Expanding the Genetic Toolbox to Improve Metabolic Engineering in the Industrial Oleaginous Yeast, \u3cem\u3eYarrowia lipolytica\u3c/em\u3e

The oleaginous yeast, Yarrowia lipolytica, is becoming a popular host for industrial biotechnology because of its ability to grow on non-conventional feedstocks and naturally accumulate significant amounts of lipids. With new genome editing technologies, engineering novel pathways to produce lipid-derived oleochemicals has become easier. The goal, however, is to expand the genetic toolbox to improve the efficiency of metabolic engineering such that production capacities could expand from proof-of-concept shake flasks to an industrial scale. Building efficient metabolic circuits require controlling strength and timing of several enzymes in a metabolic pathway. One method to do this is through transcription – using suitable promoters to control expression of genes that code for enzymes. Native promoters have limited application because of complex regulation and non-tunable expression. Engineering hybrid promoters alleviates these issues to obtain predictable and tunable gene expression. In Y. lipolytica, how to design these promoters is not fully understood, resulting in only a handful of engineered promoters to date. In this work, we aim to develop tools for gene expression by investigating promoter architecture and designing tunable systems. In addition to Upstream Activating Sequences (UAS), tuning promoter strength can be achieved by varying sequence in the core promoter, TATA motif, and adjacent proximal sequences. UASs can modulate transcription strength and inducibility, enabling controlled timing of expression. A promoter of the acyl-CoA oxidase 2 (POX2) from the β-oxidation pathway was truncated heuristically to identify oleic acid (OA) UAS sequences. By fusing tandem repeats of the OA UAS elements, tunable yet inducible fatty acid hybrid promoters were engineered. The current approaches to identify novel UAS elements in Y. lipolytica are laborious. Therefore, we investigated DNA accessibility through nucleosome positioning to determine if a relationship between POX2 UASs and DNA accessibility can be inferred. The goal is to eventually apply this approach develop newer hybrid promoters efficiently. Finally, the hybrid fatty acid inducible promoter we developed was used to rationally engineering a Y. lipolytica strain capable of producing high amounts of free fatty acids. By localizing the fatty acyl / fatty aldehyde reductase in the peroxisome, we compartmentalized fatty alcohol production. This strategy led to upwards of 500 mg/L of fatty alcohols produced. It is a promising route to eventually make short to medium chain fatty alcohols in Y. lipolytica by utilizing the native β-oxidation machinery

Carbon source dependent promoters in yeasts

Budding yeasts are important expression hosts for the production of recombinant proteins. The choice of the right promoter is a crucial point for efficient gene expression, as most regulations take place at the transcriptional level. A wide and constantly increasing range of inducible, derepressed and constitutive promoters have been applied for gene expression in yeasts in the past; their different behaviours were a reflection of the different needs of individual processes. Within this review we summarize the majority of the large available set of carbon source dependent promoters for protein expression in yeasts, either induced or derepressed by the particular carbon source provided. We examined the most common derepressed promoters for Saccharomyces cerevisiae and other yeasts, and described carbon source inducible promoters and promoters induced by non-sugar carbon sources. A special focus is given to promoters that are activated as soon as glucose is depleted, since such promoters can be very effective and offer an uncomplicated and scalable cultivation procedure

Genome Wide Analysis Identifies Sphingolipid Metabolism As A New Target Of Valproic Acid

Bipolar disorder (BD), which is characterized by depression and mania, affects about 1% of the total world population. Current treatments are effective in only 40-60% of cases and cause severe side effects. Valproic acid (VPA), a branched short-chain fatty acid, is one of the most widely used drugs for the treatment of BD. Although many hypotheses have been postulated to explain the molecular mechanism of action of this drug in BD, the therapeutic mechanism is not understood. This knowledge gap has hampered the development of new drugs to treat this disorder. To identify candidate pathways affected by VPA, I performed a genome wide expression analysis in yeast cells grown in the presence or absence of the drug. Many genes and pathways showed altered expression in response to VPA. Among these, sphingolipid metabolism genes showed altered expression in response to both chronic and acute VPA treatment. Chronic VPA caused upregulation of FEN1 and SUR4, encoding fatty acid elongases that catalyze the synthesis of very long chain fatty acids (C24 to C26) required for the synthesis of ceramide. Interestingly, fen1Δ and sur4Δ mutants exhibited VPA sensitivity. Consistent with this, VPA increased levels of ceramides, especially those that contain C24 and C26 fatty acids. As expected with an increase in ceramide, VPA decreased the expression of amino acid transporters, increased the expression of ER chaperones, and activated the unfolded protein response element (UPRE), suggesting that VPA induces the UPR pathway. These effects are rescued by supplementation of inositol and are similarly observed in inositol-starved ino1Δ cells. Starvation of ino1Δ cells increased expression of FEN1 and SUR4, increased ceramide levels, decreased expression of nutrient transporters, and induced the UPR. These findings suggest that VPA-mediated inositol depletion induces the UPR by increasing the de novo synthesis of ceramide. In response to acute VPA, the gene that exhibited the highest upregulation was RSB1, which encodes a transporter of the long chain bases (LCBs) dihydrosphingosine (DHS) and phytosphingosine (PHS). In addition to increased mRNA, acute VPA increased Rsb1 protein levels. The rsb1Δ mutant exhibited increased sensitivity to PHS in the presence of VPA, suggesting that VPA increases PHS levels. Consistent with this, acute VPA increased PHS levels, especially in rsb1Δ cells. LCBs are precursors of ceramide synthesis, which begins in the endoplasmic reticulum by the conversion of palmitoyl-CoA to PHS or DHS. These intermediates are converted to ceramide via ceramide synthase by addition of a fatty acid synthesized by the fatty acid elongation pathway. Orm proteins are negative regulators of de novo synthesis of PHS, which was shown to function as a signaling molecule. My findings indicate that acute VPA downregulates ORM and fatty acid elongases FEN1 and SUR4. This leads to increased PHS levels and increased expression of RSB1 as well as genes that transport and metabolize PHS, including YOR1 and DPL1. Inositol starvation of the ino1Δ mutant for 30 minutes increased expression of RSB1 and YOR1 and decreased expression of FEN1, SUR4, ORM1, and ORM2. This study shows for the first time that acute VPA-mediated inositol depletion increases levels of PHS. In summary, I identified sphingolipid metabolism as a new target of VPA. My studies showed that VPA exerts inositol depletion-mediated differential effects on sphingolipid species. Chronic VPA increases ceramide levels and induces the UPR pathway, whereas, acute VPA increases the levels of PHS. These findings suggest that sphingolipid metabolism is a potential target of VPA that could be important for the therapeutic action of this drug

The baker’s yeast Saccharomyces cerevisiae as a model for studying chronic diseases related to lipid-based ER-stress

The composition of biological membranes is a complex interplay of lipids and proteins. The lipid part is organized in the form of a lipid bilayer that allows integral uptake of membrane proteins as well as their peripheral association. Membranes allow the formation of compartments that ensure an optimized execution of biochemical processes. This optimization of metabolic processes is achieved through maintaining a membrane specific protein and lipid composition. The composition and function of biological membranes go hand in hand and are interdependent. For example, the barrier function of the plasma membrane in eukaryotic cells is based on an increased packing density of lipids, which is ensured by maintaining a sterol gradient along the secretory pathway. Since the molar fraction of lipids exceeds the fraction of membrane proteins within a membrane, they exert a significant impact on the determination of physicochemical properties. Furthermore, cellular lipid composition is more subject to external stimuli such as dietary changes and is more dynamically regulated. Thus, it is desirable to obtain a more detailed understanding of the impact lipid environments exert on the functionality of membrane proteins. Although knowledge about the diversity of membrane compositions is growing, mechanisms responsible for the adaptation and maintenance of these membrane compositions remain poorly understood. The endoplasmic reticulum (ER) is the central organelle for lipid biosynthesis in most eukaryotic cells and marks the entry point to the secretory pathway. It is therefore the ideal organelle to address questions of cellular regulation of membrane composition. The ER forms a continuous and branched membrane network that can be subdivided both structurally and functionally into specialized subdomains. The two most prominent functional subdomains are the rough ER (RER) and the smooth ER (SER). The naming of the domains is derived from the appearance of the membrane surface. In the case of the RER, the membrane surface is populated with translating ribosomes. The RER is thus a hotspot of protein biosynthesis. The SER, on the other hand, is devoid of ribosomes but harbors, among other things, enzymes responsible for de novo lipid synthesis. Thus, both subdomains of the ER functionally complement one another and enable a coordinated membrane biogenesis. Furthermore, the ER is largely responsible for the maturation of secretory proteins, which account for nearly one third of cellular protein synthesis. Subtle perturbations in the balance of protein production and protein folding can overwhelm the folding capacities of the ER and subsequently lead to an accumulation of unfolded or misfolded proteins. Such a condition is commonly referred to as ER-stress. Blockage or long-term impairment of ER functionality has far-reaching consequences and is suspected to be involved in the development of complex metabolic diseases such as diabetes mellitus and non-alcoholic fatty liver disease. The unfolded protein response (UPR) is of particular importance in combating and preventing ER-stress. The UPR is a highly conserved signaling pathway in eukaryotic cells that is thought to buffer short secretory stress peaks by dynamically regulating the secretory capacity of the ER. Since the discovery of the UPR, the canon of activating signals has continued to expand and now includes perturbed lipid composition of the ER membrane. Aberrant lipid compositions that result in the activation of the UPR are termed lipid bilayer stress (LBS).The present work is divided into three sections and each section is devoted to an overarching theme of an LBS-driven UPR. In the first section, the extent to which a functional UPR is involved in modeling cellular lipid composition in the steady state of a cultivation was investigated. Furthermore, the question was addressed to what extent the UPR is actively involved in an adaptation of lipid composition under ER stress. To address these questions, we took advantage of the fact that the UPR in S. cerevisiae is mediated by a single sensor protein, the inositol requiring enzyme 1 protein (Ire1p). Hence, by using a wild-type (WT) strain and the isogenic ire1∆ strain, it was possible to distinguish between UPR-dependent and UPR-independent processes in the remodeling of cellular lipid composition. The reducing agent dithiothreitol (DTT), which interferes with the formation of disulfide bridges in the lumen of the ER, and the pharmacologically active compound tunicamycin (TM), which inhibits N-glycosylation of newly synthesized proteins, were used as inducers of proteotoxic ER stress. The potency of both agents was systematically determined in a growth-based minimal inhibitory concentration (MIC) assay for both strains for complex rich culture medium as well as for synthetic defined minimal medium. Concentrations that reliably triggered the UPR were used in follow-up experiments and their effects on cell growth and lipid composition were analyzed. It was found that there were no significant differences between the strains in terms of cell growth. The greatest difference in growth rate was seen in the comparison between the two media in the case of the unstressed condition. When the strains were cultured in rich complex medium they showed a doubling rate of 86 min, opposed to a doubling rate of 107 min in the case of the synthetic-defined minimal medium. The selected concentrations of DTT and TM showed a growth inhibitory effect for both strains that occurred one hour after application regardless of the medium. Quantitative lipidomic analyses of the stressed and unstressed cells showed that for short-term treatment (1 h), both DTT and TM had negligible effects on the lipid composition of WT and ire1∆ cells in case of the synthetic-defined minimal medium. On the other hand, in the case of the rich and complex medium, there was a significant accumulation of phosphatidate (PA) in cells stressed with DTT compared to the unstressed control.Comparison of lipid composition showed, as in the case of growth rate, that the greatest differences occurred between the media. Here, significant changes were found in the lipid class of ergosterol, the complex sphingolipids, the phosphoglycerides phosphatidylethanolamine and phosphatidylinositol, and the storage lipids triacylglycerides and ergosterol ester. The study systematically addressed the question of the extent to which DTT and TM, as agents for inducing a UPR, have an effect on the cellular lipid composition. In conclusion, the UPR does not affect lipid composition in the absence of ER stress-inducing agents. Furthermore, the data imply that the UPR induced by DTT or TM in the acute phase one hour after drug application has no significant effect on the membrane composition in synthetic-defined minimal medium under the here established conditions. In the second section, the dimerization behavior of Ire1p under proteotoxic and LBS conditions was investigated. A prerequisite for Ire1p-dependent activation of the UPR is oligomerization of individual Ire1p protomers. Crystal structure analyses of the soluble sensor domain of Ire1p have revealed interaction sites for di- and oligomerization and contributed to our understanding of an activation of Ire1p by proteotoxic stress. In addition, a mechanism for membrane-mediated oligomerization by aberrant lipid composition has been described in the past. This mechanism is based on the unusual structure of the transmembrane domain of Ire1p. It consists of a short hydrophobic transmembrane helix (TMH) which transitions into an amphipathic helix (AH) at its N-terminus. This unusual architecture causes the AH to insert into the ER membrane with the hydrophobic side, thereby inducing a tilt of the short TMH. In addition, the transmembrane domain (TMD) of Ire1p causes local compression of the membrane. The energetic cost of membrane deformation is dependent on the lipid composition, which largely correlates with the prevailing lipid packing density. Ire1p can minimize the increased energetic cost of membrane stiffening via oligomerization by minimizing the membrane area to be compressed. However, whether different forms of ER stress, i.e. proteotoxic ER stress or LBS, differentially affect the architecture of Ire1p in the TMD was unresolved. To resolve structural changes in the TMD, systematic cysteine- mediated crosslinking was performed. For this purpose, a cysteine-free variant of Ire1p was first generated and genomically integrated by homologous recombination. Genomic integration and expression by the native Ire1p promoter were used to account for the oligomerization-induced activation of Ire1p by an endogenous expression level. The functionality of this cysteine-free variant was validated at the cellular level in the form of growth assays and on the molecular level via RT-qPCR of UPR specific mRNAs. Subsequently, single cysteines were introduced into the cysteine-free construct by mutagenesis of the TMD. The single cysteine variants generated in this way were reassessed for their ability to trigger a UPR. To identify residues that potentially establish an interaction surface in signal-actingclusters of Ire1p, ER membranes were obtained from cells that were either unstressed or previously treated by DTT, TM, or by inositol depletion, as a form of pure LBS. This showed that the single cysteine mutant F544C exhibited the highest cross-linking efficiency for all stress conditions tested. Furthermore, a comparable cross-linking pattern emerged for all stress conditions, such that the TMD of Ire1p appears to adopt a single conformation upon activation regardless of the type of ER-stress applied. In the third section of the presented thesis, I utilized a mutant of the OLE signaling pathway to study the role of the UPR in context of LBS. This mutant is characterized by an increased degree of saturation of membrane lipids compared to wild-type cells. In S. cerevisiae, there is only a single gene, OLE1, which encodes a ∆-9 desaturase that inserts double bonds into acyl-CoA precursors of membrane lipids. The OLE pathway describes the regulation of OLE1 expression and is mediated by the two transcription factors Mga2p and Spt23p. Deletion of the MGA2 gene can induce a dysregulation that is responsible for an increase in the degree of saturation concomitant with an activation of the UPR. The aim was to use S. cerevisiae to create a eukaryotic model system with which the role of saturated lipids in perpetuating an ER-stress condition could be investigated. This question is relevant because a variety of complex metabolic diseases, such as diabetes mellitus or non-alcoholic fatty liver disease, have been associated with elevated levels of saturated lipids. The presented results provide clues to the molecular mechanisms that may be involved in the development of such diseases. Initially, growth conditions were optimized to maximize membrane-based stress. As a result, cell viability and cell viability were strongly reduced in the mga2∆ mutant opposed to the WT. Furthermore, the proportion of saturated membrane lipids increased to 50-mol %. The significant change in lipid composition was accompanied by dramatic, morphological changes in the ER, which occurred in up to 70% of the microscopically analyzed mga2∆ cells. Fluorescence recovery after photobleaching (FRAP) experiments demonstrated that the diffusion rate of ER-derived membrane proteins within the aberrant ER was significantly reduced compared with a healthy ER. To identify modulators of this morphologically aberrant ER, a genome-wide high-content screen was performed. A variety of gene deletions were identified that either decreased ("rescue") or increased ("worse") the appearance of the aberrant structures. While gene deletions from the "rescue" class showed an increased number of mutants regulating the cell cycle and specifically mitosis, the representatives of the "worse" class were predominantly of mitochondrial origin. Validation of selected representatives of both classes at the level of lipid composition and percent distribution of aberrant ER showed that, as expected, aberrant ER formation correlated with the amount of saturated glycerophospholipids. In addition, these mutants showed significantly increased levels of ceramides, phosphatidic acids, and diacylglycerides.The formation of the aberrant ER could be narrowed down to the act of mitosis by time-lapse microscopy. This process is preceded by a significant increase in membrane mass in interphase. Further supporting data indicative of cell cycle involvement in the formation of the aberrant ER structures could be collected in a secondary targeted screen. In this secondary screen, individual genes were overexpressed that had an effect on the residence time in individual cell cycle phases. The percentages of aberrant ER harboring cells exceeded the values from the original screen of the "rescue" as well as the "worse" category. It was further investigated whether UPR-induced membrane expansion could contribute to the formation of the structures. Expression of inducible membrane expansion systems, as well as overexpression of the ICE2 gene, which leads to ER membrane expansion in wild-type cells, caused a significant increase in aberrant ER structures in the mga2∆ strain background. In a third growth-based post-screening, mutants and additives affecting the growth of the mga2∆ mutant were investigated. This is interesting because it has previously been shown that mga2∆ cells exhibit a distinct growth defect compared to wild-type ones when switching from fermentative growth to respiration. Mitochondrial involvement could thus be identified by correlating the growth defect with the generation of reactive oxygen species (ROS). Overexpression of HAP4, a transcription factor that upregulates mitochondrial activities and leads to the excessive generation of ROS, caused a deterioration of cell growth only in the mga2∆ strain background. Supplementation of the medium with the free radical scavenger vitamin C significantly reduced the growth defect of mga2∆. Consequently, it was also shown microscopically that the development of an aberrant ER in mga2∆ cells was prevented by both vitamin C and reduced glutathione. Overall, a model system was established that allows an investigation of the interplay of the unfolded protein response with cell cycle regulation, lipid metabolism and mitochondrial function.Die Komposition biologischer Membranen ist ein komplexes Zusammenspiel aus Lipiden und Proteinen. Der Lipidanteil organisiert sich in Form einer Lipiddoppelschicht die eine integrale Aufnahme von Membranproteinen erlaubt als auch deren periphere Assoziation ermöglicht. Membranen erlauben die Bildung von Kompartimenten die ein optimiertes Ablaufen von biochemischen Prozessen gewährleisten. Erreicht wird diese Optimierung von Stoffwechselprozessen, indem jede Membran eine spezifische Protein- und Lipidzusammensetzung aufweist. Die Komposition und Funktion biologischer Membranen gehen Hand in Hand und bedingen einander. So beruht die Barrierefunktion der Plasmamembran in eukaryotischen Zellen auf einer erhöhten Packungsdichte der Lipide, die durch Aufrechterhaltung eines Sterol-Gradienten entlang des sekretorischen Weges gewährleistet wird. Da der molare Anteil der Lipide den Anteil der Membranproteine innerhalb einer Membran übersteigt, kommt Ihnen eine besondere Rolle bei der Bestimmung physiochemischer Eigenschaften zu. Ferner unterliegt die zelluläre Lipidzusammensetzung stärker externen Reizen wie sie beispielsweise bei Änderungen der Diät auftreten und wird dynamischer reguliert. Demnach ist es erstrebenswert ein genaueres Verständnis über die Wirkung der Lipidumgebung auf die Funktionalität von Membranproteinen zu erhalten. Obwohl das Wissen bezüglich der Unterschiedlichkeit von Membrankompositionen stetig wächst bleiben Mechanismen, die für die Adaption und Aufrechterhaltung dieser Membrankompositionen verantwortlich sind weitestgehend unverstanden. Das endoplasmatische Retikulum (ER) ist das zentrale Organell für die Lipidbiosynthese in den meisten eukaryotischen Zellen und markiert den Eintrittspunkt zum sekretorischen Weg. Es ist daher das ideale Organell, um Fragen der zellulären Regulation von Membrankompositionen zu adressieren. Das ER bildet ein kontinuierliches und verzweigtes Membrannetzwerk aus, welches sich sowohl strukturell als auch funktionell in spezialisierte Subdomänen unterteilen lässt. Die beiden prominentesten funktionellen Subdomänen sind das raue ER (englisch rough ER (RER)) und das glatte ER (englisch smooth ER (SER)). Die Namensgebung der Domänen leitet sich aus dem Erscheinungsbild der Membranoberfläche ab. Im Fall des RER ist die Membranoberfläche mit translatierenden Ribosomen bestückt. Das RER ist somit ein Hotspot der Proteinbiosynthese. Das SER hingegen ist frei von Ribosomen, beherbergt jedoch unter anderem Enzyme die für die de novo Lipidsynthese verantwortlich sind. Beide Subdomänen des ER ergänzen sich demnach funktional und ermöglichen eine koordinierte Membranbiogenese. Ferner ist das ER maßgeblich für die Reifung sekretorischer Proteine, die knapp ein Drittel der zellulären Proteinsynthese stellen, verantwortlich. Feine Störungen im Gleichgewicht von Proteinproduktion und Proteinfaltung können dieFaltungskapazitäten des ER überfordern und in Folge zu einer Akkumulation ungefalteter oder fehlgefalteter Proteine führen. Ein solcher Zustand wird allgemein als ER-Stress bezeichnet. Eine Blockierung oder langfristige Beeinträchtigung der Funktionalität des ER hat weitreichende Folgen und steht im Verdacht an der Entstehung von komplexen metabolischen Erkrankungen wie Diabetes mellitus und der nicht-alkoholischen Fettleber beteiligt zu sein. Eine besondere Bedeutung in der Bekämpfung und Vorbeugung von ER-Stress kommt der unfolded protein response (UPR) zu. Die UPR ist ein hoch-konservierter Signalweg in eukaryotischen Zellen, der kurze sekretorische Belastungsspitzen abfangen soll indem er dynamisch die sekretorische Kapazität des ER reguliert. Seit der Entdeckung der UPR hat sich der Kanon der aktivierenden Signale stets erweitert und beinhaltet inzwischen auch eine gestörte Lipidzusammensetzung der ER-Membran. Aberrante Lipidzusammensetzung, die eine Aktivierung der UPR zur Folge haben, werden als lipid bilayer stress (LBS) bezeichnet. Die vorliegende Arbeit ist in drei Abschnitte unterteilt und jeder Abschnitt ist einem übergreifenden Thema einer LBS getriebenen UPR gewidmet. Im ersten Abschnitt wurde untersucht, inwiefern eine funktionale UPR an der Modellierung der zellulären Lipidkomposition im steady state einer Kultivierung beteiligt ist. Ferner wurde die Frage bearbeitet inwieweit die UPR aktiv an einer Adaption der Lipidzusammensetzung bei ER-Stress beteiligt ist. Um diesen Fragen gerecht zu werden, wurde sich die Tatsache zu Nutze gemacht, dass die UPR in S. cerevisiae von einem einzigen Sensorprotein, dem inositol requiring enzyme 1 protein (Ire1p) vermittelt wird. Demnach konnte durch Verwendung eines Wildtyp-Stammes (WT) und dem isogenen ire1∆ Stamm zwischen UPR-abhängigen und UPR-unabhängigen Vorgängen bei der Remodellierung der zellulären Lipidkomposition unterschieden werden. Als Auslöser eines proteotoxischen ER-Stress wurde das Reduktionsmittel Dithiothreitol (DTT), welches unter anderem mit der Ausbildung von Disulfidbrücken im Lumen des ER interferiert, sowie der Wirkstoff Tunicamycin (TM), der die N-Glykosylierung von neu synthetisierten Proteinen unterbindet, verwendet. Die Potenz beider Wirkstoffe wurde systematisch in einem wachstumsbasierten minimal inhibitory concentration (MIC) Assay für beide Stämme für komplexes reichhaltiges Nährmedium als auch für synthetisch definiertes Nährmedium bestimmt. Konzentrationen die zuverlässig die UPR auslösten wurden in Folgeexperimenten eingesetzt und deren Wirkung auf Zellwachstum und Lipidkomposition analysiert. Dabei zeigte sich im Hinblick auf das Zellwachstum, dass es keine signifikanten Unterschiede zwischen den Stämmen gab. Der größte Unterschied in der Wachstumsrate zeigte sich im Vergleich zwischen beiden Medien im Fall der ungestressten Bedingung. Wurden die Stämme in reichem, komplexem Medium kultiviert zeigten sie eine Verdopplungsrate von 86 min, im Fall des synthetisch-definiertem Medium eine Verdopplungsrate von 107 min. Die gewählten Konzentrationen von DTT und TM zeigten für beide Stämme einen wachstumshemmenden Effekt der eine Stunde nach Applikation unabhängig vom Medium eintrat. Quantitative Lipidomanalysen der gestressten und ungestressten Zellen zeigten, dass bei kurzzeitiger Behandlung (1 Stunde) sowohl DTT als auch TM in Fall des synthetisch-definiertem Medium nur einen vernachlässigbaren Effekt auf die Lipidkomposition von WT und ire1∆ Zellen hatten. Im Fall des reichen und komplexen Mediums hingegen, kam es in mit DTT gestressten Zellen zu einer signifikanten Akkumulation von Phosphatidat (PA) gegenüber der ungestressten Kontrolle. Der Vergleich der Lipidkomposition zeigte wie schon im Fall der Wachstumsrate, dass die größten Unterschiede zwischen den Medien auftraten. Hier wurden signifikante Änderungen in der Lipidklasse von Ergosterol, den komplexen Sphingolipiden, den Phosphoglyceriden Phosphatidylethanolamin und Phosphatidylinositol sowie den Speicherlipiden Triacylglyceriden u

Adaptation of Hansenula polymorpha to methanol: a transcriptome analysis

Background: Methylotrophic yeast species (e.g. Hansenula polymorpha, Pichia pastoris) can grow on methanol as sole source of carbon and energy. These organisms are important cell factories for the production of recombinant proteins, but are also used in fundamental research as model organisms to study peroxisome biology. During exponential growth on glucose, cells of H. polymorpha typically contain a single, small peroxisome that is redundant for growth while on methanol multiple, enlarged peroxisomes are present. These organelles are crucial to support growth on methanol, as they contain key enzymes of methanol metabolism. In this study, changes in the transcriptional profiles during adaptation of H. polymorpha cells from glucose- to methanol-containing media were investigated using DNA-microarray analyses. Results: Two hours after the shift of cells from glucose to methanol nearly 20% (1184 genes) of the approximately 6000 annotated H. polymorpha genes were significantly upregulated with at least a two-fold differential expression. Highest upregulation (> 300-fold) was observed for the genes encoding the transcription factor Mpp1 and formate dehydrogenase, an enzyme of the methanol dissimilation pathway. Upregulated genes also included genes encoding other enzymes of methanol metabolism as well as of peroxisomal b-oxidation. A moderate increase in transcriptional levels (up to 4-fold) was observed for several PEX genes, which are involved in peroxisome biogenesis. Only PEX11 and PEX32 were higher upregulated. In addition, an increase was observed in expression of the several ATG genes, which encode proteins involved in autophagy and autophagy processes. The strongest upregulation was observed for ATG8 and ATG11. Approximately 20% (1246 genes) of the genes were downregulated. These included glycolytic genes as well as genes involved in transcription and translation. Conclusion: Transcriptional profiling of H. polymorpha cells shifted from glucose to methanol showed the expected downregulation of glycolytic genes together with upregulation of the methanol utilisation pathway. This serves as a confirmation and validation of the array data obtained. Consistent with this, also various PEX genes were upregulated. The strong upregulation of ATG genes is possibly due to induction of autophagy processes related to remodeling of the cell architecture required to support growth on methanol. These processes may also be responsible for the enhanced peroxisomal b oxidation, as autophagy leads to recycling of membrane lipids. The prominent downregulation of transcription and translation may be explained by the reduced growth rate on methanol (td glucose 1 h vs td methanol 4.5 h).Applied Science

Cardiolipin Is Required For Optimal Acetyl-Coa Metabolism

The phospholipid cardiolipin (CL) is crucial for many cellular functions and signaling pathways, both inside and outside of mitochondria. My thesis focuses on the role of CL in energy metabolism. Many reactions of electron transport and oxidative phosphorylation, the transport of metabolites needed for these processes, and the stabilization of electron transport chain supercomplexes, require CL. Recent studies indicate that CL is required for the synthesis of iron-sulfur (Fe-S) co-factors, which are essential for numerous metabolic pathways. Activation of carnitine-acetylcarnitine translocase, which transports acetyl-CoA into the mitochondria, is CL dependent. The presence of substantial amounts of CL in the peroxisomal membrane suggests that CL may be important for peroxisomal functions. Understanding the role of CL in energy metabolism may identify physiological modifiers that exacerbate the loss of CL and underlie the variation in symptoms observed in Barth syndrome, a genetic disorder of CL metabolism. In order to identify biochemical pathways exacerbated by the loss of CL, I carried out a Synthetic Genetic Array (SGA) screen of the yeast CL mutant crd1Δ. The results indicated that crd1Δ is synthetically lethal with mutants in pyruvate dehydrogenase (PDH), which catalyzes the conversion of pyruvate to acetyl-CoA. Previous studies have shown that synthesis of acetyl-CoA depends primarily on pyruvate conversion in the mitochondria and the cytosol. The crd1Δ mutant exhibited decreased acetyl-CoA levels and decreased growth on acetate as a sole carbon source. Gene expression and protein levels of PDH were increased, but PDH specific activity remained unaltered. These findings suggest that defective ability to convert acetate to acetyl-CoA and possibly decreased enzymatic activity of PDH may account for perturbed acetyl-CoA synthesis in CL-deficient cells. Consistent with a requirement for CL in acetyl-CoA synthesis, perturbation of CL synthesis leads to decreased activity of carnitine-acetylcarnitine translocase, a transporter found in the mitochondrial membrane specific for import of acetylcarnitine into the mitochondria. Growth of crd1Δ at elevated temperature and on acetate medium is restored by supplementation of carnitine, acetylcarnitine, or oleate. Interestingly, synthetic lethality was observed between crd1Δ and mutants in the glyoxylate cycle, suggesting that this cycle is essential to replenish TCA cycle intermediates in CL-deficient cells. The studies described in this thesis are the first to demonstrate that CL is required for synthesis and transport of acetyl-CoA. To obtain an understanding of tafazzin function, an SGA screen was carried out to identify mutants that synthetically interact with taz1Δ. Interesting interactions were observed with phospholipase B, ornithine carbamoyltransferase, and genes required for mitochondrial iron homeostasis and vacuolar protein sorting. These findings suggest that tafazzin may be involved in cellular processes other than CL remodeling

Evaluation of precursor and cofactor engineering strategies influencing fatty acid metabolism in Saccharomyces cerevisiae

If humanity is to reduce the rate of climate change, it is essential that our societies switch to a more sustainable production of fuels and chemicals, which in turn depends on technological development. Oleochemical production via microbial catalysts – such as the yeast Saccharomyces cerevisiae – can use a considerably broader range of renewable substrates compared to the conventional production processes. Additionally, it enables optimization of the catalytical properties of the chosen host via metabolic engineering. Oleochemicals are derived from fatty acids (FAs), whose biosynthesis depends on the conversion of a substrate to cytosolic acetyl-CoA – the precursor for FA synthesis. FA synthesis additionally requires large amounts of the reducing cofactor NADPH. The primary aim of this thesis was to develop and evaluate metabolic engineering strategies with potential to positively influence FA production of S. cerevisiae, mediated via an increased supply of acetyl-CoA or NADPH.\ua0\ua0\ua0\ua0 A major portion of the thesis is focused around a heterologous metabolic pathway to produce acetyl-CoA based on the activity of a phosphoketolase (XFPK) and a phosphotransacetylase (PTA). This pathway theoretically allows to reduce carbon and energy losses compared to the native yeast system. We identified several efficient XFPK candidates with potential to generate a high flux through the pathway. Furthermore, we show that two endogenous proteins – Gpp1 and Gpp2 – efficiently degrade the XFPK-formed produced acetyl-phosphate (AcP) to acetate, accumulating during cultivation. We show that this limits the benefit of the heterologous pathway, likely due to increased proton decoupling and ATP consumption during acetate activation. When we co-expressed XFPK and PTA, deletion of GPP1 appeared to be required to enable a significant flux towards acetyl-CoA during growth on glucose, reducing acetate accumulation. While a 25% increase in FA production was observed at the end of the glucose phase, the final titer was reduced by 20% compared to the control. We suggest that PTA expression negatively affects FA production during ethanol consumption due to low level of AcP during such conditions, leading to net flux from acetyl-CoA to AcP. Therefore, we propose that ethanol formation should be avoided in order to optimize XFPK/PTA use.\ua0\ua0\ua0\ua0 Regarding cofactor supply, we investigated if increasing activity of Stb5 – a transcriptional activator of genes involved in the pentose phosphate pathway (PPP) and NADPH production – could influence FA synthesis positively. STB5 overexpression had a beneficial effect on FA production in the glucose phase, an effect shown to be independent of flux through the PPP. However, final titers were affected negatively, and transcriptomic analysis indicates that mechanisms were activated in cells to counteract a Stb5-imposed redox imbalance. This suggests that an effective drain of NADPH – e.g. during product formation – is required to prevent systemic negative effects of STB5 overexpression.\ua0\ua0\ua0\ua0 The results produced within the scope of this thesis will serve as an aid in future metabolic engineering strategies targeting compounds relying on acetyl-CoA or NADPH

Transcriptional profiling and Functional studies of Zinc cluster transcription factors in Candida albicans

The yeast Candida albicans is a commensal member of the gastrointestinal and urogenital tracts of most healthy humans. However, its capacity to function as an opportunistic pathogen allows it to cause systematic infections of immunocompromised individuals. Over the past two decades, the C. albicans zinc cluster transcription factor family (ZCFs) has been a fascinating subject of research – with studies identifying their roles in virulence, morphogenesis, biofilm formation, drug resistance and many other cellular processes. An understanding of these ZCFs may reveal new targets for therapeutic strategies. My work focused on generating genome-wide transcriptional profiling for a large subset of 35 ZCF gain-of-function mutants (GOF) to elucidate the transcriptional profiles among the ZCFs, and on investigating in depth the function of some specific ZCFs in the fungal pathogen. Transcriptional profiling revealed the target genes that are activated by the ZCF-GOF mutants and provided insight into the underlying roles of the factors. My study focused on establishing the transcriptional regulatory relationship among the ZCFs and understanding the function of some uncharacterized ZCFs. In chapter 2, I selected a set of 35 mostly uncharacterized ZCF, or little is known about them to explore their function using RNA-based transcriptional profiling in collaboration with professor M. Hallett lab. The network approach often shows a specific ZCF-GOF caused activation of expression of other ZCFs, which highlights the extensive interactions among ZCFs. We suggest that most expression changes can be the result of downstream longer-term adaptive responses that induce the expression of intermediate transcription factors. In chapter 3, I characterized a new element involved in hyphal development regulation as a previously unstudied Candida-specific ZCF encoded by CaORF19.1604 that I named Rha1 (Regulator of Hyphal Activity). I identified Rha1 through screening a ZCF-GOF library and noting the Rha1-GOF strain was in a filamentous form under yeast growth conditions. I have characterized Rha1 inactivation mutants and GOF alleles, and I explored the Rha1 regulatory network involving Brg1 and Ume6, which are upregulated hyphal activators that appeared in the Rha1-GOF profile to show that Rha1 affects hyphal gene expression and upregulates Brg1/Ume6 and downregulates Nrg1. In chapter 4, I investigated the role of ZCF4 in cell wall biogenesis, filamentation, biofilm formation, and drug resistance. I explored the ZCF4 function after noting its upregulation in most of the activated ZCF profiles like Rha1-GOF. Zcf4-GOF showed a severe filamentation defect on serum-based medium but exhibited normal filamentation under other cues. I have shown that ZCF4-influenced filamentation is nutrient dependent. In chapter 5, I showed the robust ability of C. albicans to use proline as a carbon and nitrogen source by describing CaPut3 as a proline catabolism regulator. The functional studies demonstrated Put3 has a conserved role in regulating proline catabolism in C. albicans and Saccharomyces cerevisiae, but CaPut3 initiates the degradation of proline even in the presence of a rich nitrogen source such as ammonium sulphate. Collectively, this study established a framework of functional study TFs and generated robust transcriptional data from an activated set of 35 ZCFs to help understand the biology of C. albicans, an important human pathogen