Molecular biology and genetics of non-conventional yeasts
Yeasts include important industrial organisms, pathogens, and popular laboratory organisms that serve as general models to understand the eukaryotic cell. For decades Saccharomyces cerevisiae baker's yeast, has been one of the best characterized organisms from the genetics and physiology point of view. Development of new yeast species into laboratory model organisms will facilitate comparative approaches to solve several basic questions on yeast (as well as on any other organisms) within genetics, biochemistry, cell biology, physiology and biotechnology (for review see Piskur and Langkjaer, 2004).
Saccharomyces complex members primarily degrade hexoses only to C3 and C2 compounds pyruvate and ethanol, even in the presence of oxygen. The occurrence of fermentation under aerobic conditions is referred to as the Crabtree effect and the yeasts exhibiting it as Crabtree-positive yeasts (Pronk et al., 1996). While a majority of yeasts cannot grow in the absence of oxygen (aerobic yeasts), several Saccharomyces complex yeasts can also survive without any oxygen (Andreasen and Stier, 1953; Pronk et al., 1996; Møller et al., 2001). The life cycle of the Saccharomyces complex yeasts is also very unique. Sexually reproducing yeasts can either undergo mating between heterothallic lines, which are self-sterile, or homothallic lines, which are self-fertile. In S. cerevisiae, homothallism occurs because cells can switch mating type. A site-specific nuclease, encoded by HO gene is responsible for the homothallic behaviour and ho mutants are heterothallic and represents a central element in the mating type switching (Haber, 1998).
How did the progenitor of Saccharomyces develop these characters and what were the molecular mechanisms operating during this yeast's evolutionary history? Are these traits unique in nature? The most elegant way to answer these questions is to study related and less related species, compare them to each other, and on this basis deduce the common progenitor and molecular mechanisms, which have operated upon separation of the lineages.
The whole-genome duplication (Wolfe and Shields, 1997; Kellis et al., 2004) and the horizontal gene transfer (Butler et al., 2004; Gojkovic et al., 2004) provided the background for the development of a facultative anaerobic lifestyle (Pronk et al., 1996; Møller et al., 2001), the petite-positive phenotype (Møller et al., 2001), homothallism (Butler et al., 2004) and an efficient glucose repression circuit (Johnston, 1999). Comparative genomics and introduction of several new yeasts for molecular studies now help to place these events at different branching points of the yeast phylogenetic tree and to estimate their relative timing. S. Saccharomyces A. Ashbya; C. Candida
In our preliminary search we have identified a group of non-conventional yeasts important in wine fermentation and brewing of lambic beer, belonging to the Brettanomyces/Dekkera group (Kurtzman and Fell, 1998), which are apparently good ethanol producers, they can grow anaerobically and generate petite mutants (Siurkus, 2004). These yeasts have so far been only poorly studied (Hoeben, 1986) and they are very far relatives of Saccharomyces yeasts (Cai et al., 1996).
We collected over 100 isolates, presumably belonging to Brettanomyces/Dekkera. The phylogenetic tree based on alignment of the partial sequences of the small ribosomal mitochondrial subunit divides these yeasts into two sub-groups (see figure above). The D. bruxellensis sub-group seems to be able to grow without oxygen, exhibits a Crabtree-positive effect and is petite-positive, while B. naardensis is a Crabtree-negative, needs aerobic conditions to promote the growth and is petite-negative. These observations are complementary to the Saccharomyces complex, where the S. cerevisiae sub-group is petite-positive and anaerobic and exhibits glucose repression, while the Kluyveromyces sub-group is petite-negative and Crabtree-negative and aerobic (Piskur and Langkjaer, 2004).
Surprisingly, D. bruxellensis chromosomal patterns exhibit a high variation in the number and sizes of their chromosomes (from 0.75 to 6.5 Mb), and nuclear genomes (from 15.5 to 39.05 Mb). Why does the apparent genome of the D. bruxellensis isolates vary so much? It could be that these genomes are partially haploid and partially polyploid or it could be that the genomes contain many variable intergenic sequences?
It is hardly possible that the number of genes is higher than in other yeasts but the number of introns could be higher. The high variation of chromosomal patterns of D. bruxellensis could suggest that the organization of the nuclear genome of this species has been and is frequently reshaped. In other words, the D. bruxellensis species could have a very relaxed control over the size of its genome.
Dekkera/Brettanomyces yeasts have several interesting phenotypes, such as the capacity for anaerobic growth, petite-positive character, and ethanol tolerance, all of which seem to have evolved in parallel with Saccharomyces yeasts. However, do these phenotypes have the same genetic background in both yeast groups? In addition, Dekkera/Brettanomyces exhibits very dynamic chromosome and genome size structures. Does this observation have a biological meaning and how could this genetic plasticity be explained?
We would like to develop Dekkera/Brettanomyces into laboratory organisms, thus providing the background for future experiments, which would help to answer the questions posed above. Therefore, we plan to:
- Develop a mutagenesis technique for isolation of auxotrophic mutants
- Test if various strains can mate or develop a protoplast fusion method
- Sequence the genome of one of the strains
- Isolate and characterize mitochondrial DNA and study the nature of the petite mutant
- Study genome and chromosome stability
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