Nucleosomes affect local transformation efficiency

Genetic transformation is a natural process during which foreign DNA enters a cell and integrates into the genome. Apart from its relevance for horizontal gene transfer in nature, transformation is also the cornerstone of today's recombinant gene technology. Despite its importance, relatively little is known about the factors that determine transformation efficiency. We hypothesize that differences in DNA accessibility associated with nucleosome positioning may affect local transformation efficiency. We investigated the landscape of transformation efficiency at various positions in the Saccharomyces cerevisiae genome and correlated these measurements with nucleosome positioning. We find that transformation efficiency shows a highly significant inverse correlation with relative nucleosome density. This correlation was lost when the nucleosome pattern, but not the underlying sequence was changed. Together, our results demonstrate a novel role for nucleosomes and also allow researchers to predict transformation efficiency of a target region and select spots in the genome that are likely to yield higher transformation efficiency

The interaction between yeast and bread dough

Fermentation and bread making are processes as old as the civilization. Yeast and biotechnology became part of the human s life even before we had any knowledge about the existence of microorganisms. Today, fermentation is still a very important part of our lives, only we have built a pile of knowledge about this process and continue learning to control and adapt it based on our needs and taste. The behavior of yeast cells during industrial processes such as the production of beer, wine and bioethanol has been extensively studied. By contrast, our knowledge about yeast physiology during solid-state processes, such as bread dough, cheese or cocoa fermentation remains limited. We investigated changes in the transcriptome of three genetically distinct Saccharomyces cerevisiae strains during bread dough fermentation. Our results show that regardless of the genetic background, all three strains exhibit similar changes in expression patterns. At the onset of fermentation, expression of glucose-regulated genes changes dramatically, and the osmotic stress response is activated. The middle fermentation phase is characterized by the induction of genes involved in amino acid metabolism. Finally, at the latest time point, cells suffer from nutrient depletion and activate metabolic pathways associated with starvation and stress response. Further analysis showed that genes regulated by the High Osmolarity Glycerol (HOG) pathway, the major pathway involved in the response to osmotic stress and glycerol homeostasis, are among the most differentially expressed genes at the onset of fermentation. More importantly, deletion of HOG1 and other genes of this pathway significantly reduces fermentation capacity. Together, our results demonstrate that cells embedded in semi-solid matrix of bread dough suffer severe osmotic stress, and that a proper induction of the HOG pathway is critical for an optimal fermentation.When faced with osmotic stress, for example during semi-solid state bread dough fermentation, yeast cells produce and accumulate glycerol (as the main compatible solute) in order to prevent dehydration by balancing the intracellular osmolarity with that of the environment. However, increased glycerol production also results in decreased CO2 production, which may reduce dough leavening. We investigated the effect of yeast glycerol production level on bread dough fermentation capacity of a commercial bakery strain and a laboratory strain. We found that ?gpd1 mutants that show decreased glycerol production show impaired dough fermentation. In contrast, overexpression of GPD1 in the laboratory strain results in increased fermentation rates in high-sugar dough and improved gas retention in the fermenting bread dough. Together, our results reveal the crucial role of glycerol production level by fermenting yeast cells in dough fermentation efficiency as well as gas retention in dough, thereby opening up new routes for the selection of improved commercial bakery yeasts.Besides leavening the dough, yeast can impact the sensory profile of bread.Therefore the choice for a different strain for dough fermentation can result in changes in the sensory profile of the end product. Saccharomyces cerevisiae is currently the predominant yeast used in food fermentation. Even though the choice of this species is associated with efficient fermentation, desirable flavor and other beneficial characteristics, it leads to limited diversity in the aroma profiles of the end product. Hence, there is currently a growing interest in industry to employ non-conventional yeast strains in order to improve product and meet the specific requirements of particular customers. Here, we selected a small set of non-conventional yeast strains to examine their capacity in the baking industry, including two other Saccharomyces species not currently used in bakery and eight non-Saccharomyces strains. Out of our 10 initial strains/species examined, we found that two (Torulaspora delbrueckii and Saccharomyces bayanus) had excellent properties. Besides displaying acceptable dough fermentation, sensory analysis and HS-SPME-CG-MS analysis showed that these strains produced an aroma profile that was very different from that produced by commercial bakery strain and was perceived as desirable by the majority of our sensory panel.ACKNOWLEDGEMENTS i SAMENVATTING iii SUMMARY v LAYOUT vii TABLE OF CONTENTS ix 1. CHAPTER I: Introduction 1 1.1. How dough as an environment affects yeast 2 1.1.1. Bread’s ingredients and bread making 2 1.1.2. Solid-state 5 1.1.3. High osmolarity 6 1.1.4. Other possible factors 8 1.1.5. Study of environmental effects 8 1.2. How yeast impacts dough’s and bread’s properties? 10 1.2.1. Carbohydrate catabolism in yeast 10 1.2.2. Leavening 12 1.2.3. Rheology 12 1.2.4. End product’s sensorial profile 14 1.3. Improving yeast for dough fermentation application 15 1.3.1. Using non-conventional strains 15 1.3.2. Improving conventional yeast 18 1.4. Context, aim and outline of the research 20 2. CHAPTER II: Dynamics of the yeast transcriptome during bread dough fermentation 22 2.1. Results and Discussion 23 2.1.1. Strain selection for dough fermentation and transcriptome analysis 23 2.1.2. Yeast cells show a transient response during dough fermentation 24 2.1.3. The majority of differentially expressed genes are involved in metabolic shifts and response to nutrient levels 27 2.1.4. The HOG pathway is required to adapt to the high osmolarity at the onset of dough fermentation 29 2.1.5. Discussion 33 2.2. Materials and Methods 36 2.2.1. Strains and microbial procedure 36 2.2.2. Flour characterisation, dough preparation and fermentation 36 2.2.3. Gas production measurement 36 2.2.4. Sampling for RNA extraction 37 2.2.5. RNA isolation 37 2.2.6. RNA-seq and data analysis 38 2.2.7. Hierarchical clustering for heat map 38 2.2.8. Categorizing of expression profiles and Gene Ontology 38 2.2.9. Physical interaction network 39 2.2.10. PheNetic 39 2.2.11. Network visualization and analysis 40 2.2.12. RNA seq Data 40 3. CHAPTER III: Glycerol production by fermenting yeast cells is essential for optimal bread dough fermentation 42 3.1. Results and Discussion 43 3.1.1. Deletion and overexpression of GPD1 changes cellular glycerol production 43 3.1.2. Glycerol production is crucial for efficient dough fermentation 44 3.1.3. Overexpression of GPD1 improves fermentation of high-sugar dough in a laboratory strain 45 3.1.4. Elevated glycerol production correlates with better gas retention in dough 46 3.1.5. Discussion 49 3.2. Materials and Methods 52 3.2.1. Strains, plasmid and microbial procedure 52 3.2.2. Intracellular glycerol extraction 53 3.2.3. Dough preparation, and fermentation 53 3.2.4. Gas production measurement 54 3.2.5. Metabolite extraction from dough and HPLC analysis. 54 3.2.6. Rheofermentometer analysis of fermenting dough 54 4. CHAPTER IV: Non-conventional yeast strains increase the aroma complexity of bread 57 4.1. Results and Discussion 58 4.1.1. Evaluation of non-conventional yeast strains for bread dough fermentation 58 4.1.2. Sensory analysis 60 4.1.3. Characterization of differentially produced aroma compounds 61 4.1.4. Discussion 70 4.2. Materials and Methods 71 4.2.1. Strain selection and microbial procedure 71 4.2.2. Population growth measurements using Bioscreen C 71 4.2.3. Biogenic amine safety tests 71 4.2.4. Dough preparation and fermentation monitoring 72 4.2.5. Bread making 72 4.2.6. Sensory analysis 73 4.2.7. Analysis of bread volatile compounds 73 5. CHAPTER V: Nucleosomes Affect Local Transformation Efficiency 76 5.1. Introduction 77 5.2. Results and Discussion 78 5.2.1. Transformation efficiency anti-correlates with nucleosome density 78 5.2.2. The effect of nucleosome positioning on transformation efficiency is independent of the local DNA sequence 82 5.2.3. Discussion 83 5.3. Material and Methods 85 5.3.1. Microbial procedure 85 5.3.2. Target selection 85 5.3.3. Transformation protocol 85 5.3.4. GC content & melting temperature 86 5.3.5. Nucleosome mapping 86 6. CHAPTER VI: General conclusion and future perspectives 88 6.1. Dynamics of the yeast transcriptome during bread dough fermentation 89 6.2. Glycerol production by fermenting yeast cells is essential for optimal bread dough fermentation 89 6.3. Non-conventional yeast strains increase the aroma complexity of bread 90 6.4. Nucleosomes Affect Local Transformation Efficiency 91 APPENDIX 93 REFERENCES 105 CURRICULUM VITAE 118nrpages: 133status: publishe

Succinic acid production during dough fermentation with Saccharomyces cerevisiae is governed by the TCA cycle and glyoxylate shunt

Succinic acid has a significant impact on the rheological properties of wheat dough. To investigate the pathways responsible for production of succinic acid by yeast during bread dough fermentation, a mutation strategy using the model S288C yeast strain was used. Deletion of the genes responsible for the oxidative pathway of the TCA cycle and the glyoxylate shunt strongly affected the fermentation rate of yeast and the succinic acid levels produced during dough fermentation. Using Δaco1 and Δaco1Δicl1 mutants in dough fermentation led to a 36 and 77 % decrease in succinic acid levels in fermented dough, respectively. Moreover, using the Δidh1Δidp1 mutant in dough resulted in 85 % decrease in succinic acid production in fermenting dough. In contrast, applying the Δsdh1Δsdh2 mutant in fermenting dough led to a two-fold higher succinic acid accumulation in dough. Despite various reports on the functionality of the reductive pathway of the TCA cycle during anaerobic fermentation, we found no evidence for the impact of this pathway on fermentation rate and succinic acid production by using Δfrd1Δosm1 in dough fermentation. The impact of mutants with decreased (Δidh1Δidp1) and increased (Δsdh1Δsdh2) levels of succinic acid production on the rheological properties of dough was investigated using the Rheofermentometer. The results showed no difference in maximum dough height and gas retention capacity of dough containing mutant strains compared to control dough prepared with S288C.status: accepte

Contribution of the tricarboxylic acid (TCA) cycle and the glyoxylate shunt in Saccharomyces cerevisiae to succinic acid production during dough fermentation

Succinic acid produced by yeast during bread dough fermentation can significantly affect the rheological properties of the dough. By introducing mutations in the model S288C yeast strain, we showthat the oxidative pathway of the TCA cycle and the glyoxylate shunt contribute significantly to succinic acid production during dough fermentation. More specifically, deletion of ACO1 and double deletion of ACO1 and ICL1 resulted in a 36 and 77% decrease in succinic acid levels in fermented dough, respectively. Similarly, double deletion of IDH1 and IDP1 decreased succinic acid production by 85%, while also affecting the fermentation rate. By contrast, double deletion of SDH1 and SDH2 resulted in a two-fold higher succinic acid accumulation compared to the wild-type. Deletion of fumarate reductase activity (FRD1 and OSM1) in the reductive pathway of the TCA cycle did not affect the fermentation rate and succinic acid production. The changes in the levels of succinic acid produced by mutants Δidh1Δidp1 (low level) and Δsdh1Δsdh2 (high level) in fermented dough only resulted in small pH differences, reflecting the buffering capacity of dough at a pH of around 5.1. Moreover, Rheofermentometer analysis using these mutants revealed no difference in maximumdough height and gas retention capacity with the dough preparedwith S288C. The impact of the changed succinic acid profile on the organoleptic or antimicrobial properties of bread remains to be demonstrated.publisher: Elsevier articletitle: Contribution of the tricarboxylic acid (TCA) cycle and the glyoxylate shunt in Saccharomyces cerevisiae to succinic acid production during dough fermentation journaltitle: International Journal of Food Microbiology articlelink: http://dx.doi.org/10.1016/j.ijfoodmicro.2015.03.004 content_type: article copyright: Copyright © 2015 Elsevier B.V. All rights reserved.status: publishe

Glycerol Production by Fermenting Yeast Cells Is Essential for Optimal Bread Dough Fermentation

Glycerol is the main compatible solute in yeast Saccharomyces cerevisiae. When faced with osmotic stress, for example during semi-solid state bread dough fermentation, yeast cells produce and accumulate glycerol in order to prevent dehydration by balancing the intracellular osmolarity with that of the environment. However, increased glycerol production also results in decreased CO2 production, which may reduce dough leavening. We investigated the effect of yeast glycerol production level on bread dough fermentation capacity of a commercial bakery strain and a laboratory strain. We find that Δgpd1 mutants that show decreased glycerol production show impaired dough fermentation. In contrast, overexpression of GPD1 in the laboratory strain results in increased fermentation rates in high-sugar dough and improved gas retention in the fermenting bread dough. Together, our results reveal the crucial role of glycerol production level by fermenting yeast cells in dough fermentation efficiency as well as gas retention in dough, thereby opening up new routes for the selection of improved commercial bakery yeasts.status: publishe

Implementing QR codes in academia to improve sample tracking, data accessibility, and traceability in multicampus interdisciplinary collaborations.

The growing number of multicampus interdisciplinary projects in academic institutions expedites a necessity for tracking systems that provide instantly accessible data associated with devices, samples, and experimental results to all collaborators involved. This need has become particularly salient with the COVID pandemic when consequent travel restrictions have hampered in person meetings and laboratory visits. Minimizing post-pandemic travel can also help reduce carbon footprint of research activities. Here we developed a Quick Response (QR) code tracking system that integrates project management tools for seamless communication and tracking of materials and devices between multicampus collaborators: one school of medicine, two engineering laboratories, three manufacturing cleanroom sites, and three research laboratories. Here we aimed to use this system to track the design, fabrication, and quality control of bioelectronic devices, in vitro experimental results, and in vivo testing. Incorporating the tracking system into our project helped our multicampus teams accomplish milestones on a tight timeline via improved data traceability, manufacturing efficiency, and shared experimental results. This tracking system is particularly useful to track device issues and ensure engineering device consistency when working with expensive biological samples in vitro and animals in vivo to reduce waste of biological and animal resources associated with device failure

Implementing QR codes in academia to improve sample tracking, data accessibility, and traceability in multicampus interdisciplinary collaborations.

The growing number of multicampus interdisciplinary projects in academic institutions expedites a necessity for tracking systems that provide instantly accessible data associated with devices, samples, and experimental results to all collaborators involved. This need has become particularly salient with the COVID pandemic when consequent travel restrictions have hampered in person meetings and laboratory visits. Minimizing post-pandemic travel can also help reduce carbon footprint of research activities. Here we developed a Quick Response (QR) code tracking system that integrates project management tools for seamless communication and tracking of materials and devices between multicampus collaborators: one school of medicine, two engineering laboratories, three manufacturing cleanroom sites, and three research laboratories. Here we aimed to use this system to track the design, fabrication, and quality control of bioelectronic devices, in vitro experimental results, and in vivo testing. Incorporating the tracking system into our project helped our multicampus teams accomplish milestones on a tight timeline via improved data traceability, manufacturing efficiency, and shared experimental results. This tracking system is particularly useful to track device issues and ensure engineering device consistency when working with expensive biological samples in vitro and animals in vivo to reduce waste of biological and animal resources associated with device failure

Implementing QR codes in academia to improve sample tracking, data accessibility, and traceability in multicampus interdisciplinary collaborations

The growing number of multicampus interdisciplinary projects in academic institutions expedites a necessity for tracking systems that provide instantly accessible data associated with devices, samples, and experimental results to all collaborators involved. This need has become particularly salient with the COVID pandemic when consequent travel restrictions have hampered in person meetings and laboratory visits. Minimizing post-pandemic travel can also help reduce carbon footprint of research activities. Here we developed a Quick Response (QR) code tracking system that integrates project management tools for seamless communication and tracking of materials and devices between multicampus collaborators: one school of medicine, two engineering laboratories, three manufacturing cleanroom sites, and three research laboratories. Here we aimed to use this system to track the design, fabrication, and quality control of bioelectronic devices, in vitro experimental results, and in vivo testing. Incorporating the tracking system into our project helped our multicampus teams accomplish milestones on a tight timeline via improved data traceability, manufacturing efficiency, and shared experimental results. This tracking system is particularly useful to track device issues and ensure engineering device consistency when working with expensive biological samples in vitro and animals in vivo to reduce waste of biological and animal resources associated with device failure

Non-Conventional Yeast Strains Increase the Aroma Complexity of Bread

Saccharomyces cerevisiae is routinely used yeast in food fermentations because it combines several key traits, including fermentation efficiency and production of desirable flavors. However, the dominance of S. cerevisiae in industrial fermentations limits the diversity in the aroma profiles of the end products. Hence, there is a growing interest in non-conventional yeast strains that can help generate the diversity and complexity desired in today's diversified and consumer-driven markets. Here, we selected a set of non-conventional yeast strains to examine their potential for bread fermentation. Here, we tested ten non-conventional yeasts for bread fermentation, including two Saccharomyces species that are not currently used in bread making and 8 non-Saccharomyces strains. The results show that Torulaspora delbrueckii and Saccharomyces bayanus combine satisfactory dough fermentation with an interesting flavor profile. Sensory analysis and HS-SPME-GC-MS analysis confirmed that these strains produce aroma profiles that are very different from that produced by a commercial bakery strain. Moreover, bread produced with these yeasts was preferred by a majority of a trained sensory panel. These results demonstrate the potential of T. delbrueckii and S. bayanus as alternative yeasts for bread dough leavening, and provide a general experimental framework for the evaluation of more yeasts and bacteria.status: publishe

Nucleosomes affect local transformation efficiency

Genetic transformation is a natural process during which foreign DNA enters a cell and integrates into the genome. Apart from its relevance for horizontal gene transfer in nature, transformation is also the cornerstone of today's recombinant gene technology. Despite its importance, relatively little is known about the factors that determine transformation efficiency. We hypothesize that differences in DNA accessibility associated with nucleosome positioning may affect local transformation efficiency. We investigated the landscape of transformation efficiency at various positions in the Saccharomyces cerevisiae genome and correlated these measurements with nucleosome positioning. We find that transformation efficiency shows a highly significant inverse correlation with relative nucleosome density. This correlation was lost when the nucleosome pattern, but not the underlying sequence was changed. Together, our results demonstrate a novel role for nucleosomes and also allow researchers to predict transformation efficiency of a target region and select spots in the genome that are likely to yield higher transformation efficiency.status: publishe