Genome transcriptomic analysis of diploid yeast strains
Transcriptomic profile of each yeast strain grown in the different medium has allowed us to compare the gene expression in the two conditions. In L71 and L62 strains, no significant changes in the transcript levels were found. This result showed that industrial strains L71 and L62 seem to be more tolerant to a modification of growth medium than the laboratory strain. However, we found than a less extended number of genes were differentially expressed in the laboratory strain BY4743 as compared to the industrial strains L69 and L60, indicating the relevance to investigate these conditions in industry. Indeed, we observed 177 and 140 genes were differentially expressed in L69 and L60 strains, but the transcription of only 39 genes was modified in laboratory strain (Figure IIIC-1).
In the case of industrial yeasts, 59 and 40 genes were respectively upregulated in L60 and L69 strains upon the modification of the growth culture. In laboratory strain, upregulated genes were mostly involved in transport, response to stress, cell wall organization and phospholipid metabolism (see table IIIC-3). In L69 strain, we observed an increase of gene expression of genes implied in protein fate, stress response and transport of siderophore and drug (table IIIC-4). In L60 strain, we also observed an upregulation of genes required for transport of siderophore as well as phosphate and metal ions. In addition, in this strain, highest levels of transcription were found for FLO genes encoded proteins with binding function and for genes involved in metabolism of lipids and maltose (see table IIIC-5).
To identify the general response to a modification of growth medium in the laboratory strain and industrials strains, we selected genes, whose expression was induced or repressed more than twofold and common to the three strains (see Figure IIIC-1).
Figure IIIC-1. Representation of the differentially expressed genes between the two growth medium composition. Venn diagram and number indicates genes differentially expressed (pvalue of 5%, Fold Change of 2).
Differences in expression levels of the 16 common genes were shown in Figure IIIC-2. We observed that the main changes in transcription concerned the synthesis of amino acids, in particular the serine synthesis (SER33) and methionine pathway (MET1, MET2, MET3, MET5, MET6, MET10, MET14, MET16, MET17 and MET28), which are both connected, but also the sulfate assimilation pathways (SUL2, SAM2). A comparison of the gene transcript profiles of L69, L60 and BY4743 cells exposed to a rich or minimum glucose medium showed that genes involved in the synthesis of methionine were overexpressed in L60 strain, whereas the expression of these genes were down-regulated in L69 strain. In addition, the repression of SAM2 and SUL2 linked to the sulfate assimilation pathway, would suggest a requirement for the L60 cells to conserve methionine (Celton et al., 2012).
Figure IIIC-2. Differences in expression levels of DE genes in function of growth medium composition (fold change is the relation between gene expression of the strain in YPD and gene expression of the strain in YNB).
Another important result is that the transcript level of YGP1 was were very low in industrial strains (L69 and L60) and highly increased in laboratory strain grown in rich medium, as compared to minimal medium. Increased expression of YGP1 in response to the limitation of nutrients, is in agreement with the fact that the levels of both YGP1 mRNA and its corresponding protein are increased in response to low nutrient levels (Destruelle et al., 1994). Moreover, this gene encodes a secretory glycoprotein related to the cell wall, which can suggest that nutrient limitation can affect the cell wall.
Therefore, we investigate how a modification of growth medium composition can affect the transcription level of genes related to the cell wall. As shown in figures IIIC-3 and IIIC-4, we selected 80 genes related to cell wall biogenesis and organization, whose expression was different between the two conditions (YPD and YNB medium).
For the L71 strain, genes induced in a rich medium are involved in cell wall organization (YPS6, CWP2, EXG1, CCW14, PUN1, KTR6, KRE6), reponse to stress such as PAU23 and TIR3, which encode cell wall mannoproteins and AFB1 whose is expressed under mating conditions. On the contrary, we observed for this same strain a repression of genes implied in flocculation (FLO5, YHR213W, FLO11) and in alcohol metabolism process (ATF1, ATF2). These two genes encode two alcohol acetyltransferase, which form volatile esters and acetate esters during wine and brewing fermentation. Comparison of L71 and BY4743 profiles showed the same regulation of genes related to the yeast cell wall organization. Additionally, analysis of the transcription levels in rich or minimal medium for the laboratory strain have shown an activation of some genes involved in cell wall organization, but also implied in the synthesis of chitin (RCR1, CHS1), mannoprotein (PAU24) and glucan (SCW10). In L62, SHC1 is upregulated in YPD medium as compared to the alkaline medium YNB. Paralog to SKT5, this gene is required for the synthesis of the chitosan layer of ascospores and activates the chitin synthase Chs3p during sporulation. In L69, genes implied in flocculation have a distinct level of transcription. FLO11 and YHR213W genes are upregulated, whereas FLO10 and FLO5 are downregulated in YPD medium. Moreover, we observed in this strain an upregulation of genes involved in chitin synthesis (SKT5, PCM1, CHS7) as well as in the cell wall growth and in its maintenance (MKC7). The expression of several genes encoding for the cell wall mannoproteins (PAU24, PIR1, TIR1, CWP2, FIT1) and a cell wall adhesin (FIG2) are also changed. To summarize, we found that the transcripts levels of several GPI-CWPs were changed when the yeast cells are grown in synthetic medium. The effect of growth conditions on transcriptional activation or repression of specific CWPs was previously report on laboratory strains by Wodicka et al. (Wodicka et al., 1997). Moreover, we could suggest that the repression of these genes is in line with a ratio of mannan in the rich medium as compared to it in the minimal medium. In contrary to them, we did not observe a large increase of the transcript level of FKS2 (GSC2) in minimal medium, but our data indicate an increased expression of genes involved in chitin synthesis in rich medium correlated with the higher level of chitin measured in cells grown on rich (YPD) medium.
IMPACT OF AUTOLYSIS PROCESS ON THE CELL WALL
The impact of packaging yeast cells as “YCW” (for Yeast Cell Wall) by autolysis and drying process on the cell wall composition and nanomechanical properties of two industrial strains were investigated. L71 and L69 from Lallemand collection were chosen in this study regarding to their particular interest in wine fermentation.
Material and methods:
Levels of chitin, β-1,3-glucan, β-1,6-glucan and mannan in the cell wall were determined by the acido-enzymatic method. The determination of the cell wall composition was performed on cells grown under standard laboratory condition, which correspond to a growth in a glucose rich medium until exponential phase. Cell wall composition was also investigated on cells propagated under industrial protocol to produce “cream”, which was subjected to autolysis (20 hr at 55°C) followed by separation and by spray-drying in order to produce the dry “YCW”. Atomic force microscopy was used to evaluate the surface properties such as average roughness measurement, stiffness and adhesion. Furthermore, AFM tips were functionalized with concanavalin A that binds α-glucose as well as α-mannose residues, which occur in mannan oligosaccharides of the yeast cell wall. Single-molecule force experiments with concanavalin A tips bring information on the distribution of mannan at the cell surface and on their extension. Transcript levels of genes in L71 and L69 strains were compared to explain the differences between the two strains at genetic level.
This study has shown that packaging yeast cells as “YCW” by autolysis and drying process does not change the composition of the cell wall but causes a change in topography and surface properties of the cell. However, AFM in this study has a great interest, because this technology brings a specific view of the effects on the cell surface. High resolution imaging of the cell surface showed a modification from a smooth surface for exponential-phase cells to a wrinkled topography for YCW cells, resulting in a 4-fold increase of the average roughness of the YCW cells. In addition, the observation of some holes of 600 nm diameter localized on the middle of YCW cells in both L69 and L71 strains, would suggest a release of intracellular constituents of the yeast. Furthermore, the L69 strain, which has the highest mannoproteins content, was also characterized by the presence of highly adhesive patches forming nanodomains. Using transcriptomic analyses, we found that the transcript levels of FLO11 and YHR213w were relatively much higher in L69 than in L71 strain. In S. cerevisiae, the sequence of the Flo11 protein presents amyloid forming motifs that can lead to partial β-aggregation. Therefore, we can suggest that an upregulation of FLO11 in L69 strain can lead to massive amounts of Flo11p, which formed β-aggregate amyloids clusters into nanodomains.
Table of contents :
Chapter I: LITERATURE REVIEW
1. Generality on yeast
1.1. Physiology: generality about growth, metabolism and genetic
1.2. Yeast and the mode of culture and production process
1.3. Cell wall as biotechnological product
2. Methods to study the yeast cell wall
2.1. Methods to determine the yeast cell wall composition
2.2. Biophysical approach: Atomic Force Microscopy
2.3. Molecular tools: transcription profile and genomic analysis
2.4. Phenotypic observations: use of drugs
3. Biosynthesis of the cell wall polysaccharides
4. Architecture of the cell wall and remodeling enzymes
4.1. Architecture of the yeast cell wall
4.2. Hydrolytic enzymes of the yeast cell wall
4.3. Signalling pathways involved in the yeast cell wall architecture
5. Conclusion of the literature review and objectives of the thesis
Chapter II: DETERMINATION OF THE YEAST CELL WALL COMPOSITION
Chapter III: EFFECT OF THE STRAIN BACKGROUND ON THE CELL WALL
Chapter IIIA: STUDY OF CELL WALL MUTANTS (gas1Δ, chs3Δ, mnn9Δ)
Chapter IIIB: STUDY OF FOUR INDUSTRIAL STRAINS
Supplementary results: Cell wall composition and transcriptomic analysis of industrial yeasts: Effect of the growth medium
Discussion of the results
Chapter IV: IMPACT OF AUTOLYSIS PROCESS ON THE CELL WALL
Chapitre V: CONCLUSION AND PERSPECTIVES
Chapter VI: REFERENCES
Chapter VII: APPENDIX
1. List of figures
2. List of tables
4. Material and methods
4.2. Proteins measurement
4.3. Analysis of sugars : HPAEC-PAD
4.4. Production of endo-β(1,6)-glucanase from Trichoderma harzianum
4.5. Atomic force microscopy
4.6. Canonical correlations of cell wall physico-chemical traits and transcriptomic profile
5. List of Publications
6. List of Communications