Browsing by Subject "Yeast"

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Bioethanol production from lignocellulosic biomass
(2023) Hoppert, Luis; Kölling, Ralf
The aim of this thesis was to develop a high gravity second-generation bioethanol process and investigate the effects of a high solid loading. The insights gained from the initial experiments helped to understand the underlying mechanism behind the limitations of a high solid loading. Based on these findings, strategies were developed to overcome these limitations.
Development of a genetically defined diploid yeast strain for the application in spirit production
(2005) Schehl, Beatus; Heinisch, Jürgen
Yeast strains of the species Saccharomyces cerevisiae currently in use for the production of consumable alcohols such as beer, wine and spirits are genetically largely undefined. This prevents the use of standard genetic manipulations, such as crossings and tetrad analysis, for strain improvement. Furthermore, it complicates the application of the majority of modern methods developed in yeast molecular biology. In this work two haploid laboratory strains with suitable auxotrophic markers were used for the construction of a genetically well defined, prototrophic diploid production strain. This strain was tested for its fermentative and sensory performances in comparison to commercially available yeasts. Different fruit mashes were fermented, subjected to distillation and analysed for fermentation parameters including growth, sugar utilization, ethanol production and generation of volatile compounds, higher alcohols, uretahne and glycerol. All spirits produced were tested for their sensory performances and the data obtained statistically consolidated. Our results clearly demonstrate that this laboratory strain does not display any disadvantage compared with commercial yeasts in spirit production for any of the parameters tested, yet it offers the potential to apply both classical breeding and modern molecular genetic techniques adjusting yeast physiology to special production schemes.
Effekt der Überproduktion von Enzymen des Glucosestoffwechsels auf das Wachstum und die Alkoholbildung in der Hefe Saccharomyces cerevisiae
(2006) Emili, Markus; Heinisch, Jürgen
The wine-, beer- and baker's yeast Saccharomyces cerevisiae is the major source in world wide alcohol production. Regarding the research in bioethanol production, the work presented here was aimed to examine the effect of the in vivo overproduction of all enzymes contributing to the conversion of glucose to ethanol in the yeast Saccharomyces cerevisiae with the prospect of increasing ethanol formation. S. cerevisiae is probably the best studied eucaryotic organism with respect to both classical and molecular genetics. It turned out to be of great advantage that two different multi-copy-vectors could be employed in these studies. Each of them was used in the first part of the work to insert half of the set of genes intended for overexpression. The first genes were inserted by restriction and ligation and later on a combination of the PCR-technique, with which the genomic fragments of interest were amplified, and the efficient homologous recombination in vivo was used. With these methods, the gene encoding a hexose transporter (HXT1), all the genes encoding glycolytic enzymes (HXK2, PGI1, PFK1, PFK2, FBA1, TPI1, TDH1 bzw. TDH2, PGK1, GPM1, ENO2, PYK1), as well as the genes encoding enzymes needed for the conversion of pyruvate to ethanol (PDC1, ADH1), were cloned. Following the isolation from yeast, the plasmids were amplified in E. coli and characterized by restriction analysis. The measurement of specific enzyme activity in crude extract of yeast transformants with such plasmids showed a slight overproduction (factor 1,5 to 3,0) for all enzymes, except for glyceraldehyde-3-phosphate dehydrogenase. For HXT1, an increased mRNA level (factor 14 in contrast to the control) was taken as evidence for overproduction. In the enzymatic determinations a clear tendency showing a lower overproduction with an increasing number of genes on the plasmids was observed. These findings suggest a negative feedback on glycolytic flux regulation. The the growth rates obtained in the second part of the work also showed a clear reduction with increasing numbers of plasmid-encoded genes. Regarding the physiological parameters, no changes in the coefficients for glucose consumption and ethanol formation could be found in comparison to a wild-type control, and the yield remained basically unchanged as well. Interestingly, abolishing the ATP-inhibition of phosphofructokinase by expression of a mutant allele of PFK1, resulted in a faster growth of transformants with an otherwise isogenic background. This result indicates the physiological relevance of the allosteric regulation at this essential glycolytic step. A lack of enzyme activity in one of the glycolytic steps in deletion mutants normally leads to growth inhibition on hexoses. On this basis, the construction of a yeast strain was initiated with the objective to obtain stable multi-copy transformants simply by growing cells on different sugars as carbon sources. In detail, this was done by crossing a strain carrying a pgi1-deletion with a strain carrying a pyk1-deletion followed by sporulation and tetrad dissection. Preliminary data with intermediate strain constructs indicate a clear increase in plasmid stability after growing cells on complex media. From the results of this thesis, valuable insights into the regulation of the glycolytic flux in vivo can be deduced, which may serve as a basis for ongoing research on the improvement of ethanol formation by yeast.
Influence of the newly identified Mos10 interaction partner Vps68on ESCRT-III function
(2021) Alsleben, Sören; Kölling, Ralf
The endosomal sorting complex required for transport (ESCRT) is a part of the heteromeric complex machinery consisting of ESCRT-0, -I, -II, and -III ensuring functional protein traffic of endocytic and biosynthetic cargo. Stepwise sorting of labeled cargo material inside the lumen of the endosome by invagination and abscission of the endosomal membrane to form intraluminal vesicles (ILV’s) is mediated by the ESCRT-III complex. The complex consists of eight members of which Vps20, Snf7, Vps2, and Vps24 are considered ESCRT-III essential subunits, and Chm7, Did2, Ist1, and Mos10/Vps60 are commonly labeled as complex associated proteins. The correct interplay between the proteins ensures cargo sorting into the MVB (multivesicular body) pathway and transport from the late endosome into the vacuolar lumen for degradation. Besides the initial function of vacuolar protein sorting (vps), the complex is involved in a multitude of cellular processes like cell abscission, virus budding, autophagy, and remaining nuclear envelope integrity. The step-wise assembly of the ESCRT-III complex is mediated after the cascade-like ESCRT-0 to ESCRT-II complex formation at the membrane budding site, collecting cargo protein for invagination into the endosomal lumen. ESCRT-III Vps20 is recruited to the membrane by the ESCRT-II member Vps25, then nucleating Snf7 association and oligomerization. Additional assembly of ESCRT-III members like Vps24 and Vps2 further drives membrane bending away from the cytosol to the final abscission event, before being recycled back to cytosolic monomers by Vps4. Although Mos10 has been implicated in the recycling step of the ESCRT-III units by interacting with the Vps4/Vta1 complex, the protein’s function remains poorly characterized. This thesis tried to find new insights in Mos10 functionality by finding yet uncharacterized interacting partners, thus connecting the protein to new putative non-endosomal functions or understanding its role in the established ESCRT-III complex. For this purpose, a series of crosslinking experiments with tagged variants of Mos10 were performed. Purification was achieved by IMAC (Immobilized Metal Ion Affinity Chromatography) after adding a poly-his sequence to the protein and by immunoprecipitation of sfGFP tagged Mos10. Both methods revealed a multitude of putative Mos10 interacting partners by MS analysis to be further reduced by applying the SILAC (stable isotope labeling with amino acids in cell culture) technique. After selecting possible Mos10 interacting partners, IP and Co-IP experiments of tagged candidate variants were used to identify an interaction between the two proteins. An interaction between Mos10-6His and Vps68-13myc besides native Mos10 and Vps68-fGFP could be verified by purification of Vps68 and co-precipitating Mos10. The influence of Vps68 on the assembly and composition of the ESCRT-III complex was examined. After Vps68 depletion, an enrichment of the core subunits Snf7, Vps2, and Vps24 in the complex was detected with a reduced number of Did2, Ist1, and Mos10 molecules. Thus, it appears that ESCRT-III disassembly is blocked in ∆vps68 mutant. The influence of VPS68 deletion on the intracellular localization of ESCRT-III proteins was examined by fluorescence microscopy with sfGFP-tagged variants. While the localization of most ESCRT-III proteins was not significantly altered, a marked relocalization was observed for Mos10. In wildtype, Mos10-sfGFP was localized at the vacuolar membrane, while in ∆vps68 it was dispersed into vesicular structures enriched at the cell cortex. Further, the impact of VPS68 deletion on the sorting of the endocytic cargo protein Ste6 was investigated. By cycloheximide chase experiments, it could be shown that Ste6 is strongly stabilized in a ∆vps68 mutant. This indicates that the transport of the protein to the yeast vacuole for degradation is blocked. The ∆vps68 block in endocytic trafficking was compared with other mutants of the vps-pathway, whose site of action has been established. These experiments show that the VPS68 deletion neither leads to a class D phenotype, as in ∆vps21, nor to a class E phenotype, as in ∆snf7. The Ste6-GFP distribution in the ∆vps68 mutant rather resembles wildtype with more pronounced accumulation of endosomal dots. The data taken together suggest that Vps68 acts after the formation of the ESCRT-III complex and is required for cargo delivery from the late endosome to the vacuolar lumen.
Powerful proteins : structure and function of catalytic subunits of electrogenic NADH:quinone oxidoreductases
(2013) Steffen, Wojtek; Fritz-Steuber, Julia
Electrogenic NADH:quinone oxidoreductases are large, membrane-embedded enzyme complexes found in the respiratory chain of prokaryotes and the mitochondria of eukaryotes. They represent the first module of the oxidative phosphorylation system which converts the energy from nutrients into an electrochemical gradient by coupling redox reactions to the translocation of cations across membranes. A long chain of events, such as the synthesis of ATP, ion homeostasis, reactive oxygen species production and bacterial motility depend on the activity of these complexes. Complex I consists of up to 45 subunits and can be found in the inner mitochondrial membrane of eukaryotes and in prokaryotes, where it is called NDH I. We investigated the isolated, hydrophobic ND5 subunit, which shows homologies to cation/proton antiporters, from human or Yarrowia lipolytica complex I. In vivo and biochemical analyses provided data on the cation translocation function and the alteration of function by disease-associated mutations. Taken together with the recently published 3D structure of bacterial complex I, these data allowed us to demonstrate that the ND5 subunit could possibly act as an antiporter module of mitochondrial complex I. Sodium ion translocating NADH:quinone oxidoreductase (Na+-NQR) is an enzyme found in many pathogenic bacteria. It consists of six subunits (NqrA - NqrF) whose 3D structures and enzymatic mechanisms were not known in detail at the time this project was initiated. By using high-resolution X-ray structures and site-directed mutagenesis, combined with biochemical studies, we proposed a model for catalysis and substrate selectivity on the atomic level of the electron input module of the complex, the NADH oxidizing domain of subunit NqrF. Furthermore, we analyzed the binding of silver ions to a cysteine residue in the NADH binding pocket and found that it leads to the inhibition of the Na+-NQR and to the killing of Vibrio cholerae in the nanomolar range. Subunit NqrA forms part of the quinone reductase module. By the use of physicochemical and biochemical methods we identified the herbicide 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone (DBMIB) as a quinone antagonist and inhibitor of the Na+-NQR complex and discovered two adjacent quinone binding sites on NqrA.