Diploid yeast strains used in this work.
Abstract
ASaccharomyces boulardii strain, which does not carry any auxotrophic markers, was transformed with knockout constructs for the genes HIS3 and ADE2 using the dominant antibiotic marker genes encoding for kanamycin/G418- and nourseothricin/NATR resistance. Thereby, homozygous derivatives that were histidine or adenine deficient were obtained. Histidine prototrophy was easily reconstituted by transforming his-defective diploid derivatives with yeast plasmids carrying the HIS3 gene. Despite different attempts, for example, by creating a rme1::KANX rme1::NATR double-deleted S. boulardii yeast strain (RME1 encodes for Regulator of Meiosis), no visible sporulation to obtain haploid derivatives could be obtained. Besides, no filamentation properties of S. boulardii were observed. As previously mentioned, this yeast strain was confirmed to thrive at 37°C, a temperature disliked by some but not all S. cerevisiae strains used in the laboratory. S. boulardii is a diploid derivative of S. cerevisiae that does not sporulates and survives at temperatures as those found in the human gut. It can be easily manipulated by using conventional yeast methods to introduce auxotrophic markers and obtain heterozygous diploid knockout derivatives that can be transformed with yeast plasmids following conventional yeast protocols, thereby it could be even suited for biochemical and genetic research purposes.
Keywords
- Saccharomyces cerevisiae
- Saccharomyces boulardii
- probiotic
- genetic properties
- yeast transformation
1. Introduction
The French microbiologist Henry Boulard isolated in 1923 a yeast strain (later named after him) after observing natives in Indochina affected by digestive disorders to chew litchi and mangosteen skins. It was said (but never proven) that those people could even protect themselves thereby against outbreaks of cholera.
Ever since, there has been an increasing body of medical reports addressing the beneficial properties of
Yeast strains used in the laboratory such as S288C are mostly derivatives of industrial yeast strains used for ages in breweries [3]. More recently, derivatives of diploid strain Σ1278b, which shows filamentous properties related to non-domesticated yeast strains [4], are used in molecular biology research. Surprisingly, the sequence identity of both yeast strains is only 46% [5] indicating considerable genetic variability due to adaptation to differing milieus and to human domestication of this eukaryotic species [6].
Thanks to molecular genetic techniques,
2. Results
2.1. Growth properties of S. boulardii
To further characterize this yeast, its growth properties at different temperatures are compared with other diploid yeast strains (all yeast strains used are summarized in Table 1).
Name and properties of diploid yeast strain | Auxotrophic properties/antibiotic resistances |
---|---|
BY4743 | Requires histidine, leucine, methionine, and uracil |
RH2585/2586 | Requires histidine and uracil |
RH2585/2586 ∆flo8::kanX ∆flo8::NATR | No requirements; G418- and NAT-resistant |
None | |
No requirements; G418- and NAT-resistant | |
No requirements; G418- and NAT-resistant | |
Requires histidine; G418- and NAT-resistant | |
Requires adenine; G418- and NAT-resistant |
Table 1.
G418/geneticin and NAT/nourseothricin are selective antibiotics for yeast strains.
![](http://cdnintech.com/media/chapter/56888/1512345123/media/F1.png)
Figure 1.
Properties of
A further investigated property is
Another interesting issue was to induce meiosis and sporulation in
As shown in Figure 1C,
2.2. Transformation of S. boulardii with conventional yeast plasmids
As an auxotrophic histidine-deficient yeast strain was now available, I decided to transform it with conventional yeast plasmids that complement for the lack of HIS3. For this purpose,
After 2–3 days of incubation at 30°C, his+-transformants were nicely observed (Figure 2; left panel), indicating that a simple yeast transformation protocol was sufficient to transform this yeast strain and to recover its prototrophy. Plasmids used for transformation (p301HIS3 GAL-p20-HA from
![](http://cdnintech.com/media/chapter/56888/1512345123/media/F2.png)
Figure 2.
Transformation of
2.3. Sequencing and comparison of S. boulardii p20 gene
p20, a small acidic protein of 161 amino acids, is encoded by the non-essential gene CAF20 which only exists in a variety of yeast species (such as
![](http://cdnintech.com/media/chapter/56888/1512345123/media/F3.png)
Figure 3.
Sequence comparison of p20 from different yeast species. Multiple sequence alignment of p20 from different yeast sources (CANGA,
The CAF20 gene from
3. Conclusions
In this work, I present data indicating that
In accordance with those properties, it is probably not detrimental for human health (at least not for immunocompetent individuals) as it will not easily establish in the gut or penetrate the intestinal blood barrier. All this does not mean that it has beneficial physiological properties and I would like to ask the question: is this not just a further conventional yeast strain?
Acknowledgments
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