addition, recent data show that overexpression of ADH1 gene leads to production of higher amounts of ethanol. Therefore we suggest that overexpression of both PDC1 and ADH1 genes will further increase the yield of ethanol from by-product glycerol. The newest data of our experiments will be provided and discussed.

<footnote n="1">¹ Department of Biotechnology and Microbiology, University of Rzeszow, Zelwerowicza 4,
35-601 Rzeszow, Poland;</footnote>

<footnote n="2">² Institute of Cell Biology, National Academy of Science of Ukraine, Drahomanov Street 14/16, 79005 Lviv, Ukraine.</footnote>
ETHANOL AND BIOGAS FROM STEAM EXPLODED ISP (INTEGRATED STORAGE AND PRE-TREATMENT) SAWDUST INOCULATED WITH *Arxula adeninivorans*

Johanna Blomqvist, Albina Bakeeva, Volkmar Passoth

Department of Microbiology, BioCenter, SLU, Box 7025, 75007 Uppsala, Sweden

E-mail: johanna.blomqvist@slu.se

To decrease the fossil fuel dependency and greenhouse gas emissions biofuels should be produced from second generation (lignocellulosic) biomass, such as forest residues. The lignocellulosic biomass needs to be pretreated, usually thermochemically, before enzymatic hydrolysis and fermentation for the production of biofuel. We have developed a method called integrated storage and pretreatment (ISP) where a biopreservation yeast is inoculated to the biomass prior storage. Ideally the yeast will both act as a biocontrol agent, inhibiting spoilage microorganisms, and start limited biomass degradation. As a result, a less harsh pretreatment is necessary leading to lower levels of inhibitors and lower energy requirements for thermochemical pretreatment (1). This has successfully been demonstrated on wheat straw but not on spruce sawdust, since the yeasts did not survive the storage (2). In this project we inoculated *Arxula adeninivorans* to birch sawdust and stored it for 6 weeks at 37°C. A. *adeninivorans* is a heat tolerant yeast that also has the capacity to degrade aromatic compounds. The storage flora of the sawdust was determined and the sawdust was pretreated using steam explosion. Then SSF (simultaneous saccharification and fermentation) with *Saccharomyces cerevisiae* as the fermentation yeast was performed followed by biogas digestion of the residue. Biogas digestion of steam exploded sawdust without prior ethanol fermentation was also performed. Preliminary results showed that A. *adeninivorans* did not have any biocontrol activity. The results from the microbial analyses and SSF and biogas digestion will be presented.

1. Passoth V, Tabassum MR, Nair HAR, Olstorpe M, Tiukova I, Stahlberg J (2013): Enhanced ethanol production from wheat straw by integrated storage and pre-treatment (ISP). Enz Microb Technol 52-105-110

2. Blomqvist J, Leong, SL, Sandgren M, Lestander T and Passoth V (2014): Temperature dependent changes of the microbial storage flora of birch and spruce sawdust. Biotechnol Appl Biochem 61:58-64
BIO-LIPID PRODUCTION FROM LIGNOCELLULOSES MATERIAL WITH
OLEAGINOUS YEASTS

Jule Brandenburg<fnref n="1" />, Johanna Blomqvist<fnref n="1" />, Jana Pickova<fnref n="2" />, Volkmar Passoth<fnref n="1" />

E-mail: jule.brandenburg@slu.se

Considering the limitation of fossil raw materials utilization of renewable resources for fuel and chemicals production is required. Biofuel production should be sustainable and not compete with food and feed production. Lignocellulose is the most abundant biomass resource on earth and its hydrolysates are commonly esteemed as promising substrates for biofuel production.

*Lipomyces starkeyi* and *Rhodotorula glutinis* are oleaginous yeasts, able to convert carbon sources present in lignocellulose to lipids, up to more than fifty percent of their biomass. We have tested lipid accumulation by these two yeasts when growing on birch hydrolysate. Using a fed batch strategy we obtained lipid contents of the biomass of more than 40%. The proportion of non-saturated fatty acids varied considerable between both species, with higher amounts of polyunsaturated fatty acids in *R. glutinis*, making this yeast apart from biodiesel production also to a good candidate for generating essential fatty acids for feed and food. However, commonly used strategies of acid lipid extraction may generate artifacts regarding the relation of saturated and non-saturated fatty acids. The impact of different lipid extraction methods on determined fatty acid composition and total lipid content are discussed.

<footnote n="1">¹ Department of Microbiology, BioCenter, SLU, Box 7025, 75007 Uppsala, Sweden</footnote>

<footnote n="2">² Department of Food Science, BioCenter, SLU, Box 7051, 75007 Uppsala, Sweden</footnote>
CONSTRUCTION OF CAT8 GENE DELETION STRAINS IN THE YEAST *Hansenula polymorpha*

Justyna Ruchala<fnref n="1" />, Iwona Kata<fnref n="1" />, Olena Kurylenko<fnref n="2" />, Konstyantyn Dmytruk<fnref n="2" />, Andriy Sibirny<fnref n="1" />,<fnref n="2" />

Contact address: justynar87@gmail.com

Efficient alcoholic fermentation of hexose and pentose sugars by yeast is essential to achieve maximal ethanol yield from lignocelluloses hydrolyzates. The yeast *Hansenula polymorpha* is capable of xylose alcoholic fermentation at elevated temperatures and therefore can be used in Simultaneous Saccharification and Fermentation Process (SSF).

The aim of this work was to isolate mutants of *H. polymorpha* with knock out of the gene *CAT8* which is known to be involved in regulation of gluconeogenesis in *S. cerevisiae* and regulation of alcoholic fermentation in the non-conventional yeast *Pichia guilliermondii*. We did hope that the mutants *H. polymorpha* defected in *CAT8* will be more efficient in ethanol production from glucose and especially from xylose. Such deletion strains were planned to be constructed in both *H. polymorpha* wild-type strain and in the previously isolated by us mutants with elevated ethanol production from xylose due to overexpression the genes encoded the key enzymes involved in xylose metabolism (xylose reductase, xylitol dehydrogenase and xylulokinase) and subsequently selected on resistance to 3-bromopyruvic acid (glycolysis inhibitor).

It is known that *CAT8* encodes carbon source-responsive transcriptional regulators that cooperatively control expression of genes involved in ethanol utilization. Near 200 genes involved in central metabolism pathways as well as several transcriptional factors are activated by *CAT8*. Deletion of *CAT8* gene affected all key gluconeogenic enzymes by defect in glucose derepression, glucose metabolism is systematically shifted in the direction of the fermentation at the transcriptional level.

Currently, the cassettes for *CAT8* gene deletion in wild-type strain and ethanol overproducing mutants have been constructed and the corresponding mutant strains have been isolated. The studying the glucose and xylose alcoholic fermentation in the constructed knock out strains is under way.

<footnote n="1">¹ Department of Biotechnology and Microbiology, University of Rzeszow, Zelwerowicza 4, 35-601 Rzeszow, Poland</footnote>

<footnote n="2">² Institute of Cell Biology, National Academy of Science of Ukraine, Drahomanov Street 14/16, 79005 Lviv, Ukraine</footnote>
INSERTIONAL TAGGING OF THE *Pichia stipitis* GENES INVOLVED IN XYLOSE ALCOHOLIC FERMENTATION

Krzysztof Berezka<fnref n="1" />, Johanna Blomqvist<fnref n="2" />, Kostyantyn Dmytruk<fnref n="3" />, Orest Hryniv<fnref n="3" />, Tomas Linder<fnref n="2" />, Volkmar Passoth<fnref n="2" />, Andriy Sibirny<fnref n="1" />,<fnref n="3" />

Contact email: kberezka@gmail.com

Among several exhaustively studied microorganism, the yeast *Pichia stipitis* seems to be particularly promising in terms of xylose alcoholic fermentation, the pentose which is the major constituent of lignocellulosic biomass hydrolyzates. Nevertheless, the ethanol yield and productivity are still insufficient to meet the requirements of a feasible industrial technology; hence the construction of the more effective *P. stipitis* ethanol producers is of great significance. Our aim was to isolate the insertional mutants with altered ethanol production from xylose and determine gene(s) affecting their fermentative capabilities. Mutants were obtained by random insertional mutagenesis, then screened for the altered growth abilities on solid media with different sugars and for their resistance to 3-bromopyruvate (3-BP). Shaking cultures in semianaerobic conditions were carried out in order to measure sugar consumption and ethanol formation rates. Subsequently, the most interesting strains were analyzed to determine the genetic background of the observed alterations.

Of more than 13,300 screened mutants, only 17 were characterized with significantly changed ethanol yields during the fermentation. More than 70% of the strains resistant to 3-BP exhibited improved fermentative capabilities. In the case of the two best fermenting strains, insertion and the corresponding structure disruption, which presumably led to the enhancement of ethanol formation, occurred within the ORFs of two genes: coding for calcium-binding protein and functionally unknown, not yet described mitochondrial transporter. Additional experiments aimed to prove the role of the mentioned genes in regulation of xylose alcoholic fermentation are in progress.

<footnote n="1">¹ Department of Biotechnology and Microbiology, University of Rzeszow, Zelwerowicza 4, Rzeszow 35-601, Poland,</footnote>

<footnote n="2">² Department of Microbiology, Uppsala BioCentrum, Swedish University of Agricultural Sciences, Box 7025, SE-750 07, Uppsala, Sweden,</footnote>

<footnote n="3">³ Department of Molecular Genetics and Biotechnology, Institute of Cell Biology, NAS of Ukraine, Drahomanov Street, 14/16, Lviv 79005 Ukraine.</footnote>
CONSTRUCTION OF GLUTATHIONE OVERPRODUCERS IN METHYLOTROPHIC YEAST *Hansenula polymorpha* BY ENGINEERING OF γ-GLUTAMYLCYSTEINE SYNTHETASE

<u>Marianna Yurkiv</u><fnref n="1" />, Olena Kurylenko<fnref n="1" />, Kostyantyn Dmytruk<fnref n="1" /> and Andriy Sibirny<fnref n="1" />,<fnref n="2" />

Contact email: mariannayurkiv1@gmail.com

Tripeptide glutathione (gamma-glutamyl-L-cysteinyl-glycine, GSH) is the most abundant non-protein thiol that protects cells from metabolic and oxidative stresses and is widely used as food additives, in medicine and cosmetic industry. Although GSH can be produced by chemical or enzymatic synthesis, microbiological production using natural or engineered microorganisms is currently the most common method for the commercial production of GSH. Synthesis of GSH includes two consecutive reactions catalyzed by gamma-glutamylcysteine synthetase (GCS) and glutathione synthetase, respectively. Microbial GSH overproduction is limited by mechanisms of feedback inhibition of GCS, the first and rate-limiting enzyme of GSH biosynthesis, by the end product. In addition the expression of gene coding for GCS is repressed by GSH.Thermotolerant methylotrophic yeast *H. polymorpha* with naturally high content of GSH and resistance to different kinds of stress is considered as promising organism for genetic modification and design of competitive GSH producer.The modified versions of *GSH2* gene, coding for GCS in *H. polymorpha*, obtained by error prone PCR were cloned under the control of strong constitutive promoter of glyceraldehyde-3-phosphate dehydrogenase in replicative plasmid pYT3. The selection of Gsh2 mutant forms was done on the basis of deletion strain Δ*gsh2 H. polymorpha*, dependent on exogenous GSH. Transformants with modified versions of *GSH2* gene were obtained on medium without GSH. Selected transformants were analyzed for their resistance to different prooxidant agents (1,2,3-triazole, diethylmaleate, ethionine) as compared to strains carrying unmodified *GSH2* gene. Selected strains possessed growth on medium supplemented with triazole. This compound is able to chemical interaction with GSH reducing intracellular concentration of the tripeptide. Strains providing more intensive growth on the selective medium revealed higher GSH accumulation as compared to strains carrying unmodified *GSH2* gene, indicating the reduction of Gsh2 feedback inhibition. Identification of the mutations in frame of modified alleles of *GSH2* gene is under the progress.

<footnote n="1">¹Department of Molecular Genetics and Biotechnology, Institute of Cell Biology, NAS of Ukraine, Drahomanov Street, 14/16, Lviv 79005, Ukraine</footnote>

<footnote n="2">²Department of Biotechnology and Microbiology, University of Rzeszow, Zelwerowicza 4, Rzeszow 35-601, Poland</footnote>
METABOLIC ENGINEERING OF YEAST *Saccharomyces cerevisiae* FOR REROUTING OF CARBON FLUX TOWARDS GLYCEROL PRODUCTION

Marta Semkiv<fnref n="1" />, Kostyantyn Dmytruk<fnref n="1" />, Andriy Sibirny<fnref n="1" />,<fnref n="2" />

Contact email: smarta0309@gmail.com

Glycerol is used in cosmetic, paint, automotive, food, tobacco, pharmaceutical industries. Currently there is an interest in development of microbial or yeast strains effectively converting cheap feedstocks to glycerol. Facultative anaerobic yeast *Saccharomyces cerevisiae* can be a good platform for development of recombinant strains overproducing glycerol under low-aeration conditions. In *S. cerevisiae* glycerol synthesis from dihydroxyacetone phosphate is catalyzed by glycerol-3-phosphate dehydrogenase (Gpd1) and glycerol-3-phosphate phosphatase (Gpp2). But, in physiological conditions the major part of intracellular dihydroxyacetone phosphate is converted to glyceraldehyde-3-phosphate by triose phosphate isomerase (Tpi1), resulting in ethanol formation. We aimed to construct recombinant strains with reduced Tpi1 activity and increased Gpd1 and Gpp2 activities to redirect Carbon flux towards glycerol production. In order to decrease *TPI1* gene expression recombinant *S. cerevisiae* strains with shortened versions of *TPI1* gene promoter were constructed. Obtained strains contain 100, 50 or 25 base pairs of native *TPI1* gene promoter before *TPI1* ORF, and revealed corresponding sequential decreases in Tpi1 activity. Glycerol production by strains with 100 bp version of *TPI1* promoter wasn't substantially higher than that by WT strain, whereas strains with 50 or 25 bp version of *TPI1* promoter revealed up to 2 times increase in glycerol production in comparison with WT strain. In order to enhance the activities of enzymes involved in glycerol synthesis, we transformed *S. cerevisiae* with vectors containing genes *GPD1*, or *GPP2*, or hybrid *GPD1-GPP2* ORF (encoding artificial fusion of both enzymes) under the control of strong constitutive promoter of the alcohol dehydrogenase gene (*ADH1*). Recombinant strains overexpressing *GPP2* gene possessed similar glycerol titer to WT strain. Recombinant strains overexpressing *GPD1* gene or *GPD1-GPP2* fusion showed diverse increase in glycerol production, probably depending on gene copies number. Glycerol production reached 4 folds increase in the best of studied strain as compared to WT.

<footnote n="1">¹Department of Molecular Genetics and Biotechnology, Institute of Cell Biology, NAS of Ukraine, Drahomanov Street, 14/16, Lviv 79005 Ukraine;</footnote>

<footnote n="2">²Department of Biotechnology and Microbiology, University of Rzeszow, Zelwerowicza 4, Rzeszow 35-601 Poland</footnote>
THE ROLE OF TRANSCRIPTION FACTOR Sef1 IN THE REGULATION OF
FLAVINOGENESIS IN YEAST *Candida famata*

Oleksii Lyzak<fnref n="1" />, Kostyantyn Dmytruk<fnref n="1" />, Andriy Sibirny<fnref n="1" />, <fnref n="2" />

Contact email: sibirny@cellbiol.lviv.ua

Riboflavin (vitamin B2) is one of the most important vitamins required for human and animals. This vitamin is metabolic precursor of flavin nucleotides, FMN (flavin mononucleotide) and FAD (flavin adenine dinucleotide) involved as coenzymes in numerous enzymatic reactions, mostly of oxidative metabolism. Riboflavin deficiency causes retardation of growth, skin, nervous system and eye diseases. Although the riboflavin biosynthesis pathway is well studied it regulation is not known well. Only one regulatory gene SEF1 was found in flavinogenic yeast C. famata so far. The SEF1 is involved in positive regulation in riboflavin synthesis as additional copies of the gene leads to improvement of riboflavin production in C. famata. One-hybrid system on base of *Saccharomyces cerevisiae* was used to identify DNA binding sites of Sef1. The strains BY4742 of S. cerevisiae with plasmid harboring SEF1 gene of C. famata under the control of galactose-inducible GAL1 promoter of S. cerevisiae and reporter plasmid bearing LAC4 gene of *Kluyveromyces lactis* under control of RIB1 and SEF1 promoters of C. famata were constructed. Shortened sequences of promoter RIB1 lacking hypothetical Sef1 binding sites was used. β-galactosidase assay was used for detection of the Sef1 binding capacity of the target sequences. To study the expression of SEF1 gene the chimeric genes were constructed. For this purpose, SEF1 gene was fused with Myc-tag or GFP sequence on 3'- or 5'- end. Obtained sequences were cloned under the control of own or TEF1 promoters. Constructed plasmids were used for transformation of C. famata Δsef1 strain to provide complementation test. The study of the constructed strains is under the progress.

<footnote n="1">¹ Institute of Cell Biology, NAS of Ukraine, Drahomanov Street 14/16, Lviv, 79005, Ukraine</footnote>

<footnote n="2">² Rzeszów University, Ćwiklińskiej 2, Rzeszów 35-601, Poland</footnote>
METABOLIC ENGINEERING OF THE METHYLOTROPHIC YEAST *Hansenula polymorpha*
FOR CONSTRUCTION OF THE HIGH-TEMPERATURE ETHANOL PRODUCERS FROM
XYLOSE

Olena Kurylenko<fnref n="1" />, Justyna Ruchala<fnref n="2" />, Kostyantyn Dmytruk<fnref n="1" /> and Andriy Sibirny<fnref n="1" />,<fnref n="2" />

Contact email: olenkakurylenko@gmail.com

The nonconventional thermotolerant yeast *Hansenula polymorpha* naturally ferments xylose, the second most abundant sugar in lignocellulosic hydrolysates, to ethanol at elevated temperatures. However, the efficiency of this process using wild-type strains is low and not sufficient for industrial ethanol production. Several metabolic engineering approaches have been successfully developed to improve ethanol production from xylose in *H. polymorpha*. Overexpression of engineered xylose reductase with decreased affinity toward NADPH (*XYL1m*) together with native genes encoding xylitol dehydrogenase (*XYL2*) and xylulokinase (*XYL3*) led to an elevation in ethanol yield from xylose in the wild-type strains during xylose alcoholic fermentation at high temperatures (45-48°C). Also multicopy overexpression of *DAS1* and *TAL2* genes, encoding peroxisomal enzymes transketolase (dihydroxyacetone synthase) and transaldolase, respectively, had positive effect on ethanol production from xylose. In the current work both approaches were combined to improve ethanol production from xylose using earlier described *H. polymorpha* an ethanol-non-utilizing mutant (2EthOH⁻) as a host. First the transformants overexpressing modified *XYL1m* and native *XYL2* genes were isolated and characterized to possess 1.5-fold higher ethanol production from xylose as compared to the parental strain. The additional overexpression of *XYL3* gene resulted in further 2.3-fold improvement of ethanol production and completely reduced xylitol formation during xylose fermentation. The best ethanol producing strain 2EthOH⁻/*XYL1m*/*XYL2*/*XYL3* obtained by metabolic engineering approaches was subjected to selection for resistance to the known inhibitor of glycolysis, the anticancer drug 3-bromopyruvate. The best isolated mutant accumulated up near 10 g/l of ethanol from xylose at 45°C. Additional overexpression of *DAS1* and *TAL2* genes revealed further increase up to 30 % in ethanol production during alcoholic fermentation of xylose as compared to the parental strain.

<footnote n="1">¹Department of Molecular Genetics and Biotechnology, Institute of Cell Biology, NAS of Ukraine, Drahomanov Street, 14/16, Lviv 79005, Ukraine</footnote>

<footnote n="2">²Department of Biotechnology and Microbiology, University of Rzeszow, Zelwerowicza 4,
Rzeszow 35-601, Poland</footnote>
DISRUPTION OF *ATG13* GENE CAUSES THE INCREAS OF ETHANOL PRODUCTION DURING XYLOSE ALCOHOLIC FERMENTATION OF THE THERMOTOLERANT YEAST *Hansenula polymorpha*

Orest Hryniv<fnref n="1" />, Dorota Grabek-Lejko<fnref n="2" />, Kostyantyn Dmytruk<fnref n="1" />,
Barbara Kshanovska<fnref n="1" />, Andriy Sibirny<fnref n="1" />,<fnref n="2" />*

Contact email: orhryniv@gmail.com

Fuel ethanol production from the renewable raw material, such as plant biomass or lignocellulose, has a great economic and ecological significance. However, the production of fuel ethanol from non-starch, lignocellulosic materials is not developed yet. The main reason is the absence of robust microorganism capable of efficient alcoholic fermentation of all main sugars of lignocellulose, most importantly, xylose. Thermotolerant yeast *Hansenula polymorpha* is a promising organism which ferments glucose and xylose even at 48°C. But for cost-effective ethanol production, molecular mechanisms of regulation of xylose fermentation need to be studied and available strains need to be substantially improved.

Method of insertional mutagenesis, based on obtaining mutation by integrating a nucleotide sequence in the genome of recipient organism leading to alteration of gene expression, was used for identification of genes involved in xylose alcoholic fermentation.

3-Bromopyruvate inhibits hexokinase, leading to the glycolysis disruption. Yeast mutants resistant to 3-bromopyruvate could possess increased glycolysis and elevated amount of synthesized ethanol. Several 3-bromopyruvate resistant mutants of yeast *H. polymorpha* were selected on xylose containing medium via insertional mutagenesis. One of the selected strains possesses double increase in ethanol production from xylose. Insertion cassette disrupted *ATG13* gene which is involved in conserved process of autophagy initiation. Disruption of the *ATG13* in mutant strain has no effect on peroxisome degradation. However the deletion of *ATG13* gene caused the block of this process. The Δ*atg13* strain of *H. polymorpha* produced elevated amount of ethanol from xylose, similar to insertional strain. *ATG1/ATG13* complex is responsible for initiation of autophagy processes. Deletion of *ATG1* gene in *H. polymorpha* has no influence on ethanol production from xylose.

Autophagy-related gene *ATG13* is somehow involved in the regulation of xylose alcoholic
fermentation pathway in the yeast *H. polymorpha*. The role of peroxisomes in xylose
alcoholic fermentation is discussed.

<footnote n="1">¹ Department of Molecular Genetics and Biotechnology, Institute of Cell Biology, NAS of Ukraine, Drahomanov Street, 14/16, Lviv 79005, Ukraine</footnote>

<footnote n="2">² Department of Biotechnology and Microbiology, University of Rzeszow, Zelwerowicza 4, Rzeszow 35-601, Poland</footnote>
IMPROVEMENT OF HIGH TEMPERATURE ALCOHOLIC FERMENTATION OF
*Saccharomyces cerevisiae*

Taras Luzhetskyi<fnref n="1" />, Semkiv Marta<fnref n="1" />, Dmytruk Kostyantyn<fnref n="1" />, Sibirny Andriy<fnref n="1" />,<fnref n="2" />

Contact email: luzhetskyi@gmail.com

Ethanol is currently the most common liquid fuel derived from biomass. In recent years, the volume of industrial production of ethanol increased by the use of alcohol in the transport sector. Ethanol is used in automobile engines as a supplement to gasoline-ethanol blends or pure in specialized engines. The yeast *Saccharomyces cerevisiae* is the dominant industrial producer of ethanol via alcoholic fermentation of sugar substrates. Efficiency of alcoholic fermentation strongly depends on the resistance of the yeast strains to the stress conditions, which can appear during fermentation, e. g. temperature increase or high ethanol concentration. Disaccharide trehalose is one of the major stress protectants in *Saccharomyces cerevisiae* cells. It is synthesized through two sequential steps. Trehalose-6-phosphate synthase (encoded by *S. cerevisiae* gene *TPS1*) catalyzes trehalose-6-phosphate synthesis from glucose derivatives glucose-1-phosphate and UDP-glucose. Trehalose-6-phosphate is further dephosphorylated by trehalose-6-phosphate phosphatase (encoded by gene *TPS2*) to produce trehalose. The aim of this work is improvement of high temperature alcoholic fermentation of yeast *S. cerevisiae* by overexpression of the genes *TPS1* and *TPS2*. For overexpression of trehalose-6-phosphate synthase and trehalose-6-phosphate phosphatase, the vector for multicopy integration was constructed, in which ORFs of *TPS1* and *TPS2* genes were placed under the control of strong constitutive *ADH1* promoter. The resulting vector was linearized and used for transformation of *S. cerevisiae* industrial strain. Recombinant strains overexpressing genes for trehalose synthesis possessed increased intracellular concentration of trehalose. Thermotolerance and tolerance to the higher ethanol concentrations of the constructed strains were also increased. Recombinant strains with higher trehalose concentration produce more ethanol during fermentation at 42°C. This difference is particularly noticeable in the first hours of the fermentation. Recombinant strains with increased intracellular concentration of trehalose produce more ethanol during alcoholic fermentation at 42°C. Constructed strains are promising for industrial implementation.

<footnote n="1">¹ Department of Molecular Genetics and Biotechnology, Institute of Cell Biology, NAS of Ukraine, Drahomanov Street, 14/16, Lviv 79005, Ukraine</footnote>

<footnote n="2">² Department of Biotechnology and Microbiology, University of Rzeszow, Zelwerowicza 4, Rzeszow 35-601, Poland</footnote>
EXPLORING THE ENDOCYTIC PATHWAY BY COMBINING HIGH-THROUGHPUT
GENETICS AND HIGH-CONTENT MICROSCOPY

Mojca Mattiazzi Ušaj¹,², Matej Ušaj¹, Marinka Žitnik³, Dana Kablawi¹, Blaž Zupan³, Brenda Andrews¹ and Charles Boone¹

¹Donnelly Centre for Cellular and Biomolecular Research, University of Toronto, Toronto, Ontario, Canada

²Department of Molecular and Biomedical Sciences, Jozef Stefan Institute, Ljubljana, Slovenia

³Faculty of Computer and Information Science, University of Ljubljana, Ljubljana, Slovenia
Contact email: m.mattiazziusaj@utoronto.ca

Endocytosis is a highly conserved fundamental cellular process that controls the lipid and protein composition of the plasma membrane, and the exchange of the majority of molecules between a cell and its environment. It is a complex process that depends on an intricate network of interacting proteins and precise coordination of molecular events, and serves as a link between many intracellular signalling networks. We have combined synthetic genetic array (SGA) analysis with high-throughput confocal fluorescence microscopy and quantitative image analysis to assess the phenotypes of cortical actin patches, endosomes and vacuoles for yeast S. cerevisiae deletion mutants and temperature sensitive (TS) essential gene mutants, covering approximately 5400 open reading frames (~90% of the yeast genome). Our systematic and automated approach confirmed previously known components of the endocytic pathway and identified new genes, including some previously uncharacterized, involved in the process of endocytosis. Here, the work pipeline, results and ongoing efforts will be presented.
COMPLEX GENETIC INTERACTION PROFILES REVEAL EVOLUTIONARY FATES
OF DUPLICATED GENES

Elena Kuzmin<fnref n="1" />,<fnref n="2" />, Benjamin VanderSluis<fnref n="4" />, Yiqun Chen<fnref n="2" />, Raamesh Deshpande<fnref n="4" />, Matej Usaj<fnref n="2" />,
Michael Costanzo<fnref n="2" />, Chad L. Myers<fnref n="4" />, Brenda J. Andrews<fnref n="1" />,<fnref n="2" />, Charles Boone<fnref n="1" />,<fnref n="2" />

Contact email: elena.kuzmin@utoronto.ca

The *Saccharomyces cerevisiae* genome retained 551 genes after whole genome duplication and the deletion of 35% of these sister paralogs results in a synthetic sick or lethal (SS/SL) genetic interaction, suggesting that they buffer each other's loss. The remaining duplicates do not show an SS/SL and may be redundant with a third gene, which masks their functional relationship. The digenic interaction profiles of individual paralogs provide insight about their degree of functional divergence; whereas, the functions that are shared between the paralogs can be interrogated through their trigenic interaction profiles. We are using an automated form of yeast genetics called Synthetic Genetic Array (SGA) analysis to explore trigenic interactions involving paralog pairs for the purpose of understanding paralog gene function and testing models of how paralog pairs are maintained through evolution. In its simplest form, the fate of duplicated genes has been either one of functional divergence, for functional specialization by sub- or neo-functionalization, or functional redundancy, for back-up compensation or dosage amplification. To differentiate between these possibilities, we are generating genetic interaction profiles for single mutants of sister paralogs and double mutants lacking both paralogs. So far we have analyzed over 250 paralog pairs and we have identified a functionally divergent set, as well as a set displaying a gradient of trigenic interaction frequency, suggesting that these paralogs retained varying degrees of functional redundancy. Physiological and evolutionary features, such as the double mutant fitness defect, average digenic interaction degree, expression level, rate of divergence and coding sequence similarity, correlate with trigenic interaction frequency and can be used for its prediction using a machine learning algorithm. Thus, the assessment of trigenic interactions offers a novel approach to study the functional relationship and buffering capacity of sister duplicates.

<footnote n="1">¹Molecular Genetics, U of Toronto</footnote>

<footnote n="2">²Donnelly Centre for Cellular & Biomolecular Research, Toronto, Canada</footnote>

<footnote n="3">³Cell & Systems Bio. Eco., Evo. Bio. & Comp. Sci., U of Toronto</footnote>

<footnote n="4">⁴Comp. Sci. & Engineering, U of Minnesota-Twin Cities, Minneapolis, USA</footnote>
## CHAPERONE OVERLOAD IS A MAJOR DETERMINANT OF DOSAGE SENSITIVITY

Zoltán Farkas, Zoltán Bódi, Dorottya Kalapis, Béla Szamecz, Gábor Boross, Csaba Pál

Synthetic and Systems Biology Unit, Institute of Biochemistry, Biological Research Center of the Hungarian Academy of Sciences, Szeged, Hungary

Contact e-mail: farkas81@gmail.com

Why are proteins harmful when produced in excess? While protein burden and associated energetic costs have long been suggested as an important cause of dosage sensitivity of highly expressed genes, the underlying molecular mechanisms have remained largely unclear in eukaryotes. Here, we ask how the costs of an unneeded fluorescent protein (yEVenus) vary across genetic backgrounds and environmental stresses. By integrating chemical perturbation assays and a genome-wide genetic interaction study in yeast *Saccharomyces cerevisiae*, we mapped three major subsystems with critical roles in buffering gene expression costs: the cellular machinery involved in the speed and accuracy of translation, amino acid metabolism and the Hsp70-prefoldin chaperone system. While protein expression cost occurs in all tested environments, it was especially pronounced under amino acid starvation and high temperature. Taken together, our work indicates two major sources of gene expression costs. First, translation of unneeded proteins tie up amino acids that could be incorporated into other beneficial proteins. Second, by perturbing cellular protein homeostasis, gratuitous gene overexpression causes both protein mistranslation and aggregation on a global scale.
GENERAL PRINCIPLES OF TRANSCRIPTOMIC CHANGES FOLLOWING SINGLE-
GENE DELETION IN *Saccharomyces cerevisiae*

Károly Kovács<fnref n="1" />, Patrick Kemmeren<fnref n="2" />, Marian J.A. Groot Koerkamp<fnref n="2" />, Frank C. P. Holstege<fnref n="2" />, Richard Notebaart<fnref n="3" />, Csaba Pál<fnref n="1" />, Balázs Papp<fnref n="1" />

Contact email: kkaroly@gmail.com

What are the general patterns and principles behind transcriptomic responses following
genetic perturbations? Our work is the first to analyse this question in a systematic manner.
What gene features determine the extent of transcriptomic changes following a single-gene
deletion? How specific are these changes to the deleted gene? Are they mostly adaptive or
rather deleterious? A recent systematic dataset, provided by the Holstege lab, allowed us to
address these questions on an unprecedented scale. This dataset contains information on
genome-wide mRNA expression changes for 1484 viable knock-out strains of *Saccharomyces*
*cerevisiae*. Our results indicate that the number of up- or downregulated genes show strong
positive correlation with both the fitness contribution and the degree of pleiotropy of the
deleted gene. While we revealed a significant overrepresentation of functional relatedness
between the deleted and the responsive genes, this effect is small and most changes do not
occur within functional modules. By overlaying the gene expression data on a genetic
interaction map of yeast, we demonstrate that most expression changes are unlikely to provide
compensation following gene deletion.

<footnote n="1">¹ Biological Research Centre Hungarian Academy of Sciences, Szeged</footnote>

<footnote n="2">² Molecular Cancer Research, University Medical Center Utrecht, Utrecht, the Netherlands.</footnote>

<footnote n="3">³ Radboud University, Nijmegen</footnote>
RNA FUNCTIONAL PROFILING BY GENE DELETION IN *S. cerevisiae*

Steven Parker, Jian Wu, Daniela Delneri and Raymond O'Keefe

Faculty of Life Sciences, The University of Manchester, Michael Smith Building, Oxford Road, Manchester M13 9PT, UK

Developments in genome-wide analysis, such as next generation sequencing technologies and high-resolution tiling arrays, have revealed that the eukaryotic genome is extensively transcribed. Recent studies have shown that a large proportion of the transcriptome contains RNAs which are not translated in to protein; non-coding RNA (ncRNA). The focus of current debate is into how many of these non-coding RNAs are functional, and what their function is. Since its release in 2000, the Yeast Knock-out (YKO) deletion collection has proven to be a valuable tool for research teams studying cellular processes and the mechanisms of drug action. However, pervasively transcribed RNA-coding genes are absent from the collection, preventing the study of ncRNA. We have constructed ~500 barcoded ncRNA gene deletion strains. These strains include annotated snoRNAs, tRNAs and recently identified stable unannotated transcripts (SUTs) and cryptic unstable transcripts (CUTs). Confirmed diploid heterozygotes were sporulated and dissected to create haploid and diploid homozygote deletion strains. For continuity, we replicated the methodology used to create the original YKO deletion collection. Any phenotypes observed during the construction process were noted. Furthermore, the ncRNA deletion strains have been used for RNA fitness profiling, utilizing next generation sequencing of the included barcodes. This will help us to understand the role of individual ncRNAs in different growth conditions.
DISCOVERY OF NOVEL GENETIC INTERACTIONS BY CHROMOSOMAL SEGMENTAL DELETIONS AND ITS APPLICATION TO ETHANOL FERMENTATION IN *Saccharomyces cerevisiae*

Yu Sasano, Saeed Kaboli, Takuya Yamakawa, Tao Takagaki, Keisuke Sunada, Minetaka Sugiyama, Yoshinobu Kaneko and Satoshi Harashima

Department of Biotechnology, Graduate School of Engineering, Osaka University, Suita, Osaka 565-0871, Japan

Contact email: sasano@bio.eng.osaka-u.ac.jp

It has been recognized these days that most of the industrially useful traits are controlled not by a single gene but by multiple genes. Thus, a technology that allows large scale manipulation of a genome and chromosomes is strongly required. In this context, we have developed chromosome engineering technology called *PCR-mediated chromosome splitting* (PCS) in *Saccharomyces cerevisiae*. PCS enables splitting of yeast chromosome at any desired site and generates two functional chromosomes. PCS can be applied to many derivative technologies. One such application is *PCR-mediated chromosomal deletion* (PCD), which enables deletion of any genomic regions as we desire. Using PCD technology, we deleted 110 genomic regions in a haploid *S. cerevisiae* strain. These 110 regions were selected not to contain any essential genes and are larger than 9 kb. Surprisingly, we discovered that deletion of almost half of the regions lead to lethality, suggesting that these regions contain synthetic lethal combinations. In many cases, there were no known synthetic lethal combinations in such regions. This result implied that there are a lot of unidentified synthetic lethal combinations in adjacent genomic regions beyond expectations predicted from large scale genome analysis conducted so far. Hence, our result will uncover a novel landscape of genetic interactions in *S. cerevisiae* genome. Furthermore, we demonstrated that the PCD technology can also be applied to breeding of yeast strains since some segmental deletion mutants showed improved tolerance to bioethanol fermentation-related stresses such as high temperature, high concentration of ethanol, and weak acid. In conclusion, the PCD technology can be exploited not only for revealing genomic interactions but also for breeding of industrially useful yeast strains.
USE OF PROTOTROPHIC DELETION LIBRARIES AS A TOOL TO IDENTIFY
NITROGEN RELATED GENES IN LAB AND WINE YEAST

Josephine Jasmine Peter, Tommaso Liccioli, Michelle Walker and Vladimir Jiranek

School of Agriculture, Food and Wine, The University of Adelaide, PMB1, Glen Osmond SA 5064, Australia.

Contact email: Josephine.peter@adelaide.edu.au

Nitrogen present in grape juice promotes yeast biomass formation and shortens fermentation duration. Insufficient nitrogen often causes slow or sluggish fermentation and can result in wine with microbial contamination. Problems due to nitrogen deficiency can be rectified by optimizing vineyard fertilization or more commonly supplementing the fermentation with ammonium salts. An alternative to supplementation is to use wine yeast that can efficiently assimilate nitrogen and complete fermentation in low nitrogen conditions. In order to generate “nitrogen efficient” strains it is important to investigate gene functions related to nitrogen assimilation under fermentative conditions. Several genome wide studies in the past used of lab yeast deletion libraries (collection of strains each with a single gene deleted) to identify gene functions. However, these libraries contain auxotrophic markers (requiring amino acid supplementation) that make quantitative physiological studies harder especially under nitrogen limited conditions. Therefore, to reduce the bias caused by auxotrophic markers we have used the prototrophic deletion mutant collection in both lab and wine yeast backgrounds to identify genes related to nitrogen assimilation. The methodology of this project involves firstly the screening of the prototrophic lab yeast deletion library (BY4741) and the partial wine yeast library (AWRI 1631) for growth, nitrogen and sugar consumption rates under limiting and non-limiting nitrogen concentrations. The second part of the study aims to identify the function of the candidate genes highlighted in the screen using bioinformatic tools. The last part of the study seeks to compare the identified genes in both laboratory and wine yeast backgrounds and to understand the role of the identified genes in cell growth and metabolism. The outcomes of this study will increase the knowledge on the yeast genome in relation to nitrogen assimilation. This knowledge will then be used to formulate new strategies to produce improved strains with high nitrogen efficiency to benefit the wine making process.
DEVELOPMENT OF *Kluyveromyces marxianus* FOR CELL FACTORY APPLICATIONS

John P Morrissey

School of Microbiology, University College Cork, Ireland

The yeast *Kluyveromyces marxianus* is widely used in biotechnology for applications that include production of ethanol, enzymes and single cell protein. The ability to produce ethanol is of particular interest because of the growing bioethanol sector. Certain traits, namely thermotolerance, high secretory capacity, GRAS/QPS status, and the fastest growth rate of any eukaryotic microbe render *K. marxianus* especially suitable for industrial exploitation. Industrially and for research applications, a range of different wild-type strains are used and research from our group, and by others, has found that there is extensive phenotypic variation between strains. At one level, this is advantageous as it allows screening to identify natural variants with specific traits of interest. It also creates challenges, however, as variation in stress response and other parameters can hinder strain development for commercial application. Recent years has seen significant advances, especially with regard to the development of molecular and genomic tools. In addition, there has been renewed interest in using *K. marxianus* as a cell factory for the production of specific chemicals, especially those with applications as flavour or fragrance metabolites. This presentation will review the progress in the field in general towards developing *K. marxianus* as a cell factory and will discuss specific work from our group in the areas of stress tolerance and bioflavour production.
ENGINEERING THE YEAST mRNA METABOLISM FOR FISHING THE DESIRED PHENOTYPE

Paola Branduardi, Francesca Martani, Francesca Marano and Danilo Porro

Department of Biotechnology and Biosciences, Piazza della Scienza 2, 20126 Milano - Italy
Contact email: paola.branduardi@unimib.it

Biotechnological processes are of increasing significance for industrial production of fine and bulk chemicals, including biofuels. Under operative conditions microorganisms meet multiple stresses such as non optimal pH, temperature, oxygenation and osmotic stress, among others. Moreover, they have to face inhibitory compounds released during the pre-treatment of lignocellulosic biomasses, which constitute the preferential substrate for second generation production processes. All together these factors impair cellular metabolism and growth and, as a consequence, reduce the productivity of the process. The highly desirable evolution of robust cell factories is rarely ascribable to a single molecular element, since it requires a complex cellular reprogramming, implying the simultaneous modification of many regulatory and operative elements. In the last years, the cellular engineering approach termed “global transcription machinery engineering” (gTME) has been proposed, in association with specific screening protocols, as a method for reaching the above mentioned purpose. In addition to transcription, cells can modulate their complex phenotype by controlling mRNA metabolism and trafficking, translation and finally post-translational modifications. Here we present our approach for manipulating post-transcriptional events in the yeast cell factory *Saccharomyces cerevisiae* by modulating and mutagenizing one of the key regulatory element, PAB1. Data related to one of the improved mutant selected from the screening will be also presented and discussed.
CHARACTERIZATION OF BEER-RELATED PHYSIOLOGICAL PROCESSES IN
*Saccharomyces cerevisiae*, *Saccharomyces eubayanus* AND THEIR INTERSPECIFIC
HYBRID, THE BEST OF BOTH WORLDS

Marit Hebly<fnref n="1" />, Anja Brickwedde<fnref n="1" />, Maureen R.M. Driessen<fnref n="1" />, Erik A.F. de Hulster<fnref n="1" />, Marcel van den Broek<fnref n="1" />, Peter J.T. Verheijen<fnref n="2" />, Jack T. Pronk<fnref n="1" />, Jean-Marc Daran<fnref n="1" /> and Pascale Daran-Lapujade<fnref n="1" />

Contact email: A.Brickwedde@tudelft.nl

The yeast strains used for industrial fermentation of lager beers are taxonomically classified as *Saccharomyces pastorianus* natural, aneuploid species arising from the hybridization of *S. cerevisiae* and a non-*cerevisiae* *Saccharomyces* species. The first complete genome sequence of a lager brewing strain, *S. pastorianus* Weihenstephan 34/70 (WS34/70) was released in 2009 confirming the hybrid nature of lager yeast genome. The recent discovery and genome analysis of *S. eubayanus* revealed that this cryotolerant yeast, whose genome sequence shows a 99.5% identity with that of the ‘*bayanus*’ part of lager strains, contributed the non-*cerevisiae* part of the lager brewing strain *S. pastorianus* genome.

The hybrid genomes of lager brewing strains raise intriguing questions about the relative
contribution and regulation of genes in the *S. cerevisiae* and *S. eubayanus* subgenomes
involved in cellular processes relevant for lager brewing. It is, for example, unclear whether
the production of specific flavours and off-flavours is predominantly encoded by one of the
subgenomes. To study in more details how each subgenome may contribute respectively to
specific phenotypic traits of lager strains, a de novo interspecies hybrids between the two
proposed *S. pastorianus* parents, *S. cerevisiae* and *S. eubayanus*, was created. We have
subsequently investigated and characterized the performance of the hybrid strain in
environmental conditions relevant for the brewing process. The physiological characteristic of
the hybrid strain was then compared with those of the parental strains as well as with the one
of a *S. pastorianus* strain. The analysis of these data provided a deeper understanding of the
relative contribution of *S. cerevisiae* and *S. eubayanus* in *S. pastorianus* phenotypic behavior.

<footnote n="1">¹ Industrial Microbiology Section, Department of Biotechnology, Delft University of Technology, Julianalaan 67, 2628 BC Delft, The Netherlands</footnote>

<footnote n="2">² Cell System Engineering Section, Department of Biotechnology, Delft University of Technology, Julianalaan 67, 2628 BC Delft, The Netherlands</footnote>
PRODUCTION OF SPECIALIZED FATTY ACIDS IN *Saccharomyces cerevisiae*

Ângela Carvalho, Klavs Riishede Hansen, Michael Dalgaard Mikkelsen,
Richard J. S. Baerends

Evolva Biotech A/S, Lersø Parkallé 42-44, DK-2100, København Ø, Denmark
Contact email: angelac@evolva.com

The primary aim of this project is to design strains of *S. cerevisiae* to produce specialized fatty acids that are precursors for biosynthesis of commercially interesting compounds. In the first stage of the project metabolic enzymes that are required to redirect fatty acid metabolism will be identified. Simultaneous expression of large gene sets using eYACs (expression Yeast Artificial Chromosomes) will then be applied to functionally clone new enzymes for fatty acid synthesis. Using this information rational engineering will be employed to tailor make novel GM strains that produce relevant fatty acids.
DECIPHERING THE GENETIC AND METABOLIC BASES OF YEAST AROMA
PROPERTIES

Matthias Eder¹, Jean-Luc Legras¹, Carole Camarasa¹, Sylvie Dequin¹

¹ UMR1083 SPO, Institut National de la Recherche Agronomique, F-34060 Montpellier, France

Contact mail: matthias.eder@supagro.inra.fr

The yeast species *Saccharomyces cerevisiae* plays a vital role in the formation of aroma compounds during wine fermentation. Wine aroma includes volatile compounds that are produced de novo by yeast, like esters, higher alcohols and organic acids, and compounds that are formed from precursors present in the must, like terpenols or DMS.

The main aim of this work is to identify the genomic and metabolic bases for the formation of these molecules. For this purpose a crossing will be performed between two wine yeast strains selected because of their difference in the need for nitrogen during fermentation. Approximately 130 F2-segregants will be genotyped by next-generation sequencing and individually phenotyped by measuring extracellular metabolites using high-performance liquid chromatography (HPLC) and gas chromatography-mass spectrometry (GC-MS). Based on the metabolic data, the intracellular fluxes in central metabolism will be estimated using a constraint-based model of yeast metabolism. Quantitative trait locus (QTL)-mapping will be used to identify specific allele variations influencing the aroma profile and the metabolic fluxes. Finally the role of detected alleles will be validated by allele replacement and studied by functional genomics.

This will lead to a more profound knowledge about the correlation between genetic properties and phenotypic characteristics and will enable the exploitation of the natural diversity of yeast to generate novel aromatic wine yeast strains.
MULTI-STRESS RESISTANCE AND LAG-PHASE TIME REDUCTION IN WINE
FERMENTATION

David Ferreira<fnref n="1" />,<fnref n="2" />, Sylvie Dequin<fnref n="2" />, Virginie Galeote<fnref n="2" />, Anne Ortiz-Julien<fnref n="1" />

Contact email: david.ferreira@supagro.inra.fr

Saccharomyces cerevisiae has been used for several millennia to perform wine
fermentation due to its endurance and unmatched qualities. Nevertheless, at the moment
of inoculation, wine yeasts must cope with specific stress factors that still challenge
wine-makers nowadays by slowing down or even compromising the fermentation
process. The objective of the present work is to improve the multi-stress resistance of
commercial yeast strains specifically during the lag-phase in order to reduce
fermentation start period and possibly allow direct wine inoculation. Red and white
wine fermentations present distinguished sets of stress factors at the moment of
inoculation: for instance high osmotic pressure and lack of vitamins (e.g. thiamine) in
red and considerable SO2 concentrations, lack of important lipid components due to
wine clarification and low temperatures (14°C) in white. Due to these differences,
several commercial yeast strains will be preliminary tested and the best candidates
chosen according to their best suitability for each type of fermentation. Next, two
parallel approaches will be conducted: an evolutionary engineering approach where for
several generations specific selective pressures will be applied in order to promote yeast
adaptation to stressful conditions as well as a decrease in lag-phase duration.
Concomitantly a QTL approach will also be conducted with the aim of identifying
important genes and loci responsible for stress resistance and/or for yeast improved
growth during lag-phase. It is expected to obtain optimized wine yeasts with improved
stress resistance and reduced lag-phase while keeping the desirable metabolic and
fermentation traits. A deeper knowledge about the genetic and molecular bases
responsible for yeast adaptation and multi-stress resistance is intended as well.

<footnote n="1">¹Lallemand SAS, F-31700 Blagnac, France</footnote>

<footnote n="2">²INRA, UMR1083 Sciences pour l'oenologie, 2, place Viala, 34060 Montpellier, France</footnote>
REPROGRAMMING OF *Saccharomyces cerevisiae* SHIKIMATE PATHWAY FOR THE PRODUCTION OF BULK CHEMICALS

Manuela Gottardi, Christine Brueckner, Mislav Oreb and Eckhard Boles

Institute of Molecular Biosciences, Goethe University Frankfurt, Frankfurt am Main,
Germany

Contact email: Gottardi@bio.uni-frankfurt.de

The production of bulk chemicals is often associated to energy waste and it is not an
environmental friendly process. Therefore, finding new alternatives to produce commodity
chemicals is now a major interest.

*S. cerevisiae* can be engineered as a microbial cell factory in order to produce interesting compounds and it has many advantages if compared to other microbial hosts.

In particular, we are focusing on the Shikimate pathway, which is the central pathway involved in the production of aromatic amino acids.

Through metabolic engineering approaches we intend to reprogram *S. cerevisiae* and redirect
its metabolism towards the production of bulk chemicals that might be derived from
Shikimate pathway intermediate metabolites.
EVALUATION OF Zygosaccharomyces bailii FOR LACTIC ACID
TOLERANCE

Nurzhan Kuanyshev, Danilo Porro and Paola Branduardi

Università degli Studi di Milano – Bicocca, Dipartimento di Biotecnologie e Bioscienze, Piazza della Scienza 2, 20126 Milano, Italy

Contact email: n.kuanyshev@unimib.campus.it

Nowadays an industry searches for a cheap and environmentally friendly way to produce fine chemicals. The cell biofactories are an attractive system to fulfill modern requirements for an industrial production of chemicals and among those lactic acid is considered as an oldest and high-volume produced chemical. However, bacterial strains used in lactic acid fermentation suffer from low acid tolerance making downstream purification of final product troublesome and expensive. Exploitation of acid tolerant yeast strains can effectively eliminate the obstacle. Zygosaccharomyces bailii is a well-known food spoilage yeast usually found in can and wine industry. Z. bailii possess a significant low pH tolerance and organic acid tolerance which can be considered as an advantage in lactic acid production. Therefore we would like to elucidate and understand the mechanism underlying lactic acid tolerance in Z. bailii not thoroughly investigated up to now. Data resulting from several fermentations using both flasks and bioreactor with different pH, oxygen supply and organic acid concentrations will be presented and discussed, together with evaluations regarding viability profile measured at single cell level.
ELUCIDATING MECHANISMS THAT DETERMINE DIFFERENTIAL
MALTOTRIOSE UTILIZATION BETWEEN STRAINS OF THE HYBRID
YEAST *Saccharomyces pastorianus*

Frederico Magalhães, Virve Vidgren, Jukka Ekberg, Laura Ruohonen and Brian Gibson
VTT Technical Research Centre of Finland, FI-02044 VTT, Finland.
Contact email: brian.gibson@vtt.fibrian.gibson@vtt.fi

The lager brewing yeast *Saccharomyces pastorianus* is an allotetraploid hybrid of *S. cerevisiae* and *S. eubayanus*. Its hybrid condition allows it to benefit from the strong fermentative capacity of *S. cerevisiae* and the cryotolerance of *S. eubayanus*. These properties make it suitable for the production of lager beer which requires low fermentation temperatures. Possibly as a result of different hybridization events, two genetically distinct groups are present within the *S. pastorianus* taxon, Saaz and Frohberg. The groups display some functional differences, e.g. cold tolerance is more evident in the Saaz group, while the Frohberg group has greater fermentation performance. It has been observed that the Saaz group (as well as the parental *S. eubayanus*) has limited ability to utilize the maltotriose present in brewer's wort and this may be the cause of its relatively poor fermentation performance. The mechanisms responsible for this limitation are still to be uncovered. It is hypothesized that the inability to utilize maltotriose is related to insufficient transmembrane transport. To test this hypothesis a comprehensive study of wort fermentation performance, sugar utilization and membrane transport activity in Saaz strains is being conducted. The expression of maltotriose transporter genes (namely *MTT1*, *MPH2/3* or *AGT1*) in Saaz strains will determine whether transmembrane transport is the main factor limiting fermentation performance of Saaz strains or if other factors, yet to be identified, are contributing to this property.
GALLIC ACID PRODUCTION BY THE TWO TANNASES Atan1p AND Atan2P USING
THE YEAST *Arxula adeninivorans*

<u>Anna Malak</u>, Eric Böer, Gotthard Kunze

Leibniz Institute of Plant Genetics and Crop Plant Research (IPK), Corrensstr.3, D-06466 Gatersleben, Germany

Contact email: malak@ipk-gatersleben.de

Tannin acyl hydrolase (tannase, EC 3.1.1.20) is an inducible enzyme and belongs to the group of hydrolases. It hydrolyses ester bonds between sugar and gallic acid, as well as dep sides bonds between two molecules of gallic acid present in tannins. The most wanted product of this reaction is gallic acid that is used as additive for colorants, as an antioxidant in food, as well as precursor for trimethoprim production and others. The presented studies focus on the development of more efficient and cheaper methods of tannase production by genetically modified *Arxula adeninivorans*. This dimorphic, non-pathogenic, thermo- and osmotolerant yeast, which exhibits a very broad spectrum of usable substrates, is the only organism known, which synthesizes two different tannases. Atan1p (encoded by *ATAN1*) is secreted and the other one Atan2p (encoded by *ATAN2*) is localized in the cell wall. Two vectors containing *ATAN1* and *ATAN2* genes with hexahistidine encoding sequence were constructed and transformed into an *A. adeninivorans* yeast strain. The respective recombinant proteins expressed by the trangsgenic strain were purified by IMAC and enzymatic parameter were determined. Furthermore the large scale protein production was optimized. The characterization of Atan2p protein is in progress. Deletion mutants generated by gene disruption: *A. adeninivorans* G1236 [*Δatan1*], G1237 [*Δatan2*], and a double mutant MS1031 [*Δatan1*, *Δatan2*]) were produced for comparison of the function of both enzymes in the cell.
AUTOMATED ANNOTATION, STRUCTURAL VALIDATION, AND METABOLIC
MODELING OF INDUSTRIAL YEAST GENOMES

Raúl A. Ortiz-Merino, Kenneth H. Wolfe.

UCD Conway Institute, School of Medicine & Medical Science, University College

Dublin, Belfield, Dublin 4, Ireland

Contact email: rul.ohm@gmail.com

Unicellular fungi (yeasts) have been used for the production of food and beverages since long before we possessed a detailed description of the cellular processes underlying such practices. Today, a vast amount of knowledge has been accumulated and utilized with both academic and industrial implications, shaping the field of yeast biotechnology. Modern yeast biotechnology depends heavily on genome sequencing technologies and the bioinformatic methods that are used to interpret genome sequences. Therefore, we will optimize and improve a computational system for automated gene identification and *de novo* genome sequence annotation, and apply it to industrially-relevant yeasts sequenced using next-generation genome sequencing technologies. The system (Yeast Genome Annotation Pipeline; YGAP) was developed in the Wolfe lab in 2011 and is based on using interspecies synteny data from the Yeast Genome Order Browser. A second aim of this project is to develop automated methods for validating the structural integrity of genome sequence assemblies. Commonly, next-generation *de novo* genome sequence assemblies consist of multiple contigs that are physically linked together into scaffolds. Our goal is to obtain scaffolds that correspond as closely as possible to the structures of complete chromosomes. A third aim is to use newly-annotated yeast genomes to construct metabolic models of the corresponding species, by cross-referencing from existing metabolic models for other species. It will be of particular interest to focus on interspecies differences in predicted metabolic networks. As a starting point, we will use new public genomic sequences for two strains of *Kluyveromyces marxianus* to test the performance of the YGAP annotation pipeline and identify its current weaknesses relative to author-supplied annotations and benchmark gene sets from other species. This research project receives funding from the People Programme (Marie Curie Actions) of the European Union's Seventh Framework Programme FP7/2007-2013/ under REA grant agreement n° 606795, project acronym: YEASTCELL.
TOWARDS A GENOME SCALE METABOLIC MODEL OF *K. marxianus*: A KEY TOOL
TO IMPROVE ITS INDUSTRY-RELEVANT PROPERTIES.

Javier Varela¹ and John Morrissey¹

¹ Microbiology Department, University College Cork, Cork, Ireland.

The redirection of metabolic fluxes towards the production of a desired compound usually requires an overall understanding of cell metabolism. One effective strategy to obtain this view is by constructing a Genome Scale Metabolic (GSM) model. For yeasts, different GSM models have been built and were shown to be effective in designing metabolic engineering experiments. The yeast *Kluyveromyces marxianus* has special traits (lactose utilization, thermotolerance and high growth rate) that make it attractive for different biotechnological applications. Although this yeast has been already used in industry, there is a growing interest in enhancing its special properties. The main aim of this research project is to generate a GSM model for *K. marxianus*. For this purpose, first a metabolic-based genome annotation will take place using the tool MERLIN. Then, the metabolic reconstruction and model building will be carried out using RAVEN. To facilitate this process, previous GSM models from *S. cerevisiae* and *K. lactis* will be taken into account. In parallel, comparative genomics analyses will be done to serve as a general tool to assist the orthology-based reconstruction. Finally, in order to obtain a final high quality model, manual curation and experimental data will be considered. The generated model will be used to compare the *K. marxianus* metabolic network with those of other yeasts to understand the basis of its unique traits. At the same time the model will serve as a framework for metabolic engineering of this yeast enhancing its potential as a cell factory for the production of industry-relevant compounds.
SYNTHESIS OF WAX ESTERS IN *Saccharomyces cerevisiae* BY ENZYMATICALLY
CATALYZED ESTERIFICATION OF LONG-CHAIN FATTY ACIDS AND PRIMARY
LONG-CHAIN FATTY ALCOHOLS

<u>Leonie Wenning</u>, Verena Siewers, Jens Nielsen

Department of Chemical and Biological Engineering, Systems Biology, Chalmers University
of Technology, Kemivägen 10, SE-412 96 Göteborg, Sweden

Contact email: wenning@chalmers.se

The objective of this project is the enzymatic synthesis of wax esters by the esterification of primary, long-chain fatty alcohols and long-chain fatty acids with both components varying in their carbon chain length. Naturally, wax esters occur in pro- as well as eukaryotes where they fulfill different purposes, e.g. they can serve as a storage compound or have a protective or structural function (Röttig et al. 2013). Wax esters have a broad application range and can be used for many commercial purposes. These applications include e.g. personal care products, lubricants, varnishes, inks, waxes, detergents, resins & plastics. Wax esters can also be used for coatings (for fruits & pills), as an oil phase in formulas containing active compounds to enhance the efficiency of topical drugs or even as a replacement for diesel fuel. Unfortunately, so far most of the possible applications are limited to cosmetic and medical products due to the high price of production (Hassan et al. 2009). Because of this fact, a renewable approach for wax ester synthesis in a well-studied organism like *S. cerevisiae* is desirable. Just recently it was shown that wax ester synthases of different origin can be used for the production of wax esters in *S. cerevisiae* (Shi et al. 2012). Dependent on the enzyme source, different alcohols were preferred as a substrate when testing the esterification of palmitoyl coenzyme A. Because of this promising observation, further enzymes shall be tested for their ability to enable wax ester biosynthesis in *S. cerevisiae*.

References

Hassan El-Mallah M, El-Shami SM: Investigation of liquid wax components of Egyptian jojoba seeds. *J. Oleo. Sci.* 2009, 58:543-548.

Röttig A, Steinbuechel A: Acyltransferases in bacteria. *Microbiol. Mol. Biol. Rev.* 2013, 77:277-321.

Shi S, Valle-Rodriguez JO, Khoomrung S, Siewers V, Nielsen J: Functional expression and characterization of five wax ester synthases in *Saccharomyces cerevisiae* and their utility for biodiesel production. *Biotechnol Biofuels* 2012, 5:7-16.
KINETIC ANALYSIS AND GAS-LIQUID BALANCES OF THE PRODUCTION OF FERMENTATIVE AROMAS DURING WINEMAKING FERMENTATIONS

Jean-Roch Mouret<fnref n="1" />, Carole Camarasa<fnref n="1" />, Evelyne Aguera<fnref n="2" />, Stephanie Rollero<fnref n="1" />, Marc Perez<fnref n="1" />, Vincent Farines<fnref n="1" /> and Jean-Marie Sablayrolles

Contact email: mouretj@supagro.inra.fr

In winemaking, higher alcohols and esters produced during the alcoholic fermentation have a major impact on wine quality, especially for young wines. Accessing to their kinetics of synthesis is therefore of primary importance. To reach this objective, we developed an online monitoring system of the concentrations of the main volatile compounds in the off gas during the alcoholic fermentation. The high acquisition frequency of this specific device makes it possible to calculate the rates and specific rates of production, which are of direct interest for a better understanding of yeast metabolism. We also determined the gas-liquid balances of aroma production to take losses into account and to distinguish between microbial and physicochemical phenomena. We have thus demonstrated that the lower liquid concentrations of esters at high temperature resulted mostly from large losses by evaporation, with only limited changes in yeast metabolism. Then, we investigated the impact of both initial nitrogen concentration and temperature on the kinetics of production of the fermentative aromas. Nitrogen availability appeared as the main parameter affecting the kinetics of higher alcohol and ester synthesis, but the magnitude of this effect depended on temperature. The production of fermentative aromas was closely linked to central carbon metabolism, except for propanol. Indeed, two successive linear production phases (with yields dependent on initial nitrogen content and temperature) were obtained when plotting volatile compound synthesis versus sugar consumption. Rates and specific rates of isobutanol and isoamyl alcohol production were affected differently by nitrogen and temperature, indicating major differences in the regulation of synthesis for these two compounds, despite their partially shared metabolic pathways. Finally, propanol was produced exclusively during the nitrogen consumption phase, and the amount produced increased with initial nitrogen concentration. This compound was, therefore, identified as a quantitative, metabolic marker of the availability of intracellular nitrogen.

<footnote n="1">¹ INRA, UMR1083, 2 Place Viala, F-34060 Montpellier Cedex 1, France</footnote>

<footnote n="2">² INRA, UE999, F-11430 Gruissan, France</footnote>
<figure><img src="image_12.png" /></figure>
Saturday, October 11 – Vipava

08:00 – 08:45 Transportation to Vipava – buses leave from in front of the Perla Hotel

09:00 – 12:30

Parallel sessions (Vipava)

Beverages 1 & 2 (chairs: Florian Bauer / Sylvie Dequin) see p. 91 for details

Biodiversity for Applications (chairs Ian Roberts and Cene Gostinčar) see p. 92 for details

2nd Generation Biofuels (chairs Akihiko Kondo and Takayuki Mizuno) see p. 92 for details

10:30 – 11:00 Coffee Break & Meeting Local Food/Wine Producers

Regulation of and by Lipids (chairs Peter Gajdoš and Martin Kavšček) see p. 93 for details

Non-conventional Yeasts in Bioproduction
(chairs Gotthard Kunze and Janja Zajc) see p. 93 for details

12:30 – 14:00 Lunch / Vipava sightseeing & Meeting local food/wine producers

14:00 – 19:00

Parallel sessions (Vipava)

Ecology and Genomics (chairs: Duccio Cavalieri and Joseph Schacherer) see p. 95 for details

Cornucopia EU project session (chair Hana Sychrova) see p. 95 for details

Regulation of Metabolism and Nutrient Sensing
(chairs: Hyun Ah Kang and Marta Maria Rubio-Texeira) see p. 97 for details

16:10 – 17:00 Coffee break & Meeting local food/wine producers & Vipava sightseeing
Biofactories: Tools, Physiology & Products

(chairs: Hiroshi Takagi and Anne Gschaedler) see p. 98 for details

YeSVitE EU project session (chair: Ileana Vigentini) see p. 98 for details

Polygenic Traits (chairs: Gianni Liti and Ed Louis) see p. 99 for details

19:15 – 20:00 Transportation to Nova Gorica – buses leave from in front of the Grammar School
Saturday, October 11 morning sessions in Vipava – parallel sessions in:

Aula Magna and Aula Parva – Lanthieri Palace

Grammar School Hall (5 min walk from the Lanthieri Palace)

Beverages 1 / in Aula Magna

<table><tr><td>09:00 - 09:30 Florian Bauer: Environmental perturbations, nutrients and the yeast microbiome: can we predict the outcome of wine fermentations?</td><td>101</td></tr><tr><td>09:30 - 09:40 Ramon Gonzalez: Lowering ethanol content of wines by sugar respiration</td><td>102</td></tr><tr><td>09:40 - 09:50 Neil Jolly: Chenin blanc wine production with <i>Torulaspora delbrueckii</i> and <i>Saccharomyces cerevisiae</i> yeasts</td><td>103</td></tr><tr><td>09:50 - 10:00 Laura Canonico: Selected <i>Saccharomyces cerevisiae</i> and <i>Torulaspora delbrueckii</i> strains to improve the analytical profile of craft beer</td><td>104</td></tr><tr><td>10:00 - 10:10 Jure Zupan: Laboratory brewing under large-scale pressure vs. industrial brewing: microarray phenotyping of yeast during serial repitching</td><td>105</td></tr><tr><td>10:10 - 10:20 Sofia Dashko: Exploration of yeast biodiversity for potential improvements of Ribolla Gialla wine aroma profile</td><td>106</td></tr></table>

Beverages 2 / in Aula Magna

<table><tr><td>11:00 - 11:30 Sylvie Dequin: Evolutionary and reverse engineering of commercially important metabolic traits in wine yeast</td><td>107</td></tr><tr><td>11:30 - 11:40 Anna Lisa Coi: Characterization of genes involved in the adaptation of flor strains to biological ageing conditions of sherry wines</td><td>108</td></tr><tr><td>11:40 - 11:50 Huseyin Erten: The influence of different yeast inoculum levels on the quality of cider</td><td>109</td></tr><tr><td>11:50 - 12:00 Manuel Kirchmayr: Impact of inoculum preparation on microbial consortia, fermentation kinetics and product composition in traditional mezcal fermentations</td><td>110</td></tr></table>
12:00 - 12:10 Montserrat Alcazar: Effect of the agave saponins on the cell wall composition of *Saccharomyces cerevisiae* and *Kluyveromyces marxianus* yeasts strains in continuous culture 111

12:10 - 12:20 Marin Berović: New method for fast separation of magnetized yeast *Saccharomyces bayanus* in sparkling wine production 112

## Biodiversity for Applications / in Aula Parva

09:00 - 09:20 Ian Roberts: Exploring a yeast genetic resource collection to aid biorefinery production of biobased chemicals from agrifood feedstocks 113

09:20 - 09:35 Cecilia Geijer: Characterization of a novel non-GMO yeast for future lignocellulosic bioethanol production 114

09:35 - 09:50 Virve Vidgren: Maltotriose utilization in brewer's yeast with respect to transporter gene provenance 115

09:50 - 10:05 Claudia Colabella: Delimitation of *Meyerozynma guilliermondii* food isolates for improved safety in food industry 116

10:05 - 10:20 Cene Gostinčar: Opportunities arising from the genome sequencing of four *Aureobasidium* varieties 117

## 2nd Generation Biofuels / in Grammar School Hall

09:00 - 09:20 Akihiko Kondo: Development of microbial cell factories for biorefineries 118

09:20 - 09:30 Yue-Qin Tang: Breeding industrial *Saccharomyces cerevisiae* strain for lignocellulosic ethanol production 119

09:30 - 09:40 Peter Adeboye: Detoxification as a strategy for developing tolerance in *Saccharomyces cerevisiae* to phenolic compounds 120

09:40 - 09:50 Christian Marx: Engineering glutathione biosynthesis to enhance redox robustness of *Saccharomyces cerevisiae* 121
09:50 - 10:00 Takayuki Mizuno: Candida glabrata shows very high resistance for highly concentrated sulfuric acid and the tolerance is useful for ethanol production 122

10:00 - 10:20 Lisbeth Olsson: Challenges and possibilities with high solids lignocellulose fermentation 123

## Regulation of and by Lipids / in Aula Parva

11:00 - 11:15 Peter Gajdoš: Yarrowia lipolytica – useful tool for engineering lipid biosynthesis pathways 124

11:15 - 11:30 Martin Kavšček: Optimization of cultivation conditions for lipid production with a genome-scale model of Yarrowia lipolytica 125

11:30 - 11:45 Govindprasad Bhutada: Metabolic engineering to maximize lipid content in Yarrowia lipolytica 126

11:45 - 12:00 Lina Lindahl: The influence of membrane compostion on acetic acid permeability and potentially acetic acid tolerance 127

12:00 - 12:10 Katja Uršič: Tum1 affects lipid metabolism in Saccharomyces cerevisiae, however not through tRNA thiolation 128

## Non-conventional yeasts in bio-production / in Grammar School Hall

11:00 - 11:15 Gotthard Kunze: Blastobotrys (Arxula) adeninivorans – a suitable biocatalyst for new biotechnological products 129

11:15 - 11:25 Janja Zajc: Black yeast Aureobasidium pullulans: a source of genes for the improvement of industrially important traits of Saccharomyces cerevisiae 130

11:25 - 11:35 Catherine Saint-Hubert: Optimization of glucose oxidase production by recombinant Kluyveromyces lactis 131

11:35 - 11:45 Monika Kovacs: Interaction of Pseudomonas fluorescens and different spoilage yeasts 132
<table>
  <tbody>
    <tr>
      <td>11:45 - 11:55 Sara Landolfo: Is β-carotene involved in the antioxidant response of carotenogenic yeasts? An answer from three <i>Rhodotorula spp.</i> strains</td>
      <td>133</td>
    </tr>
    <tr>
      <td>11:55 - 12:05 Ilaria Mannazzu: Differential proteomic analysis of <i>Rhodotorula spp.</i> strains for the identification of enzymes involved in carotenogenesis</td>
      <td>134</td>
    </tr>
    <tr>
      <td>12:05 - 12:15 Jana Tkačova: <i>Rhodotorula glutinis</i> as a model for study of carotenoids and lipid biosynthesis</td>
      <td>135</td>
    </tr>
  </tbody>
</table>
Saturday, October 11 afternoon sessions in Vipava – parallel sessions in:

Aula Magna and Aula Parva – Lanthieri Palace

Grammar School Hall (5 min walk from the Lanthieri Palace)

## Ecology and Genomics / in Aula Magna

14:00 - 14:20 Duccio Cavalieri: Population genomics of *Saccharomyces cerevisiae* human isolates: passengers, colonizers and invaders 136

14:20 - 14:40 Joseph Schacherer: Population genomics reveals the evolutionary fate of a large-scale introgression in a protoploid yeast 137

14:40 - 15:00 Ursula Bond: Distinctive chromosomal end loss and lager specific genes define two different *Saccharomyces cerevisiae* lineages for *S. pastorianus* group I and II strains 138

15:00 - 15:20 Jürgen Wendland: Genome sequence of *Saccharomyces carlsbergensis*, the world's first pure culture lager yeast 139

15:20 - 15:35 Samina Naseeb: Role of chromosomal inversions on yeast fitness and gene expression 140

15:35 - 15:50 Qamar Almutava: Evolutionary impact and environmental adaptation of natural yeast isolates bearing karyotypic rearrangements 141

15:50 - 16:05 Kobchai Dungrattanalert: Evolutionary effect of mitochondrially encoded proteins on global gene retention in yeast hybrids 142

## Cornucopia EU project session / in Aula Parva

14:00 - 14:20 Hana Sychrova: Opening remarks

14:10 - 14:22 Benjamin Offei: Polygenic analysis of low pH tolerance in *Saccharomyces cerevisiae var. boulardii* 143

14:22 - 14:34 Honeylet Ochangco: Intracellular pH of potential probiotic yeasts during the passage through an in vitro gastrointestinal tract model 144
<table>
  <tbody>
    <tr>
      <td>14:34 - 14:46 Jana Zemančíková: Physiology and osmotolerance of two <i>Dekkera bruxellensis</i> strains</td>
      <td>145</td>
    </tr>
    <tr>
      <td>14:46 - 14:58 Md Moktaduzzaman: Studies on acetic acid stress response and galactose metabolism in <i>Dekkera bruxellensis</i></td>
      <td>146</td>
    </tr>
    <tr>
      <td>14:58 - 15:10 Anna Schifferdecker: Genotyping and phenotyping of hundreds of <i>Dekkera bruxellensis</i> isolates</td>
      <td>147</td>
    </tr>
    <tr>
      <td>15:10 - 15:22 Raquel Quintilla: MALDI-TOF as an identification tool for <i>Saccharomyces sensu stricto</i> species and food-related yeasts</td>
      <td>148</td>
    </tr>
    <tr>
      <td>15:22 - 15:34 Nerve Zhou: Nonconventional yeasts can be evolved to be efficient ethanol producers under bacterial selection pressures</td>
      <td>149</td>
    </tr>
    <tr>
      <td>15:34 - 15:46 Alicia Gutiérrez: Bioflavor production of non-<i>Saccharomyces</i> yeasts in fermented beverages</td>
      <td>150</td>
    </tr>
    <tr>
      <td>15:46 - 15:58 Dorota Radecka: Molecular basis of superior stress tolerance in nonconventional yeasts</td>
      <td>151</td>
    </tr>
    <tr>
      <td>15:58 - 16:10 Jiří Stříbný: The effect of the nitrogen sources on the formation of volatile aroma compounds by non-conventional <i>Saccharomyces</i> species (coffee break)</td>
      <td>152</td>
    </tr>
    <tr>
      <td>17:00 - 17:12 Ida Mosbech Smith: Mechanisms behind immunoregulatory effects of probiotic yeasts</td>
      <td>153</td>
    </tr>
    <tr>
      <td>17:12 - 17:24 Davide Ravasio: High throughput screening of non-conventional yeasts in fermentation performance and flavour production</td>
      <td>154</td>
    </tr>
    <tr>
      <td>17:24 - 17:36 Amparo Gamero Lluna: Development of a new fermented beverage employing cashew apple juice and non-conventional yeasts</td>
      <td>155</td>
    </tr>
    <tr>
      <td>17:36 - 17:55 Closing remarks</td>
      <td></td>
    </tr>
  </tbody>
</table>
Regulation of Metabolism and Nutrient Sensing / in Grammar School Hall

<table><tr><td>14:00 - 14:15 Hyun Ah Kang: Novel cysteine-centered sulfure metabolic pathway and its regulation in the methylotrophic yeast Hansenula polymorpha</td><td>156</td></tr><tr><td>14:15 - 14:30 Jessica Noble: Combining genetic and functional approaches to decipher the molecular bases of low sulfite production by wine yeasts</td><td>157</td></tr><tr><td>14:30 - 14:40 Lina Riego-Ruiz: A comparative approach to study nitrogen catabolite repression in yeasts</td><td>158</td></tr><tr><td>14:40 - 14:55 Marta Maria Rubio-Texeira: Transceptor signaling and endocytosis are independent events which may be triggered in parallel depending on specific substrate-transceptor interactions</td><td>159'</td></tr><tr><td>14:55 - 15:05 Eglè Lastauskiené: Ras/PKA signal transduction pathway participates in the regulation of Saccharomyces cerevisiae cell apoptosis in an acidic environment</td><td>160</td></tr><tr><td>15:05 - 15:15 Agustin Aranda: Nutrient sensing pathways control wine yeast lifespan</td><td>161</td></tr><tr><td>15:15 - 15:25 Elia Tomas: Influence of the propagation strategy in an ethanol production process from glucose and xylose: proteomics and gene expression analysis</td><td>162</td></tr><tr><td>15:25 - 15:35 Matic Legiša: Inducing pseudo-Warburg effect in yeast Saccharomyces cerevisiae</td><td>163</td></tr><tr><td>15:35 - 15:45 Laurinda Steyn: Phenotypic analysis of multiple ADH deletion mutants of Saccharomyces cerevisiae</td><td>164</td></tr><tr><td>15:45 - 15:55 Carla Gonçalves: Unravelling the molecular basis of fructophily in yeasts: a comparative genomics approach</td><td>165</td></tr><tr><td>15:55 - 16:05 Ileana Vigentini: Identification and molecular characterization of vinyl phenol reductase from Dekkera bruxellensis CBS4481</td><td>166</td></tr></table>
Biofactories: Tools, Physiology & Products / in Aula Magna

<table><tbody><tr><td>17:00 - 17:25</td><td>Hiroshi Takagi: Nitric oxide-mediated antioxidative mechanism in <i>Saccharomyces cerevisiae</i> and its application to industrial baker's yeast</td><td>167</td></tr><tr><td>17:30 - 17:42</td><td>Alejandro Munoz: Engineering of <i>Saccharomyces cerevisiae</i> for the production of poly-3-d-hydroxybutyrate from xylose</td><td>168</td></tr><tr><td>17:42 - 17:54</td><td>Patrick Fickers: Optimization of pAOX1 induction in a methanol/sorbitol co-feeding process in <i>Pichia pastoris</i></td><td>169</td></tr><tr><td>17:54 - 18:06</td><td>Oleg Tyurin: FLD gene disruption leads to an enhanced AOX1 promoter induction in response to either methanol or formate</td><td>170</td></tr><tr><td>18:06 - 18:18</td><td>Koji Yamada: A wide variety of developmental yeast from OYC</td><td>171</td></tr><tr><td>18:18 - 18:30</td><td>Jun Ishii: Genetic engineering for altering metabolic flow in <i>Saccharomyces cerevisiae</i> to produce higher alcohols</td><td>172</td></tr><tr><td>18:30 - 18:45</td><td>Anne Gschaedler: Study of the physiological state of <i>Kluyveromyces marxianus</i> using non-conventional methods</td><td>173</td></tr><tr><td>18:45 - 19:00</td><td>Emma Karlsson: Metabolic engineering of <i>Saccharomyces cerevisiae</i> for production of adipic acid from renewable sources: One route towards a bioeconomy</td><td>174</td></tr></tbody></table>

YeSVitE EU project session / in Aula Parva

<table><tbody><tr><td>17:55 - 18:15</td><td>Ileana Vigentini: Yeasts for the sustainability in viticulture and oenology: the "YeSVitE" project</td><td>175</td></tr><tr><td>18:15 - 18:25</td><td>Florian Bauer: The YeSVitE program: research focus and contribution of the institute for wine biotechnology @ Stellenbosch University</td><td>176</td></tr><tr><td>18:25 - 18:35</td><td>Osvaldo Failla: Exploitation of biodiversity in viticulture and oenology to allow a sustainable winemaking</td><td>177</td></tr><tr><td>18:35 - 18:45</td><td>Laura Corte: Yeast metabolomics and taxonomy in wine microbiology research</td><td>178</td></tr><tr><td>18:45 - 18:55</td><td>Mojca Brložnik: Quantitative phenotypic measurements for polygenic trait analysis</td><td>179</td></tr></tbody></table>
18:55 - 19:05 David Maghradze: Bio-protection of grape quality in vineyard and post-harvest withering 180

Polygenic Traits / in Grammar School Hall

17:00 - 17:20 Gianni Liti: Sequencing natural yeast populations for functional insights 181

17:20 - 17:40 Ed Louis: Linked QTLs of mixed effects in complex traits of yeast 182

17:40 - 17:50 Esther Meersman: High-throughput phenotyping and selective breeding as an effective tool to generate novel beer yeasts 183

17:50 - 18:00 Stijn Mertens: Development of interspecies yeast hybrids to introduce aromatic diversity in lager beers 184

18:00 - 18:10 Olena Ishchuk: Replication elements in the wine yeast *Dekkera bruxellensis* 185

18:10 - 18:20 Valentina Tosato: Yeast cell evolution through post-translocational adaptation (PTA) 186

18:20 - 18:30 Ana Mangado: Adaptive evolution of *Saccharomyces cerevisiae* to early stage of an alcoholic fermentation 187

18:30 - 18:40 Maria Raga: Genetic basis of phenotypic variation within *S. cerevisiae* strains during second fermentation 188

18:40 - 18:50 Antonio David Moreno Garcia: Isolation and evolution of a novel non-*Saccharomyces* xylose-fermenting strain for lignocellulosic bioethanol production 189
<figure><img src="image_13.png" /></figure>
ENVIRONMENTAL PERTURBATIONS, NUTRIENTS AND THE YEAST
MICROBIOME: CAN WE PREDICT THE OUTCOME OF WINE
FERMENTATIONS?

Debra Rossouw, Dan Jacobson and Florian F. Bauer

Institute for Wine Biotechnology, Department of Viticulture and Oenology, University of Stellenbosch, South Africa
Contact Email: fb2@sun.ac.za

Wine fermentations are continuously evolving ecosystems that are adapted to juices of variable nutritional composition and respond to continuously changing environmental conditions. The species and strains of microorganisms that constitute this ecosystem, some of which will conduct alcoholic fermentation, are primarily responsible for many of the aromatic features of the final product. For the most relevant fermentative microorganism in wine, the yeast *Saccharomyces cerevisiae*, many of the genetic and metabolic regulations that lead to the fermentation-derived aromatic and flavour profile are relatively well understood. However, the complexity of interactions between microorganisms, the differing chemical composition of grapes and grape musts across cultivars and vintages, and the variable and changing fermentation conditions, have not previously allowed for the prediction of a specific aromatic outcome for any given fermentation. Here we present data on the environmental factors that control the fermentation performance of mixed culture fermentations, and the impact of changes in nutritional composition and fermentation conditions on the aromatic features of wine made from synthetic must. The data show that simple nutrient compositions lead to predictable outcomes, with frequently approximately linear correlations between a precursor and a derived aromatic compound, but that more complex nutrient compositions result in non-linear and strain-dependent changes to the aromatic output. Changes to specific environmental factors such as temperature and osmotic pressure similarly lead to sometimes unexpected changes in aroma composition. However, the data suggests that multivariate models will be able to predict some of the relevant aromatic features of a wine based on the nutritional composition of the must, the fermentation conditions and the relevant yeast microbiome.
## LOWERING ETHANOL CONTENT OF WINES BY SUGAR RESPIRATION

Pilar Morales<fnref n="1" />, Virginia Rojas<fnref n="1" />,<fnref n="2" />, Manuel Quirós<fnref n="1" />,<fnref n="3" /> and Ramon Gonzalez<fnref n="1" />

Contact email: rgonzalez@icvv.es

During the last decade, the wine industry has been demanding the development of new approaches to reduce alcohol content of wines, which had been steadily increasing due to global climate change and consumer preferences towards well structured, full body wines. We recently proposed respiration of sugars by non-*Saccharomyces* yeasts for lowering ethanol yields. As a first step in order to develop an industrial process based in this proposal we characterized a collection of non-*Saccharomyces* yeast strains according to respiratory quotient under aerated conditions, at low pH and high sugar concentrations, yields of ethanol and other relevant metabolites, and growth responses to the main stress factors found during the first stages of alcoholic fermentation. One strain of *Metschnikowia pulcherrima* was selected for a proof of concept. The optimized procedure involved the simultaneous inoculation of one strain of *Saccharomyces cerevisiae* and that strain of *M. pulcherrima*, and controlled aeration during the first two days after inoculation of a natural white grape must. One essential optimization criterion was controlling acetic acid production, which tended to increase with increasing aeration of the must. We have demonstrated the crucial role of oxygen availability and respiratory metabolism in order to reduce alcohol levels. By choosing and optimizing the appropriate gassing conditions we managed to find a good balance between alcohol level reduction (3-4 degrees), the increase in volatile acidity mostly associated to growth of *S. cerevisiae* under aerobic conditions, and levels of dissolved oxygen during the process. The implementation at the industrial level of a strategy to lowering ethanol content of wine based on the respiratory breakdown of sugars by non-*Saccharomyces* yeasts would probably require further optimization involving yeast species and strain selection, inoculation strategies, oxygenation conditions, or fermentation nutrients.

<footnote n="1">¹ Instituto de Ciencias de la Vid y del Vino (CSIC, Universidad de la Rioja, Gobierno de La Rioja). Spain</footnote>

<footnote n="2">² Facultad de Ciencias Químicas. Universidad de Autónoma de San Luis Potosí. Mexico.</footnote>

<footnote n="3">³ Evolva Biotech A/S, Copenhagen, Denmark</footnote>
CHENIN BLANC WINE PRODUCTION WITH *Torulaspora delbrueckii* AND
*Saccharomyces cerevisiae* YEASTS

Neil Jolly<fnref n="1" />, Valmary van Breda<fnref n="1" /> and Mardé Booyse<fnref n="2" />

Contact email: jollyn@arc.agric.za

Non-*Saccharomyces* yeasts can have a positive role to play in wine fermentation and *Torulaspora delbrueckii* was one of the first species to be commercialised. In this study, two *T. delbrueckii* isolates were compared to commercial *T. delbrueckii* strains for small-scale production of Chenin Blanc wines over four vintages. The fermentation treatments included a *S. cerevisiae* reference, *T. delbrueckii* only, and *T. delbrueckii* and *S. cerevisiae* co-inoculated at time zero and 24 hours into fermentation. Fermentations were monitored by CO₂ mass loss and yeast cell counts. Fermentation speeds were the fastest for the co-inoculated fermentations and slowest for the *T. delbrueckii* on their own. Analyses on wines included standard chemical, sensory and volatile aromatic compounds. The *T. delbrueckii* only fermentations usually had higher residual sugar levels than the co-inoculated fermentations. Principle component and discriminant analyses of the data showed that vintage was a greater driver of differences between wines than fermentation treatments. However, per vintage, single, co-inoculated wines at zero hours, and co-inoculated wines at 24 hours grouped separately, indicating stylistic difference between fermentation treatments.

<footnote n="1">¹ ARC Infruitec-Nietvoorbij, Private bag X5026, Stellenbosch, South Africa</footnote>

<footnote n="2">² Biometry Unit, Private Bag X5013, Stellenbosch, 7600, South Africa</footnote>
SELECTED *Saccharomyces cerevisiae* AND *Torulaspora delbrueckii* STRAINS TO
IMPROVE THE ANALYTICAL PROFILE OF CRAFT BEER

Canonico L., Comitini F. and Ciani M.

Università Politecnica delle Marche, Department of Life and Environmental Sciences,
Ancona Italy l.canonico@univpm.it

In the present study it was investigated on the influence of *Saccharomyces cerevisiae* and *Torulaspora delbrueckii* strains on the analytical and sensorial profiles of craft beer.
In the first part of the work we investigated on the influence of the yeast cultures used in the refermentation process. After a preliminary screening of 33 S. *cerevisiae* strains, four were selected and used in bottle refermentation trials. Molecular typing, carried out to attribute the flavour profile of the refermented beers to the inoculated strains, confirmed the dominance of these inoculated strains. While the main analytical characters did not show differences among the bottle-refermented trials, distinctive and significant variations in the volatile compounds were shown. Subsequently in the second part of work, we evaluated the potential use of *Torulaspora delbrueckii* selected strains to give a peculiar bioflavoring to craft beer.
Indeed this species exhibits some positive features particularly in mixed fermentations under winemaking condition (Bely et al. 2008; Comitini et al. 2011). From a bunch of 28 strains of *T. delbrueckii*, eight (able to ferment maltose) were evaluated for their fermentative aptitude on wort. A strain that exhibited the better fermentative performance was selected and used in mixed fermentation with S. *cerevisiae* starter strain S-05 (Fermentis Lesaffre, France) at different ratio (1:1, 1:10 and 1:20) to evaluate the influence of this yeast on the analytical profile of beer. Preliminary results showed that *T. delbrueckii* in mixed fermentation positively influence the analytical profile of beer.

Bely M., Stoeckle I., Masneuf-Pomarede I., Dubourdieu D. (2008) Impact of mixed *Torulaspora delbrueckii*-*Saccharomyces cerevisiae* culture on high-sugar fermentation. Int J Food Microbiol, 122: 312-320.

Comitini F., Gobbi M., Domizio P., Romani C., Lencioni L., Mannazzu I., Ciani M. (2011) Selected non-Saccharomyces wine yeasts in controlled multi starter fermentations with Saccharomyces cerevisiae. Food Microbiol, 28: 873-882.