LABORATORY BREWING UNDER LARGE-SCALE PRESSURE VS. INDUSTRIAL BREWING: MICROARRAY PHENOTYPING OF YEAST DURING SERIAL REPITCHING

Jure Zupan<fnref n="1" />, Aleš Ogrinec<fnref n="1" />, Neža Čadež<fnref n="1" />, Nataša Kočar<fnref n="2" />, Sonja Smole Možina<fnref n="1" />, Peter Raspor<fnref n="3" />

Serial recycling of yeast biomass, i.e. repitching, has been a common practise in brewing, mainly due to economic reasons. Repitching is usually performed 6 to 12 times, because of possible yeast aging effects on beer quality. However, the rationale behind these aging effects is not understood completely. There have been reports of extensive repitching in laboratory conditions, which did not show any significant cell damage in yeast. Our aim was therefore to monitor any changes in yeast physiology along the repitching cycles. Moreover, the monitoring was performed in a large-scale fermenter up to 3250 hL along 15 repitching cycles and in small-scale laboratory fermenters of 35 ml along 20 repitching cycles. The laboratory fermenter system was constructed with the aim to mimic the industrial process as much as possible. Fermentations were performed in conical Falcon tubes at 14 °C. To mimic hydrostatic and CO₂ pressure of the industrial fermenter, the system was connected to a gas supply (20 % CO₂ in nitrogen) at the pressure of 1.1 atm. One day after each repitching, the following tests on yeast biomass were performed: cultivability (CFU), viability (methylene-blue staining), vitality (Live-Dead kit), optical density, cell counting (haemocytometer and ImageJ software), and at the selected repitching cycles also the phenotypic microarray analysis using the Omnilog system. At the end of fermentations the level of fermented sugars, pH and flocculation were determined. Regarding the cautiousness around the eighth repitching in breweries, we observed no significant changes in yeast physiology in either industrial or laboratory experiments at this point. In fact, first deficiencies in metabolism were observed after the fifteenth repitching.

<footnote n="1">¹ Biotechnical Faculty, University of Ljubljana, Jamnikarjeva 101, 1000, Ljubljana, Slovenia</footnote>

<footnote n="2">² Pivovarna Laško d. d., Laško, Slovenia</footnote>

<footnote n="3">³ Faculty of Health Sciences, University of Primorska, Polje 42, 6310 Izola, Slovenia
Jure Zupan, jure.zupan@bf.uni-lj.si</footnote>
EXPLORATION OF YEAST BIODIVERSITY FOR POTENTIAL IMPROVEMENTS OF
*RIBOLLA GIALLA* WINE AROMA PROFILE

Sofia Dashko<fnref n="1" />, Tinkara Tinta<fnref n="2" />, Paolo Sivilotti<fnref n="1" />, Melita Sternad Lemut<fnref n="1" />, Kajetan Trošt<fnref n="1" />,<fnref n="3" />,
Amparo Gamero<fnref n="4" />, Urska Vrhovsek<fnref n="3" />, Lorena Butinar<fnref n="1" />, Helena Volk<fnref n="5" />, Nerve Zhou<fnref n="5" /> and Jure
Piškur<fnref n="1" />,<fnref n="5" />

Contact email: Sofia Dashko [sofiadashko@gmail.com]

During the last decades only a limited number of yeast species have been used in commercial wine fermentations. The EUITN project Cornucopia has recently attempted to reverse this trend and screened a few hundred yeast isolates to identify novel species with a promising aroma potential, which could be employed in wine fermentations. We applied some of these yeasts in *Ribolla Gialla* must fermentations, with the future aim to intensify the aroma profile of this wine. In our experiments, we performed sequential micro-fermentations starting with a novel yeast species, and after 60 hours a commercial *Saccharomyces cerevisiae* wine strain was added. Growth rate, competition between the inoculated yeasts, sugar consumption, ethanol accumulation and aroma profile were monitored during these experimental fermentations. Sensory analyses of young wine showed that particularly one of the novel yeasts, *Kazachstania gamospora*, enriched the aroma profile of the fermented *Ribolla* must, and has also fermentative properties, which qualify it for future large-scale wine fermentation trials.

<footnote n="1">¹ University of Nova Gorica, Slovenia;</footnote>

<footnote n="2">² National Institute of Biology, Slovenia;</footnote>

<footnote n="3">³ Fondazione Edmund Mach, Italy:</footnote>

<footnote n="4">⁴ NIZO Food Research, the Netherlands;</footnote>

<footnote n="5">⁵ Lund University, Sweden</footnote>
EVOLUTIONARY AND REVERSE ENGINEERING OF COMMERCIALLY IMPORTANT
METABOLIC TRAITS IN WINE YEAST

Axelle Cadiere, Valentin Tilloy, Carole Camarasa, Frédéric Bigey, Stéphane Guézenec, Brigitte Cambon, Virginie Galeote and Sylvie Dequin

INRA, UMR1083 SPO, F-34060 Montpellier, France

Contact email: sylvie.dequin@supagro.inra.fr

The wine industry faces a series of challenges related to globalization, new market trends, technological and climate change. In this context, there is a high demand for the development of microbial strategies to develop *Saccharomyces cerevisiae* yeast strains with novel traits. Non-targeted approaches such as evolutionary engineering are alternative strategies to genetic engineering for reprogramming metabolic networks. Using this approach, we obtained evolved wine yeasts with improved gluconate utilization. The evolved strains had increased flux through the pentose phosphate pathway and produced higher amount of esters, which contribute to the fruity and floral aroma of wine. Another example was the selection, by a combination of experimental evolution and conventional breeding, of an evolved strain that produce substantially more glycerol and less ethanol. In pilot-scale experiments, this strain efficiently decreases the ethanol content of wines by 0.9 to 1.3%, while producing less acetate and more 2,3-butanediol. These evolved strains represent novel tools to improve the sensorial quality of fermented beverages. Besides, a major challenge is to elucidate the genetic bases of the evolved phenotypes, which will enable their transfer to other industrial strains. Using genome-wide approaches, we identified the evolutionary genomic variations of evolved strains with improved gluconate utilization. We showed that pleiotropic metabolic phenotypes have been acquired by a dominant, loss-of-function mutation of *BCY1*, a gene controlling the cAMP-dependent protein kinase (PKA) nutrient signaling pathway that play major roles in yeast growth, metabolism and stress response.
CHARACTERIZATION OF GENES INVOLVED IN THE ADAPTATION OF FLOR
STRAINS TO BIOLOGICAL AGEING CONDITIONS OF SHERRY WINES

<u>Anna Lisa Coi</u><fnref n="1" />, Jean-Luc Legras<fnref n="2" />, Frédéric Bigey<fnref n="2" />, Virginie Galeote<fnref n="2" />, Ilaria Mannazzu<fnref n="1" />,
Marilena Budroni<fnref n="1" /> and Sylvie Dequin<fnref n="2" />

Contact email: acoi@uniss.it

*Saccharomyces cerevisiae* is responsible of a lot of biotechnological processes essential for human feeding, as fermentation of wine, beer and bread. In contrast with these fermentation lifestyles, during the biological ageing of Sherry wines *S. cerevisiae* flor strains grow aerobically at the wine surface forming a biofilm. This distinctive feature allows cellular population to overcome stress conditions, like nitrogen and sugar depletion, after the end of fermentation. With the aim to detect alleles involved in flor yeast adaptation to biological ageing, the genomes of 7 flor and 9 wine strains have been sequenced. The comparison of flor and wine genomes revealed several divergent regions. These regions include genes from different functional categories such as cell wall proteins, *FLO11* regulation, homeostasis of metal ions which present numerous allelic variations, including SNPs and recombination. Strikingly, the many genes involved in metal ions homeostasis suggest an important role in the adaptive evolution of flor strains. We have performed the allelic replacement of different flor and wine alleles for several divergent genes and evaluated the effect of the allelic replacement on their phenotypes, respectively, in wine and flor haploid strains.

Acknowledgements:

Anna Lisa Coi gratefully acknowledges Sardinia Regional Government for the financial support of her PhD scholarship (P.O.R. Sardegna F.S.E. Operational Programme of the Autonomous Region of Sardinia, European Social Fund 2007-2013 - Axis IV Human Resources, Objective 1.3, Line of Activity 1.3.1.).

<footnote n="1">¹ Università degli studi di Sassari, Sassari, Italy</footnote>

<footnote n="2">² INRA SupAgro UMR1083 SPO, Montpellier, France</footnote>
THE INFLUENCE OF DIFFERENT YEAST INOCULUM LEVELS ON THE QUALITY
OF CIDER

<u>Hüseyin Erten</u><fnref n="1" />, Hasan Tangüler<fnref n="1" />,<fnref n="2" />, Bilal Ağırman<fnref n="1" />, Cennet Pelin Boyacı Gündüz<fnref n="1" /> and Turgut Cabaroğlu<fnref n="1" />

Contact email: herten@cu.edu.tr

In this study, the impact of the different inoculum levels of commercial dry yeast on the yeast growth, fermentation rate, ethyl alcohol and flavour compounds formation was investigated. Increasing the inoculum level increased the fermentation rate. Higher inoculation rate led to higher maximum yeast cell number in the culture. By using commercial Actiflore PM and Uvaferm CM yeasts, ethyl alcohol levels ranged from 5.84% (v/v) to 6.27% (v/v) and from 6.08 % (v/v) to 6.41% (v/v), respectively. The concentrations of flavour compounds of ciders depend on the commercial yeast strain and inoculum levels. The highest amounts of higher alcohols were found in the experiments inoculated with 1x10<sup>7</sup> cells/mL using Actiflore PM commercial yeast, esters inoculated with 1x10<sup>6</sup> and 1x10<sup>7</sup> cells/mL and carbonyl compounds pitched with 5x10<sup>6</sup>cells/mL. The highest concentrations of higher alcohols, esters and carbonyl compounds were determined in the ciders inoculated with 1x10<sup>5</sup> cells/mL using Uvaferm CM commercial yeast.

Acknowledgements: This study was supported by Cukurova University Academic Research projects Unit (Project No: ZF2007BAP44).

<footnote n="1">¹ Cukurova University, Faculty of Agriculture, Department of Food Engineering, Adana, 01330, Turkey</footnote>

<footnote n="2">²Nigde University, Faculty of Engineering, Department of Food Engineering, Nigde, 51240, Turkey</footnote>
IMPACT OF INOCULUM PREPARATION ON MICROBIAL CONSORTIA,
FERMENTATION KINETICS AND PRODUCT COMPOSITION IN TRADITIONAL
MEZCAL FERMENTATIONS

Manuel Kirchmayr¹, Melchor Arellano¹, Laura Delage¹ and Anne Gschaedler¹

¹ Centro de Investigación y Asistencia en Tecnología y Diseño del Edo. de Jalisco A.C.,
Área de Biotecnología Industrial, Av. Normalistas 800, 44270 Guadalajara, Mexico
Contact email: mkirchmayr@ciatej.mx

Mezcal, a traditional Mexican spirit obtained by distillation of fermented cooked agave juices is nowadays gaining more popularity on the market of beverages. The principal problem of most mezcal producers consists in carrying out spontaneous fermentations that last sometimes more than two weeks affecting strongly the quality of the product. These fermentations are characterized by the presence of complex microbial consortia, where bacterial populations exceed up to 100 times the concentration of yeasts. As a consequence of this fact it is commonly observed that the acidity of the final product exceeds the maximum levels fixed by the official Mexican Norm for mezcal.

In order to control the contribution of the bacteria to the chemical composition of the agave musts, a simple “artisan” inoculum was prepared with the native microflora applying conditions that favor the growth of yeast rather than bacteria. In three different mezcal factories in Guerrero state, two fermentations were carried out in parallel (with and without inoculum) in order to study the effect of this practice on the microbial consortia, fermentation kinetics and chemical composition of the fermented agave musts. The evolution of bacterial and yeast populations and of the generated aroma compounds as organic acids, esters and alcohols was monitored with PCR-DGGE and HPLC/GC, respectively.

Although the maximum yeast concentrations could apparently not be increased by this practice and the bacterial populations still were dominant, clear differences were observed in the production of organic acids and other compounds important for the organoleptic profile of the mezcal, particularly, the production of lactic acid could be clearly reduced. Moreover the fermentation time was shorter and the conversion rates of sugars into ethanol were improved when an inoculum was prepared. This work exemplifies the possibility to improve traditional fermentations introducing modern practices without ruining the artisan image of these processes.
EFFECT OF THE AGAVE SAPONINS ON THE CELL WALL COMPOSITION OF
*Saccharomyces cerevisiae* AND *Kluyveromyces marxianus* YEASTS STRAINS IN
CONTINUOUS CULTURE

Montserrat Alcazar¹, Anne Gschaedler¹, Javier Arrizon¹, Amairani Alonzo¹ and Eugenia Lugo¹

¹ Centro de Investigación y Asistencia en Tecnología y Diseño del Estado de Jalisco A.C., Normalistas 800 Col. Colinas de la Normal, Guadalajara, Jalisco, C.P 44270, México
Contact email: agschaedler@ciatej.mx

The main secondary metabolites isolated from the *Agave* leaves, are steroidal saponins, these
metabolites are also found in less proportion in the agave juice that is used in the
fermentation process for the production of alcoholic beverages in Mexico. Although the
saponins are present in less proportion in the agave juices they inhibit the growth of yeast
during the fermentation process, however the mechanisms of the cell growth inhibition are
poorly understood. The aim of this work is to study the effect of saponins on the cell wall
composition (β1-3 glucan, β1-6 glucan and mannoproteins) of the fermentative yeast
*Saccharomyces cerevisiae* and *Kluyveromyces marxianus* isolated from the tequila and
mezcal fermentations process respectively. The analysis was performed in a continuous
culture with saponins pulses (0.75, 1.5 and 3 mg/ml final concentration in reactor medium)
extracted from agave leaves of *Agave durangensis* and *A. salmiana* ssp. *crassispina*. It was
observed changes on the cell wall composition in *S. cerevisiae* at 3 mg/ml of *A. salmiana*
saponin extract where the ratio of mannan/glucan change from 1:15 to 1:6, and in *K.
marxianus* at 3 mg/ml of *A. durangensis* saponin extract where the ratio of mannan/glucan
change from 1:8 to 1:50, these difference compare with scanning eletron microscopy
observation shown that *K. marxianus* is more tolerant and have the ability to growth in
stressful environment.
NEW METHOD FOR FAST SEPARATION OF MAGNETIZED YEAST
*Saccharomyces bayanus* IN SPARKLING WINE PRODUCTION

Marin Berovič<fnref n="1" /> and Darko Makovec<fnref n="2" />

A novel method for fast magnetic separation of *Saccharomyces bayanus* yeast cells from sparkling wine was developed. The wine yeast cells were made responsive to a magnetic field with absorption of superparamagnetic nanoparticles of iron oxide maghemite (γ-Fe₂O₃) coated with a thin layer of silica and grafted with (aminoethylamino) propylmethyldimethoxy silane (APMS). The terminal amino groups of the APMS molecules provides a positive charge at the nanoparticles' surfaces and promotes their electrostatic absorption onto the negatively-charged surfaces of the yeast cells. The influence of the magnetic nanoparticles on yeast metabolism, as well as separation of magnetic yeast cells biomass in the magnetic field was studied. The optimal mass ratio between the magnetic nanoparticles and the wine yeast was determined to be 1:10. Scanning and transmission electron microscopy showed that the magnetic nanoparticles remained fixed at the microbial cell surfaces even after fermentation. The results of chemical analysis showed that besides faster microbial kinetics there were no other influences on the cell metabolism. The same results were confirmed in sensorial analyses of sparkling wine. Separation of the magnetized waste yeast biomass into the neck of the bottle using a relatively weak magnetic-field gradient can be successfully completed in approximately 15 minutes.

<footnote n="1">¹ Faculty of Chemistry and Chem.Technnol., University of Ljubljana, Aškerčeva 5, 1001 Ljubljana, Slovenia</footnote>

<footnote n="2">² Institut Jožef Stefan, 1001 Ljubljana, Slovenia
Contact e-mail: marin.berovic@fkkt.uni-lj.si</footnote>
EXPLORING A YEAST GENETIC RESOURCE COLLECTION TO AID BIOREFINERY PRODUCTION OF BIOBASED CHEMICALS FROM AGRIFOOD FEEDSTOCKS

Ian Roberts<fnref n="1" />, Adam Elliston<fnref n="2" />, Steve James<fnref n="1" />, Jo Dicks<fnref n="1" /> and Keith Waldron<fnref n="2" />

Contact email: ian.roberts@ifr.ac.uk

Biorefinery operations have generally focussed on production of biofuels to reduce dependency on fossil fuels. However, replacement fuels, whilst produced in high volume, are currently of low financial value. A further biorefining objective therefore focusses on achieving economic sustainability through co-production of higher value biobased chemicals. Production of such chemicals using yeast has already proved economically viable. Various organic acids, industrial surfactants and carotenoids are already produced commercially. However, the challenge remains to improve production efficiency and develop a wider range of biorenewable chemicals and feedstocks using approaches that do not conflict with food production. Here, we explore whether the UK National Collection of Yeast Cultures (NCYC) contains hitherto untapped genes and pathways that could play a role in yeast strain development for such purposes. We describe methodologies for screening the entire NCYC collection to make improved use of agrifood feedstocks such as Oil Seed Rape Straw (OSRS) and comparable bio-waste material.

<footnote n="1">¹National Collection of Yeast Cultures, Institute of Food Research, Norwich Research Park, Colney, Norwich, NR4 7UA United Kingdom</footnote>

<footnote n="2">²Biorefinery Centre, Institute of Food Research, Norwich Research Park, Colney, Norwich, NR4 7UA United Kingdom</footnote>
CHARACTERIZATION OF A NOVEL NON-GMO YEAST FOR FUTURE
LIGNOCELLULOSIC BIOETHANOL PRODUCTION

Cecilia Geijer<fnref n="1" />, David Moreno<fnref n="1" />, Elia Tomas Pejo<fnref n="1" />, <fnref n="2" />, Lisbeth Olsson<fnref n="1" />

Contact details: cecilia.geijer@chalmers.se

Concerns about climate change and the uncertainty about future fuel supply make renewable biofuels, such as bioethanol, attractive alternatives to fossil fuels in the short/medium term. Lignocellulosic biomass (for example spruce, wheat straw and corn stover) is an abundant raw material that can be utilized to produce ethanol with the help of a fermenting microorganism. Traditionally the yeast *Saccharomyces cerevisiae* is used for industrial ethanol production. *S. cerevisiae* can be metabolically engineered to consume xylose (the second to glucose most prevalent monosaccharide in lignocellulose). However, despite many years of intensive research, it can still not ferment xylose in a satisfying way which affects the overall ethanol yield negatively. We have isolated a non-genetically modified (non-GMO) yeast species (here called C5-yeast) that has the natural ability to efficiently produce ethanol from glucose and xylose. The aim of the project is to further characterize the growth and fermentation capacities of this novel microorganism to elucidate its' potential for lignocellulosic bioethanol production. We can show that besides glucose and xylose, the C5-yeast can also consume the pentose arabinose and the disaccharide cellobiose; both present in lignocellulosic hydrolysates. The C5-yeast rapidly converts the inhibitory sugar degradation products HMF and furfural formed during the conversion of lignocellulosic material into fermentable sugars.

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

<footnote n="2">²Unit of Biotechnological Processes for Energy Production, IMDEA Energy, Móstoles
(Madrid), Spain</footnote>
MALTOTRIOSE UTILIZATION IN BREWER'S YEAST WITH RESPECT TO TRANSPORTER GENE PROVENANCE

Virve Vidgren¹, John Londesborough¹ and Brian Gibson¹

¹ VTT Technical Research Centre of Finland, P.O. Box 106, FI-20521, VTT, Finland
Email: virve.vidgren@vtt.fi

During wort fermentation, transmembrane transport is the rate limiting factor for efficient utilization of maltotriose. Quite often residual maltotriose remains after fermentation, causing economic losses and compromising quality. The lager yeast (*Saccharomyces pastorianus*) taxon apparently arose through interspecific hybridization of an ale (*S. cerevisiae*) and an *S. eubayanus* strain and possesses sugar transporter genes derived from both parents. However, usually only one of the sugar transporter orthologues is functional, whereas the other has gained mutations leading to pseudogenization. Transporters able to carry maltotriose are therefore distributed differently between ale and lager yeast strains as well as between the two subgroups within the lager yeast, namely Saaz and Frohberg. This affects the strains' fermentation capacity, e.g. in ale strains ScAgt1 type transporters, which are efficient maltotriose carriers, dominate, whereas in lager strains of the Frohberg type the presence of Mtt1 and SbAgt1 transporters is crucial for maltotriose utilization. Lager strains of the Saaz type do not possess any functional maltotriose transporters and leave this sugar unfermented. Fermentation capacity of brewer's yeast strains can be significantly improved by modifying characteristics of maltotriose transporters. Characterization of properties of individual maltotriose transporters and their distribution among brewer's yeast is helping to elucidate the factors that limit maltotriose utilization as well as suggesting methods to improve strains for superior performance.
DELIMITATION OF *Meyerozynma guilliermondii* FOOD ISOLATES FOR IMPROVED SAFETY IN FOOD INDUSTRY

Claudia Colabella<fnref n="1" />, Luca Roscini<fnref n="1" />, Raffaella Di Cagno<fnref n="2" />, Laura Corte<fnref n="1" />, Marco Gobbetti<fnref n="2" /> and Gianluigi Cardinali<fnref n="1" />

Contact email: claudia.colabella@gmail.com

*Meyerozyma guilliermondii* is a yeast species widely isolated from several natural environments and from fruits. In medical microbiology it is known as the telomorph of the opportunistic pathogen *Candida guilliermondii*, which causes about 2% of the human blood infections. This yeast is promising in a variety of biotechnological applications as vitamins production and post-harvest control. The question if isolates from different sources are physiologically and genetically similar, or if the various environments induced significant differences, is crucial for the understanding of this species structure and to select strains appropriate for each application. This question was addressed using LSU and ITS sequencing for taxonomic assignment, i-SSR (GACA₄) for the molecular characterization and FTIR for the metabolomic fingerprint. All data showed that fruit and environmental isolates cluster separately and a general good agreement between metabolomics and molecular analysis. An additional RAPD analysis was able to discriminate strains according to the isolation position within the pineapple fruit. Although all strains are members of the *M. guilliermondii* species according to the current standards, the distribution of large variability detected suggests that some specialization occurred in the niches inhabited by this yeast and that food related strains can be differentiated from the medical isolates.

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

<footnote n="2">² Dipartimento di Scienze del suolo, della pianta e degli alimenti, University of Bari, Via Amendola 165/A Bari, Italy</footnote>
OPPORTUNITIES ARISING FROM THE GENOME SEQUENCING OF FOUR
*Aureobasidium* VARIETIES

Cene Gostinčar<fnref n="1" />,<fnref n="2" />, Robin A. Ohm<fnref n="3" />, Tina Kogej<fnref n="1" />, Silva Sonjak<fnref n="1" />, Martina Turk<fnref n="1" />, Janja Zajc<fnref n="1" />, Polona Zalar<fnref n="1" />, Martin Grube<fnref n="4" />, Hui Sun<fnref n="3" />, James Han<fnref n="3" />, Aditi Sharma<fnref n="3" />, Jennifer Chiniquy<fnref n="3" />, Chew Yee Ngan<fnref n="3" />, Anna Lipzen<fnref n="3" />, Kerrie Barry<fnref n="3" />, Igor V. Grigoriev<fnref n="3" />, Nina Gunde-Cimerman<fnref n="1" />,<fnref n="5" />

Contact email: cene.gostincar@nib.si

*Aureobasidium pullulans* is a black yeast from the order Dothideales. The species is of considerable interest due to several reasons: (i) polyextremotolerance and occurrence in numerous habitats from plant surfaces and household dust to coastal ponds of hypersaline water and Arctic glaciers; (ii) many biotechnological applications (production of polysaccharides, enzymes, antimicrobials) and use in agriculture as a biocontrol agent; (iii) emerging medical relevance.

We performed a *de novo* genome sequencing and analysis of the four recognised varieties of the species (*A. pullulans* var. *pullulans*, var. *melanogenum*, var. *subglaciale*, var. *namibiae*). Annotation of the genes was performed with the help of the transcriptome sequencing. The differences between varieties and with other phylogenetically related fungi were analysed on the genomic and (predicted) proteomic levels. The 25.43-29.62 Mb genomes encode between 10266 and 11866 predicted proteins. These include genes for various enzymes involved in the degradation of the plant material (and possibly also of plastic and aromatic compounds) and many sugar transporters. Proteins believed to be involved in the synthesis of the polysaccharide pullulan and of the siderophores are predicted. Putative stress-tolerance genes include those encoding several membrane channels and transporters, melanin synthesis enzymes, all of the components of the high-osmolarity glycerol pathway, and bacteriorhodopsin-like proteins.

The structure of the mating-type locus is homothallic, although no sexual reproduction has been observed in this species before. The differences between the genomes are large enough to justify their redefinition as separate species.

The availability of the genomic sequence of *A. pullulans* is expected to facilitate research into this interesting species and promote its use in the fields of biotechnology and agriculture.

<footnote n="1">¹ Department of Biology, Biotechnical Faculty, University of Ljubljana, Večna pot 111, SI-1000 Ljubljana, Slovenia</footnote>

<footnote n="2">² National Institute of Biology, Večna pot 111, SI-1000 Ljubljana, Slovenia</footnote>

<footnote n="3">³ US Department of Energy Joint Genome Institute, 2800 Michell Drive, Walnut Creek, CA 94598, USA</footnote>

<footnote n="4">⁴ Institute of Plant Sciences, Karl-Franzens-University Graz, Holteigasse 6, A-8010 Graz, Austria</footnote>

<footnote n="5">⁵ Centre of Excellence for Integrated Approaches in Chemistry and Biology of Proteins (CIPKeBiP), Jamova 39, SI-1000 Ljubljana, Slovenia</footnote>
DEVELOPMENT OF MICROBIAL CELL FACTORIES FOR BIOREFINERIES

Akihiko Kondo

Department of Chemical Science and Engineering, Graduate School of Engineering, Kobe
University, 1-1 Rokkodai, Nada, Kobe 657-8501, Japan

Contact email: akondo@kobe-u.ac.jp

To build an energy and material secure future, a next generation of renewable fuels produced from lignocellulosic biomass is required. Although lignocellulosic biomass, which represents an abundant, inexpensive and renewable source for bioethanol production, is of great interest as a feedstock, the complicated ethanol production processes involved make the cost of producing bioethanol from it higher compared to corn starch and cane juice. Therefore, consolidated bioprocessing (CBP), which combines enzyme production, saccharification and fermentation in a single step, has gained increased recognition as a potential bioethanol production system. CBP requires a highly engineered microorganism developed for several different process-specific characteristics. The dominant strategy for engineering a CBP biocatalyst is to express multiple components of a cellulolytic system from either fungi or bacteria in the yeast Saccharomyces cerevisiae. The development of recombinant yeast strains displaying cellulases and hemicellulases on the cell surface represents significant progress toward realization of CBP. Regardless of the process used for biomass hydrolysis, CBP-enabling microorganisms encounter a variety of toxic compounds produced during biomass pretreatment that inhibit microbial growth and ethanol yield. Systems biology approaches including disruptome screening, transcriptomics, and metabolomics have been recently exploited to gain insight into the molecular and genetic traits involved in tolerance and adaptation to the fermentation inhibitors. In this review, we focus on recent advances in development of yeast strains with both the ability to directly convert lignocellulosic material to ethanol and tolerance in the harsh environments containing toxic compounds in the presence of ethanol.
BREEDING INDUSTRIAL *Saccharomyces cerevisiae* STRAIN FOR LIGNOCELLULOSIC ETHANOL PRODUCTION

Yue-Qin Tang, Kenji Kida

College of Architecture and Environment, Sichuan University, Chengdu, Sichuan
610065, China

Contact email: tangyq@scu.edu.cn

Using an industrial *Saccharomyces cerevisiae* strain as the parental strain, strain for lignocellulosic bioethanol production was constructed. By introducing *XYL1*, *XYL2*, and *XKS1* genes, strain with xylose-fermenting ability was constructed. The strain could grow using xylose as the sole carbon source. Under normal batch fermentation condition, 50 g/L of xylose could be consumed within 36 h by the strain. By overexpression of *HXT7* and *GXS1* genes, xylose consumption rate of the strain was improved. Continuous fermentation fed with 75 g/L of xylose kept stable at a dilution rate of 0.1 h⁻¹. The xylose-fermenting capacity maintained stable during one-year continuous operation. By site directed mutagenesis, the affinity of xylose reductase to NADH was increased and strains with improved xylose consumption and ethanol yield were obtained. Tolerance of these strains to inhibitors (weak acids, furans, aldehydes, phenols) generated during pretreatment process was assessed systematically. The inhibitor-tolerance of strains was improved by overexpression of key genes in PPP pathway and acclimation under inhibitor-stressed conditions.
DETOXIFICATION AS A STRATEGY FOR DEVELOPING TOLERANCE IN
*Saccharomyces cerevisiae* TO PHENOLIC COMPOUNDS

Peter Temitope Adeboye, Maurizio Bettiga, and Lisbeth Olsson

Industrial Biotechnology, Dept. of Chemical and Biological Engineering, Chalmers University of Technology, Gothenburg.

E-mail: adeboye@chalmers.se

Several phenolic compounds are formed as products of lignin breakdown during pretreatment of lignocellulosic biomass. These phenolic compounds are inhibitory to cell growth and function as biocatalysts in the production of second generation biofuels from degraded lignocellulosic biomass. Our research is focused on developing a *Saccharomyces cerevisiae* strain with improved resistance to phenolic compounds.

As part of our study, we have focused on understanding the ability of *S. cerevisiae* to tolerate and convert phenolic compounds. We aim to understand the conversion mechanisms of phenolic compounds and adapt the knowledge to the engineering and use of *S. cerevisiae* on a biotechnological platform for bioethanol production and prospective, novel bio-based chemicals.

We have investigated toxicity of various phenolic compounds against *S. cerevisiae*. Our results showed that phenolic compounds have varied toxicity against *S. cerevisiae* and the toxicity may be dependent on the structure of the compound involved. Under aerobic batch cultivation conditions, we have also studied the conversion of phenolic compounds by *S. cerevisiae* using coniferyl aldehyde, ferulic acid and *p*-coumaric acid as representative phenolic compounds. We compiled a list of conversion products of the three starting compounds under investigation and we proposed a possible conversion pathway, currently being investigated.

In this talk, we present the proposed conversion pathway through which *S. cerevisiae* converts and detoxifies coniferyl aldehyde, ferulic acid and *p*-coumaric acid under aerobic cultivation condition.
ENGINEERING GLUTATHIONE BIOSYNTHESIS TO ENHANCE REDOX
ROBUSTNESS OF *Saccharomyces cerevisiae*

<u>Christian Marx</u>¹, Lisbeth Olsson¹ and Maurizio Bettiga¹

¹ Dept. of Chemical and Biological Engineering, Chalmers University of Technology, Göteborg, Sweden

Contact email: christian.marx@chalmers.se

The focus for biofuel production shifts to using lignocellulose biomass from forest and
agricultural by-products since it does not compete with food and feed production.
Polysaccharides must be pretreated to be made accessible to hydrolytic enzymes to generate
monomeric sugars for the following fermentation. In this pretreatment step inhibitors of
fermenting microorganisms are generated, mainly furan derivates, weak acids and phenolics.
Although *Saccharomyces cerevisiae* is more robust than bacteria, there is demand for
improvement and the development of novel yeast strains with increased inhibitor tolerance is
highly desirable.

Furan derivates and other inhibitors have been shown to induce the formation of reactive oxygen species. Engineering of the redox metabolism of *S. cerevisiae* in terms of increasing the intracellular levels of glutathione by overexpressing glutathione synthetase *GSH1* resulted in increased strain robustness in a simultaneous saccharification and fermentation (SSF) process. Cell survival and final ethanol concentrations were increased in the recombinant strains compared to the wild type in industrial media [Ask et al. 2013].

To show a correlation between the intracellular concentration of glutathione and the resulting effect on robustness, strains accumulating different amounts of glutathione will be created. GshF is a bi-functional enzyme found in several bacterial species, that catalyzes the formation of glutathione from its precursors without accumulation of the intermediate product γ-glutamylcysteine and without any relevant feedback inhibition. GshF will be overexpressed in a CEN.PK strain, followed by deletion of the native GSH1 and GSH2 enzymes catalyzing the two-step reaction in *S. cerevisiae*.

Reference:

Ask, M., Mapelli, V., Höck, H., et al., 2013, Microbial Cell Factories 12, 87-97.
*Candida glabrata* SHOWS VERY HIGH RESISTANCE FOR HIGHLY CONCENTRATED SULFURIC ACID AND THE TOLERANCE IS USEFUL FOR ETHANOL PRODUCTION

Takayuki Mizuno<fnref n="1" />, Masanori Bun-ya<fnref n="1" />, Hiroji Chibana<fnref n="2" />, Atsushi Maeda<fnref n="1" /> and Koichi Mita<fnref n="1" />

Contact email: mizuno@fe.bunri-u.ac.jp

*Candida glabrata*, an imperfect fungus, resembles *Saccharomyces cerevisiae* in morphology and gene sequencing. However, *C. glabrata* is only a haploid cell and is unable to mate with another. It is known that *C. glabrata* is yeast bringing opportunistic infection. There are increasing concerns over *Candida*, because it causes not only mucocutaneous but also systemic infections in transplant and immunosuppressed patients. The other side of *C. glabrata*, is a superior strain for fermentative production of pyruvate.

*C. glabrata* has loud fermentation power and is very useful industrially. The growth rate is extremely fast in comparison with *S. cerevisiae*. *C. glabrata* can also grow under high temperature condition (42-44 degrees). Furthermore, surprisingly *C. glabrata* shows strong tolerance for the sulfuric acid. When the ethanol is made from cellulose, highly-concentrated sulfuric acid is used for saccharification of the cellulose. We found that *C. glabrata* has tolerance to strong sulfuric acid, and at the same time, increase of the production of ethanol efficiency was seen. Furthermore, the *C. glabrata* can produce ethanol without being neutralized using the cellulose which I saccharified with strong sulfuric acid.

<footnote n="1">¹Nano Material & Bio Engineering, Tokushima Bunri Univ, Sanuki, Kagawa, Japan.</footnote>

<footnote n="2">²Medical Mycology Research Center (MMRC), Chiba University, Chiba, Japan</footnote>
CHALLENGES AND POSSIBILITIES WITH HIGH SOLIDS LIGNOCELLULOSE
FERMENTATION

Rakesh Koppram<sup>1,2</sup>, Charis Xiros<sup>1</sup>, Elia Tomás-Pejó<sup>1,3</sup>, <u>Lisbeth Olsson</u><sup>1</sup>

Contact e-mail: lisbeth.olsson@chalmers.se

One current research trend in lignocellulose based ethanol production is to increase the solid substrate loading in order to increment the final ethanol concentration, leading to decreased distillation costs and improved water economy. Even though several challenges have to be addressed, this is an important research direction with broad implications towards development of lignocellulose based biorefineries.

At high solids loading of typically 15-30 (w/w) %, high viscosity prevails leading to poor mixing and therefore large deviation occur in local conditions in the fermentor. The high solid content has a direct influence on the enzymes and microorganisms. Furthermore, high concentrations of inhibitory compounds will typically be present leading to poor cellular performance and cell viability problems. In our research group we address these problems by gaining detailed physiological insights, followed by developing fermentation strategies and applying strategies for strain improvement. In this presentation, insights from physiological behavior at high solids condition will be discussed in light of designing fermentation strategies suitable for high solid conditions. We have in particular focused on how to increase the cell viability in lignocellulose based processes and to gain understanding on the influence of solid material on cellular performance.

<footnote n="1"><sup>1</sup> Chalmers University of Technology, Department of Chemical and Biological Engineering, Industrial Biotechnology Group, Göteborg, Sweden.</footnote>

<footnote n="2"><sup>2</sup> ACIB - Austrian center for Industrial Biotechnology,
Muthgasse 107, 1220 Wien, Austria</footnote>

<footnote n="3"><sup>3</sup> Unit of Biotechnological Processes for Energy Production, IMDEA Energy, Móstoles (Madrid), Spain.</footnote>
*Yarrowia lipolytica* – USEFUL TOOL FOR ENGINEERING LIPID BIOSYNTHESIS PATHWAYS

Peter Gajdoš<fnref n="1" />,<fnref n="2" />, Milan Čertík<fnref n="1" />, Jean-Marc Nicaud<fnref n="2" />

Contact email: milan.certik@stuba.sk

*Yarrowia lipolytica* is oleaginous yeast with metabolism adapted for life in protein and oil rich environments. It possesses protease and lipase activities that enable it to utilize such substances as a source of nutrients and energy. On the other hand *Y. lipolytica* lacks amylolytic and invertase activities, which are common for other microorganisms from Fungi kingdom. Its orientation towards hydrophobic substrate utilization is underlined by presence of both *DGAT1* and *DGAT2* enzymatic activities. This makes that *Y. lipolytica* is a yeast with unique storage lipid formation machinery. Taking an advantage of *Y. lipolytica*'s natural ability of lipid accumulation strains with improved substrate flow towards lipid biosynthesis and storage were constructed. In this study strains derived from Q4 strain (Δ*dga1* Δ*dga2* Δ*lro1* Δ*are1*) were constructed. Because Q4 strain is unable to synthesize storage lipids it provides us a good platform for studying impact of single acyltransferase gene on lipid synthesis. Thus was shown that proteins encoded by *DGA1* and *DGA2* genes have major TAG forming potential. Both proteins are able to mediate accumulation of significant quantities of TAGs in yeast cells, but with slightly different fatty acid composition. Based on this findings potential single cell oil producing strains were constructed. These strains were constructed from Q4 by reintroduction of either *DGA1* or *DGA2* under TEF promoter. Newly constructed strains accumulated lipids up to 50% of biomass, which was more than two times more compared to wild type. By heterologous expression of *SUC2* from *Saccharomyces cerevisiae* an invertase activity was introduced into *Y. lipolytica* which enables it to grow on sucrose based substrates such as molasses.

The work was supported by grants VEGA 1/0975/12, APVV-0662-11 and "University Science Park of STU Bratislava" ITMS 26240220084. Peter Gajdoš was supported by SAIA National Scholarship Programme.

<footnote n="1">¹ Faculty of Chemical and Food Technology, Slovak University of Technology, Bratislava,
Slovak Republic</footnote>

<footnote n="2">² INRA, Micalis, Jouy-en-Josas, France</footnote>
OPTIMIZATION OF CULTIVATION CONDITIONS FOR LIPID PRODUCTION WITH
A GENOME-SCALE MODEL OF *Yarrowia lipolytica*

Martin Kavšček¹, Govindprasad Bhutada¹, Sepp D. Kohlwein¹ and Klaus Natter¹

¹ Institute of Molecular Biosciences, University of Graz, NAWI, Humboltstrasse 50, 8010 Graz, Austria
Contact email: klaus.natter@uni-graz.at

In recent years *Yarrowia lipolytica* has become an important non-conventional yeast for basic research and for biotechnologically relevant processes. As an oleaginous yeast, it might be used for single cell oil production. Besides, it is a good host for the production of citric acid and other intermediates of the tricarboxylic acid cycle. The availability of a sequenced genome and tools for genetic engineering are promising even greater possibilities for usage of this GRAS species in metabolic engineering. By using a validated genome scale model of baker's yeast as a scaffold and information from two recent network reconstructions of *Y. lipolytica*, we reconstructed a new metabolic network of this yeast, named iMK799, that describes the metabolism of *Y. lipolytica* very accurately. After qualitative validation, we used flux balance analysis (FBA) to confirm the accuracy of our model regarding growth performance on glucose and glycerol and product formation kinetics for lipid and citrate. Experimental and simulated growth rates differed only by 8% and 1.6% on glucose and glycerol, respectively. In addition, dynamic FBA showed good correlation of biomass production with fermentations. We simulated oxygen limitation conditions and formation of citrate and designed two new fermentation strategies to induce lipid accumulation. As for the growth characteristics, computational predictions and experimental results for neutral lipid synthesis were in good agreement. We expect that further optimization of fermentation techniques together with metabolic engineering strategies will additionally enhance lipid accumulation, provide a better understanding of lipid metabolism in *Y. lipolytica* and will contribute to the development of cost efficient strategies for lipid production for biodiesel.
METABOLIC ENGINEERING TO MAXIMIZE LIPID CONTENT IN *Yarrowia lipolytica*

Govindprasad Bhutada¹, Martin Kavšček¹, Sepp D. Kohlwein¹and Klaus Natter¹

¹ Institute of Molecular Biosciences, NAWI Graz, University of Graz, Humboldtstrasse 50/II, 8010 Graz, Austria

Contact email: klaus.natter@uni-graz.at

The oleaginous yeast *Yarrowia lipolytica* is an attractive non-conventional yeast that is used in basic research to study dimorphism and lipid metabolism. In addition, it is intensively investigated for its potential in industrial applications such as bioremediation, production of single cell protein, single cell oil and citric acid. It is known that, under optimal growth conditions *Y. lipolytica* can store about 36-40% neutral lipid in the form of triacylglycerol (TAG). The trans-esterification of TAG yields alkyl esters and glycerol, wherein alkyl esters can be used as major biodiesel component. The development of a genome scale model (GSM) and the available genetic tools provide the possibility to exploit *Y. lipolytica* as a platform for the production of TAG by metabolic engineering of the required pathways. The aim of our research is to optimize and maximize TAG content in this yeast. The classical approach is to target the *de novo* lipid synthesis pathways. In addition, channeling carbon flux towards lipid synthesis by engineering upstream pathways has a potential to achieve higher lipid storage, as predicted by computational simulations with a GSM of *Y. lipolytica*. The first targets are metabolites from glycolysis. During glucose catabolism, some carbon flux is also channeled to the formation of storage bodies. We eliminated this pathway to achieve a higher carbon flux into the tricarboxylic acid cycle for biomass and/or *de novo* lipid synthesis. Further modifications to increase the production of precursor metabolites for the *de novo* lipid synthesis pathway as well as the deletion of genes coding for TAG lipases and the β-oxidation pathway will result in an additional improvement of lipid content in *Y. lipolytica*. Hence, the engineered strains will be optimized for maximum lipid yield on glucose or glycerol as carbon source.
THE INFLUENCE OF MEMBRANE COMPOSTION ON ACETIC ACID
PERMEABILITY AND POTENTIALLY ACETIC ACID TOLERANCE

Lina Lindahl<fnref n="1" />, Helen Olsson<fnref n="1" />, Samuel Genheden<fnref n="2" />, Leif A Eriksson<fnref n="3" />, Aline X S Santos<fnref n="4" />,
Howard Riezman<fnref n="4" />, Lisbeth Olsson<fnref n="1" />, Maurizio Bettiga<fnref n="1" />

Contact email: linali@chalmers.se

Compounds entering the cell do so either by passive diffusion over the plasma membrane or through transporters in the membrane. The specific lipid composition of the plasma membrane influences both the passive diffusion rate but also the activity of membrane proteins. Acetic acid, a major hurdle in fermentation processes using lignocellulosic material, is believed to pass through the membrane in its protonated from mainly by passive diffusion [1].

Sterols and sphingolipids are lipid classes thought to contribute to membrane rigidity. Sterols are often found to be involved in stress resistance [2, 3] and in our previous work sphingolipids were pointed at as an important constituent of the plasma membrane of the yeast *Zygosaccharomyces bailii*, known to be very tolerant to acetic acid, suggesting a possible link between acetic acid tolerance and sphingolipid relative abundance in the membrane [4].

Here we will provide supporting evidence of the importance of sphingolipids and sterols in acetic acid membrane permeability. We have combined biochemistry techniques with *in silico* membrane modeling to answer the question how membrane engineering can be used to decrease acetic acid membrane permeability.

[1] Verduyn et al. Yeast (1992) 501-517.

[2] Alexandre et al. FEMS Microbiology Letters (1994) 124:17-22.

[3] Liu et al. Journal of Applied Microbiology (2013) 114:482-491.

[4] Lindberg et al. PlosONE (2003) 8(9): e73936.

<footnote n="1">¹ Department of Chemical and Biological Engineering, Industrial Biotechnology, Chalmers
University of Technology, Kemivägen 10, Gothenburg, Sweden</footnote>

<footnote n="2">² School of Chemistry, University of Southampton, Highfield, Southampton, UK</footnote>

<footnote n="3">³ Department of Chemistry and Molecular Biology, University of Gothenburg, Gothenburg, Sweden</footnote>

<footnote n="4">⁴ Department of Biochemistry, University of Geneva, Geneva, Switzerland</footnote>
Tum1 AFFECTS LIPID METABOLISM IN *Saccharomyces cerevisiae*, HOWEVER NOT THROUGH tRNA THIOLATION

Katja Uršič<fnref n="1" />, Mojca Brložnik<fnref n="1" />, Klaus Natter<fnref n="2" /> and Uroš Petrovič<fnref n="1" />

Contact email: katja.ursic@siol.com

Most, but not all genes involved in lipid metabolism have been functionally characterized. Our research group has been studying new genes that regulate lipid metabolism in *S. cerevisiae*. We found decreased amount of triacyglycerols (TAG) and increase of sterol esters (SE) in *tum1Δ* deletion mutant. The only hitherto known biological role of Tum1 is that it is one of the five proteins in the tRNA wobble uridine modification pathway. Three tRNAs specific for Gln (tQUUG), Lys (tKUUU), and Glu (tQUUC) have the 2-thiouridine derivative mcm⁵s²U as the wobble nucleoside. Tum1 is not essential for this thiolation, but has been described as a component involved in this pathway. We showed that in *tum1Δ* strain a small fraction of tRNAs still remained thiolated, whereas no thiolation of tRNA was observed in the control *uba4Δ* strain. Uba4 is downstream of Tum1 in the pathway and is essential for tRNA 2-thiolation. This result indicates that not the whole sulfur flux passes through the Tum1-mediated pathway branch. The amount of TAG and SE in *uba4Δ* cells was the same as in the wild type strain, indicating that the role of Tum1 in tRNA modification is not linked to its role in lipid metabolism. To test this further, we over-expressed tKUUU, tEUC and tQUUG which have been shown to partially suppress the inhibitory effect of the lack of mcm⁵s²U in these tRNAs in *uba4Δ* strain. Elevated levels of these three tRNA did not suppress the *tum1Δ* lipid phenotype. We thus conclude that Tum1 affects lipid metabolism through another, as yet unknown pathway, and thus has an additional molecular function that remains to be discovered.

<footnote n="1">¹Department of Molecular and Biomedical Sciences, Jožef Stefan Institute, Ljubljana, Slovenia</footnote>

<footnote n="2">²Institute of Molecular Biosciences, University of Graz, Austria</footnote>
*Blastobotrys (Arxula) adeninivorans* – A SUITABLE BIOCATALYST FOR NEW BIOTECHNOLOGICAL PRODUCTS

Urs Hähnel<fnref n="1" />, Jan Riechen<fnref n="1" />, Alexandre Chamas<fnref n="1" />, Jakub Kasprzak<fnref n="1" />, Marion Rauter<fnref n="2" />, Martin Giersberg<fnref n="1" />, Felix Bischoff<fnref n="1" />, Sebastian Worch<fnref n="1" />, Kristina Florschütz<fnref n="1" />, Anke Trautwein<fnref n="1" />, Dagmara Jankowska<fnref n="1" />, Keith Baronian<fnref n="3" /> and <u>Gotthard Kunze</u><fnref n="1" />

Contact email: kunzeg@ipk-gatersleben.de

The industrially important yeast *Blastobotrys (Arxula) adeninivorans* is an asexual hemiascomycete phylogenetically very distant from *Saccharomyces cerevisiae*. Its unusual metabolic flexibility allows it to use a wide range of carbon and nitrogen sources, while being thermotolerant, xerotolerant and osmotolerant. Based on the completely sequenced and annotated *Arxula* genome combined with gene expression data, numerous so far non-described pathways in yeasts were explored and exploited such as the metabolism of n-butanol, tannic acid and purine. The obtained data provide new knowledge to the exceptional broad substrate spectrum and robustness of this yeast. In addition *A. adeninivorans* is used as suitable host for synthesis of special products such as recombinant functional human receptors or glycosylated secretory tannases and it serves as a suitable biocatalyst for the synthesis of biotechnologically interesting products such as n-butanol and polyhydroxyalcanoate (PHA), because all essential prerequisites and components for heterologous gene expression are available. A lot of special protocols have been established (transformation/expression platform Xplor®2, gene disruption, protoplast fusion, mitotic segregation) and industrial strains were constructed. These strains are suitable producers of enzymes and enzyme mixes to degrade plastic material and lignocellulose (1), to synthesize enantiometrically pure alcohols (2) and to produce food with low purine content (3). Other biotechnological application fields for *Arxula* cells are bioremediation by accumulation of metal ions and production of biobutanol. Furthermore *A. adeninivorans* is used as microbial sensor compound to detect hormone activities (estrogenic, androgenic, glucocorticoidic, gestagenic activities), dioxins as well as pharmaceuticals in tap water, mineral water, waste water, urine and blood serum.

<footnote n="1">¹ Leibniz Institute of Plant Genetics and Crop Plant Research (IPK), Corrensstr.3, D-06466 Gatersleben, Germany;</footnote>

<footnote n="2">²Orgentis Chemicals GmbH, Bahnhofstr. 3, D-06466 Gatersleben, Germany;</footnote>

<footnote n="3">³ School of Biological Sciences, University of Canterbury, Private Bag 4800, Christchurch, New Zealand.</footnote>
BLACK YEAST *Aureobasidium pullulans*: A SOURCE OF GENES FOR THE IMPROVEMENT OF INDUSTRIALLY IMPORTANT TRAITS OF *Saccharomyces cerevisiae*

Janja Zajc<fnref n="1" />, Cene Gostinčar<fnref n="2" />, Nina Gunde-Cimerman<fnref n="1" />,<fnref n="3" />

Contact e-mail: zajc.janja@bf.uni-lj.si

Black yeast *Aureobasidium pullulans* (de Bary) G. Arnaud is characterized by an exceptional stress tolerance to a wide variety of stresses (i.e. polyextremotolerance), such as hypersaline, acidic, basic, cold and oligotrophic. It is of substantial biotechnological importance (production of pullulan, enzymes, antimicrobials) and has a potential for numerous novel applications in medicine, pharmacy, the food industry, and other fields. The recent availability of genomes of four *A. pullulans* varieties opened exciting new possibilities for harvesting its rich resources. For example it offers an opportunity for a fresh approach to improving the stress tolerance of *S. cerevisiae*. In this study, we have cloned selected genes (cation transporters, extracellular enzymes, and metabolic enzymes) from *A. pullulans*, expressed them in *S. cerevisiae* and finally evaluated the performance of the transformants at increased concentrations of salts or other relevant conditions. Yeast strains with improved capability to degrade complex substrates and/or with improved stress tolerance will be valuable to biofuel industry and agriculture.

<footnote n="1">¹ Department of Biology, Biotechnical Faculty, University of Ljubljana, Večna pot 111, SI-1000 Ljubljana, Slovenia;</footnote>

<footnote n="2">²National Institute of Biology, Večna pot 111, SI-1000 Ljubljana, Slovenia;</footnote>

<footnote n="3">³Centre of Excellence for Integrated Approaches in Chemistry and Biology of Proteins (CIPKeBiP), Jamova 39, SI-1000 Ljubljana, Slovenia</footnote>
OPTIMIZATION OF GLUCOSE OXIDASE PRODUCTION BY RECOMBINANT

*Kluyveromyces lactis*

C. Saint-Hubert<fnref n="1" />*, C-H. Biron<fnref n="2" />, R.Cuvelier<fnref n="3" />, C-M. Bols<fnref n="4" />, J-P. Perraudin<fnref n="5" />, I. Georis<fnref n="6" />, R. Dutoit<fnref n="6" />, A. Durieux<fnref n="6" />

Successful production of recombinant protein does not rely on genetic engineering alone, and often requires the optimization of fermentation processes. Here, we describe the conjugate effect of both approaches on the heterologous production of *Aspergillus niger* glucose oxidase (GOD) by *Kluyveromyces lactis*. The secretion signal peptide of KlPho5p led to a higher secretion of GOD compared to that of the □-subunit of zymocin complex. The control of fermentation processes had an extended influence on GOD production. Indeed, contrary to a complex broth with any fermentable carbon source, a selective mineral medium with lactose greatly increased the yield of GOD production from 18.9 to 341.6 U g DW⁻<fnref n="1" /> in flask cultures. The optimization of fermentation processes in bioreactor led to 501.8 U g DW⁻<fnref n="1" /> of GOD production. The control of *K. lactis* growth rate has proven essential to improve GOD secretion.

<footnote n="1">¹ Unité de BioTechnologie, Institut Meurice, Campus CERIA, Av. E. Gryson 1, 1070 Brussels, Belgium ; email : c.sainthubert@mrd.ubt.be</footnote>

<footnote n="2">² Meurice R&D asbl, Av. E. Gryson 1, 1070 Brussels, Belgium</footnote>

<footnote n="3">³ Celabor, Zoning de Petit-Rechain, Av. du Parc 38, 4650 Chaineux, Belgium</footnote>

<footnote n="4">⁴ Wetlands Engineering, Rue du Laid Burniat 5, 1348 Louvain-La-Neuve, Belgium</footnote>

<footnote n="5">⁵ Taradon Laboratory, Av. Léon Champagne 2, 1480 Tubize, Belgium</footnote>

<footnote n="6">⁶ Institut de Recherches Microbiologiques Jean-Marie Wiame, Campus CERIA, Av. E. Gryson 1, 1070 Brussels, Belgium</footnote>
INTERACTION OF *Pseudomonas fluorescens* AND DIFFERENT SPOILAGE YEASTS

Mónika Kovács, Bianka Szakács, Ágnes Belák, Anna Maráz

Department of Microbiology and Biotechnology, Corvinus University of Budapest, Somlói út
14-16, H-1118 Budapest, Hungary

Contact e-mail: monika.kovacs@uni-corvinus.hu

Metabolic activity of microorganisms plays an important role in food spoilage. Bacteria
predominate mainly in the food spoiling microbiota but yeasts are also present. Population
dynamics of the bacteria during storage is well documented, though less is known about the
behaviour of yeast species. Investigation about interactions of bacteria and yeasts during
spoilage is sparse. Our aim was to study the interaction of *Pseudomonas fluorescens* of
different origin and yeast strains isolated from spoiled food. Several strains of the tested *P.
fluoerscens* inhibited the growth of certain yeast species but the antagonistic effect was strain
dependent. Isolates of *Rhodotorula mucilaginosa* and *Cryptococcus curvatus* were highly
sensitive to the effect of *P. fluoerscens*, while the growth of *Metschnikowia pulcherrima*
strains was not affected by this bacterium. Testing the influence of yeasts on the growth of *P.
fluorescens* only *M. pulcherrima* had inhibiting activity. When co-culturing of *P. fluorescens*
and *M. pulcherrima* was performed we observed that the inhibitory effect of *M. pulcherrima*
was still detectable if it was inoculated in two orders of magnitude smaller than the bacterium.
We concluded that the antagonistic effect is due to some extracellular metabolic products
because cell free supernatants showed also inhibition. Although *M. pulcherrima* can produce
low amount of ethanol, *P. fluorescens* was not sensitive to this alcohol content. It has already
been published that both *Pseudomonas* and *M. pulcherrima* can produce iron chelating
compounds (siderophores and pulcherrimin, respectively); therefore we tested the effect of
elevated iron concentrations on the interaction. In case of iron excess the inhibitory effect of
*M. pulcherrima* against *P. fluorescens* abolished, which indicated that iron availability is one
of the key factors in the interaction. Acknowledgement: This work was supported by the
Hungarian project OTKA 101716.
IS β-CAROTENE INVOLVED IN THE ANTIOXIDANT RESPONSE OF CAROTENOGENIC YEASTS? AN ANSWER FROM THREE *Rhodotorula* spp. STRAINS

Sara Landolfo, Marilena Budroni, Annalisa Coi, Ilaria Mannazzu

Dipartimento di AGRARIA, Università degli Studi di Sassari, Viale Italia 39, Sassari, Italy
Contact email: imannazzu@uniss.it

Red yeasts ascribed to the genus *Rhodotorula* produce carotenoids, mainly β-carotene, torulene and torularhodin. The production of β-carotene may restore hydrogen peroxide resistance in yeast cells lacking cytosolic catalase. However, β-carotene works as a prooxidant in the presence of high oxygen concentrations and determines an increase in the sensitivity to reactive oxygen species when it accumulates inside yeast cell. Thus β-carotene ROS scavenging activity *in vivo* is rather controversial. Here, to further elucidate the involvement of β-carotene in yeast cellular response to oxidative stress, a wild type strain of *Rhodotorula* spp. (C2.5t1) and two of its primary mutants, that differ in the amount and type of carotenoids produced, were treated with H₂O₂ without and with diphenylamine (DPA), a known inhibitor of phytoene desaturase and the progression of carotenoids biosynthesis. While the parental strain C2.5t1, that produces the three main carotenoids, and the colorless mutant 200A6, unable to produce β-carotene, torulene and torularhodin, showed comparable resistance to H₂O₂, the β-carotene overproducing mutant 400A15 was more sensitive to this prooxidant. DPA-mediated inhibition of carotenoids biosynthesis restored growth of the three strains in the presence of H₂O₂ and resulted in a decrease in the intracellular content of superoxide anion, also in the albino mutant 200A6. H₂O₂ treatment determined comparable oxidative damage to proteins in the three strains, in spite of the different carotenoid content. In addition catalase activity induced by H₂O₂ was comparable in cells grown without and with DPA. Thus that β-carotene, but also torulene and torularhodin, do not seem to be directly involved in H₂O₂ detoxification in *Rhodotorula* spp..

Project granted to IM by Regione Autonoma della Sardegna (Legge Regionale 7-2007 Annualità 2010).
DIFFERENTIAL PROTEOMIC ANALYSIS OF RHODOTORULA SPP. STRAINS FOR THE IDENTIFICATION OF ENZYMES INVOLVED IN CAROTENOGENESIS

Raffaela Cutzu<fnref n="1" />, Sara Landolfo<fnref n="1" />, Alessandro Tanca<fnref n="2" />, Marcello Abbondio<fnref n="2" />, Maria Filippa Addis<fnref n="2" />, Sergio Uzzau<fnref n="2" />, Marilena Budroni<fnref n="1" />, Ilaria Mannazzu<fnref n="1" />

With the aim of identifying the enzymes involved in red yeasts carotenogenic pathway, a wild strain of *Rhodotorula* spp. (C2.5t1) and two mutants (400A15 and 200A6), were subject to comparative proteomic analysis by means of 2D-DIGE. These three strains differ in the amount and type of carotenoids produced. In particular, C2.5t1 produces β-carotene, torulene and torularhodin, the yellow mutant 400A15 overproduces β-carotene, and the colorless mutant 200A6 does not produce any of these carotenoids. Comparison of the proteomic maps of the three strains after 24 growth on glycerol containing medium, highlighted 38 spots as differentially abundant in the three strains. Spots showing a higher abundance in C2.5t1 and/or 400A15 in respect to 200A6 were analyzed by Tandem Mass Spectrometry and seven different databases were utilized for their identification. Sixteen peptide sequences were identified upon database searching and blastp alignment as likely belonging to carotenogenic enzymes; unfortunately, however, no more than one sequence per enzyme could be detected, possibly due to differences in the amino acid sequences of these proteins and those available in the databases utilized for the identification. In perspective, the recent publication of the genome of *Rhodotorula glutinis* and the assembly and the annotation of C2.5t1 genome is expected to provide useful tools to re-analyze the dataset generated by this study. Such analysis could result in a significant increment in the number of identifications as already experienced by other authors on the red yeast *Rhodosporidium toruloides* (Zhou et al. 2012, Nature Communications 3:1112).

Project granted to IM by Regione Autonoma della Sardegna (Legge Regionale 7-2007 Annualità 2010).

<footnote n="1">¹ Dipartimento di AGRARIA, Università degli Studi di Sassari, Viale Italia 39, Sassari Italy</footnote>

<footnote n="2">²Porto Conte Ricerche, S.P. 55 Porto Conte/Capo Caccia Km 8.400, Loc. Tramariglio, Alghero, Italy</footnote>
*Rhodotorula glutinis* AS A MODEL FOR STUDY OF CAROTENOIDS AND LIPID BIOSYNTHESIS

Jana Tkáčová, Júlia Čaplová, Milan Čertík

Faculty of Chemical and Food Technology, Slovak University of Technology, Bratislava,
Slovak Republic

Contact email: jane.tkacova@gmail.com

*Rhodotorula glutinis* as an oleaginous yeast accumulates large quantity of lipids. *R. glutinis* is also known as a producer of various pigments, especially β-carotene and unusual torulene and torularhodin. Carotenoids are natural antioxidants with significant immunostimulatory and anticancer effects with the capacity to protect cells against free oxygen and peroxide radicals. These benefits allow their widespread application in pharmaceutical, food and feed industry. Due to high demand for these biologically active molecules, the work was focused on mechanisms of carotenoid overproduction by yeasts. One of the main physiological target for regulation of pigment accumulation is selection of optimal C:N ratio of the cultivation medium. Therefore, four different C:N ratios, 20:1, 50:1, 70:1 and 100:1 have been applied to study their impact on carotenoid biosynthesis and profile. In addition, further important factor for pigment production is carbon starvation. It was found that yeasts after complete carbon assimilation utilized storage lipids as an energy sources and converted fatty acids into acetyl-CoA via the β-oxidation. Such acetyl-CoA served as a substrate for pigment biosynthesis in mevalonate pathway. C:N ratio 50:1 was found to be optimal for the highest carotenoid yield (21.0 mg/L) and their accumulation in cells (1.9 mg/g cells). It should be noted that β-carotene and torularhodin were the main pigments during the first growth stage. However pigment profile was rapidly changed during carbon starvation where rapid accumulation of -carotene and torulene was detected. Moreover, the yeast was able to accumulate over 30% storage lipids in cells (4.3 g/L) that consisted of mainly oleic and palmitic acids.

*The work was supported by grants VEGA 1/0975/12, APVV-0662-11 and "University Science Park of STU Bratislava" ITMS 26240220084.*
POPULATION GENOMICS OF *Saccharomyces cerevisiae* HUMAN ISOLATES:
PASSENGERS, COLONIZERS AND INVADERS

Carlotta De Filippo<fnref n="1" />,¹⁰, Monica Di Paola<fnref n="2" />,¹⁰, Irene Stefanini<fnref n="1" />, Lisa Rizzetto<fnref n="1" />, Luisa Berná<fnref n="2" />, Matteo Ramazzotti<fnref n="3" />, Leonardo Dapporto<fnref n="4" />, Damariz Rivero<fnref n="2" />, Ivo G Gut<fnref n="5" />, Jean-Luc Legras<fnref n="6" />,<fnref n="7" />,<fnref n="8" />, Luigina Romani<fnref n="9" />, Paolo Lionetti<fnref n="2" />, Duccio Cavalieri<fnref n="1" />,¹¹

The quest for the ecological niches of *Saccharomyces cerevisiae* has led to research in areas as diverse as wineries, oak and more recently the gut of *Crabro* wasps. Here we propose the role of the human gut in shaping *S. cerevisiae* evolution, presenting the genetic structure of a previously unknown population of yeasts, associated with Crohn's disease, providing evidence for clonal expansion within the human gut. To understand the role of immune function in human-yeast interaction we classified strains according to their immunomodulatory properties, discovering two sets of isolates: the first inducing anti-inflammatory signals via regulatory T cell proliferation and the latter, made up of strains with a mosaic genome, eliciting inflammatory, IL-17 driven immune response. The approach integrating genomics with immune phenotyping showed selection in genes involved in sporulation and cell wall remodeling to be central in the evolution of *S. cerevisiae* Crohn strains from passengers to commensals and then to pathogens.

<footnote n="1">¹ Fondazione E. Mach, Research and Innovation Centre, Via E. Mach 1, 38010 San Michele all' Adige (Trento), Italy.</footnote>

<footnote n="2">² Department of Neuroscience, Psychology, Drug Research and Child Health, Meyer Children Hospital, University of Florence, Viale G. Pieraccini 6, 50139 Florence, Italy.</footnote>

<footnote n="3">³ Department of Experimental and Clinical Biomedical Sciences, University of Florence, viale Morgagni 50, 50134 Florence, Italy.</footnote>

<footnote n="4">⁴ Department of Biological and Medical Sciences, Oxford Brookes University, Headington, Oxford, OX30BP, UK.</footnote>

<footnote n="5">⁵ Centro Nacional d'Anàlisi Genòmica, CNAG, Parc Científic de Barcelona 08028 Barcelona, Spain.</footnote>

<footnote n="6">⁶ Institut National de la Recherche Agronomique, INRA, Unité Mixte de Recherche Sciences pour l'Oenologie, UMR1083, F-34060 Montpellier, France.</footnote>

<footnote n="7">⁷ Montpellier SupAgro, Unité Mixte de Recherche Sciences pour l'Oenologie, UMR1083, F-34060 Montpellier, France.</footnote>

<footnote n="8">⁸ Université Montpellier I, Unité Mixte de Recherche Sciences pour l'Oenologie, UMR1083, F-34060, Montpellier, France.</footnote>

<footnote n="9">⁹ Department of Experimental Medicine and Biochemical Sciences, Polo Unico Sant'Andrea delle Fratte, University of Perugia, piazza Università 1, 06132 Perugia, Italy.</footnote>
POPULATION GENOMICS REVEALS THE EVOLUTIONARY FATE OF A LARGE-
SCALE INTROGRESSION IN A PROTOPLOID YEAST

Anne Friedrich<fnref n="1" />, Paul Jung<fnref n="1" />, Cyrielle Reisser<fnref n="1" />, Gilles Fischer<fnref n="2" />,<fnref n="3" /> and Joseph Schacherer<fnref n="1" />

Contact email: schacherer@unistra.fr

Yeast species represent an ideal model system for population genomics but large-scale polymorphism surveys have only been reported for two closely related yeast species, *Saccharomyces cerevisiae* and *Saccharomyces paradoxus* so far. Hence, little is known about intraspecific diversity and evolution in yeast. To obtain a new insight into the evolutionary forces shaping natural populations, we sequenced the genomes of a expansive worldwide collection of isolates of a distant species relative to *S. cerevisiae: Lachancea kluyveri*. We identified 6.5 million SNPs and showed that a large introgression event of 1-Mb of GC-rich sequence in the chromosomal arm occurred in the last common ancestor of all *L. kluyveri* strains. Our population genomic data clearly revealed that the introgressed region underwent a molecular evolution pattern very different from the rest of the genome. It is characterized by a higher recombination rate, with a dramatically increased A:T→G:C substitution rate, which is the signature of a increased GC-biased gene conversion. The presence of two distinct recombination and substitution regimes within the same genome demonstrates that the chromosome-scale compositional heterogeneity will persist after the genome has reached mutational equilibrium. Altogether, the data presented herein suggest that large DNA introgressions resulting from interspecific hybridizations lead to different evolutionary patterns within a given genome.

<footnote n="1">¹ Department of Genetics, Genomics and Microbiology, Université de Strasbourg / CNRS, UMR7156, Strasbourg, France</footnote>

<footnote n="2">² Sorbonne Universités, UPMC Univ Paris 06, UMR 7238, Biologie Computationnelle et Quantitative, Paris, France</footnote>

<footnote n="3">³ CNRS, UMR7238, Biologie Computationnelle et Quantitative, Paris, France</footnote>
DISTINCTIVE CHROMOSOMAL END LOSS AND LAGER SPECIFIC GENES
DEFINE TWO DIFFERENT *Saccharomyces cerevisiae* LINEAGES FOR S.
*pastorianus* GROUP I AND II STRAINS

Chandre Monerawela and Ursula Bond

Moyne Institute, School of Genetics and Microbiology, Trinity College Dublin,
Ireland

Contact email:ubond@tcd.ie

The lager yeast species *Saccharomyces pastorianus* is a natural hybrid arising from the fusion of two parental species, a *Saccharomyces cerevisiae*-like yeast and the newly discovered *Saccharomyces eubayanus*. Genome sequencing of the prototype Group I lager yeast strain, Weihenstephan 34/70, identified eight telomere-associated lager-specific (LgS) genes that were not present in other *Saccharomyces* species. We re-evaluated the origins of the LgS genes through bioinformatic analysis of 32 sequenced *Saccharomyces* genomes combined with PCR analysis of a further 48 un-sequenced strains. Nearest neighbour and SNP analyses confirm that the *S. cerevisiae* portion of the lager yeast genome originates from Ale-like yeasts. The analysis also revealed that four of the LgS genes originate from *S. cerevisiae* while the other four originate from *S. eubayanus*. Furthermore, a pattern of presence or absence of the LgS-genes was identified within the *S. cerevisiae* strains. We analysed the sub-telomeric regions of all 16 *S. cerevisiae*-like chromosomes in the *S. pastorianus* genomes and in the 32 sequenced *S. cerevisiae* strains and identified a pattern of loss of chromosome end loss that allows distinction between Group I and II lager yeasts and between *S. cerevisiae* strains. Based on this analysis we identify two distinct classes of Ale yeasts with patterns of LgS-gene loss and chromosome end loss identical to *S. pastorianus* Group I and II yeasts respectively. These findings lead us to speculate that *S. pastorianus* Group I and II strains originated from separate hybridisation events with two distinct Ale yeasts. Using the combined bioinformatics and PCR data, we describe a potential classification map for lager, wine, distillers, coconut, laboratory and sake yeasts.
GENOME SEQUENCE OF SACCHAROMYCES CARLSBERGENSIS, THE WORLD'S FIRST PURE CULTURE LAGER YEAST

Andrea Walther, Ana Hesselbart and Jürgen Wendland

Lager yeast beer production was revolutionized by the introduction of pure culture strains. The first established lager yeast strain is known as the bottom fermenting *Saccharomyces carlsbergensis*, which was originally termed *Unterhefe* No.1 by Emil Chr. Hansen and used in production in since 1883. *S. carlsbergensis* belongs to group I/Saaz-type lager yeast strains and is better adapted to cold growth conditions than type II/Frohberg-type lager yeasts, e.g. the Weihenstephan strain WS34/70. Here, we determined the draft genome sequence of *S. carlsbergensis* based on Illumina sequencing of 8 kb mate-pair libraries. Lager yeasts such as *S. carlsbergensis* are descendants from hybrids formed between a *S. cerevisiae* parent and a parent similar to *S. eubayanus*. Accordingly, the *S. carlsbergensis* 19.5 Mb genome is substantially larger than the 12 Mb *S. cerevisiae* genome. Based on the sequence scaffolds, synteny to the *S. cerevisiae* genome, and by using directed PCRs for gap closure we generated a chromosomal map of *S. carlsbergensis* consisting of 29 unique chromosomes. We present evidence for genome and chromosome evolution within *S. carlsbergensis* via chromosome loss and loss of heterozygosity specifically of parts derived from the *S. cerevisiae* parent. Based on our sequence data and via FACS analysis we determined the ploidy of *S. carlsbergensis*. This inferred that this strain is basically triploid with a diploid *S. eubayanus* and haploid *S. cerevisiae* genome content. In contrast the Weihenstephan strain is essentially tetraploid composed of two diploid *S. cerevisiae* and *S. eubayanus* genomes. Evolutionary implications for type I and type II lager yeasts will be discussed.
ROLE OF CHROMOSOMAL INVERSIONS ON YEAST FITNESS AND GENE
EXPRESSION

<u>Samina Naseeb</u>¹, Zorana Carter¹, David Minnis¹, Leo Zeef¹, Ian Donaldson¹ and
Daniela Delneri¹

¹ Faculty of Life Sciences, University of Manchester, Manchester, United Kingdom

The non-random gene organisation in eukaryotes is thought to play an important role in
genome evolution and function, however the effect of rearrangements on global transcription
and fitness is still controversial and not yet fully understood. To investigate the effect of gene
order and centromere location on phenotype, fertility and global gene expression, we
engineered 16 *S. cerevisiae* strains carrying different pericentric and paracentric inversions
between TY1 elements, since such repetitive regions are a natural substrate for
rearrangements. We found that four inversions were lethal, while the others did not have a
detectable impact on mitotic fitness. In meiosis spore viability didn't correlate with either size
or type of inversion, however lower fertility was seen in heterozygote carriers with 2 or 3
recombination hotspots present in the inverted region. The amount of transcriptional changes
varied among the strains and altered expression was observed throughout the whole genome
rather than being over-represented within the inversions or around the breakpoints. However,
in the strain XIV-inv, the chromatin remodeling gene *ARP5*, positioned near the inversion
break point, was significantly up-regulated. This strain showed the largest expression changes
and an up-regulation of genes involved in nuclear pore complex. Interestingly, such genome-
wide transcriptional alteration was not translated in a detectable fitness change, highlighting
the robustness of the expression network in yeast and supporting the notion that
transcriptome rewiring may compensate for changes introduced by rearrangements in order to
maintain a constant phenotype.
EVOLUTIONARY IMPACT AND ENVIROMENTAL ADAPTATION OF NATURAL
YEAST ISOLATES BEARING KARYOTYPIC REARRANGMENTS

Qamar Almutawa, Samina Naseeb, Daniela Delneri

Faculty of Life Sciences, University of Manchester, Manchester, UK
Qamar.Almutawa@postgrad.manchester.ac.uk

Yeast is a powerful eukaryotic model organism that has been studied extensively and successfully in the cell biology and physiology fields as well as in evolution studies, population genetics and speciation. *Saccharomyces* 'sensu strico' species are reproductively isolated via post-zygotic barriers; hybrids are readily formed but they are sterile upon meiosis. The major reproductive isolation barrier is due to sequence divergence and the inability of homologous chromosomes to pair up in meiosis. Surprisingly, *Saccharomyces paradoxus* and *Saccharomyces cariocanus* have low sequence divergence but are two different species. They differ in their genome by four chromosomal translocations and it is possible that such rearrangements are the cause of the very low spore viability. In fact, hybrid heterozygote for just one translocation produces only 50% of viable progeny. This project aims to understand the impact chromosomal translocations have on reproductive isolation and fitness in these two yeast isolates. Two out of the four chromosomal translocations present in *S. cariocanus* were created in *S. paradoxus* YPS138 strain to render this part of the genome collinear to *S. cariocanus* by PCR-mediated gene replacement system and Cre-*loxP* mediated recombination event. Translocated and non-translocated *S. paradoxus* strains were crossed with *S. cariocanus*. Hybrids were selected on minimal medium and meiosis was induced to score fertility. Our results showed that there is significant increase in the total spore viability from 3.3 % in the engineered hybrids carrying 4 translocations (4T) to 12% in the engineered hybrids carrying 2 heterozygote translocations (2T) (p<0.05). While the 3T engineered hybrids shows an intermediate spores viability (6.9%). These results suggested that chromosomal translocation could be the source behind the reproductive isolation between *S. paradoxus* and *S. cariocanus* species. However, from these data the expectation for complete collinear crossing could be around 40%, indicating that multiple factors may contribute to spore viability.
EVOLUTIONARY EFFECT OF MITOCHONDRIALLY ENCODED PROTEINS ON
GLOBAL GENE RETENTION IN YEAST HYBRIDS

Sarah Hewitt, [Kobchai Dungrattanalert](mailto:Kobchai_Dungrattanalert), Daniela Delneri

Faculty of Life Sciences, University of Manchester, Manchester, UK
Contact email: kobchai.dungrattanalert@postgrad.manchester.ac.uk

In eukaryotes, mitochondria take important roles in aerobic respiration, apoptosis and cellular
metabolism of several metabolites such as fatty acids, amino acids and heme. Mitochondria
DNA is inherited since it cannot be synthesised anew. In yeast hybrids, it has been observed
that mitochondrial inheritance is uniparental that only the mitochondrial DNA of one parent
is maintained. This is perhaps to avoid incompatibility which reduces overall fitness. The
mitochondrion is composed mainly by nuclear-encoded proteins, and there are only 8
mitochondrial genes. Here we investigate the impact of different types of mitochondria on the
transcriptome in interspecific *Saccharomyces* hybrids. We want to understand whether the
expression of the nuclear parental alleles changes according to the mitotype. We produced
independent lines of hybrids between *S. cerevisiae* strain BY4741 and *S. uvarum* strain
NCYC2669 that possess a single type of mitochondria: either *S. cerevisiae* or *S. uvarum*
mitochondria. The hybrids were cultured in rich YPD medium or YP medium with glycerol
and at two different temperatures (28C and 16C). Their total RNAs were extracted for
transcriptome analysis by sequencing on Illumina platform. Preliminary results in YP-
glycerol at 28C shows that the twice as many *S. cerevisiae* alleles changes expression in the
hybrids containing *S. uvarum* mitochondria compared to the hybrids with *S. cerevisiae*
mitochondria. Further experiments are on-going to study interactions and incompatibilities
between nucleus and mitochondria in different nutritional media and temperatures.
POLYGENIC ANALYSIS OF LOW PH TOLERANCE IN

*Saccharomyces cerevisiae var. boulardii*

Benjamin Offei¹,², Maria Foulquié-Moreno¹,², Johan M. Thevelein¹,²

Laboratory of Molecular Cell Biology, Institute of Botany and Microbiology, KU Leuven¹;
Department of Molecular Microbiology, VIB, Kasteelpark Arenberg 31, B-3001 Leuven-
Heverlee, Flanders, Belgium².

Contact e-mail: johan.thevelein@mmbio.vib-kuleuven.be

*Saccharomyces cerevisiae var. boulardii* (S. boulardii) is a probiotic yeast strain. The ability of S. boulardii to withstand low pH environments better than other S. cerevisiae strains has been demonstrated through various studies and this phenotype is believed to contribute directly to S. boulardii's ability to act as a probiotic. Our results have also confirmed the superiority of S. boulardii strains in terms of tolerance to extreme low pH environments, when compared to S. cerevisiae. To date no genetic element has clearly been linked to this phenotype in S. boulardi. A possible bottleneck that may hamper genomic studies to unravel the genetic basis of low pH tolerance in S. boulardii is its inability to sporulate. We have obtained results indicating that an S. boulardii strain, isolated directly from a pharmaceutical product, becomes mating competent through overexpression of an early sporulation gene. Through propidium iodide staining and flow cytometry, we have established the ploidy of the mating competent cells as diploid. However, being mating competent, we have succeeded in generating a triploid hybrid strain after crossing our S. boulardii with ER7A, a haploid low pH sensitive strain isolated from a bioethanol production wildtype S. cerevisiae strain. We are currently evaluating offspring from the triploid strain exhibiting similar levels of low pH tolerance to the superior S. boulardii strain. Such a segregant will be crossed further with a laboratory S. cerevisiae strain to obtain segregant pools for Quantitative Trait Loci (QTL) analysis and ultimately identify the causative alleles/mutations, behind the low pH tolerance in S. boulardii. Successful identification of such alleles can have important applications in improving probiotic yeast strains that are sensitive to low pH environments similar to that of the gut as well as industrial strains that are frequently subjected to acid wash to combat bacteria contamination.
INTRACELLULAR pH OF POTENTIAL PROBIOTIC YEASTS DURING THE PASSAGE THROUGH AN *IN VITRO* GASTROINTESTINAL TRACT MODEL

Honeylet Sabas Ochangco, Line Lindegaard Pedersen, Lene Jespersen and Nils Arneborg

Department of Food Science, Faculty of Science, University of Copenhagen, 1958

Frederiksberg C, Denmark

Contact email: hsoc@food.ku.dk

The aim of our study was to investigate the different strategies of yeast to regulate intracellular pH (pHi) upon exposure to gastrointestinal stresses. Fluorescence ratio imaging microscopy and CDCFDA-SE dye was employed to determine the pHi of yeasts. Two strains of *Debaryomyces hansenii* DI02 and D18335, with respectively strong and weak survival capacities under *in vitro* gastrointestinal conditions, were compared to the known probiotic yeast *Saccharomyces boulardii* with regard to pHi response using an *in vitro* gastrointestinal tract model. Our findings suggest different approaches in regulating pHi in response to gastrointestinal stresses. While *D. hansenii* DI02 adapt to high (pH 6.5) or low (pH 2.5) extracellular pH by respectively increasing or decreasing its pHi, both *D. hansenii* D18335 and *S. boulardii* maintained pHi in the range of 4.5-6 and 4-6.5 respectively. Interestingly, *D. hansenii* DI02 had wide range of pHi in sub-populations which correlated to a better survival of yeast upon exposure to gastrointestinal stress whereas *D. hansenii* D18335 had narrow range of pHi in sub-populations which correlated to a poor survival of yeast upon exposure to gastrointestinal stress. These findings give insight to survival strategies of yeasts exposed to gastrointestinal stress.
PHYSIOLOGY AND OSMOTOLERANCE OF TWO *Dekkera bruxellensis* STRAINS

Jana Zemančíková, Hana Sychrová

Department of Membrane Transport, Institute of Physiology ASCR, v.v.i, Vídenská
1083, Prague, CZ-14220, Czech Republic
Contact email: zemancikova@biomed.cas.cz

Yeast *Dekkera bruxellensis* is associated with various fermentation systems like bioethanol, beer and cider production. In winemaking process, *Dekkera* is one of the non-*Saccharomyces* yeasts which contributes to the final quality of red wine. *D. bruxellensis* produces various phenolic compounds and their concentration influences the organoleptic properties of wine, sometimes improving them, sometimes causing spoilage and large economic losses. *D. bruxellensis* genome is very dynamic, several chromosome sets can be present and copy number of genes can determine physiological properties. In this study we compare basic physiological parameters and osmotolerance of two *D. bruxellensis* strains Y879 (diploid/polyploid?) and Y881 (ploidy is not known), both isolated from wine. Though the cells have similar morphology and size, they differ in growth parameters, tolerance to various stresses, plasma-membrane potential and survival upon hyperosmotic conditions. An important property for survival of yeasts during the fermentation is their osmotolerance in which the intracellular accumulation of glycerol is involved. Our aim is to characterize *Dekkera* systems for glycerol uptake which are supposed to play a key role in counterbalancing high extracellular osmotic pressure. On the basis of homology with a transporter from *S. cerevisiae* (encoded by the *STL1* gene) we identified two genes encoding putative glycerol transporters in *Dekkera* genome, cloned them and expressed them in an osmosensitive *S. cerevisiae* mutant strain to verify their function and role in osmotolerance.

Acknowledgement. This work was supported by PITN-GA-2010-264717 Cornucopia
STUDIES ON ACETIC ACID STRESS RESPONSE AND GALACTOSE METABOLISM
IN *Dekkera bruxellensis*

Md Moktaduzzaman¹, Silvia Galafassi¹, Ileana Vigentini¹, Claudia Capusoni¹, L. Corte²,
Zhihao Ling³, Gianluigi Cardinali², Roberto Foschino¹, Jure Piškur³, Concetta Compagno¹

¹Department of Food, Environmental and Nutritional Sciences, University of Milan, Milan, Italy.

²Department of Pharmaceutical Sciences-Microbiology, University of Perugia, Perugia, Italy

³Department of Biology, Lund University, Lund, Sweden.

Contact email: md.moktaduzzaman@unimi.it

Recent studies have highlighted how *D. bruxellensis* shares with *S. cerevisiae* the ability to survive in challenging environments, such as industrial bioethanol production processes and wine. Beside the adaptability to conditions of low pH and high ethanol concentration, *D. bruxellensis* exhibits a broader spectrum of consumable carbon and nitrogen sources in comparison to *S. cerevisiae*, and this contributes to its ecological distribution. The effect of acetic acid has been widely studied in *S. cerevisiae* and several food spoilage yeasts, but not yet studied in *D. bruxellensis*. We observed that the presence of acetic acid deeply affected its growth in glucose-based media. At metabolomics level, the effect of acetic acid was found mainly in relation to some cellular components, like carbohydrates and amides. *D. bruxellensis* is known to exhibit the Crabtree-positive effect on glucose. We found that in galactose-based media this yeast utilized a respiratory metabolism, in contrast to *S. cerevisiae* which can ferment also this sugar. Interestingly, the respiratory metabolism was shifted towards fermentative after addition of glucose, as well as when nitrate was the only nitrogen source. The expression of genes involved in different metabolic pathways was also analyzed in *D. bruxellensis*. We observed that genes involved in galactose utilization, respiratory metabolism, TCA cycle, glyoxylate cycle and gluconeogenesis were repressed in glucose-based media. These results indicate that in *D. bruxellensis* glucose repression operates similarly to what occurs in *S. cerevisiae*.
GENOTYPING AND PHENOTYPING OF HUNDREDS OF *Dekkera bruxellensis*
ISOLATES

Anna J. Schifferdecker¹, Joseph Schacherer², Michael Katz³, Jure Piškur¹

¹ Department of Biology, Lund University, Sölvegatan 35, Lund 22362, Sweden

² Department of Genetics, Genomics and Microbiology, Université de Strasbourg,
28 rue Goethe, 67083, Strasbourg Cedex, France

³ Applied Yeast Research, Carlsberg Research Center, Gamle Carlsbergvej 10, 1799 Kopenhagen, Denmark

Anna. Schifferdecker@biol.lu.se

The non-conventional yeast *Dekkera bruxellensis* is worldwide known as a spoilage
organism in the wine and ethanol industry and as an “indispensable“ contributor to the
flavour profile of Belgium lambic and gueuze beers, some red wines and sourdough.
Furthermore, it became a model organism to investigate yeast evolutionary history.
Hundreds of native *D. bruxellensis* isolates have been collected from must, wine and
soft drinks all over the world (Italy, Spain, England, Mozambique, Belgium, Chile,
Portugal, Australia and South Africa). To get a better understanding of the genotype-
phenotype-paradigm, these isolates have been characterized in view of their phenotypic
traits and genome structure. Microtiter plate screenings for several different traits allow
the identification of strains that exhibit high sensitivity or resistance towards ethanol
and acetate, pH, osmotolerance or the ability to grow under relatively high or low
temperatures. In addition, the genomes of fifty strains are now sequenced and allow us
a closer look at the genomic level to highlight differences due to the strain origin and to
get an insight into the genotype-phenotype-relationship.
MALDI-TOF AS AN IDENTIFICATION TOOL FOR *Saccharomyces sensu stricto*
SPECIES AND FOOD-RELATED YEASTS

Raquel Quintilla<fnref n="1" />,<fnref n="2" />,<fnref n="3" />, Marizeth Groenewald<fnref n="1" />, Anna Kolecka<fnref n="1" />, Luuk van Haren<fnref n="1" />, Keisha Royer<fnref n="1" /> and Teun Boekhout<fnref n="1" />.

Currently, the most common method for the identification of yeasts is the sequence analysis of the internal transcribed spacers (ITS1 and 2) and the D1/D2 domains of the large subunit of the ribosomal DNA. This method provides reliable identifications, but can also be time-consuming and tedious. Here we present the Matrix Assisted Laser Desorption Ionization Time-of-Flight Mass Spectrometry (MALDI-TOF MS) as a quick, accurate and easy to use method for identification of industrial yeasts. An in-house database was created of a high quality Main Spectra (MSP) from type and reference strains of representing species belonging to 25 genera, economically relevant in the food industry. These include species from the *Saccharomyces sensu stricto* complex, spoilage yeast such as *Zygosaccharomyces* species and additional food-related yeasts such as *Debaryomyces hansenii*. A blind set of strains was tested to evaluate the accuracy and precision of the MALDI-TOF MS identifications. Here we represent data obtained with this rapid and cost-effective method. Within this set, the majority of strains were correctly identified indicating that MALDI-TOF MS is a reliable mean for the identification of food related yeast isolates.

<footnote n="1">¹ CBS-KNAW Fungal Biodiversity Centre, Uppsalalaan 8, 3584 CT Utrecht, the Netherlands</footnote>

<footnote n="2">² Laboratory of Molecular Cell Biology, Institute of Botany and Microbiology, KU Leuven, B-3001 Leuven-Heverlee, Flanders, Belgium</footnote>

<footnote n="3">³ Department of Molecular Microbiology, VIB, B-3001 Leuven-Heverlee, Flanders, Belgium
Contact email: t.boekhout@cbs.knaw.nl</footnote>
NONCONVENTIONAL YEASTS CAN BE EVOLVED TO BE EFFICIENT ETHANOL PRODUCERS UNDER BACTERIAL SELECTION PRESSURES

Nerve Zhou<fnref n="1" />, Krishna B. S. Swamy<fnref n="2" />, Jun-Yi Leu<fnref n="2" />, Silvia Galafassi<fnref n="3" />, Concetta Compagno<fnref n="3" />, Jure Piškur<fnref n="1" />

Correspondence email: Nerve.Zhou@biol.lu.se

Saccharomyces yeasts have evolved a fermentative life style even under fully aerobic conditions. However, the ability to metabolise sugars into ethanol is poor in pre-WGD yeasts when compared to post-WGD yeasts, such as *Saccharomyces cerevisiae*. Though, the events that have shaped the evolutionary history of budding yeasts have been widely explored, our understanding of the genetic factors responsible for variability in inter-species fermentative ability remain poorly understood. We performed an evolution experiment with *Lachancea kluyveri*, a pre-WGD yeast and a relatively poor ethanol producer, in an attempt to transform them into better fermenters and identify the mechanism underlying this transformation. A modified serial dilution transfer method was used to mimic the natural environment, where *L. kluyveri* had to compete for the available carbon source with co-habiting microbes such as bacteria. *L. kluyveri* was co-cultivated with six species of bacteria with increasing ethanol resistance, which served as a selection pressure and had to compete with bacteria to defend their niche. After 720 generations, the evolved strains of *L. kluyveri* could out-compete the co-cultured bacteria within 21 hours, while in the ancestral strain; the competing bacteria successfully thrived until its natural death. The evolved strains demonstrated a 55.8% increase in ethanol production, 30% increase in glucose consumption and quadrupled its glycerol production. This was coupled by a complete decline in acetate production and lowered dependency (27%) on oxygen in its cellular economics, suggesting *L. kluyveri's* transformation to fermentative metabolism yeast. Whole genome and RNA sequencing results has led us to speculate homozygotisation of chromosome III in evolved strains as a plausible cause for this transformation. We are currently performing experimental and computational analysis to confirm this speculation and further dissect the changes in genetic factors responsible for the evolution of *L. kluyveri* into an efficient ethanol producer.

<footnote n="1">¹Department of Biology, Lund University, Lund, Sweden.</footnote>

<footnote n="2">²Institute of Molecular Biology, Academia Sinica, Taipei, Taiwan.</footnote>

<footnote n="3">³Department of Food, Environmental and Nutritional Sciences, University of Milan, Milan, Italy.</footnote>
BIOFLAVOR PRODUCTION OF NON-*Saccharomyces* YEASTS IN FERMENTED BEVERAGES

Alicia Gutiérrez<fnref n="1" />, Michael Katz<fnref n="1" />, Silvia Carlin<fnref n="2" />, Urska Vrhovsek<fnref n="2" /> and Zoran Gojkovic<fnref n="1" />

Flavors produced by biological methods (bioflavors) are now more and more requested by consumers, increasingly concerned with health and environmental problems caused by synthetic chemicals. Currently, the use of different microorganisms for natural flavor synthesis is a fast growing sector due to interest in the development of new products, such as alcohol-free beers, reduced alcohol wines and fruity aroma drinks. Non-*Saccharomyces* yeasts constitute an untapped reservoir with a huge industrial potential in this sector. In this work, 60 non-*Saccharomyces* yeasts were screened, covering 40 different genera, for their capacity to produce pleasant novel aromas. Apart from the choice of yeast, several factors such as medium composition, contribute to flavor production. For this reason, three important beverage sectors were studied (wine, beer, cider) and their effect on fermentation activity and volatile/non-volatile production. We will present results that show that different combinations of strains and media can be used to produce new products with divergent aroma profiles.

<footnote n="1">¹ Applied Yeast Research, Carlsberg Research Center, Gamle Carlsberg Vej 4-6, 1799, Copenhagen V, Denmark</footnote>

<footnote n="2">² Food Quality and Nutrition Department, Fondazione Edmund Mach – Research and Innovation Centre, Via Edmund Mach 1, 38010, San Michele all'Adige, Italy
Contact email: AliciaGutierrez.Linares@carlsberg.com</footnote>
MOLECULAR BASIS OF SUPERIOR STRESS TOLERANCE IN NONCONVENTIONAL YEASTS

Dorota Radecka, Maria Remedios Foulquie y Moreno, Johan M. Thevelein

Lab. Molecular Cell Biology, Institute of Botany and Microbiology, Department of Biology, KU Leuven and Department of Molecular Microbiology, VIB, Kasteelpark Arenberg 31, bus 2438 B-3001 Leuven-Heverlee, Flanders, Belgium

Contact email: Dorota.Radecka@mmbio.vib-kuleuven.be

Stress resistance is a desirable characteristic of industrial yeast strains since they encounter a plethora of sequential and simultaneous stress conditions, such as high osmolarity and temperature fluctuations, in the course of their production and usage. The availability of highly stress resistant baker's and brewer's yeast strains would be beneficial in this respect. A thorough exploration of the largely untapped resource of highly stress resistant nonconventional yeast species could reveal a promising road to further improve industrial yeast strains. This research focuses on two species, *Zygosaccharomyces rouxii* and *Kluyveromyces marxianus*, which were selected based on previous reports for their high osmo- and thermotolerance, respectively. In this study, phenotypic evaluation of the CBS yeast strain collection was performed to select the most sugar tolerant and temperature tolerant strains. *Zygosaccharomyces rouxii* (CBS 741) displayed tolerance to high glucose concentrations (up to 90%) while *Kluyveromyces marxianus* (CBS 2080) managed to grow at high temperature (up to 50°C). Identification of individual unknown genes from those nonconventional yeast species responsible for their extreme phenotype is the first step to further improve traits of interest in industrially used *Saccharomyces cerevisiae*. In order to do so, a cDNA library of both species was constructed in a customized vector. The libraries were screened in the *S. cerevisiae* S288c background, aiming to identify genes that are responsible for the extreme phenotype of unconventional yeast species and capable of improving the performance of closely related species such as *S. cerevisiae*.
THE EFFECT OF THE NITROGEN SOURCES ON THE FORMATION OF VOLATILE AROMA COMPOUNDS BY NON-CONVENTIONAL *Saccharomyces* SPECIES

Jiří Stříbný, Roberto Pérez-Torrado and Amparo Querol

Food Biotechnology Department, Institute of Agrochemistry and Food Technology (IATA-CSIC), Avda. Agustín Escardino, 7 46980 Paterna (Valencia), Spain
Contact email: stribnyjiri@gmail.com

Nitrogen is one of the most important yeast nutrient which significatnly influences
fermentation. The use of different nitrogen sources to avoid its deficiency in the fermentation
process has become a common practice in the wine industry. Another common practice in
winemaking of the recent years is introducing of some non-conventional species from
*Saccharomyces* genus with unusual abilities. The species *S. kudriavzevii* and *S. uvarum* have
shown very interesting properties leading to higher production of certain aromatic
compounds. Since ammonium and amino acids are commonly used as a nitrogen supplement,
we aimed to determine how *S. kudriavzevii* and *S. uvarum* manage these nitrogen sources and
how they affect the production of volatile aroma compounds. The results show differences
especially in the total volatile compounds composition giving *S. uvarum* as a great producer
of acetate esters while *S. kudriavzevii* dominates in the production of higher alcohols. Based
on this information we decided to compare the homologous genes from *S. cerevisiae*, *S. kudriavzevii* and *S. uvarum* involved in the flavour compounds synthesis. The in silico
analysis identified the most radical substitutions in *ARO10*, *ATF1* and *ATF2* genes. When
expressed in winery *S. cerevisiae* T73 strain, the increased amount of several aromatic
compounds was observed. We detected increase of the production of banana and fruity
flavours, such as isoamyl alcohol, isobutanol and their esters. Enzymatic assay also revealed
interesting differences among the Aro10 proteins where Aro10p from *S. kudriavzevii* shows
no preference to measured substrates while for *S. cerevisiae* Aro10p phenylpyruvate is the
preffered substrate. Our results and the fact of close phylogenetic relationship among the
three species suggest that the modification of overall wine aroma could be reached only by
substitution of a few nucleotides.
Ida M. Smith¹, ², Adam Baker¹, Nils Arneborg², and Lene Jespersen²

¹Chr. Hansen A/S, Health & Nutrition Division, Hørsholm, Denmark

²University of Copenhagen, Department of Food Science, Frederiksberg, Denmark

Contact email: dkims@chr-hansen.com

The concept of individual microorganisms influencing the makeup of T cell subsets via interactions with intestinal dendritic cells (DCs) appears to constitute the foundation for immunoregulatory effects of probiotics, and several studies have reported probiotic strains resulting in reduction of intestinal inflammation through modulation of DC function. Consequent to a focus on *Saccharomyces boulardii* as the fundamental probiotic yeast, very little is known about non-*Saccharomyces* yeasts in terms of their interaction with the human gastrointestinal immune system.

We evaluated the immune stimulating capabilities of a diverse selection of non-*Saccharomyces* yeasts by incubation with human monocyte-derived DCs followed by DC incubation with autologous naive T cells. Quantification of secreted cytokine levels led to the identification of yeasts with highly reproducible and distinct DC and T cell cytokine induction profiles, as compared to the established probiotic *S. boulardii*. The observed differences in induced cytokine profiles indicate that certain yeasts are capable of inducing an immune response dominated by $T_{reg}$ cells, whereas others appear to induce a more complex adaptive immune response involving $T_{H1}$, $T_{H17}$, and $T_{reg}$ cells.

To explore the mechanisms behind the observed cytokine induction, we blocked relevant DC pattern recognition receptors and investigated the cytokine inducing properties of yeast cell wall extracts. Our data identify the β-glucan receptor Dectin-1 as key for DC recognition of *S. boulardii* as well as non-*Saccharomyces* yeasts, initiating downstream signaling pathways leading to the observed DC cytokine profiles. In contrast, TLR2 and DC-SIGN do not appear involved in the recognition. As expected based on the identification of Dectin-1 as involved in yeast recognition, β-glucan containing yeast cell wall extracts induced robust DC cytokine secretion, an observation that parallels recent *in vivo* findings and appears to support a hypothesis that yeast cell wall components are responsible for the observed immune cell stimulation.
HIGH THROUGHPUT SCREENING OF NON-CONVENTIONAL YEASTS IN
FERMENTATION PERFORMANCE AND FLAVOUR PRODUCTION

Davide Ravasio, Jürgen Wendland, and Andrea Walther

Carlsberg Laboratory. Yeast Genetics

The yeast biodiversity harbours many thousand species. This represents a yet untapped
natural resource of strains that could contribute with novel flavors to industrial
fermentations. Our approach to cover this biodiversity was based on the previous
identification of strains in fermentations, for example of beverages, fruits or meat. Our list
of several dozen strains encompasses a wide variety of species within the *Saccharomyces*
clade. These strains were tested via plate assays for various responses to stresses, e.g.
ethanol, osmolarity and temperature. Then all strains were subjected to defined
fermentation conditions and their flavour profiles were analyzed by GC/MS. As could have
been expected the list of aroma compounds we detected covered a wide variety of
substances and compound groups. Data mining and statistical analysis software were
employed to cluster and highlight species related flavors. Interestingly, significant
differences in the production of volatile compounds were observed even within the same
species. Esters were the major volatile compounds detected. Strong producers of sweet and
fruity fatty acid ethylesters such as ethyl butanoate, ethyl hexanoate, ethyl octanoate and
ethyl decanoate were found within *Schwanniomyces* sp., *Torulaspora* sp. and
*Wickerhamomyces* sp. Strains of the genus *Wickerhamomyces* also produced significant
amounts of acetic acid and ethyl acetate also in comparison to other species. In addition to
the traditional volatiles, e.g. isoamyl alcohol, we also discovered strains rich in specific
flavors. *Candida* sp. and *Pichia* sp., for example, were able to produce small amounts of
terpenes (isogeraniol and geraniol), flavors that are usually derived from grapes. This work
provides further insight into the flavour characteristics of non-*Saccharomyces* yeasts and
their suitability e.g. for mixed fermentations.
DEVELOPMENT OF A NEW FERMENTED BEVERAGE EMPLOYING CASHEW
APPLE JUICE AND NON-CONVENTIONAL YEASTS

<u>Amparo Gamero</u><fnref n="1" />, Xiao Ren<fnref n="2" />,<fnref n="3" />, Leo Lamboni<fnref n="2" />,<fnref n="3" />, Wynand Alkema<fnref n="1" />, Eddy Smid<fnref n="2" />,
Anita Linnemann<fnref n="3" />

Contact email: amparo.gamerolluna@gmail.com

Cashew apples are considered low cost products or a waste stream in the cashew nut
processing industry. Around 90% of the cashew apple production is left on the field after
removing the nuts. However, such an amount of biomass presents a high potential as
fermentation substrate, since it is rich in vitamins (especially vitamin C), carbohydrates and
minerals. The objective of this study, as part of Cornucopia project, was to investigate the
use of cashew apple juice as a low cost substrate for developing a process for the production
of a novel beverage fermented by non-conventional yeasts. A number of modifications were
done to improve the juice quality, decreasing its typical sourness and astringency due to the
high tannin contents. Yeast strains were selected and subsequently tested for their growth
potential at various conditions of temperature and oxygen and the aroma profiles of the
fermented juices were determined using GC-MS. Statistical analysis was applied to evaluate
the impact of oxygen, temperature and strains on volatile aroma production. The final
product was slightly yellowish, acidic in taste, low in alcohol and maintained a high level
ascorbic acid and total polyphenols.

<footnote n="1">¹Health department, NIZO Food Research B.V., Ede, The Netherlands.</footnote>

<footnote n="2">²Laboratory of Food Microbiology, Wageningen University, The Netherlands.</footnote>

<footnote n="3">³Laboratory of Food Quality and Design, Wageningen University, The Netherlands.</footnote>
NOVEL CYSTEINE-CENTERED SULFURE METABOLIC PATHWAY AND ITS
REGULATION IN THE METHYLOTROPHIC YEAST *Hansenula polymorpha*

Hyun Ah Kang

Department of Life Science, Chung-Ang University, Seoul 156-756, Korea

Contact email: hyunkang@cau.ac.kr

Sulfur, an essential element for all living organisms, is present in two amino acids, cysteine and methionine. In yeast and filamentous fungi, sulfide can be condensed either with *O*-acetylhomoserine to generate homocysteine, the precursor of methionine, or with *O*-acetylserine to directly generate cysteine. We systematically analyzed the sulfur metabolic pathway of the thermotolerant methylotrophic yeast *Hansenula polymorpha*, which has attracted much attention as an industrial yeast strain for various biotechnological applications. Quite interestingly, the detailed sulfur metabolic pathway of *H. polymorpha*, which was reconstructed based on combined analyses of the genome sequences and validation by systematic gene deletion experiments, revealed the absence of *de novo* synthesis of homocysteine from inorganic sulfur in this yeast. Thus, the direct biosynthesis of cysteine from sulfide is the only pathway of synthesizing sulfur amino acids from inorganic sulfur in *H. polymorpha*, despite the presence of both directions of transsulfuration pathway between cysteine-homocysteine-methionine. Moreover, only cysteine, but no other sulfur amino acid, was able to repress the expression of a subset of sulfur genes, suggesting its central and exclusive role in the control of *H. polymorpha* sulfur metabolism. Our results revealed the unique features of *H. polymorpha* sulfur metabolic pathway and its regulation, which are noticeably distinct from those of other yeast and filamentous fungal species.
COMBINING GENETIC AND FUNCTIONAL APPROACHES TO DECIPHER THE MOLECULAR BASES OF LOW SULFITE PRODUCTION BY WINE YEASTS

Jessica Noble<fnref n="1" />, Isabelle Sanchez<fnref n="2" />, Anne Ortiz-Julien<fnref n="1" /> and Bruno Blondin<fnref n="2" />

Contact email: jnoble@lallemand.com

Enological yeasts contribute greatly to the final aromatic balance of wines through the production of volatile compounds of interest; nevertheless, they can also be responsible for the production of negative off-flavours, such as sulfur compounds. Sulfite and sulfide are two of those compounds whose production has to be controlled. The sulfate assimilation pathway has been widely studied, however, little is known about the molecular basis responsible for the differences in sulfite and/or sulfide production between yeasts strains. In order to address the genetic determinism of such properties, we implemented a QTL mapping approach along with phenotypic and transcriptomic characterization of a couple of wine yeast strains highly differing in their sulfite production. This global study resulted in the identification of two new allelic variants of the *MET2* and *SKP2* genes. Functional validations demonstrated their involvement in the control of the production of sulfite, sulfide and acetaldehyde. The combination of both alleles of the low sulfite producer strain is responsible for a strong control of the entire sulfur assimilation pathway and we show that it is strong enough to control the production levels of sulfur compounds in other wine yeast. Transfer of those alleles in a high sulfite producer strain has already been performed through backcrossing cycles using marker-assisted selection and allowed us to construct an optimized strain low SO₂, low H₂S and low acetaldehyde producer.

<footnote n="1">¹ Lallemand SAS, 19 rue des Briquetiers, BP59, 31702 Blagnac Cedex, France</footnote>

<footnote n="2">² INRA, UMR1083, 2 Place Viala, F-34060 Montpellier Cedex 1, France</footnote>
A COMPARATIVE APPROACH TO STUDY NITROGEN CATABOLITE REPRESSION
IN YEASTS

Francisco Pérez de los Santos, Ángel Jiménez and Lina Riego-Ruiz

División de Biología Molecular, Instituto Potosino de Investigación Científica y Tecnológica.

San Luis Potosí, SLP, México.

Contact email: lina@ipicyt.edu.mx

Fungi are ubiquitous free living cells and are able to use a wide range of compounds as carbon and nitrogen sources. The yeast *Saccharomyces cerevisiae*, in a mechanism known as Nitrogen Catabolite Repression (NCR), is able to preferably utilize good nitrogen sources (e. g. glutamine) instead of bad nitrogen sources (e. g. proline). When good nitrogen sources are available, NCR-sensitive genes are repressed. In *S. cerevisiae*, NCR is regulated by the Ure2 protein and by four GATA family proteins: two activators (Gln3 and Gat1) and two repressors (Dal80 and Gzf3). Our interest is to study, using a comparative approach, how different yeasts use diverse nitrogen sources and how this process is regulated. *Candida glabrata* is a human pathogen yeast that is more phylogenetic related to *S. cerevisiae* than to the *Candida* species. *Lachancea kluyveri* is a budding yeast that was first isolated within the yeast flora from the intestinal canal of *Drosophila*. In none of these yeasts a nitrogen repression mechanism has been described yet. For *C. glabrata* and *L. kluyveri*, we have generated a collection of single and double mutant strains in the orthologous GATA factor genes (*GLN3* and *GAT1*) and in the *URE2* gene in order to evaluate its role in nitrogen assimilation when cells were grown on glutamine, ammonia or proline as sole nitrogen sources. In *C. glabrata*, Gln3 but not Gat1, is involved in the uptake of ammonium and proline. In contrast for *L. kluyveri*, Gat1 has a principal role in ammonium and proline utilization. To elucidate if there is a NCR-like mechanism in *L. kluyveri* and in *C. glabrata*, we are evaluating, by qRT-PCR, the transcription of some of the genes involved in the transport and catabolism of the nitrogen sources tested.
TRANSCEPTOR SIGNALING AND ENDOCYTOSIS ARE INDEPENDENT EVENTS
WHICH MAY BE TRIGGERED IN PARALLEL DEPENDING ON SPECIFIC
SUBSTRATE-TRANSCEPTOR INTERACTIONS

Marta Rubio-Texeira, Harish Nag Kankipati, Griet Van Zeebroeck and Johan M. Thevelein

In the last few years, work in our laboratory has shown the existence of plasma membrane
nutrient transporters with signal transduction activity, which have been designated as
transceptors. These transceptors are usually present in a high concentration at the plasma
membrane under conditions in which cells are starved for the nutrient they transport. Upon re-
addition of the missing nutrient to the medium, transceptors transport the nutrient and
simultaneously transduce a signal that results in protein kinase A activation and resumption of
cell growth. When the transceptors are exposed to sufficient amounts of the nutrient they
transport, they are usually endocytosed and sorted to the vacuole for degradation and then
replaced by another set of specific plasma membrane transporters without signaling function,
which remain present during normal growth. This is for example the case for the amino acid
permease, Gap1, and the sulfate transporters, Sul1,2. We wanted to ascertain whether
transceptor signaling triggers endocytosis of the transceptor. Through work with the
transceptors Gap1 and Sul1,2, we have found that different substrates can cause signaling
without triggering endocytosis and vice versa. Moreover, we have found conditions in which
oligo-ubiquitination (a primary signal until now considered sufficient to target a plasma
membrane protein for endocytosis) is triggered by interaction of the transceptor with a non-
transported competitive inhibitor for signaling, without triggering endocytosis. On the other
hand, we have found specific mutants blocking transport and endocytosis but not signaling
which indicate that the substrate interaction sites/trajectory for signaling and for triggering
endocytosis may be partially overlapping but not always coincident. We are also gathering
increasing evidence that signaling and endocytosis occurring during interaction and transport
of extracellular substrates with the transceptor do not require previous intervention of
intracellular metabolic pathways in order to take place. Our results rather support the concept
that different substrates bind to partially-overlapping binding sites in the same general
substrate-binding pocket of Gap1, triggering divergent conformations, resulting in different
conformation-induced downstream processes.
RAS/PKA SIGNAL TRANSDUCTION PATHWAY PARTICIPATES IN THE
REGULATION OF *Saccharomyces cerevisiae* CELL APOPTOSIS IN AN ACIDIC
ENVIRONMENT

Eglė Lastauskienė<fnref n="1" />, Auksė Zinkevičienė<fnref n="2" />

Contact email: egle.lastauskiene@gf.vu.lt

The acidification of the medium is observed during yeast cell growth. This process contributes to the emission of organic acids, mainly acetic acid. Acetic acid is known as the inducer of apoptosis in the yeast *Saccharomyces cerevisiae*. In this study, we showed that hydrochloric acid can also induce apoptosis in yeast cells, and the apoptotic phenotype triggered by treating yeast cells with hydrochloric acid is modulated by the Ras/PKA pathway. The Ras/PKA pathway is highly conserved between all eukaryotic organisms, as well as cell processes that are related to apoptosis and aging. Apoptosis of the yeast cell is the source of molecules such as amino acids and peptides. In this research, we demonstrated that the activation of the Ras/PKA pathway by insertion of Ras2Val19 allele or deletion of PDE2 gene increases cell death, displaying the markers of apoptosis

in an acidic environment. Downregulation of the pathway by deletion of RAS2, RAS1, PDE1, and insertion of the Ha-ras gene increases the cell viability and diminishes cell death with the apoptotic phenotypes. The deletion of PDE1 gene and double deletion of both phosphodiesterase genes prevent the induction of apoptosis in the cells. Modulations in the Ras/PKA pathway affect cell viability and apoptosis during natural gradual acidification of the medium as well as in acid stress conditions.

<footnote n="1">¹Department of Microbiology and Biotechnology, Faculty of Natural Sciences, Vilnius University, M.K. Ciurlionio str. 21/27, LT-03101 Vilnius, Lithuania.</footnote>

<footnote n="2">²Department of Immunology, State Research Institute Centre for Innovative Medicine, Zygimantų str. 9, LT-01102 Vilnius, Lithuania</footnote>
NUTRIENT SENSING PATHWAYS CONTROL WINE YEAST LIFESPAN

Cecilia Picazo<fnref n="2" />, Helena Orozco<fnref n="2" />, Emilia Matallana<fnref n="2" />, <u>Agustín Aranda</u><fnref n="1" />

Contact email: arandaa@iata.csic.es

Nutrient sensing pathways promote growth and protein translation when nutrients are abundant, while repressing stress response. When food is scarce (dietary restriction), growth stops and stress response mechanisms are activated leading to an increased longevity on stationary phase, or an expanded chronological lifespan (CLS). We have previously studied the genetic determinants of CLS in wine yeast on grape juice fermentation, when nitrogen is scarce and in laboratory media, where glucose if the limiting factor. We noticed that some mechanism that control longevity are sensitive to growth condition, as the transcriptional regulator member of the SAGA complex GCN5, whose deletion shortens CLS in laboratory conditions, while extending it in winemaking conditions. Others components of the SAGA complex were studied. The most important pathway devoted to the response to nitrogen source is TOR/Sch9. Sch9 is a kinase that acts below the TOR kinases controlling protein translation and metabolism. Its deletion in wine yeast leads to an extended CLS in laboratory synthetic medium SC. This effect is partially dependent of GCN5, as it deletion blocks the lifespan extension of SCH9 mutation. Surprisingly SCH9 deletion shortens maximum CLS during grape juice fermentation. This effect is not related to respiration, but to the response to low nitrogen concentration. Surprisingly the amount of glycerol at the end of fermentation of this mutant is highly increased. Therefore, modification of longevity genes is a way to modify metabolite production during winemaking. The relationship between GCN5 and SCH9 and the retrograde response and the mitochondria is studied. Another interesting nitrogen-sensing pathway is the general amino acid control ruled by the Gcn2 kinase and the transcription factor Gcn4. The effect of deleting those genes is quite different. GCN4 deletion leads to a shortened CLS, while deletion of GCN2 promotes a mild extension.

<footnote n="1">¹ Institute of Agrochemistry and Food Technology IATA-CSIC, Valencia, Spain</footnote>

<footnote n="2">²Departament of Biochemistry and Molecular Biology, University of Valencia, Spain</footnote>
INFLUENCE OF THE PROPAGATION STRATEGY IN AN ETHANOL PRODUCTION PROCESS FORM GLUCOSE AND XYLOSE: PROTEOMICS AND GENE EXPRESSION ANALYSIS

Elia Tomás-Pejó<fnref n="1" />,<fnref n="2" />, Lucile Jeusset<fnref n="1" />,<fnref n="3" /> and Lisbeth Olsson<fnref n="1" />

Contact email: elia.tomas@imdea.org

The replacement of fossil fuels with renewable fuels, like bioethanol, is decisive for diminishing CO₂ emissions and reducing the oil dependency. The current industrial bioethanol production relies on sugar- and starch-based raw materials and it is crucial to shift to more sustainable resources like lignocellulosic biomass (e.g. wheat straw, corn stover, etc.). Yeast strains used in lignocellulosic bioethanol production have to cope with challenging conditions, such as high concentration of inhibitors produced during pretreatment and simultaneous use of different carbon sources. A very important step in every fermentation process is the yeast propagation, in which it is crucial to obtain high number of cells with the best fermentative efficiency. In the last years, the possibility of exposing the cells to lignocellulosic hydrolysate with inhibitors during the propagation step has given good results in terms of cell viability and high ethanol concentrations in the following fermentation. The yeast molecular adaptation to different growth conditions during the propagation is, however, poorly characterized. In this work we used the xylose-fermenting strain *Saccharomyces cerevisiae* KE6-12 to study the impact of the propagation strategy in the ethanol production process. We proved that the propagation strategy influences significantly the ethanol production. In fermentation tests with wheat straw hydrolysate, cells propagated in minimal media with 15% (v/v) hydrolysate produced 25% more ethanol and consumed 50% more xylose than cells propagated without hydrolysate (0% v/v). Furthermore, cells fermentation performance varies when the hydrolysate is added at early, mid or late exponential phase during propagation. The different hydrolysate addition points implied changes in gene expression of *ADH6*, *ZWF1*, *GSY2*, *TPS1*, *ERG2* and *ALD6*. The whole proteome of *S. cerevisiae* KE6-12 after hydrolysate addition at mid exponential phase was analyzed and compared with the proteome of cells subjected to heat shock to unravel the beneficial effect of the hydrolysate addition.

<footnote n="1">¹ Industrial Biotechnology, Dept. of Chemical and Biological Engineering, Chalmers University of Technology, Gothenburg, Sweden.</footnote>

<footnote n="2">² Unit of Biotechnological Processes for Energy Production, IMDEA Energy, Móstoles (Madrid), Spain.</footnote>

<footnote n="3">³ Department of Sciences and Processes for Food and Bioproducts, AgroParisTech, Paris, France.</footnote>
INDUCING PSEUDO-WARBURG EFFECT IN YEAST *Saccharomyces cerevisiae*

Tadeja Ambrožič and Matic Legiša

National Institute of Chemistry, Hajdrihova 19, SI-1000 Ljubljana, Slovenia
Contact e-mail: matic.legisa@ki.si

Human cancer cells are characterised by rapid glucose consumption, with most of the glucose-derived carbon being secreted as lactate despite abundant oxygen availability. This deviant energetic metabolism known as the »Warburg effect« has been designated as one of the hallmarks of the cancer. We were first to show that 6-phosphofructo-1-kinase (PFK1), the key regulatory enzyme of glycolysis might be posttranslationally modified in cancers. After proteolytic cleavage of the C-terminal portion of the enzyme, an active, shorter fragment was formed that was insensitive to citrate and ATP inhibition and could cause un-restricted metabolic flow over glycolysis leading to lactate excretion. Excretion of a primary metabolite – ethanol under the aerobic conditions is characteristic also for the yeast cells during the growth at high specific growth rates. To study the role of human shorter PFK1 fragments on the deregulated glycolytic flow, yeast cells might be the most appropriate model organism. For this purpose HD114-8D *pfk* nul strain has been chosen as a recipient for the modified truncated *pfkM* gene encoding 47 kDa fragment of human muscle type PFK-M isoenzyme. Transformants enabling different levels of gene expression were designed and tested under different growth conditions. Initially no growth of transfromants could be detected in the supplemented minimal media (SSM) with different fermentative sugars as a sole C-source. Detailed analyses revealed that deregulated glycolytic flux caused by highly active shorter fragments led to unbalanced NADH/NADPH ratio in transformants growing on fermentative sugars. By optimising the growth conditions a medium was selected that enabled faster growth of the transformant with the truncated *pfkM* gene in respect to those with the native PFK-M enzyme. Recombinant yeast cells could be eventually used for high throughput screening of drugs that might inhibit the activity of shorter fragments in cancer cells and restrict their proliferation.
PHENOTYPIC ANALYSIS OF MULTIPLE *ADH* DELETION MUTANTS OF
*Saccharomyces cerevisiae*

Laurinda Steyn¹, James C. du Preez¹, Jacobus Albertyn¹ and Olga de Smidt²

¹Department of Microbial, Biochemical, and Food Biotechnology, University of the Free State, PO Box 339, Bloemfontein 9300, South Africa

²School of Agriculture and Environmental Sciences, Central University of Technology, Bloemfontein, South Africa

Contact e-mail: steynl@ufs.ac.za

Five alcohol dehydrogenase (ADH) genes are associated with ethanol metabolism in *Saccharomyces cerevisiae*. Five single deletion mutants, five quadruple deletion mutants and a null mutant were engineered to investigate the physiological role of each of these alcohol dehydrogenase (Adh) isozymes. A complement of alcohol dehydrogenase (Adh) isozymes are required for normal metabolic function in *S. cerevisiae*. However, even with multiple *ADH* deletions the mutant strains were still able to grow, albeit with different degrees of impaired growth.

The objective of this report was to elucidate the metabolic effects of these multiple *ADH* gene deletions, using a high-throughput analytical approach. The metabolic profiles were determined using an OmniLog® Phenotype MicroArray™ system, which is based on redox chemistry and employs cell respiration as a universal reporter. The assay facilitates the rapid and precise quantitation of phenotypes. Phenotypic analysis provided a deeper understanding of the possible functional substitution of these five Adh isozymes by comparing the lost or gained ability of the mutant strains to utilise substrates compared to the reference strain.
UNRAVELLING THE MOLECULAR BASIS OF FRUCTOPHILY IN YEASTS: A
COMPARATIVE GENOMICS APPROACH

Carla Gonçalves<fnref n="1" />, Sara Cabral<fnref n="2" />, Maria José Leandro<fnref n="2" />, Catarina Prista<fnref n="2" />, Madalena Oom<fnref n="1" />,<fnref n="3" />,
Paula Gonçalves<fnref n="1" />

Contact e-mail: carla.goncalves@fct.unl.pt

*Saccharomyces cerevisiae* consumes glucose slightly faster than fructose when both sugars are present, which may lead to problems during wine production due to slow or incomplete fermentations. Other yeasts, like many *Zygosaccharomyces* species, exhibit instead a fructophilic behaviour at high glucose and fructose concentrations. This phenotype seems to be linked to the presence of a particular type of high capacity, low affinity fructose transporters related to Ffz1, which was first identified in *Z. bailii*<fnref n="1" />. Outside the *Zygosacharomyces* genus, Ffz1-like transporters have been found only in the distantly related *Candida magnolia* clade<fnref n="2" />, where it also seems to play a role in fructophily.

We adopted a comparative genomics approach to uncover other genetic requirements
underlying fructophily and are presently focusing on the yeast *Hanseniaspora guilliermondii*,
which exhibits fructophilic behaviour under some conditions, but lacks Ffz1-like transporters.
We identified 10 putative hexose transporters in the draft genome sequence of *H. guilliermondii* and determined which were expressed under conditions that trigger fructophilic
behaviour. Some of these transporters were expressed in a *S. cerevisiae* strain devoid of
hexose transporters and their kinetic properties and substrate specificity were determined.

We are also exploring the possibility that the kinetic properties and specificity of hexokinases,
that normally accept both glucose and fructose as substrates, may play a role in fructophily.
To that end, hexokinase genes were identified in the genomes of fructophilic yeasts and the
kinetics of hexokinase activity was compared between fructophilic and non-fructophilic
yeasts.

Novel genetic determinants of fructophily will be expressed in *S. cerevisiae* and the ability of
resulting strains to ameliorate imbalances in the consumption of fructose during wine
fermentation will be evaluated.

Pina, C. et al., 2004. Ffz1, a new transporter specific for fructose from *Zygosaccharomyces bailii*,
*Microbiology* 150(7):2429-2433

Lee, D.H. et al., 2014. Molecular cloning and characterization of two novel fructose-specific transporters from the osmotolerant and fructophilic yeast *Candida magnoliae* JH110, *Appl Microbiol Biotechnol.* 98(8): 3569-3578

Acknowledgments:

This work was supported by research grants: PTDC/AGR-ALI/112802/2009, PEst-
OE/BIA/UI0457/2011 and SFRH/BD/89489/2012 from Fundação para a Ciência e a Tecnologia,
Portugal.

<footnote n="1">¹Centro de Recursos Microbiológicos, Faculdade de Ciências e Tecnologia UNL, Portugal</footnote>

<footnote n="2">²CBAA, Instituto Superior de Agronomia,UL, Tapada da Ajuda, 1349-017 Lisboa, Portugal</footnote>

<footnote n="3">³ Instituto Superior Ciências da Saúde Egas Moniz, Centro de Investigação Interdisciplinar Egas Moniz (CiiEM), Caparica, Portugal</footnote>
IDENTIFICATION AND MOLECULAR CHARACTERIZATION OF VINYL PHENOL REDUCTASE FROM *Dekkera bruxellensis* CBS4481

Tiziana M. Granato<fnref n="1" />, Diego Romano<fnref n="1" />, Ileana Vigentini<fnref n="1" />, Roberto Foschino<fnref n="1" />, Daniela Monti<fnref n="2" />, Gianfranco Mamone<fnref n="3" />, Pasquale Ferranti<fnref n="4" />, Chiara Nitride<fnref n="4" />, Stefania Iametti<fnref n="1" />, Francesco Bonomi<fnref n="1" />, Concetta Compagno<fnref n="1" />, Francesco Molinari<fnref n="1" />

Contact email: ileana.vigentini@unimi.it

In enology “Brett” character refers to the wine spoilage caused by the yeast *Dekkera/Brettanomyces bruxellensis* and its production of volatile phenolic off-flavours. This peculiarity is due to the activity of the vinylphenol reductase enzyme, that catalyses the conversion from vinyl- to ethyl-phenols and it is both strain- and cultural condition dependent. Nineteen strains of *D. bruxellensis* were screened for vinyl phenol reductase (VPR) activity. Presence/absence of the precursors in the growth medium gave no significant differences in VPR activity, confirming that this enzyme is constitutively expressed. A purified protein with VPR activity was extracted from *D. bruxellensis* CBS4481 cells. The amino acidic sequence, achieved by trypsinolysis and mass spectrometry, revealed a high homology with a Cu/Zn superoxide dismutase (SOD) in the *D. bruxellensis* AWRI1499 genome. Actually, the isolated protein possessed both vinyl phenol reductase and superoxide dismutase activities. The bioinformatics analysis showed the presence of cofactor-binding sites, that are absent or severely altered in sequences of superoxide dismutases from other wine-relevant yeast species, which do not display vinyl phenol reduction. This moonlighting activity of *D. bruxellensis* SOD as a VPR is related to its capacity of catalyzing NADH-dependent reduction of vinyl phenols.

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

<footnote n="2">² Istituto di Chimica del Riconoscimento Molecolare, National Research Council (C.N.R.), Milan, Italy</footnote>

<footnote n="3">³ Institute of Food Science and Technology, National Research Council (C.N.R.), Avellino, Italy</footnote>

<footnote n="4">⁴ Dipartimento di Agraria, University of Naples ‘Federico II’, Portici, Italy</footnote>
NITRIC OXIDE-MEDIATED ANTIOXIDATIVE MECHANISM OF *Saccharomyces*
*cerevisiae* AND ITS APPLICATION TO INDUSTRIAL YEAST

Hiroshi Takagi

Graduate School of Biological Sciences, Nara Institute of Science and Technology, 630-0192
Nara, Japan

Contact email: hiro@bs.naist.jp

The yeast *Saccharomyces cerevisiae* possesses various defense mechanisms against environmental stresses that generate reactive oxygen species (ROS), leading to growth inhibition or cell death. Our recent study showed a novel antioxidative mechanism mediated by nitric oxide (NO) in yeast cells, but the mechanism underlying the oxidative stress tolerance remained unclear. We report here one of the downstream pathways of NO involved in stress-tolerance mechanism in yeast. Our microarray and real-time quantitative PCR analyses revealed that exogenous NO treatment induced the expression of genes responsible for copper metabolism under the control of the transcription factor *Mac1*, including the *CTR1* gene encoding high-affinity copper transporter. We also found that NO produced under high-temperature stress conditions increased the transcription level of the *CTR1* gene, intracellular copper content, the activity of Cu,Zn-superoxide dismutase *Sod1*, and cell viability after exposure to high-temperature in a manner dependent on *Mac1*. NO did not affect the expression of the *MAC1* gene, indicating that NO activates *Mac1* through its post-translational modification. Based on the results shown here, we propose a novel NO-mediated antioxidative mechanism through the activation of *Mac1*. During bread-making processes, baker's yeast mostly *S. cerevisiae* cells are exposed to baking-associated stresses, such as air-drying, high-sugar concentrations and freeze-thaw stresses. These stresses in common induce protein misfolding and mitochondrial damage leading to ROS generation. To develop the commercial production and fermentation process, it is necessary to construct yeast strains with higher tolerance to these stresses. We showed that engineered baker's yeast strains with enhanced NO synthetic ability are tolerant to multiple baking-associated stresses by reducing intracellular ROS level. The increased NO level also improved the fermentation ability after air-drying and freeze-thaw stress treatment in baker's yeast. Hence, appropriate NO production could be promising for breeding novel industrial yeast strains that are tolerant to various stresses.
ENGINEERING OF *Saccharomyces cerevisiae* FOR THE PRODUCTION OF POLY-3-D-HYDROXYBUTYRATE FROM XYLOSE

Alejandro Muñoz de las Heras, Anders G. Sandström, Diogo Portugal Nunes and Marie F. Gorwa-Grauslund

Division of Applied Microbiology, Lund University, SE-222 41 Lund, Sweden
Contact email: alejandro.munoz_de_las_heras@tmb.lth.se

Poly-3-D-hydroxybutyrate (PHB) is a promising biopolymer produced naturally by several bacterial species. However PHB production from undetoxified lignocellulosic hydrolysate, that represent a renewable and cheap substrate, has not been demonstrated. Since baker's yeast *Saccharomyces cerevisiae*, is known to be efficient in fermenting this type of material to ethanol, it could represent an interesting alternative host for PHB production. However, S. cerevisiae needs to be engineered for the utilization of xylose, the main pentose found in lignocellulosic biomass and for PHB production.

In the present study, we evaluated the capacity of metabolically engineered *Saccharomyces cerevisiae* to produce PHB from xylose. In-house engineered *S. cerevisiae* strains, capable of pentose utilization were transformed with the PHB pathway genes from the well-characterized PHB-producer *Cupriavidus necator*. The two host strains carried genes for xylose utilization from the yeast *Scheffersomyces stipitis*, but had different NADH/NADPH preference ratio for the xylose reductase (XR) enzyme, the initial enzymatic step in the *S. stipitis* xylose pathway.
We will present the results of the strain evaluation for PHB production under aerobic conditions.
OPTIMIZATION OF pAOX1 INDUCTION IN A METHANOL/SORBITOL CO-FEEDING PROCESS IN *Pichia pastoris*

Frederic Carly<fnref n="1" />, Hongxing Niu<fnref n="2" />, Patrick Fickers<fnref n="1" />

Contact email: pfickers@ulb.ac.be

The methylotrophic yeast *Pichia pastoris* is currently one of the most interesting hosts for production of heterologous proteins thanks to its short doubling time, its strong methanol-induced pAOX1 promoter and the availability of glycoengineered strains. In the context of heterologous protein production optimization, recent studies have highlighted the benefits of using methanol/sorbitol mixture as a feeding medium. However, very few studies have been performed to quantitatively analyze the cellular physiology during co-feeding. Most of them focused only on the final product (i.e. heterologous protein) without any insights into pAOX1 regulation. In the present work, we report on the quantitative characterization of *P. pastoris* cell metabolism, with special emphasis on the quantification of pAOX1 induction during a methanol/sorbitol co-feeding process by the mean of a *LacZ* reporter gene and transient continuous culture. Our results demonstrated that the cell-specific oxygen consumptions (qO2) could be reduced by decreasing the methanol fraction in the feeding media and that optimal pAOX1 induction was achieved and maintained in the range of 0.45 to 0.75 C-mol/C-mol of methanol fraction. In addition, the qO2 was reduced by 30% at most in those conditions. The effect of the feeding rate on pAOX1 induction was also investigated in fed-batch culture for a 0.6 C-mol/C-mol of methanol fraction. Our results clearly show a significant improvement of the induction level with an optimal feeding rate of 6,6 mmolC.gDCW.h⁻<fnref n="1" />. The results also showed that *P. pastoris* tends to use methanol as the main carbon source in non-limiting oxygen conditions, while sorbitol seems to be the main carbon source in oxygen-limiting conditions.

<footnote n="1">¹ Unité de Biotechnologies et Bioprocédés, Université libre de Bruxelles, Av. Roosevelt 50, CP165/61, 1050 Bruxelles, Belgium</footnote>

<footnote n="2">² Center for Process Systems Engineering, Department of Chemical Engineering Imperial College London, South Kensington Campus SW7 2AZ, London, UK</footnote>
FLD GENE DISRUPTION LEADS TO AN ENHANCED AOX1 PROMOTER
INDUCTION IN RESPONSE TO EITHER METHANOL OR FORMATE

Oleg V Tyurin¹, Dmitry G Kozlov¹

¹ Research Institute for Genetics and Selection of Industrial Microorganisms, Moscow, Russia
Contact email: oleg_tyurin@genetika.ru

The understanding of mechanisms of methanol induction in methylotrophic yeasts, targeting increase in pAOX activity and inducers variety as well, has valuable biotechnological application. Recently we showed that not only toxic methanol but non-toxic and safe formate as well can effectively induce pAOX1 in yeast *K.phaffii* (GS115) and *K.kurtzmanii* Y727 [Naumov et al. 2013]. However, the nature of intracellular inducer of pAOX1 is still unknown. The data obtained allowed us to consider two scenarios of induction: either formed of formate methanol itself can act as a direct inducer, or one of the intermediate of methanol utilization pathway can induce pAOX1. In order to check the first "methanol" hypothesis, we derived the mutant strains GS115 *fldA* and Y727 *fldA* with FLD gene coding for formaldehyde dehydrogenase deleted to subject them to formate induction. It was expected that there would be no induction in these cells in response to formate due to the reason that FLD disruption breaks the methanol utilization pathway, so no methanol can be formed of formate. But in fact, that assay revealed that FLD disruption, on the contrary, led to an even enhanced induction of pAOX1 in the above mentioned strains in response to formate as well as to methanol. In particular, the activity of beta-galactosidase under control of pAOX1 increased 2-3-fold in mutant strains with disrupted FLD compared to the one in the wild strains. Thus, these data suggest that methanol is not likely to be a direct inducer of pAOX1. Accordingly, there should be yet non-identified intermediate directly involved in the mechanism of induction or there might be an alternative, yet unknown, pathway of transformation of formate into methanol.
A WIDE VARIETY OF DEVELOPMENTAL YEAST FROM OYC

Koji Yamada, Shuhei Matsuo, Shuhei Furukawa, Tomoo Isobe, Hideyo Fukuhara, Ryoichi Nakajima and Masayasu Ando

Laboratory of Yeast & Fermentation, Food Division, Oriental Yeast Co., Ltd, 3-6-10 Azusawa, Itabashi-Ku, Tokyo 174-8505, Japan

Contact Email: yamada.koji.cj@nisshin.com

Various bakery markets have been formed in Japan. Each yeast maker has developed various yeasts that combine various abilities to meet market needs. In Japan, Oriental Yeast Co., LTD (OYC) has been developed various yeast and supported the market.

OYC has yeasts which combine the unique abilities such as for ultra-sweet dough, cold sensitive fermentation, fungicidal property, cold dough, and so on. We will report each of the ability in this conference, and discuss about the factors to exert the abilities.
GENETIC ENGINEERING FOR ALTERING METABOLIC FLOW IN
*Saccharomyces cerevisiae* TO PRODUCE HIGHER ALCOHOLS

Jun Ishii<fnref n="1" />, Fumio Matsuda<fnref n="2" />,<fnref n="3" />, and Akihiko Kondo<fnref n="3" />,<fnref n="4" />

Contact E-mail: junjun@port.kobe-u.ac.jp

The production of higher alcohols including C3-C5 alcohols in engineered bacteria has received significant attention, because they can be used as fuels, fuel additives, or commodity chemicals. The budding yeast, *Saccharomyces cerevisiae*, has considerable potential as a producer of higher alcohols because of its capacity to naturally fabricate fusel alcohols, in addition to its robustness and tolerance to low pH. However, because its natural productivity is not significant, we considered a strategy of genetic engineering to increase production of higher alcohols. For example, the branched-chain higher alcohol isobutanol, which is involved in valine biosynthesis, offers higher octane values than their straight-chain counterparts with equivalent carbon numbers and can be used with current infrastructure in addition to possessing almost the same capabilities (energy density, hygroscopicity and octane number) as gasoline. To produce isobutanol, we first overexpressed two genes encoding 2-ketoacid decarboxylase (KDC) and alcohol dehydrogenase (ADH) in *S. cerevisiae* and then tried the gene deletions involved in pyruvate metabolisms. We further tested the overexpressions of a number of endogenous genes to alter the metabolic flow for improving isobutanol production. To achieve this, we constructed the autonomous replication plasmid for expressing three genes simultaneously, and prepared several sets with several selection markers. Using the constructed vectors, we overexpressed nearly 10 genes and successfully improved the isobutanol production in *S. cerevisiae*. This work was supported by Special Coordination Funds for Promoting Science and Technology, Creation of Innovation Centers for Advanced Interdisciplinary Research Areas (Innovative Bioproduction Kobe; iBioK), MEXT, Japan, and in part by Industrial Technology Research Grant Program in 2011 from New Energy and Industrial Technology Development Organization (NEDO) of Japan.

<footnote n="1">¹Organization of Advanced Science and Technology, Kobe University, Japan</footnote>

<footnote n="2">²Graduate School of Information Science and Technology, Osaka University, Japan</footnote>

<footnote n="3">³RIKEN Biomass Engineering Program, Japan</footnote>

<footnote n="4">⁴Department of Chemical Science and Engineering, Graduate School of Engineering, Kobe University, Japan</footnote>
STUDY OF THE PHYSIOLOGICAL STATE OF *Kluyveromyces marxianus* USING
NON-CONVENTIONAL METHODS

Alejandro Arana Sánchez<fnref n="1" />, Emilie Amillastre<fnref n="1" />, Enrique Herrera López<fnref n="1" />, Javier Arrizon Gaviño<fnref n="1" />, Laurence Preziosi-Belloy<fnref n="2" />, Nicolas Gay<fnref n="2" />, <u>Anne Gschaedler</u><fnref n="1" /> and Charles Ghommidh<fnref n="2" />

In bioprocesses and particularly in yeast fermentation the activity and the physiological state of microorganisms are variables still difficult to assess. Most of the information is obtained from delayed off-line measurements and remains insufficient for the development of real time control strategies to optimize the potential of microorganisms and design high performance processes. On-line quantification of the physiological state of cells is of paramount importance for the understanding and improvement of cell metabolism and thus to control pathways of interest.

In the project, Spectroscopies for the evaluation and control in real time of biological cells and physiological state (SPECTRE), innovative techniques such as: optical and dielectric spectroscopy are combined to estimate and measure the instantaneous biomass and the physiological state of microorganisms during fermentation. The yeast *Kluyveromyces marxianus* was chosen as a study model due to its capacities for enzyme production (fructanase) and aromatic compounds like esters. The Fogale® dielectric spectroscopy probe allows determine the viability of cells and the Casy® cell counter permit to determine single cell size and volume. The first results obtained in batch culture with different carbon substrates, with/without induction for enzyme production using the dielectric spectroscopy and the Casy cell counter showed that additionally to the measurement of number of cells and cell viability, the selected tools permitted to unravel additional physiological data of the cells, like the evolution of cell volume, membrane capacitance and cellular conductivity reflecting the physiological state of the cell. From the strategies developed it will be possible to set optimization and automatic control strategies to guarantee high performance processes.

<footnote n="1">¹ Centro de Investigación y Asistencia en Tecnología y Diseño del Edo. de Jalisco A.C., Área de Biotecnología Industrial, Av. Normalistas 800, 44270 Guadalajara, Mexico.</footnote>

<footnote n="2">² Université Montpellier II, IATE, Place E. Bataillon, 34095 Montpellier, France.</footnote>
METABOLIC ENGINEERING OF *Saccharomyces cerevisiae* FOR PRODUCTION OF ADIPIC ACID FROM RENEWABLE SOURCES - ONE ROUTE TOWARDS A BIOECONOMY

Emma Karlsson<sup>a</sup>, Luigi D'Avino<sup>a</sup>, Lisbeth Olsson<sup>a</sup>, Valeria Mapelli<sup>a</sup>

<sup>a</sup>Industrial Biotechnology, Chalmers University of Engineering, Kemigården 4, 412 96 Gothenburg, Sweden

Contact email: Emma.Karlsson@chalmers.se

Adipic acid is a six carbon long dicarboxylic acid, considered to be the most important synthetic dicarboxylic acid annually produced, according to the International Energy Agency (IEA). The global production of adipic acid had in 2010 a volume of 2.8 million tonnes, for a total market price of 4.9 billion USD. The current production of adipic acid relies on non-renewable fossil raw materials, leading to emission of the greenhouse gases carbon dioxide and N<sub>2</sub>O. In addition, the production starts from benzene, whose use has several health related negative implications. This project aims to create a greener process for production of adipic acid developing a fermentation-based process using Swedish domestic renewable raw materials, such as forest residues and/or algae. These materials will be used to establish a biorefinery, wherein the fermentation process for the biosynthesis of adipic acid will represent the core process. Our current strategy is based on the generation of genetically modified strains of the yeast *Saccharomyces cerevisiae*, harbouring heterologous enzymatic activities allowing the conversion of lysine into adipic acid. This system is our first choice and will also work as proof-of-concept for bio-based production of adipic acid. Here we present the metabolic engineering strategy we are pursuing, based on two possible metabolic pathways for conversion of lysine into adipic acid. Preliminary results on the effect of adipic acid on *S. cerevisiae* physiology, lysine uptake, the expression of the heterologous genes of choice, and the conversion of lysine into adipic acid precursors are presented.
YEASTS FOR THE SUSTAINABILITY IN VITICULTURE AND OENOLOGY: THE
“YESVITE” PROJECT

Ileana Vigentini¹, Concetta Compagno¹, Osvaldo Failla¹, Roberto Foschino¹, Gianluigi Cardinali², Laura Corte², Cristina Pelliccia², Luca Roscini², Jure Piškur³, Olena P. Ishchuk³, Uroš Petrovič⁴, Janez Kokosar⁴, Mojca Brloznik⁴, Amy A. Caudy⁵, Frederick P. Roth⁵, Florian F. Bauer⁶, Benoit Divol⁶, Maret Du Toit⁶, Evodia Setati⁶, David Maghradze⁷, Ekaterine Abashidze⁷, Ramash Chipashvili⁷

¹ Department of Food, Environmental and Nutritional Sciences and Department of Agricultural and Environmental Sciences, Università degli Studi di Milano, Milan, Italy

²Department of Applied Biology, Università degli Studi di Perugia, Perugia, Italy

³Department of Biology, Lund University, Lund, Sweden

⁴Department of Molecular and Biomedical Sciences, Jožef Stefan Institute, Ljubljana, Slovenia

⁵Donnelly Centre for Cellular and Biomolecular Research, University of Toronto, Toronto, Canada

⁶Institute for Wine Biotechnology and Department of Viticulture and Oenology, Stellenbosch University, Stellenbosch, South Africa

⁷Institute of Horticulture, Viticulture and Oenology, Agricultural University of Georgia, Tbilisi, Georgia

Contact email: ileana.vigentini@unimi.it

The YeSVitE project faces the ongoing process of innovation in winemaking taking into account the continuous challenge of the development of sustainable production practices and new food products. The relevance of the programme stems in the exploitation of the genetic and metabolic potential of yeasts providing answers to questions such as how to produce economically and environmentally sustainable quantities of wines of high quality, or “typical” and traditional wines, and how to produce a healthier product. The main activities of YeSVitE address to the safety and quality of wine with the aim to the development of strains characterised by genetic and phenotypic traits that can modify the final product and the investigation of natural interactions between grape and wine-related microbes to propose alternative solutions for a sustainable viticulture and oenology. YeSVitE has seen the birth of a consortium composed by seven research units with relevant expertise in the study of yeasts as model organisms and as key tools for wine and biotechnological applications. All the involved partners of the network improve the programme with novel knowledge and skills since they operate in areas of unique interest and are characterised by a strong know-how in winemaking. The project is organised in four work packages that are expected to be completed in four years of joint activity.
THE YESVITE PROGRAM: RESEARCH FOCUS AND CONTRIBUTION OF THE
INSTITUTE FOR WINE BIOTECHNOLOGY @ STELLENBOSCH UNIVERSITY

Benoit T. Divol, Mathabatha E. Setati, Maret du Toit and Florian F Bauer

¹ Institute for Wine Biotechnology, Department of Viticulture and Oenology, University of Stellenbosch, South Africa

Contact Email: fb2@sun.ac.za

The IWBT promotes an integrated research program that covers the biology of the full wine research value chain from grape production to wine fermentation, wine chemistry and sensory properties. Research related to the YESVitE program at the IWBT focusses on broader microbiological questions related to wine making, and includes studies on yeast diversity, the fermentation properties of yeast and bacteria, the interactions between species, and the genetic and metabolic regulation of these processes as well as the production of aroma and other impact compounds. In 2014, the YESVitE program focuses on the cellular and molecular response to sulphur dioxide exposure in *Brettanomyces bruxellensis*. The potential role of specific nitrogen sources present in wine is particularly investigated, and molecular responses will be investigated using a transcriptomic approach. This work is performed in collaboration with the University of Milan. For 2015 and 2016, exchanges will focus on interactions between different wine relevant yeast and bacterial species, as well as on their impact on fermentation kinetics and aromatic properties
EXPLOITATION OF BIODIVERSITY IN VITICULTURE AND OENOLOGY TO
ALLOW A SUSTAINABLE WINEMAKING

Osvaldo Failla<fnref n="1" />, Concetta Compagno<fnref n="2" />, Roberto Foschino<fnref n="2" />, Ileana Vigentini<fnref n="2" />

Contact email: osvaldo.failla@unimi.it

Universally recognized as a resource of inestimable value, biodiversity is becoming a recurring topic in several roundtables. As one of the key theme of the next World Expo, biodiversity has nowadays to face several concepts, such as innovation and technology transfer, which aspire to “zero impact” on ecosystems but that inherently remain related to the productivity meaning. Thus, sustainability becomes the "chance" to maintain all the future human activities and the biodiversity preservation the "tool" to limit the environmental impact of industrial productions. Main contribution to the YeSVitE project of the Italian Partner in Milan will be to handle this topic from a qualitative point of view, focusing on the theme of biodiversity analysis and its safeguard, and from an economic/social side, through a wine optimization that promotes the use of yeasts in non-conventional ways. For example, yeast spoilage in beverages has become of increasing importance because of the use of less-severe processing, the great variety of new formulations and the tendency to reduce the use of preservatives. In this context, a better control in the sulphur dioxide manage during winemaking could avoid both the yeast spoilage and, in a sustainable perspective, to limit sulphite in bottled wines. Finally, to face the market, the wine industry should consider the development of innovative wine styles, suitable for the modern consumer needs. Wines with peculiar, distinguishable and intense varietal aroma should be projected with specific grapes varieties and yeast strain, able to amplify the varietal quality.

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

<footnote n="2">² Department of Food, Environmental and Nutritional Sciences, Università degli Studi di Milano, Milan, Italy</footnote>
Laura Corte, Luca Roscini, Claudia Colabella, and Gianluigi Cardinali

Department of Pharmaceutical Sciences, University of Perugia, Via Borgo 20 Giugno, 74, Perugia - Italy

Contact email: gianluigi.cardinali@unipg.it

Most of the efforts in yeast taxonomy during the last three decades have been deployed in the application of molecular DNA techniques, particularly focused on a handful of markers such as LSU, ITS and few others. In spite of the great advancement in phylogenetic terms, this strategy has necessarily overlooked most of the phenotypic description of strains and isolates. The impact of this choice from a general microbiological point of view is questionable, whereas it has brought about a general disinterest in knowing what a strain can effectively “do”, which is at the basis of the industrial and biotechnological application of these organisms.

Wine microbiology is coping with a general crisis of the sector in Europe, with an increasing interest on the microbial biodiversity potential and with the challenges launched by modern life styles, such as the alcohol reduction, the aroma characterization etc. In this scenario, yeast diversity must be necessarily studied with both the molecular and the phenotypic approaches, taking advantage of the technological opportunities available.

From the viewpoint of molecular taxonomy, an important target is to produce effective concepts and efficient algorithms to delimitate species and to allow for an objective definition of the biodiversity present in various geographic areas, wines and winemaking operations.

FTIR (Fourier Transform InfraRed spectroscopy) offers a great tool for a general, rapid and inexpensive way to characterize strains not only for the species they belong to but especially to seek correlations between FTIR bands and relevant phenotypic characters.
QUANTITATIVE PHENOTYPIC MEASUREMENTS FOR POLYGENIC TRAIT
ANALYSIS

<u>Mojca Brložnik</u>¹, Sofia Dashko¹,², Petra Kaferle¹, Justin Fay³, Janez Kokošar¹, Jure Piškur²,⁴,
Klaus Natter⁵ and Uroš Petrovič¹

¹ Jožef Stefan Institute, Department of Molecular and Biomedical Sciences, Ljubljana,
Slovenia

² University of Nova Gorica, Wine Research Center, Vipava, Slovenia

³Washington University School of Medicine, Center for Genome Sciences, St. Louis, Missouri, United States of America

⁴ Lund University, Department of Biology, Lund, Sweden

⁵ Institute of Molecular Biosciences, University of Graz, Graz, Austria
Contact email: mojca.brloznik@ijs.si

The laboratory and natural *Saccharomyces cerevisiae* and other yeast strains are phenotypically very diverse. Some of their phenotypes have biotechnologically and industrially important characteristics. In our laboratory we have been developing growth rate based and fluorescent marker based methods for phenotyping of yeast strains. An example of the latter is being used to determine the lipid content of different strains with flow cytometer or plate reader, where this trait is determined on a single cell or on the population level. The growth rate based method can be performed either in liquid or on agar based media. For the latter, we have developed a designated robotic manipulator that enables quantitative measurements of fitness of a very large number of different strains, based on images acquired using transmission scanner and R package Gitter automated analysis pipeline. As an example, we have been using this approach to identify traits that are potentially interesting for the wine industry from a new collection of natural yeast isolates. For instance, we determined growth under different chemical or physical stresses, such as different carbon and nitrogen sources, or growth inhibitory compounds (acetate, ethanol, sulfites etc.). Such quantitative phenotypic measurements are especially useful for polygenic trait analysis, as they provide important information for inference of causal genetic elements.
BIO-PROTECTION OF GRAPE QUALITY IN VINEYARD AND POST-HARVEST WITHERING

David Maghradze¹, Ekaterine Abashidze¹, Ramash Chipashvili¹

¹Institute of Horticulture, Viticulture and Oenology, Agricultural University of Georgia, Tbilisi, Georgia

Contact email: david.maghradze@gmail.com

The control of fungal diseases and mycotoxins contamination during grape maturation and post-harvesting is currently based on treatments with chemical fungicides. However the environmental dispersion, the progressive loss of effectiveness, the emergence of resistant strains and the increasing level of residues in wine, overall straw wines, have led the European Union to the restrict the use of these compounds (UE Directive 128/2009), addressing the researchers towards innovative and eco-friendly protocols to face the problem. A sustainable approach consists of exploiting the natural antagonistic potential of various yeasts against molds that occurs by different mechanisms such as nutrient competition, killer toxins character, iron depletion, ethyl-esters production or inducing host-plant resistance. Inhibition capabilities on mycelial growth or conidia germination of *Botrytis* and *Penicillium* spp. have been reported by some strains of species living in vineyard and cellar ecosystem, like *Criptococcus laurentii*, *Metschnikowia fructicola*, *Pichia guilliermondii*, *P. membranifaciens*, *P. ohmeri*, *Rhodotorula glutinis*. In this background, the Georgian Partner of the YeSVitE project will carried out a deepen investigation on antagonism patterns that can constitute a promising source of new knowledge to set biological control strategies in order to prevent or reduce harvested commodity damages.
Anders Bergström<fnref n="1" />,<fnref n="2" />, Jared T. Simpson<fnref n="2" />, Francisco Salinas<fnref n="1" />, Benjamin Barré<fnref n="1" />, Agnès Llored<fnref n="1" />, Leopold Parts<fnref n="2" />,<fnref n="3" />, Amin Zia<fnref n="4" />,<fnref n="5" />, Alex N. Nguyen Ba<fnref n="4" />, Alan M. Moses<fnref n="4" />, Paul Coupland<fnref n="2" />, Joseph Schacherer<fnref n="6" />, Edward J. Louis<fnref n="7" />, Ville Mustonen<fnref n="2" />, Jonas Warringer<fnref n="8" />, Richard Durbin<fnref n="2" /> and Gianni Liti<fnref n="1" />

Much is still unknown about the relative phenotypic importance of different kinds of genomic variation, and about the evolution of the genotype-phenotype relationship. We address these and related questions by population level sequencing in baker's yeast. By Illumina sequencing of 42 natural yeast genomes, we found that while genetic divergence in terms of SNPs is much lower in *Saccharomyces cerevisiae* than its sister species *S. paradoxus*, copy number and genome content variation is higher. We hypothesize that this might explain the observation that the domesticated *S. cerevisiae* displays much higher phenotypic diversity than its wild relative. The subtelomeres were highly enriched for functionally relevant variation, highlighting their importance to phenotypic evolution. We are now expanding to sequence 1002 *S. cerevisiae* genomes from a wide range of locations and habitats, greatly widening the population genomics repertoire for the species. Additionally, we are sequencing 10 strains on the long-read PacBio platform to overcome some of the technical limitations of short read sequencing, which particularly affect the repetitive and variable subtelomeres.

<footnote n="1">¹Institute for Research on Cancer and Ageing, Nice (IRCAN), University of Nice, Nice, France</footnote>

<footnote n="2">²Wellcome Trust Sanger Institute, Cambridge, United Kingdom</footnote>

<footnote n="3">³Donnelly Centre for Cellular and Biomolecular Research, University of Toronto, Toronto, Canada</footnote>

<footnote n="4">⁴Department of Cell & Systems Biology, University of Toronto, Toronto, Canada</footnote>

<footnote n="5">⁵Stanford Center for Genomics and Personalized Medicine, Stanford University School of Medicine</footnote>

<footnote n="6">⁶Department of Genetics, Genomics and Microbiology, University of Strasbourg, Strasbourg, France</footnote>

<footnote n="7">⁷Centre of Genetic Architecture of Complex Traits, University of Leicester, Leicester, United Kingdom</footnote>

<footnote n="8">⁸Department of Chemistry and Molecular Biology, University of Gothenburg, Gothenburg, Sweden</footnote>
LINKED QTLs OF MIXED EFFECTS IN COMPLEX TRAITS OF YEAST

Ed Louis¹, Dave Barton¹, Tom Walsh¹, Alexander Gorban², Evgeny Mirkes² and Gianni Liti³

¹Centre for Genetic Architecture of Complex Traits, University of Leicester, UK

²Mathematical Modeling Centre, University of Leicester, UK

³Institute for Research on Cancer and Ageing of Nice, Université de Nice Sophia Antipolis, France

Contact email: ejl21@le.ac.uk

The use of backcrosses and multigenerational crosses in linkage analysis of complex traits in yeast has revealed that many QTLs identified in F1 progeny are actually composed of several linked QTLs. The linked sets generally are comprised of loci with QTLs with effects in the same phenotypic direction as the original, as well as antagonistic QTLs with opposite effects. Initially thought to be a surprising result, this observation fits expectations of how yeast populations adapt and evolve. A re-examination of the high resolution mapping of over 20 QTLs involved in heat tolerance from a 12-generation cross reveals such a linked set with very rare haplotypes of 3 linked segregating variants found in the heat tolerant population. Other non-additive associations are found between unlinked loci as well. We are now generating large numbers of arrayed individuals from multigenerational mapping populations to determine how much of the complex phenotypic variation has this underlying genetic architecture. This has implications as to how QTL analysis should be done in the future.
HIGH-THROUGHPUT PHENOTYPING AND SELECTIVE BREEDING AS AN
EFFECTIVE TOOL TO GENERATE NOVEL BEER YEASTS

Meersman *Esther*<fnref n="1" />,<fnref n="2" />, Steensels Jan<fnref n="1" />,<fnref n="2" />, Stijn Mertens<fnref n="1" />,<fnref n="2" />, Saels Veerle<fnref n="1" />,<fnref n="2" />, Snoek Tim<fnref n="1" />,<fnref n="2" />, Picca Nicolino Martina<fnref n="1" />,<fnref n="2" />, Verstrepen Kevin<fnref n="1" />,<fnref n="2" />

Fermented foods and beverages have been consumed by humans for over 8000 years. The shift from spontaneous to inoculated fermentations, which was started in the late 19th century, greatly increased the quality and reproducibility of the fermentations. However, the main difficulty was to select a yeast strain with all beneficial characteristics necessary for an efficient and high-quality fermentation. Until recently, the appropriate tools and knowledge were lacking to make a well-considered and scientifically found choice. In some industrial foodstuff fermentations, yeast strains are used as starter cultures because of historical rather than scientific reasons. In this study, we screened a large collection of feral and industrial *Saccharomyces* yeast strains (>600), the majority being particularly suited for industrial purposes, such as the production of beer and wine. Our high throughput screening and subsequent data analysis showed an enormous geno- and phenotypic diversity among different *Saccharomyces* strains. Next, hybrids with superior beneficial characteristics were constructed using several different breeding approaches. For example, new hybrid strains with an increased production of fruity aromatic compounds (producing up to 45% more isoamyl acetate) or a higher tolerance to ethanol (producing up to 18,5% ethanol in rich growth medium) were developed. This research provides a unique insight in the variability of industrial and wild yeast strains. It enables us to identify yeast strains for specific production tasks and allows the development of custom-made hybrid strains with specific characteristics. Furthermore, it paves the way for fundamental insight in complex, polygenic phenotypes such as aroma production and ethanol tolerance, which can lead to the identification of target genes for metabolic engineering.

<footnote n="1">¹ Laboratory for Genetics and Genomics, Centre of Microbial and Plant Genetics (CMPG), KU Leuven, Kasteelpark Arenberg 22, 3001 Leuven, Belgium.</footnote>

<footnote n="2">² Laboratory for Systems Biology, VIB, Gaston Geenslaan 1, 3001 Leuven, Belgium.
Contact email: esther.meersman@biw.vib-kuleuven.be</footnote>
DEVELOPMENT OF INTERSPECIES YEAST HYBRIDS TO INTRODUCE AROMATIC DIVERSITY IN LAGER BEERS

Stijn Mertens<fnref n="1" />,<fnref n="2" />, Jan Steensels<fnref n="1" />,<fnref n="2" />, Veerle Saels<fnref n="1" />,<fnref n="2" /> & Kevin Verstrepen<fnref n="1" />,<fnref n="2" />

Despite the enormous diversity in ale yeasts, the diversity of yeasts used in the lager beer
industry (*Saccharomyces pastorianus*) is very limited. In fact, only two archetypes of lager
yeasts (Saaz and Frohberg-type) are currently used in breweries. These two types are the
result of two independent interspecies hybridization events, presumably originating from the
15th or 16th century, between the traditional brewer's yeast *Saccharomyces cerevisiae* and the
*Saccharomyces eubayanus*. This confined genetic (and as a consequence phenotypic) diversity
of lager yeasts is reflected in the relatively limited influence of the yeast strain on the aroma
profile of lager beers. Because product innovation has become more and more important to
breweries, new lager yeasts with a diverse aroma production opens the window for the
production of new lager beers with different flavors. In this study, we generated a large set of
new lager yeasts by crossing carefully selected S. cerevisiae and cold tolerant *Saccharomyces*
species like *S. eubayanus*, *Kudriavzevii* and *mikatae*. We obtained more than 250 new and
unique lager strains with a diversified aroma production profile. Next, these newly developed
hybrids were genetically stabilized and screened for their fermentation capacity and aroma
production in lager beer fermentations and their temperature tolerance. The applied approach
resulted in a large set of new lager yeasts that display an enormous phenotypic diversity, with
different lineages able to produce a whole new spectrum of lager beers with diverse aroma
profiles. Moreover, these strains are directly applicable in industry, since the experimental
procedure only relies on natural crossing and not on genetic modification.

<footnote n="1">¹ Laboratory for Genetics and Genomics, Centre of Microbial and Plant Genetics (CMPG), KU Leuven, Kasteelpark Arenberg 22, 3001 Leuven, Belgium.</footnote>

<footnote n="2">² Laboratory for Systems Biology, VIB, Gaston Geenslaan 1, 3001 Leuven, Belgium.
Contact E-mail: Stijn.Mertens@biw.vib-kuleuven.be</footnote>
REPLICATION ELEMENTS IN THE WINE YEAST *Dekkera bruxellensis*

Olena P. Ishchuk<fnref n="1" />, Tanja Vojvoda Zeljko<fnref n="1" />, Sofia Mebrahtu Wisén<fnref n="1" />, Åsa K. Hagström<fnref n="1" />, Elżbieta Rozpędowska<fnref n="1" />, Linda Hellborg<fnref n="1" />, Juozas Siurkus<fnref n="2" />, Mikael Rørdam Andersen<fnref n="2" />, Dorte Joerck-Ramberg<fnref n="2" />, Zhihao Ling<fnref n="1" />, Anna J. Schifferdecker<fnref n="1" />, Andrei A. Sibirny<fnref n="3" />,<fnref n="4" /> & Jure Piškur<fnref n="1" />

Contact e-mail: Olena.Ishchuk@biol.lu.se

The wine yeast *Dekkera bruxellensis* is adapted to environmental conditions that are particularly harsh and limiting. It is asexual, and its different strains' chromosomes greatly vary in number and organization. In the present study we isolated and described the replication elements of *D. bruxellensis*. For this purpose we first created auxotrophic strains, prepared genomic libraries, and developed a transformation protocol. Surprisingly, from two different strains, only three different loci (named *CIGO1*, *CIGO2* and *CIGO3*) were sub-cloned and supported autonomous replication. The *CIGO* loci differ from the known yeast *CEN* elements, and their biological activity was retained within only the 600-900 bp segments. For the three loci, the plasmid loss in the non-selective medium was between 20-75%. The reporter gene expression indicated that the copy number exceeded ten per cell. In the tested strains having very different karyotypes, each *CIGO* loci hybridized to at least one chromosome, but each chromosome hybridized to only one of the three CIGO loci. In the recently sequenced genome of Y879, two *CIGO* loci are over-represented in the genome sequence and thereby present in several copies. These properties suggest that the *CIGO* loci could function as a base for a new replication unit, for example a new chromosome, which thereby could increase genome dynamics. An enhanced ability to rearrange the genome could be an efficient tool to exploit new and demanding niches.

<footnote n="1">¹ Department of Biology, Lund University, Sölvegatan 35, Lund SE-223 62, Sweden</footnote>

<footnote n="2">² Department of Systems Biology, Technical University of Denmark, DK-2800 Lyngby, Denmark</footnote>

<footnote n="3">³ Institute of Cell Biology, NAS of Ukraine, Drahomanov Street 14/16, Lviv 79005, Ukraine</footnote>

<footnote n="4">⁴ Department of Biotechnology and Microbiology, University of Rzeszow, Zelwerowicza 4, Rzeszow 35-601 Poland</footnote>
YEAST CELL EVOLUTION THROUGH POST-TRANSLOCATIONAL ADAPTATION
(PTA)

Valentina Tosato, Nicole West, Jason Sims and Carlo V. Bruschi

International Centre for Genetic Engineering and Biotechnology (ICGEB), Trieste, Italy

We developed a system named BIT (Bridge-Induced Translocations) that allows to link
together two different chromosomes of *S. cerevisiae* exploiting the homologous
recombination system of the yeast cells [1, 2]. The BIT mechanism is strongly Rad52, and
Rad54 dependent, while Pol32-independent [3] and its efficiency is related to the length and
the features of the chosen DNA homologies. The main consequence of BIT is an increase of
gene expression that spreads up to 160 Kb from the translocation breakpoints [2]. The same
BIT event may lead to different ploidy of the translocants and therefore generates a
heterogeneous population by the genotypic and phenotypic point of view [4]. In this process,
yeast cells may undergo an adaptation phase during which they acquire not only different
gene expression patterns, but also complex genomic re-arrangements [4,5].

As cellular stress we imposed the high temperature (37°C) reasoning that, since BIT might
de-regulate up to 30% of all gene expression, PTA could result in temperature-resistance
phenocopies. We verified that the adapted translocants able to grow at 37°C, over-expressed
some of the genes coding for heat shock proteins and glycogen metabolism.

In conclusion, PTA produces a phenotypic variability that can be selected upon by particular
environmental conditions, contributing to cellular evolution.

[1] Tosato V, Waghmare S, Bruschi CV (2005) CHROMOSOMA vol. 114, p. 15-27

[2] Nikitin D, Tosato V, Zavec AB, and Bruschi CV (2008) PNAS, vol. 105, p. 9703-08

[3] Tosato V, Sidari S, Bruschi CV (2013) PLOS ONE vol. 8(4):e60926

[4] Rossi B, Noel P, Bruschi CV (2010) GENETICS. vol. 186 p. 775-90

[5] Nikitin D, Bruschi CV, Sims J, Breitenbach M, Rinnerthaler M, Tosato V (2014)
EUR J CELL BIOL, doi: 10.1016/j.ejcb.2014.03.003.
ADAPTIVE EVOLUTION OF *Saccharomyces cerevisiae* TO EARLY STAGE OF AN ALCOHOLIC FERMENTATION

Ana Mangado¹, Pilar Morales¹, Jordi Tronchoni and Ramon Gonzalez¹

¹ Oenologic department, ICVV (CSIC-UR-CAR), Logroño, La Rioja, Spain.
Contact email: ana.mangado@icvv.es.

Experimental evolution was used to identify genes involved in the adaptation to the early stages of wine fermentation. Evolution experiments were performed in continuous culture for 150-250 generations, in conditions emulating the initial stages of alcoholic fermentation. We performed three independent experimental evolution experiments of a haploid laboratory strain BY4741 and one evolution with a haploid strain (not adapted to winemaking growth conditions) obtained by meiotic segregation of the wine yeast EC1118. By the end of the experiments, colonies were phenotypically characterized, and strains that showed improved initial growth rates were selected. We used next generation sequencing techniques in order to find the genetic changes. Four strains were selected from the evolution of BY4741. The mutations found, pointed to the Rsp5p-Bull/2p ubiquitin ligase complex as the preferred evolutionary target under these experimental conditions. Rsp5p is a multifunctional enzyme able to ubiquitinate target proteins participating in different cellular processes, while Bullp is an Rsp5p substrate adaptor involved in the ubiquitin-dependent internalization of Gap1p and other plasma membrane permeases. Our results might be related to increased halftime of plasma membrane amino acid permeases. Apparently the genetic background of the laboratory strain is conditioning the result, however, we have shown the strength of this approach. The evolution of the haploid segregant of EC1118 was run for 250 generations, three adapted strains were selected. Preliminary bioinformatic analysis highlighted changes in chromosome numbers in mutant strains. We are currently testing this results by karyotyping, qPCR, flow cytometry and other techniques.
GENETIC BASIS OF PHENOTYPIC VARIATION WITHIN *S. cerevisiae* STRAINS
DURING SECOND FERMENTATION

<u>Maria Martí-Raga</u><fnref n="1" />,<fnref n="2" />, Gemma Beltran<fnref n="1" />, Albert Mas<fnref n="1" />, Philippe Marullo<fnref n="2" />,<fnref n="3" />

Contact email: maria.martir@urv.cat

The production of sparkling wine by the traditional method (champagne, cava) implies a
second fermentation that takes place inside the bottle. Prior the inoculation into the base
wine, yeasts need to go through an adaptation process called "pied-de-cuve", which will
allow yeast cells to maintain their metabolic activity in the restrictive conditions of the
second fermentation (high ethanol, low oxygen availability, increasing CO2 pressure, low
temperature). With the aim of understanding the genetic basis causing the phenotypic
diversity of *Saccharomyces cerevisiae* strains during the second fermentation, we performed
a QTL mapping analysis. Different QTL mapping strategies can be applied to identify natural
variations explaining complexes traits. The classical F1-hybrid design allows the QTL
mapping on several different traits for one subset of progeny but requires the genetic map of
hundreds of individuals to be actually efficient. The development of NGS technics allow the
construction of the genetic map for hundreds of progenies by sequencing their genome at low
coverage with a reduced cost. In this work we carried out this strategy by genotyping 190
progenies using Illumina technology. A genetic map of more than 5000 reliable markers was
obtained for each progeny. We evaluated the phenotypic landscape of sparkling wine
production by measuring 47 phenotypes involved in both the "pied-de-cuve" and second
fermentation, including single cell, population and fermentation traits. Linkage analysis on
the phenotypes of 117 segregants resulted in the mapping of over 60 QTLs, including the
population size at the final point of the "pied-de-cuve", the fermentative performance, or the
formation of pseudo-hypha-growing cells. To decipher the molecular basis of the phenotypic
variation observed, we are currently testing the candidate genes by reciprocal hemizygosity.
Overall, the molecular dissection of the QTLs can provide useful information about the
adaptation mechanisms of the yeast during sparkling wine production.

<footnote n="1">¹ Dept. Bioquímica i Biotecnologia, Universitat Rovira i Virgili, Tarragona, Spain.</footnote>

<footnote n="2">² EA Oenologie ISVV, Université Bordeaux,Villenave d'Ornon, France.</footnote>

<footnote n="3">³ Biolaffort, Bordeaux, France.</footnote>
ISOLATION AND EVOLUTION OF A NOVEL NON-SACCHAROMYCES
XYLOSE-FERMENTING STRAIN FOR LIGNOCELLULOSIC BIOETHANOL
PRODUCTION

Antonio D. Moreno<fnref n="1" />, Cecilia Geijer<fnref n="1" />, Elia Tomás-Pejó<fnref n="1" />,<fnref n="2" />, Lisbeth Olsson<fnref n="1" />

Contact e-mail: davidmo@chalmers.se

The economic success of lignocellulosic bioethanol requires the fermentation of all available sugars from biomass. Being the major pentose sugar in lignocellulose, the fermentation of xylose is, therefore, considered essential. The fermentative yeast *Saccharomyces cerevisiae* is the most promising candidate for lignocellulosic bioethanol production due to its very effective glucose fermentation, high ethanol tolerance and resistance to inhibitors present in lignocellulosic streams. Nevertheless, wild type S. *cerevisiae* is not able to ferment xylose and all of the genetically modified *Saccharomyces* strains (GMO) still need further improvements to fulfill all requirements for an economically viable lignocellulosic ethanol production. We have discovered a non-*Saccharomyces* xylose-fermenting yeast (here called C5-yeast), which shows a great potential to be used for bioethanol production from lignocellulosic streams. Unlike xylose-fermenting *Saccharomyces* strains, the C5-yeast is not genetically modified and its application in the industry would find less legislative problems when reaching the market. In the present work, the C5-yeast was isolated from a xylose-fermenting population and evolutionary engineered to enhance its fermentation abilities and robustness. During the isolation process, three different morphologies (smooth, flat and wrinkled) of the C5-yeast were found when growing the xylose-fermenting population in plates with minimal media and xylose as a sole carbon source. Among all morphologies, flat-C5-yeast showed the highest xylose consumption rates (>90% after 72 h) and the highest ethanol conversion yields (≈50% of the theoretical considering glucose and xylose) during the fermentation of wheat straw hydrolysates. The isolated flat-C5-yeast was selected for evolutionary engineering in order to enhance its sugar conversion yields and the tolerance towards the inhibitory compounds that are present in the hydrolysate. Although further characterization is needed, an evolved C5-yeast could be considered a suitable fermentative strain for lignocellulosic bioethanol production.

<footnote n="1">¹ Chalmers University of Technology, Department of Chemical and Biological Engineering, Industrial Biotechnology Group, Göteborg, Sweden.</footnote>

<footnote n="2">²Unit of Biotechnological Processes for Energy Production, IMDEA Energy, Móstoles (Madrid), Spain.</footnote>
<figure><img src="image_14.png" /></figure>
Sunday, October 12, Nova Gorica – Plenary Sessions

Novo Nordisk session on Yeast Evolution

Part I (chair: Jørgen Hansen)

09:00 - 09:30 Concetta Compagno: Evolution of yeast ethanol fermentation 193

09:30 - 10:00 Daniela Delneri: Genomic analysis of cold tolerant traits in yeast species 194

10:00 - 10:20 Gabor Boross: The genomic landscape of compensatory evolution 195

10:20 – 10:40 Coffee Break

Part II (chair: Daniela Delneri)

10:40 - 11:10 Ken Wolfe: Evolution of mating-type switching systems in nonconventional yeasts 196

11:10 - 11:35 Alicia Gonzalez: Functional duplication of paralogous genes in *S. cerevisiae*: Role of transcriptional regulation and isozyme oligomeric organization 197

11:35-11:55 Matthew Goddard: Niche construction initiates the evolution of a mutualistic interaction between *S. cerevisiae* and Drosophilid flies 198

12:00 – 13:30 Lunch

Genialis lectures (chair: Gianni Liti)

13:30 - 14:00 Brenda Andrews: From phenotypes to pathways: global exploration of cellular networks using yeast functional genomics 199

14:00 - 14:30 Charlie Boone: Latest developments in genetic network analysis 200
14:30 – 15:00 Announcement of the best young investigator

15:00 – 15:30 Coffee break

Carlsberg session – Closing plenary lectures (chair: Johan Thevelein)

<table><tr><td>15:30 - 16:05</td><td>Sakkie Pretorius: Designing and building wine yeast for the future</td><td>201</td></tr><tr><td>16:05 - 16:40</td><td>Justin Fay: Population genomics of wild yeast and the origin of wine strains</td><td>202</td></tr><tr><td>16:40 - 17:15</td><td>Jens Nielsen: Acquiring novel phenotypes of yeast through adaptive laboratory evolution</td><td>203</td></tr><tr><td>17:15 – 18:00</td><td>Presentation of the new ICY and ISSY meetings & Closing Ceremony</td><td></td></tr><tr><td>20:00</td><td>Conference Dinner</td><td></td></tr></table>
EVOLUTION OF YEAST ALCOHOLIC FERMENTATION

Concetta Compagno<fnref n="1" /> and Jure Piškur

Correspondence email: concetta.compagno@unimi.it

One of the most prominent and unique features of *Saccharomyces cerevisiae* is its ability to rapidly convert sugars to ethanol at both anaerobic and aerobic conditions. This characteristic is called the “Crabtree effect” and represents the background for the “make-accumulate-consume” strategy, which in natural habitats rich of sugars helps this yeast to out-compete other microorganisms. Why, when and how did yeasts remodel their carbon metabolism to be able to accumulate ethanol under aerobic conditions and at the expense of decreasing biomass production? Recent data on the carbon metabolism in Saccharomycetaceae species, allowed us to reconstruct the ancient environment, which could promote the evolution of alcoholic fermentation. The coming out scenario suggests that the first step towards the so-called “fermentative lifestyle” was the exploration of anaerobic niches, resulting in an increased metabolic capacity to degrade sugar to ethanol. The strengthened glycolytic flow produced in parallel a beneficial effect on the microbial competition outcome, and later evolved as a “new” tool promoting the yeast competition under aerobic conditions. The basic aerobic alcoholic fermentation ability was subsequently “upgraded” in several lineages by evolving additional regulatory steps, like glucose repression in the *S. cerevisiae* clade, in order to achieve a stricter metabolic control.

<footnote n="1">¹ Dipartimento di Scienze Biomolecolari Biotecnologie, Universita' degli Studi di Milano, Italy</footnote>
GENOMIC ANALYSIS OF COLD TOLERANT TRAITS IN YEAST SPECIES

Daniela Delneri

Faculty of Life Sciences University of Manchester, UK

Contact e-mail: D.Delneri@manchester.ac.uk

Temperature is one of the leading factors that drive adaptation of organisms and ecosystems. Remarkably, many sister species share the same habitat because of their different temporal or micro-spatial thermal adaptation. In this study, we seek to find the underlying molecular mechanisms of the cold-tolerant phenotype of closely related yeast species adapted to grow at different temperatures, namely *S. kudriavzevii* CA111 (cryo-tolerant) and *S. cerevisiae* 96.2 (thermo-tolerant). Using two different systems approaches, i. thermodynamic-based analysis of a genome-scale metabolic model of *S. cerevisiae* and ii. large-scale competition experiment of the yeast heterozygote mutant collection, genes and pathways important for the growth at low temperature were identified. In particular, defects in lipid metabolism, oxidoreductase and vitamin pathways affected yeast fitness at cold. Combining the data from both studies a list of candidate genes was generated and mutants for two predicted cold favoring genes, *GUT2* and *ADH3*, were created in two natural isolates. Compared to the parental strains, these mutants showed lower fitness at cold temperatures, with *S. kudriavzevii* displaying the strongest defect. Strikingly, in *S. kudriavzevii* these mutations also significantly improve the growth at warm temperatures. In addition, overexpression of *ADH3* in *S. cerevisiae* increased its fitness at cold. These results suggest that temperature-induced redox imbalances could be compensated by increased glycerol accumulation or production of cytosolic acetaldehyde through the deletion of *GUT2* or *ADH3*, respectively.
THE GENOMIC LANDSCAPE OF COMPENSATORY EVOLUTION

Bela Szamecz<fnref n="1" />, Gabor Boross<fnref n="1" />, Dorottya Kalapis<fnref n="1" />, Karoly Kovacs<fnref n="1" />, Gergely Fekete<fnref n="1" />, Zoltan Farkas<fnref n="1" />, Viktoria Lazar<fnref n="1" />, Monika Hrtyan<fnref n="1" />, Patrick Kemmeren<fnref n="2" />, Marian J.A. Groot Koerkamp<fnref n="2" />, Edit Rutkai<fnref n="3" />, Frank C. P. Holstege<fnref n="2" />, Balazs Papp<fnref n="1" />, Csaba Pal<fnref n="1" />

Contact email: borossg@brc.hu

Adaptive evolution is generally assumed to progress through the accumulation of beneficial mutations. However, as deleterious mutations are common in natural populations, they generate a strong selection pressure to mitigate their detrimental effects through compensatory genetic changes. This process can potentially influence directions of adaptive evolution by enabling evolutionary routes which are otherwise inaccessible. Therefore, the extent by which compensatory mutations shape genomic evolution is of central importance. Here, we studied the capacity of the baker's yeast genome to compensate the complete loss of genes during evolution, and explored the long-term consequences of this process. We initiated laboratory evolutionary experiments with over 180 haploid yeast genotypes, all of which initially displayed slow growth due to the deletion of a single gene. Compensatory evolution following gene loss was rapid and pervasive: 68% of the genotypes reached near wild-type fitness through accumulation of adaptive mutations elsewhere in the genome. Genomic analysis revealed that as compensatory mutations were generally specific to the functional defect incurred, convergent evolution at the molecular level was extremely rare. Moreover, the majority of the gene expression changes due to gene deletion remained unrestored. Accordingly, compensatory evolution promoted genomic divergence of parallel evolving populations. However, these different evolutionary outcomes are not phenotypically equivalent, as they generated diverse growth phenotypes across environments. Taken together, these results indicate that gene loss initiates adaptive genomic changes that rapidly restore fitness, but this process has substantial pleiotropic effects on cellular physiology and viability upon environmental change. Our work also implies that gene content variation across species could be partly due to the action of compensatory evolution rather than the passive loss of genes.

<footnote n="1">¹ Synthetic and Systems Biology Unit, Biological Research Center, Szeged, Hungary.</footnote>

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

<footnote n="3">³ Institute for Biotechnology, Bay Zoltán Non-Profit Ltd., Szeged, Hungary.</footnote>
EVOLUTION OF MATING-TYPE SWITCHING SYSTEMS IN NONCONVENTIONAL
YEASTS

Sara J. Hanson and Kenneth H. Wolfe

UCD Conway Institute, School of Medicine & Medical Science, University College Dublin,
Dublin 4, Ireland

Contact email: kenneth.wolfe@ucd.ie

The yeast *Saccharomyces cerevisiae* has a well-known system that allows haploid cells to switch mating-types between MATa and MATalpha, but how this complex system originated is unknown. Switching is a programmed chromosomal rearrangement event in which a segment of DNA is removed from the active (expressed) MAT locus, and replaced by an unrelated sequence copied from one of two silent (unexpressed) loci, HMLalpha or HMRa. Switching is probably evolutionarily adaptive because it enables isolated haploid cells to form spores if growth conditions are poor. The *S. cerevisiae* system requires a lot of components: 3 MAT-like loci in the genome (MAT, HML, HMR); triplication of two sequences (X and Z) that flank these 3 loci to guide the DNA exchanges; an endonuclease (HO) to make the dsDNA break that initiates the process; and a mechanism (Sir proteins) to prevent transcription and HO cleavage at HML and HMR. We have characterized the switching systems of *Hansenula polymorpha* and *Pichia pastoris*, two methylotrophic yeasts that are very distantly related to *S. cerevisiae*. The MAT loci of these species are orthologous and syntenic with the *S. cerevisiae* MAT locus, but they switch mating-types by a mechanism that is simpler than that of *S. cerevisiae* and which shows how the *S. cerevisiae* system may have evolved from simpler components.
FUNCTIONAL DUPLICATION OF PARALOGOUS GENES IN *S. cerevisiae*:
ROLE OF TRANSCRIPTIONAL REGULATION AND ISOZYME OLIGOMERIC
ORGANIZATION

Geovani López, James Gonzalez, Mijail Lezama, Maritrini Colon, Ximena Martinez de la Escalera and Alicia Gonzalez

Departamento de Bioquímica y Biología Estructural, Instituto de Fisiología Celular, Universidad Nacional Autónoma de México, Apartado Postal 70-242, México D. F. 04510, México

The genome of the yeast *Saccharomyces cerevisiae* (*S. cerevisiae*) contains duplicated regions. This redundancy phenomenon is the result of single whole genome duplication, as well as the occurrence of sporadic independent short-segment repeats. Approximately 8% of the genes in the pre-duplication *S. cerevisiae* genome have been retained in duplicate, so that duplicate pairs formed by polyploidy account for approximately 16% of the current gene set. The products of most of the duplicated genes seem to have conserved their original cellular role, although diversification has resulted in sub-fuctionalization; so that retention of the paralogous pair is need to completely fulfill the original function. We have previously proposed that diversification the of *GDH1* and *GDH3*-encoded NADP-dependent glutamate dehydrogenases and the *LYS20-LYS21*-encoded homocitrate synthases could result in the formation of hetero-oligomeric isozymes showing peculiar biochemical properties, which could play an important role under certain environmental conditions. Our results indicate that Leu4 and Leu9 mitochondrial monomers, can form a Leu4-Leu9 α-isopropilmalate synthase hetero-oligomeric isozymes “in vivo”, displaying leucine sensitivity which is intermediate to that present in the resistant Leu9-9 and the sensitive Leu4-4 homo-dimers, indicating *LEU4* and *LEU9* retention allows the existence of three isoforms with varying degrees of leucine-sensitivity. Our results also show that the “ancestral type” yeast *Kluyveromyces lactis* has retained two paralogous genes encoding α-isopropilmalate synthases.
NICHE CONSTRUCTION INITIATES THE EVOLUTION OF A MUTUALISTIC
INTERACTION BETWEEN *S. cerevisiae* AND DROSOPHILID FLIES

Claudia C. Buser<fnref n="1" />, Richard D. Newcomb<fnref n="1" />,<fnref n="2" />, Anne C. Gaskett<fnref n="1" />, and Matthew R. Goddard<fnref n="1" />

Contact email: m.goddard@auckland.ac.nz

Niche construction theory explains how organisms' niche modifications may feed back to affect their evolutionary trajectories. In theory, the evolution of other species accessing the same modified niche may also be affected. A general mechanism driving the evolution of mutualisms has not been identified. Drosophilid flies benefit from accessing yeast-infested fruits, but the consequences of this interaction for yeasts are unknown. We reveal high levels of variation among strains of *Saccharomyces cerevisiae* in their ability to modify fruits and attract *Drosophila simulans*. More attractive yeasts are dispersed more frequently, both in the lab and in the field and flies associated with more attractive yeasts have higher fecundity. These results suggest that the yeast-fly interaction is mutualistic, facilitated by the yeast's niche modification. Overall our results provide support for the idea that niche construction can initiate the evolution of mutualisms.

<footnote n="1">¹The School of Biological Sciences, University of Auckland, Auckland, New Zealand,</footnote>

<footnote n="2">²The New Zealand Institute for Plant & Food Research Ltd, Auckland, New Zealand.</footnote>
FROM PHENOTYPES TO PATHWAYS: GLOBAL EXPLORATION OF CELLULAR
NETWORKS AND PATHWAYS USING SYSTEMATIC YEAST CELL BIOLOGY

Brenda J Andrews

The Donnelly Centre, University of Toronto, Toronto, Ontario Canada M5S 3E1
contact email: Brenda.andrews@utoronto.ca

We have developed two powerful pipelines which combine array-based yeast genetics and automated microscopy for systematic and quantitative cell biological screens or phenomics. Our first pipeline uses the Synthetic Genetic Array (SGA) method to introduce fluorescent markers of key cellular compartments, along with sensitizing mutations, into yeast mutant collections. We then perform live cell imaging on the mutant arrays using HTP confocal microscopy to quantitatively assess the abundance and localization of our fluorescent reporters, providing cell biological readouts of specific pathways and cellular structures in response to thousands of genetic perturbations. Our second pipeline exploits the yeast GFP collection, a unique resource consisting of thousands of strains with different genes uniquely tagged with GFP. This remarkable collection has been arguably underutilized for systematic analysis of the proteome, largely due to the challenges associated with analysis of large sets of cell biological data. We addressed this challenge by adopting a high-content screening approach to measure protein abundance and localization changes in an automated fashion on a genome scale. Our general approach, in particular our network analysis and visualization methods, are readily extensible to other systems.
THE GENETIC LANDSCAPE OF A CELL

Charles Boone

Donnelly Centre, Department of Molecular Genetics, 160 College St, Toronto, ON, Canada, M5S 3E1

Contact email: charlie.boone@utoronto.ca

Genetic interactions can play an important role in determining the relationship between genotype and phenotype and may underlie a significant component of the “missing heritability” in current genome-wide association (GWAS) studies. To explore the general principles of genetic networks, we’ve taken an unbiased and global approach to map digenic interactions in the budding yeast *Saccharomyces cerevisiae*. We developed Synthetic genetic array (SGA) analysis, which automates yeast genetics, enabling the combinatorial construction of defined mutants and the mapping of genetic interactions quantitatively. We are generating a comprehensive genetic landscape for yeast, examining networks derived from an analysis of all gene pairs, covering both nonessential and essential genes. Constructing a higher order network driven by genetic interaction profiles reveals the roles of specific genes and traces a global functional wiring diagram of the cell.