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Projects / Programmes source: ARIS

Ecology and mycotoxin production of fungi of the genus Wallemia, contaminants of sweet and salted food

Research activity

Code Science Field Subfield
4.06.00  Biotechnical sciences  Biotechnology   

Code Science Field
B230  Biomedical sciences  Microbiology, bacteriology, virology, mycology 
Keywords
Wallemia, halophilic fungi, food, mycotoxins, compatible solutes, food preservation, salt, sugar, honey, taxonomy, physiology, biochemistry, differential gene expression, cell wall, plasma membrane
Evaluation (rules)
source: COBISS
Researchers (14)
no. Code Name and surname Research area Role Period No. of publicationsNo. of publications
1.  11155  PhD Damjana Drobne  Biology  Researcher  2008 - 2011  865 
2.  29348  PhD Martin Fettich  Biochemistry and molecular biology  Junior researcher  2008 - 2011  11 
3.  25974  PhD Cene Gostinčar  Biotechnology  Researcher  2008 - 2010  338 
4.  05935  PhD Nina Gunde-Cimerman  Biotechnology  Head  2008 - 2011  1,263 
5.  17279  Barbara Kastelic Bokal    Technical associate  2008 - 2011 
6.  28193  PhD Marjetka Kralj Kunčič  Microbiology and immunology  Researcher  2008 - 2010  45 
7.  18845  Milena Marušič    Technical associate  2008 - 2011 
8.  06777  PhD Ana Plemenitaš  Biochemistry and molecular biology  Researcher  2008 - 2011  379 
9.  24580  PhD Hans-Josef Schroers  Plant production  Researcher  2008 - 2011  197 
10.  15328  PhD Kristina Sepčić  Biochemistry and molecular biology  Researcher  2008 - 2011  731 
11.  23963  PhD Silva Sonjak  Biotechnology  Researcher  2008 - 2011  58 
12.  29599  PhD Rok Tkavc  Biotechnology  Junior researcher  2008 - 2011  45 
13.  18510  PhD Martina Turk  Biochemistry and molecular biology  Researcher  2008 - 2011  193 
14.  16103  PhD Polona Zalar  Microbiology and immunology  Researcher  2008 - 2011  463 
Organisations (3)
no. Code Research organisation City Registration number No. of publicationsNo. of publications
1.  0381  University of Ljubljana, Faculty of Medicine  Ljubljana  1627066  48,417 
2.  0401  Agricultural institute of Slovenia  Ljubljana  5055431  20,088 
3.  0481  University of Ljubljana, Biotechnical Faculty  Ljubljana  1626914  66,769 
Abstract
The applicants have since year 2000 pioneered the characterisation of fungal species that are thriving in global hypersaline natural environments. Few fungal species are able to survive and grow at very high concentrations of salt or sugar (ie very low water activities). Genus Wallemia is one of these extremophilic eukaryotes, which also populate salterns and salt lakes world-wide. Wallemia sebi has for a long time been known only to be an important food contaminant. However, the applicants have shown in 2007 that W. sebi in reality is composed of three distinctly different species: W. sebi, W. muriae and W. ichthyophaga, the latter one being the most extreme of all eukaryotes that can cope with high salinity. The applicants have discovered that all Wallemia spp. produce a toxin with a remarkably strong haemolytic effect. Its synthesis is stimulated with high concentrations of salt. This toxin may or may not be identical to the previously described toxin in W. sebi, Walleminol, the mechanism of action of which is not known today. Salt and sugar intended for food preservation may contain Wallemia spp. and thus reduce the quality or safety of (Slovenian) food. The main elements and deliverables of the proposed research are: 1. To identify the habitats and ecology of the three Wallemia species including their occurrence in food. 2.To characterise which toxins are produced by which of the three species under which conditions. These results will help industry keep food standards high, and to develop or anticipate new testing procedures. Additionally the toxins may find interesting applications for medical research. 3.To study the genetic and physiological parameters that allow the species to adapt to extreme environments, particularly on the level of cell wall, plasma membrane and mycotxin production. The results may help to breed or modify industrially relevant yeasts as well as agriculturally relevant plants that better resist low-water activity conditions.
Significance for science
Until 2005, the fungal genus Wallemia contained only one species, W. sebi, known as a contaminant of food preserved with high concentrations of salt or sugar and as the causative agent of allergic farmers' lung disease. In 2005, we described three species: W. sebi, W. muriae and W. ichthyophaga and resolved the phylogenetic position of the genus Wallemia, which enabled unraveling of the natural ecology of individual species and their relation to food. Particularly important for science is the discovery that W. ichthyophaga populates waters rich with MgCl2, so far considered as »sterile«. Strains are extremely chaophilic, since they can grow even on media with 2.3 M MgCl2. Before this discovery only a few species of bacteria were known as being able to grow on 1.5 M MgCl2. We have also discovered that the three known species are not food-borne. It appears that food is contaminated by a so far undescribed complex of species W. aff. sebi. Additionally, a new species Wallemia sp. was found, which represents the phylogenetically missing link between W. ichthyophaga and the more related species W. sebi and W. muriae. Our preliminary results showed that all three species of the genus Wallemia synthesize an unknown haemolytic metabolite, more pronounced at low water activity, of great importance for human health and food-processing industry. Also other tested halophilic and halotolerant species of fungi, isolated from salterns, show haemolytic activity at increased salt concentrations. In W. sebi, the haemolitic activity was due to a mixture of sterols and long chain fatty acids that acted as detergents on membranes of erythrocytes. W. ichthyophaga is so far the most halophilic eukaryotic microorganism known to date, since it does not grow without at least 10% of NaCl in the medium. It represents an important new model organism for investigations of adaptations to life at high salinity. W. ichthyophaga adapts on the level of cell morphology by growing as meristematic clumps, on the level of cell wall with extreme thickening and multi-layered structure, on the level of membranes by changing its composition and fluidity. At high salinities, the cells of W. ichthyophaga maintain low intracellular ion content and they counteract differences in osmotic pressure by the synthesis of compatible solutes, particularly glycerol. We have identified some differentially expressed genes, such as the enzyme glycerol-3-phosphate dehydrogenase. When we heterologously introduced it into brewers' yeast, we significantly increased its tolerance to salt. Research of extremophilic adaptations in fungi contributed to the involvement into a new Center of excellence CipKeBip. One of its main activities is the investigation of proteins of extremophilic fungi. Collaborators of the project have started activities, which will lead to the determination of genomic and transcriptomic sequences of W. ichthyophaga. This represents one of the three genome projects lead by our group. The three projects represent the first sequencing of genome of any extremely halotolerant or halophilic fungus on a global scale and at the same time the first projects of sequencing of an eukaryotic genome lead by Slovenian scientists. The determination of genomic and transcriptomic sequences (at three different salt concentrations) of W. ichthyophaga is conducted with the Chinese institute BGI. Collected data, with the help of comparative genomics, will help in our understanding of the extreme tolerance to salt in W. ichthyophaga. The final intention is to acquire transgenic yeasts and plants, adapted to osmotic (saline) stress. Halotolerant yeasts are important in different industrial processes, which are characterized by elevated concentrations of salt, such as fermentations in food industry, production of bioethanol from waste materials, while halotolerant plants represent the potential solution to global salinization of agricultural soil.
Significance for the country
In the future years, most probably climatic changes will occur that will lead to increased temperatures, drought, consequential irrigation and soil salinisation. Agricultural land in different parts of the world located in warmer climatic zones, such as China and India, are being increasingly salinized. With climatic change, we can expect salinisation of land also in more temperate zones, such as in Slovenia. Investigations of adaptations of halophilic fungi are thus important for the development of tolerance to salt and drought of industrially important yeasts and plants and for the acquirement of new information and knowledge. The importance of this field was proved by signing a common project between Slovenia and China (BGI institute). Within its framework, the first sequencing ever of a genome of a halophilic fungus will be performed on the global basis. This will also be the first project of sequencing of an eukaryotic genome that will be conducted by Slovenian scientists. The assembled data analyzed with the aid of comparative genomics will help us to understand the exceptional tolerance to salt of W. ichthyophaga. The importance of research of extremophilic fungi for the development of Slovenia has been additionally confirmed also by joining the newly established Center of Excellence for integrated approaches in chemistry and biology of proteins CipKeBIP. One of the main areas of research within the Center is the investigation of proteins from extremophilic microorganisms. Another evidence of actuality are the organization of two international conferences: Black yeast conference under the auspices of ISHAM in May 2010 in Ljubljana, Slovenia and the 4th Polar and Alpine Microbiology Conference in September 2011 in Ljubljana, Slovenia. The importance of research of extremophilic fungi is also evident by presentation of results of the project at different international forums and conferences, as plenary lectures (5), invited lectures (5) and posters (2), which all promoted achievements of Slovenian scientists. Strains of fungi that were isolated during the research project (60) have been included in the gene bank of Extremophilic fungi (EX), within the Infrastructural Centre Mycosmo. With the inclusion of the strains originating from habitats that are difficult to access into the gene bank, the gene bank was importantly enriched, and strains became available to the scientific and industrial community. An important achievement represent isolates of the so far extremely rare species W. ichthyophaga and the discovery of new species within the genus Wallemia. Strains of these new species are so far preserved exclusively in the Slovene gene bank EX. Since fungi of the genus Wallemia are mycotoxic contaminants of food preserved with high concentrations of salt and sugar, the finding that food-borne species are still undescribed is particularly important. The finding that fungi of the genus Wallemia are able to synthesize a haemolytic metabolite (s) is important for Slovenian and for international food industry. The synthesis of this metabolite has been discovered during the project also in other halophilic and halotolerant food-borne fungi. It appears that it represents an adaptation of fungi to hypersaline conditions. Several young researchers and undergraduate students were involved in the project. Two doctoral theses (dr. Cene Gostinčar and dr. Marjetka Kralj Kunčič) and 4 diploma theses have been successfully completed. Another Ph.D. thesis will be completed this year. Since several collaborators of the project are habilitated at the University of Ljubljana, they were able to transfer their knowledge to undergraduate and graduate students also during lectures and practical courses.
Most important scientific results Annual report 2008, 2009, final report, complete report on dLib.si
Most important socioeconomically and culturally relevant results Annual report 2008, 2009, final report, complete report on dLib.si
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