Metabolic engineering approaches reveal widespread physiological functions of membrane lipids for Saccharomyces cerevisiae

The lipid composition of biological membranes can differ significantly between organisms and even between organelles of the same cell in terms of lipid compounds and specific ratios of lipid classes. Referring to this, every membrane features a characteristic lipid composition that is thought to regulate its physicochemical properties and cellular function by providing lipid environments supporting the integrity of membrane-localized protein machinery and membrane-associated processes. Chapter I gives a brief overview of the interlinkage between the chemical nature of membrane lipids, the structural and functional organization as well as the physicochemical properties of lipid bilayers and their influence on membrane-embedded proteins. Studies to gain detailed knowledge on how membrane lipid composition influences the physiology of cells and regulates cellular processes require tools to manipulate lipid composition in vivo. By employing metabolic engineering approaches based on titratable gene expression tools, sets of Saccharomyces cerevisiae strains in which membrane lipid composition is under experimental control were engineered. The study described in Chapter II addresses OLE1, encoding for the sole fatty acid desaturase of budding yeast, to control the extent of acyl unsaturation of fatty acids incorporated in phospholipids. This approach revealed cellular roles for the physical state of cell membranes, so called membrane fluidity, on yeast flocculation and hypoxic growth. It is shown, how the endogenous lipid homeostasis machinery of budding yeast is adapted to carry out a broad response to oxygen limitation (hypoxia) and how it activates a non-canonical yeast flocculation pathway involving FLO1, which encodes for cell wall glycoproteins that mediate cell-cell-interactions by binding cell wall mannose residues of adjacent cells. In Chapter III, the previously generated strain in which expression of OLE1 is under experimental control was used as a cellular platform to assay the activity of heterologously expressed stearoyl-CoA desaturases (SCDs). Putative SCDs from human pathogens T. brucei and T. cruzi were functionally expressed in S. cerevisiae, thereby additionally confirming their SCD activity in vivo. The presented assay might also provide a tool to screen for inhibitors of SCDs, which are interesting drug targets in the treatment of bacterial and parasitic infections in humans. The study presented in Chapter IV addresses ERG9, an essential gene involved in the ergosterol biosynthetic pathway and used a metabolic engineering approach to achieve control over the total sterol biosynthetic activity of the cell. Cells that allowed for manipulating the native sterol homeostasis were employed to unveil physiological effects of ergosterol and total sterol depletion on the cell’s general viability as well as on fundamental membrane associated processes such as protein sorting and endo- and exocytosis. By combining this metabolic engineering approach and the powerful method of marker-free CRISPR/Cas9-mediated gene tagging, it was possible to establish a cellular system for investigating the impact of sterol depletion on the lateral distribution pattern of lipid-raft associated GFP-tagged membrane proteins within the plasma membrane of yeast. Chapter V introduces a novel set of all-in-one constitutive and inducible CRISPR/Cas9 vectors that allow for a very easy and highly convenient application of the technology in S. cerevisiae. The simplicity of the inducible system is based on the possibility of introducing a desired gRNA targeting sequence with homologous recombination-mediated assembly of overlapping single-stranded oligonucleotides. The inducible Cas9 expression approach also introduces the novel concept of chronologically separating the cloning procedure from the actual genome editing step by preloading cells with an all-in-one CRISPR/Cas9 plasmid. This way, CRISPR/Cas9-supported genome editing can be obtained with high efficiency and effectivity by just transforming a desired preloaded target strain with donor DNA to be genomically integrated without the need of co-introducing any of the CRISPR system components. These novel CRISPR/Cas9 systems will help to overcome limitations often observed for challenging metabolic and genetic engineering approaches that can be e.g. used for following studies to reveal physiological roles of membrane lipids for budding yeast

Untersuchungen zum Gasaustausch : Entwicklung und Applikation eines zeitlich aufgelösten Massenbilanzverfahrens

Ein neues Massenbilanzverfahren zur zeitlich aufgelösten Messung des Gastransfers an der Wasseroberfläche wird vorgestellt. Die neue Methodik bietet den entscheidenden Vorteil, dass unter Evasionsbedingungen in Laborexperimenten nur die zeitliche Änderung in der Luftkonzentration der Tracergase gemessen werden muss. Die Erfordernis einer Absolutmessung der Luftkonzentration entfällt ebenso wie die einer Bestimmung der Wasserkonzentration. Die hohe zeitliche Auflösung der Messmethode erlaubt eine rasche Abfolge von Messungen bei veränderten Versuchsbedingungen. Systematische Gasaustauschmessungen können nun binnen weniger Stunden durchgeführt werden. Durch vergleichende Konzentrationsmessungen in der Luft- sowie in der Wasserphase von volatilen Aromaten mittels UV-Spektroskopie konnte die entwickelte Methode validiert werden. Im Rahmen der UV-Messungen wurde ferner ein Verfahren entwickelt, das es ermöglicht, auch bei geringer Auflösung der verwendeten Spektrometer, die Luftkonzentration der Aromaten mittels Differentieller Optischer Absorptionsspektroskopie über einen weiten Konzentrationsbereich korrekt zu bestimmen. Mit den entwickelten Messmethoden sind nun auch zuverlässige Messdaten für sehr kleine Wind- und Schubspannungsgeschwindigkeiten verfügbar. Hiermit konnte der Übergang des Schmidtzahlexponenten beim Wechsel von einer glatten zu einer rauen Wasseroberfläche nachvollzogen werden. Der Umschlag setzt bereits bei unerwartet geringen Windgeschwindigkeiten ein und vollzieht sich über einen weiten Bereich von Windgeschwindigkeiten. Erstmals konnte im Labor der Effekt des chemisch beschleunigten Gasaustausches unter einer großen Variation an chemischen und physikalischen Bedingungen gezeigt werden

Koordination und Integration von Umweltfachplanungen und ihr Verhältnis zur Stadtplanung

KOORDINATION UND INTEGRATION VON UMWELTFACHPLANUNGEN UND IHR VERHÄLTNIS ZUR STADTPLANUNG Koordination und Integration von Umweltfachplanungen und ihr Verhältnis zur Stadtplanung / Degreif, Stefanie (Rights reserved) ( -

Personal art vs. public school assignments: Case studies of disengaged high school art students

The qualitative case studies focused on four students enrolled in high school beginning art classes who were motivated to produce artwork outside of class but did not engage with the class art projects. Each of these four subjects produced personal artwork outside of the school that differed from art produced for assignments in class. The study explored the motivation and work habits of these subjects. The study utilized observation of in class student work habits and behavior, and analyzed both in class and personal art work. Data was gathered from three sources: observations of students while they worked in class, the personal and class artwork of students, and interviews with students. The study arrived at common themes and issues in these students' work

The Sport-Treff in Stadecken-Elsheim : the use of a restaurant as an event location

In dieser Bachelorarbeit geht es um eine Gaststätte in Stadecken-Elsheim und wie diese als Event Location genutzt werden kann. Die ersten Kapitel drehen sich um die Grundlagen des Eventmanagements und der Veranstaltungsstätten. Im letzten Teil dieser Arbeit werden die Gemeinde Stadecken-Elsheim und die jetzige Situation des Sport-Treffs beschrieben und welche Maßnahmen es zur Optimierung gibt

Metabolic engineering approaches reveal widespread physiological functions of membrane lipids for Saccharomyces cerevisiae

The lipid composition of biological membranes can differ significantly between organisms and even between organelles of the same cell in terms of lipid compounds and specific ratios of lipid classes. Referring to this, every membrane features a characteristic lipid composition that is thought to regulate its physicochemical properties and cellular function by providing lipid environments supporting the integrity of membrane-localized protein machinery and membrane-associated processes. Chapter I gives a brief overview of the interlinkage between the chemical nature of membrane lipids, the structural and functional organization as well as the physicochemical properties of lipid bilayers and their influence on membrane-embedded proteins. Studies to gain detailed knowledge on how membrane lipid composition influences the physiology of cells and regulates cellular processes require tools to manipulate lipid composition in vivo. By employing metabolic engineering approaches based on titratable gene expression tools, sets of Saccharomyces cerevisiae strains in which membrane lipid composition is under experimental control were engineered. The study described in Chapter II addresses OLE1, encoding for the sole fatty acid desaturase of budding yeast, to control the extent of acyl unsaturation of fatty acids incorporated in phospholipids. This approach revealed cellular roles for the physical state of cell membranes, so called membrane fluidity, on yeast flocculation and hypoxic growth. It is shown, how the endogenous lipid homeostasis machinery of budding yeast is adapted to carry out a broad response to oxygen limitation (hypoxia) and how it activates a non-canonical yeast flocculation pathway involving FLO1, which encodes for cell wall glycoproteins that mediate cell-cell-interactions by binding cell wall mannose residues of adjacent cells. In Chapter III, the previously generated strain in which expression of OLE1 is under experimental control was used as a cellular platform to assay the activity of heterologously expressed stearoyl-CoA desaturases (SCDs). Putative SCDs from human pathogens T. brucei and T. cruzi were functionally expressed in S. cerevisiae, thereby additionally confirming their SCD activity in vivo. The presented assay might also provide a tool to screen for inhibitors of SCDs, which are interesting drug targets in the treatment of bacterial and parasitic infections in humans. The study presented in Chapter IV addresses ERG9, an essential gene involved in the ergosterol biosynthetic pathway and used a metabolic engineering approach to achieve control over the total sterol biosynthetic activity of the cell. Cells that allowed for manipulating the native sterol homeostasis were employed to unveil physiological effects of ergosterol and total sterol depletion on the cell’s general viability as well as on fundamental membrane associated processes such as protein sorting and endo- and exocytosis. By combining this metabolic engineering approach and the powerful method of marker-free CRISPR/Cas9-mediated gene tagging, it was possible to establish a cellular system for investigating the impact of sterol depletion on the lateral distribution pattern of lipid-raft associated GFP-tagged membrane proteins within the plasma membrane of yeast. Chapter V introduces a novel set of all-in-one constitutive and inducible CRISPR/Cas9 vectors that allow for a very easy and highly convenient application of the technology in S. cerevisiae. The simplicity of the inducible system is based on the possibility of introducing a desired gRNA targeting sequence with homologous recombination-mediated assembly of overlapping single-stranded oligonucleotides. The inducible Cas9 expression approach also introduces the novel concept of chronologically separating the cloning procedure from the actual genome editing step by preloading cells with an all-in-one CRISPR/Cas9 plasmid. This way, CRISPR/Cas9-supported genome editing can be obtained with high efficiency and effectivity by just transforming a desired preloaded target strain with donor DNA to be genomically integrated without the need of co-introducing any of the CRISPR system components. These novel CRISPR/Cas9 systems will help to overcome limitations often observed for challenging metabolic and genetic engineering approaches that can be e.g. used for following studies to reveal physiological roles of membrane lipids for budding yeast

PAS c als signaltransduzierende Domäne des Sensorproteins DcuS von Escherichia coli

Bakterien besitzen membranintegrierte Sensoren für die Reaktion auf verändernde Umweltbedingungen.rnViele der Sensoren sind Zweikomponenten-Systeme bestehend aus einer Sensorhistidinkinase und einem Responseregulator der die zellulare Antwort auslöst. DcuS, der C4-Dicarboxylat-Sensor von DcuS ist eine membranintegrierte Histidin-Kinase. DcuS ist ein Multidomänen-Protein mit einer sensorischen periplasmatischen PASP (Per-Arnt-Sim) Domäne, zwei Transmembranhelices, eine cytoplasmatische PASC-Domäne und eine C-terminale Kinase-Domäne. PAS-Domänen sind ubiquitäre Signalmodule die in allen Reichen des Lebens zu finden sind. PAS-Domänen detektieren eine Vielfalt von Reizen wie Licht, Sauerstoff, Redoxpotential und verschiedene kleine Moleküle so wie die Modulation von Protein-Protein Interaktionen. PAS-Domänen sind strukturell homolog und besitzen eine charakteristische α/β-Faltung. Eine große Anzahl der sensorischen PAS-Domänen wurden identifiziert, aber viele der PAS-Domänen besitzen keinen apparenten Cofaktor und die Funktion ist unbekannt.rnEine Kombination aus gerichteter und ungerichteter Mutagenese, Protein-Protein-Interaktionsstudien und Festkörper-NMR (ssNMR) Experimente mit strukturellem Modelling wurde zur Untersuchung der Struktur und Funktion der cytoplasmatischen PAS-Domäne des membranintegrierten Sensors DcuS verwendet. Die Experimente zeigen, dass PASC eine wichtige Rolle in die Signaltransduktion von PASP zur C-terminalen Histidin-Kinase von DcuS spielt.rnBacteria contain membrane integral sensors for response to changing environmental conditions. Many of the sensors are two-component systems consisting of a sensor histidine kinase and a response regulator that triggers the cellular response. DcuS, the C4-dicarboxylate sensor of E. coli is a membrane integral periplasmic sensing histidine kinase. DcuS is a multidomain protein consisting of a sensing periplasmic PASP (Per-Arnt-Sim) domain, two transmembrane helices, a cytoplasmic PASC and the C-terminal kinase domain. PAS domains are ubiquitous signalling modules found in all kingdoms of life.They can detect a vast quantity of stimuli including light, oxygen, redox potential and various small molecules as well as modulating protein-protein interactions. rnPAS domains are structurally homologous and characterised by a conserved α/β-fold. A large number of sensory PAS domains have been identified but many of the PASC domains contain no apparent cofactor and their function is unknown.rn A combination of random and side-directed mutagenesis, protein-protein-interaction studies and solid-state NMR (ssNMR) experiments with structural modelling was used to study the structure and function of the cytoplasmic PASC domain of the membrane embedded construct of DcuS. The experiments show an important role for PASC for signal transduction from the PASp to the C-terminal histidine kinase of DcuS.rnr

Novel auto-selection systems for transformation selection of Saccharomyces cerevisiae in rich complex media

The most widely used strategy for selection of yeast transformed with episomal plasmids comprises the use of auxotrophic yeast strains in combination with vectors containing complementing prototrophic marker genes. Another approach uses heterologous genes or cassettes which, if present in the vector, render the otherwise sensitive yeast strain resistant to antibiotics. In addition, auto-selection systems for Saccharomyces cerevisiae have been developed that eliminate the requirement for synthetic drop-out media or the use of antibiotics for transformation selection and subsequent plasmid maintenance in expression cultures. Here we describe a combination of host strain and vector system introducing a novel concept of auto-selection systems that allows for easy and robust propagation of host cells deleted in essential genes in supplemented media before being transformed with rescuing plasmids. With that, our approach is favorable over commonly used selection strategies and has major advantage over other auto-selection systems. Our approach complements the auto-selection toolbox already available for S. cerevisiae, thus contributing a novel system that enables the use of complex peptone-based media for protein expression and metabolic engineering approaches. We therefore expect that this new strategy will be of general interest to the yeast research community in academia and industry