Lehrstuhl Technische Biochemie
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Item Signalverstärkende Methoden der Raman-Mikrospektroskopie für die Analytik niedermolekularer Naturstoffe(2022) Ebersbach, Paul; Kayser, Oliver; Franzke, JoachimItem Opportunities and limits of ozonation as pre-treatment method for production waste water from pharmaceutical manufacturing(2022) Daoud, Fares; Kayser, Oliver; Wintgens, ThomasDuring production of active pharmaceutical ingredients, process waste water is generated at several stages of manufacturing. Whenever possible, waste water will be processed by conventional waste water treatment plants. However, due to low biodegradability of pharmaceuticals or toxicity towards aquatic organisms, some waste water streams must be processed differently. For those, incineration constitutes the current method of choice. A main disadvantage of incineration is high consumption of primary energy sources leading to substantial emission of carbon dioxide (CO2). Thus, ozone treatment followed by biological waste water treatment was tested as an alternative method. In 2009, preliminary laboratory experiments were conducted to evaluate the elimination of DTPA in process waste water. Based on the initial results, the responsible authorities granted approval for a large-scale ozonation. Additional experiments at laboratory scale were performed with different waste water streams to assess the elimination of the target compound and the generation of its main transformation products. These were determined by high performance liquid chromatography – high-resolution mass spectrometry (HPLC-HRMS). All waste streams originated from production facilities of Bayer Corp. in Wuppertal. Four waste water streams from the ciprofloxacin, moxifloxacin, rivaroxaban and DTPA production have been investigated. The obtained results demonstrated that the concentration of moxifloxacin and its metabolites can be effectively reduced (> 99.7%) prior entering the receiving water. Applying the same ozonation time the concentration of ciprofloxacin and its metabolites remained too high for safe discharge. The concentration of rivaroxaban in the ozonated waste water was effectively lowered under acidic conditions. DTPA concentration was reduced to levels assuring safe discharge into the receiving water. Additionally, the ecotoxicity of the ozonated waste water for all target compounds was investigated using three trophic levels. A comparison of CO2 emissions showed that ozonation in some cases is an ecological alternative to incineration.Item Entwicklung eines mikrofluidischen Systems zur kontinuierlichen elektrophoretischen Auftrennung und Analyse von Enzymreaktionen(2021) Jender, Matthias; Nett, Markus; Franzke, JoachimThis thesis describes the results of a research project as part of the Leibniz Research Cluster (LRC). The aim of this thesis was the development of a microfluidic free-flow electrophoresis (μFFE) separation unit for the integration into a microfluidic multistage reactor. The research of the LRC project was driven by the vision of a continuous, cell-free and enzyme-catalysed synthesis of polyketide building blocks for the development of bioactive substances. The role of the μFFE chips developed in this work was to continuously separate mixtures of substances into their single components. In the course of this work two different microfluidic FFE chips were developed: one based on poly(dimethylsiloxan) (PDMS) and another one based on poly(methyl metacrylate) (PMMA). The initially manufactured PDMS chips were soon replaced by PMMA chips, which allowed for more reproducible separations and long-term stable usage. With the functional FFE chips, methods for the separation of the substances being part of the enzymatic reactions were developed and optimised. With ammonium acetate in particular concentrations a background electrolyte was found, which enabled a good separation with μFFE and at the same time was compatible with electrospray ionisation mass spectrometry (ESI-MS), the conceived tool for control analytics. After initially analysing samples from the FFE chip with capillary electrophoresis, the focus was put on online analysis via ESI-MS. The coupling of μFFE with MS using a switching valve made an online monitoring of the FFE separation at multiple μFFE outlets possible. With the development of a novel ESI-multi-emitter, the speed of analysis and the number of analysed μFFE outlets were further increased. Overall, the initially defined goals of this project were reached largely. However, for the seamless integration of the developed μFFE system into the conceived multistage reactor, further optimisation and specific adaption to the other involved systems would be necessary.Item Analyse mikrobiologischer Proben auf Basis der Ionenmobilitätsspektrometrie(2021) Drees, Carolin; Kayser, Oliver; Franzke, JoachimDie vorliegende Dissertation konzentriert sich zum einen auf experimentelle Anwendungen der Ionenmobilitätsspektrometrie im Bereich der Mikrobiologie und zum anderen auf gezielte instrumentelle Entwicklungen, um die Akzeptanz der Methode zu verbessern. Zunächst konnte gezeigt werden, dass durch Headspace-Analysen unterschiedlicher Bakterienkulturen die Identifizierung verschiedener Spezies und die Überwachung deren bakteriellen Wachstums möglich war. Aufbauend auf diese Arbeiten wurde als neue Ionisierungsmethode das Flexible Microtube Plasma vorgestellt, um damit die üblichen radioaktiven β-Strahlungsquellen mit ihren regulatorischen Einschränkungen zu ersetzen. Nach gezielten Optimierungsschritten konnte Helium durch Stickstoff als Arbeitsgas ersetzt werden ohne Sensitivität einzubüßen. Hierzu wurde unter anderem eine Ionisierungskammer mittels 3D-Druck gefertigt. Darauf aufbauend wurde im Folgenden erstmals ein vollständig, mittels 3D-Druck Verfahren gefertigtes Ionenmobilitätsspektrometer vorgestellt und dessen Performance mit einer Referenz aus PTFE verglichen. Veränderte Materialeigenschaften machten es zunächst notwendig weitere Designoptimierungen durchzuführen. Letztendlich wurde das optimierte, vollständig 3D gedruckte Ionenmobilitätsspektrometer der Referenz qualitativ angeglichen. Somit konnte gezeigt werden, dass sich der 3D-Druck als alternatives Herstellungsverfahren für Ionenmobilitätsspektrometer eignet. Für die Analyse flüssiger Proben wurde abschließend ein miniaturisierter Thermodesorptionschip vorgestellt. Dieser Chip ermöglichte erstmals in Kombination mit dem Flexible Microtube Plasma nicht-flüchtige Stoffe, wie den Autoinducer N-hexanoyl-L Homoserinlacton, direkt aus einer Lösung heraus zu adsorbieren, anschließend zu thermodesorbieren und mittels Ionenmobilitätsspektrometer nachzuweisen.Item In vitro production and exudation of 20-hydroxymaytenin from Gymnosporia heterophylla (Eckl. and Zeyh.) Loes. cell culture(2021-07-21) Pitakbut, Thanet; Spiteller, Michael; Kayser, OliverThe metabolite 20-Hydroxymaytenin (20-HM) is a member of the quinone-methide pentacyclic triterpenoids (QMTs) group. This metabolite group is present only in Celastraceae plants, and it has shown various biological activities from antioxidant to anticancer properties. However, most QMTs metabolites including 20-HM cannot be synthesized in a laboratory. Therefore, we optimized a plant tissue culture protocol and examined the potential of Gymnosporia heterophylla (synonym. Maytenus heterophylla) to produce 20-HM in an in vitro experiment. For the first time, we reported the optimum callus induction medium with a high percentage success rate of 82% from the combination of 1 mg/L indole-3-butyric acid and 5 mg/L naphthalene acetic acid. Later, our cell suspension culture cultivated in the optimum medium provided approximately 0.35 mg/g fresh weight of 20-HM. This concentration is roughly 87.5 times higher than a concentration of 20-HM presenting in Elaeodendron croceum (Celastraceae) leaves. In addition, we also found that 20-HM presented in a cultivation medium, suggesting that G. heterophylla cells secreted 20-HM as an exudate in our experiment. Noticeably, 20-HM was missing when Penicillium cf. olsonii occurred in the medium. These findings hint at an antifungal property of 20-HM.Item Bioengineering studies and pathway modeling of the heterologous biosynthesis of tetrahydrocannabinolic acid in yeast(2020-10-12) Thomas, Fabian; Schmidt, Christina; Kayser, OliverHeterologous biosynthesis of tetrahydrocannabinolic acid (THCA) in yeast is a biotechnological process in Natural Product Biotechnology that was recently introduced. Based on heterologous genes from Cannabis sativa and Streptomyces spp. cloned into Saccharomyces cerevisiae, the heterologous biosynthesis was fully embedded as a proof of concept. Low titer and insufficient biocatalytic rate of most enzymes require systematic optimization of recombinant catalyst by protein engineering and consequent C-flux improvement of the yeast chassis for sufficient precursor (acetyl-CoA), energy (ATP), and NADH delivery. In this review basic principles of in silico analysis of anabolic pathways towards olivetolic acid (OA) and cannabigerolic acid (CBGA) are elucidated and discussed to identify metabolic bottlenecks. Based on own experimental results, yeasts are discussed as potential platform organisms to be introduced as potential cannabinoid biofactories. Especially feeding strategies and limitations in the committed mevalonate and olivetolic acid pathways are in focus of in silico and experimental studies to validate the scientific and commercial potential as a realistic alternative to the plant Cannabis sativa.Item Molecular biological elucidation of late tropane alkaloid biosynthesis(2019) Kohnen-Johannsen, Kathrin Laura; Kayser, Oliver; Sickmann, AlbertTropane alkaloids (TA) are important and valuable secondary plant metabolites which occur in the families of Solanaceae, Erythroxylaceae, as well as Convolvulaceae, Moraceae, and Brassicaceae. These plant families produce the TAs hyoscyamine and scopolamine, cocaine, or calystegines, respectively. Due to the medicinal and pharmacological properties of hyoscyamine, scopolamine and cocaine, TA containing plants have been used for thousands of years. Even today, many drugs are derived from TAs and are used in the treatment of various diseases. Of the TAs, scopolamine is the most in demand due to its pharmacological effects and legal status. The market supply of scopolamine is still met by conventional field cultivation of Duboisia hybrids. Climate change is resulting in less-stable growth conditions for traditional farming, which limits the reliability of agricultural yields and has warranted investigation of alternative TA production processes. One proposed approach is to transfer biosynthesis into biotechnological host which would enable TA production in containable and scalable fermentative infrastructure using low-cost compounds like tropine and phenyllactic acid. All TA production approaches require profound knowledge of the biochemical pathways. To date, these pathways have not been fully elucidated, which limits their greater biotechnological application. This thesis seeks to increase the fundamental understanding of the molecular processes of the late stages of TA biosynthesis. This thesis deals with the TA biosynthesis in Duboisia plants which is highly specialized and specifically localized. The spatial distribution of TA in planta, the expression of genes involved in the biosynthesis and the TA pattern during plant development are investigated and described. Moreover, it presents investigations towards identification of the littorine synthase, a currently unknown key enzyme in TA biosynthesis. The results of this research add to our understanding of TA biosynthesis and provides important insights for the future development of alternative bio-production processes.Item Characterizing mitochondrial signaling networks by quantitative proteomics(2020) Gonczarowska Jorge, Humberto; Sickmann, Albert; Kayser, OliverDie Mitochondrien der Hefe besitzen über 1000 Proteine, von denen mehr als 99% durch nukleäre DNA und nur der Rest durch mitochondriale DNA kodiert wird. Um das Mitochondrium erreichen zu können, ist eine effiziente Protein-Zell-Signalweiterleitung notwendig, um die Moleküle nicht nur an das Organell selbst, sondern auch an eines der vier Subkompartimente des Mitochondriums korrekt zu adressieren. Mitochondriale Protein-Fehlzuordnung ist mit verschiedenen Krankheiten wie Alzheimer und Parkinson assoziiert. Um den Proteintransport in die Mitochondrien besser zu verstehen, wurde ein wichtiger Komplex namens Mitochondrial Processing Peptidase (MPP) gestört, um die proteomische Dynamik unter diesen Bedingungen zu untersuchen. Die MPP ist notwendig, um Proteine fertig zu stellen, die nukleär codiert sind und die innere Membran oder die Matrix der Mitochondrien als Ziel haben. Mit einem funktional eingeschränktem MPP war es möglich, Proteine, die diesem Weg folgen, sicher zuzuordnen und die Lokalisierung dieser Proteine in einem Subkompartiment zu bestimmen. Da auch Phosphorylierungen eine wichtige Rolle bei der zellulären Signalübertragung spielen, wurde das Phosphoproteom untersucht, was mögliche Phosphorylierungsstellen, die am zellulären Wachstum von Hefe beteiligt sind, offenbarte. Mit dem Ziel die (Phospho-)Proteomabdeckung der mitochondrialen Proben [HG-1] zu erhöhen, wurde eine neue Methode der Bottom-up Proteomik entwickelt, da die klassische Methode durch Lücken in der Proteomabdeckung die Möglichkeit reduzierte, dynamische biologische Systeme zu verstehen. Subtilisin wurde aufgrund seiner breiten Schnittstellen-Spezifität als proteolytisches Enzym ausgewählt, was die Wahrscheinlichkeit erhöhte, verborgene Bereiche des (Phospho-)Proteoms zu enthüllen. In der Tat war es mit Subtilisin möglich, die Proteomabdeckung zu erhöhen und Phosphorylierungsstellen zu erreichen, die noch nicht mit herkömmlichen proteolytischen Enzymen abgedeckt waren. Subtilisin wurde weiter untersucht und die Kompatibilität mit Label-Techniken (TMT, iTRAQ und label-free) und In-depth Proteomik-Untersuchungen wurde bestätigt, wobei Subtilisin sich als ein leistungsfähiges Werkzeug erwies, um dynamische Systeme in Mitochondrien und zelluläre Signalweiterleitung besser zu verstehen.Item Nicotine‐free, nontransgenic tobacco (Nicotiana tabacum L.) edited by CRISPR‐Cas9(2019-06-17) Schachtsiek, Julia; Stehle, FelixItem Establishment and development of methods for genetic modifications in plants and their effect on the metabolism(2019) Schachtsiek, Julia; Kayser, Oliver; Schwab, WilfriedItem Virus-induced gene silencing (VIGS) in Cannabis sativa L.(2019-12-26) Schachtsiek, Julia; Hussain, Tajammul; Azzouhri, Khadija; Kayser, Oliver; Stehle, FelixBackground:The raised demand of cannabis as a medicinal plant in recent years led to an increased interest in understanding the biosynthetic routes of cannabis metabolites. Since there is no established protocol to generate stable gene knockouts in cannabis, the use of a virus-induced gene silencing (VIGS) method, resulting in a gene knockdown, to study gene functions is desirable.Results:For this, a computational approach was employed to analyze the Cannabis sativa L. transcriptomic and genomic resources. Reporter genes expected to give rise to easily scorable phenotypes upon silencing, i.e. the phy-toene desaturase (PDS) and magnesium chelatase subunit I (ChlI), were identified in C. sativa. Subsequently, the targets of specific small interfering RNAs (siRNAs) and silencing fragments were predicted and tested in a post-transcriptional gene silencing (PTGS) approach. Here we show for the first time a gene knockdown in C. sativa using the Cotton leaf crumple virus (CLCrV) in a silencing vector system. Plants transiently transformed with the Agrobacterium tumefaciensstrain AGL1, carrying the VIGS-vectors, showed the desired phenotypes, spotted bleaching of the leaves. The success-ful knockdown of the genes was additionally validated by quantitative PCR resulting in reduced expression of tran-scripts from 70 to 73% for ChlI and PDS, respectively. This is accompanied with the reduction of the chlorophyll a and carotenoid content, respectively. In summary, the data clearly demonstrate the potential for functional gene studies in cannabis using the CLCrV-based vector system.Conclusions:The applied VIGS-method can be used for reverse genetic studies in C. sativa to identify unknown gene functions. This will gain deeper inside into unknown biosynthetic routes and will help to close the gap between avail-able genomic data and biochemical information of this important medicinal plant.Item The phytochemical and biological investigation of Jatropha pelargoniifolia root native to the Kingdom of Saudi Arabia(2018-07-28) Aati, Hanan; Kayser, Oliver; El-Gamal, Ali; Ahmed, Atallah F.Extensive phytochemical analysis of different root fractions of Jatropha pelargoniifolia Courb. (Euphorbiaceae) has resulted in the isolation and identification of 22 secondary metabolites. 6-hydroxy-8-methoxycoumarin-7-O-β-d-glycopyranoside (15) and 2-hydroxymethyl N-methyltryptamine (18) were isolated and identified as new compounds along with the known diterpenoid (1, 3, 4, and 7), triterpenoid (2 and 6), flavonoid (5, 11, 13, 14, and 16), coumarinolignan (8–10), coumarin (15), pyrimidine (12), indole (17, 18), and tyramine-derived molecules (19–22). The anti-inflammatory, analgesic, and antipyretic activities were evaluated for fifteen of the adequately available isolated compounds (1–6, 8–11, 13, 14, 16, 21, and 22). Seven (4, 6, 10, 5, 13, 16, and 22) of the tested compounds showed a significant analgesic effect ranging from 40% to 80% at 10 mg/kg in two in vivo models. Compound 1 could also prove its analgesic property (67.21%) when it was evaluated on a third in vivo model at the same dose. The in vitro anti-inflammatory activity was also recorded where all compounds showed the ability to scavenge nitric oxide (NO) radical in a dose-dependent manner. However, eight compounds (1, 4, 5, 6, 10, 13, 16, and 22) out of the fifteen tested compounds exhibited considerable in vivo anti-inflammatory activity which reached 64.91% for compound 10 at a dose of 10 mg/kg. Moreover, the tested compounds exhibited an antipyretic effect in a yeast-induced hyperthermia in mice. The activity was found to be highly pronounced with compounds 1, 5, 6, 10, 13, and 16 which decreased the rectal temperature to about 37 °C after 2 h of the induced hyperthermia (~39 °C) at a dose of 10 mg/kg. This study could provide scientific evidence for the traditional use of J. pelargoniifolia as an anti-inflammatory, analgesic, and antipyretic.Item Dataset on nicotine-free, nontransgenic tobacco (Nicotiana tabacum l.) edited by CRISPR-Cas9(2019-08-23) Schachtsiek, Julia; Stehle, FelixThis dataset in brief is related to the research letter entitled "Nicotine-free, nontransgenic tobacco (Nicotiana tabacuml.) edited by CRISPR-Cas9" [1]. Cured tobacco products with a significantly reduced nicotine content helps people to overcome their nicotine addiction. Here we summarize additional data and method descriptions of the generation process of a nicotine-free, nontransgenic tobacco plant. This included the cloning, transformation and regeneration of transgenic tobacco plants, followed by the analysis of the nicotine content and genomic modifications caused by CRISPR-Cas9 mediated gene editing. Subsequently, nicotine-free plants were screened for loss of T-DNA cassette, i.e. nontransgenity. Finally, a metabolic footprint was recorded by 1H NMR analysis.Item Tropane alkaloids: chemistry, pharmacology, biosynthesis and production(2019-02-22) Kohnen-Johannsen, Kathrin Laura; Kayser, OliverTropane alkaloids (TA) are valuable secondary plant metabolites which are mostly found in high concentrations in the Solanaceae and Erythroxylaceae families. The TAs, which are characterized by their unique bicyclic tropane ring system, can be divided into three major groups: hyoscyamine and scopolamine, cocaine and calystegines. Although all TAs have the same basic structure, they differ immensely in their biological, chemical and pharmacological properties. Scopolamine, also known as hyoscine, has the largest legitimate market as a pharmacological agent due to its treatment of nausea, vomiting, motion sickness, as well as smooth muscle spasms while cocaine is the 2nd most frequently consumed illicit drug globally. This review provides a comprehensive overview of TAs, highlighting their structural diversity, use in pharmaceutical therapy from both historical and modern perspectives, natural biosynthesis in planta and emerging production possibilities using tissue culture and microbial biosynthesis of these compounds.Item Identification of putative precursor genes for the biosynthesis of cannabinoid-like compound in Radula marginata(2018-05-09) Hussain, Tajammul; Kayser, Oliver; Plunkett, Blue; Ejaz, Mahwish; Espley, Richard, V.The liverwort Radula marginata belongs to the bryophyte division of land plants and is a prospective alternate source of cannabinoid-like compounds. However, mechanistic insights into the molecular pathways directing the synthesis of these cannabinoid-like compounds have been hindered due to the lack of genetic information. This prompted us to deep sequencing, de novo assembly and annotation of R. marginata transcriptome, which resulted in the identification and validation of the genes for cannabinoid biosynthetic pathway. In total, we have identified 11,421 putative genes encoding 1554 enzymes from 145 biosynthetic pathways. Interestingly, we have identified all the upstream genes of the central precursor of cannabinoid biosynthesis, cannabigerolic acid (CBGA), including its two first intermediates, stilbene acid (SA) and geranyl diphosphate (GPP). Expression of all these genes was validated using quantitative real-time PCR. We have characterized the protein structure of stilbene synthase (STS), which was identified as a homologue of olivetolic acid in R. marginata. Moreover, the metabolomics approach enabled us to identify CBGA-analogous compounds using electrospray ionization mass spectrometry (ESI-MS/MS) and gas chromatography mass spectrometry (GC-MS). Transcriptomic analysis revealed 1085 transcription factors (TF) from 39 families. Comparative analysis showed that six TF families have been uniquely predicted in R. marginata. In addition, the bioinformatics analysis predicted a large number of simple sequence repeats (SSRs) and non-coding RNAs (ncRNAs). Our results collectively provide mechanistic insights into the putative precursor genes for the biosynthesis of cannabinoid-like compounds and a novel transcriptomic resource for Radula marginata. The large-scale transcriptomic resource generated in this study can further serve as the reference transcriptome to explore the Radulaceae family.Item Phytochemical and biological investigation of Jatropha pelargoniifolia roots native to the Kingdom of Saudi Arabia(2018) Aati, Hanan Yahya; Kayser, Oliver; Sickman, AlbertItem Evaluation of C-prenylating enzymes for the heterologous biosynthesis of cannabigerolic acid(2018) Degenhardt, Sara Friederike; Kayser, Oliver; Warzecha, HeribertAromatic prenyltransferases catalyze the prenylation of an electron-rich aromatic compound by a prenyl diphosphate by transfer a prenyl residue to carbon, nitrogen or an oxygen atom of the aromatic acceptor molecule. The bioactivity of the resulting products is often increased, compared to the non-prenylated substrates. Prenylation of aromatic compounds is important for the diversification of secondary metabolites like phytocannabinoids. The membrane-bound cannabigerolic acid synthase from Cannabis sativa catalyzes the prenylation of olivetolic acid (OA) with geranyl diphosphate (GPP) to form cannabigerolic acid (CBGA). CBGA is the central precursor of the main pharmacologically active compounds of C. sativa: Δ9-tetrahydrocannabinol (THC) and cannabidiol (CBD). A biotechnological production using heterologous yeast presents a promising alternative to meet the increasing pharmaceutical demand and interest in these cannabinoids. In this regard, this study aimed for the identification of potential aromatic prenyltransferases able to produce CBGA and their heterologous expression. Three different membrane-bound aromatic prenyltransferases, isolated from C. sativa, were expressed in Saccharomyces cerevisiae, but were not able to produce CBGA. Furthermore, the soluble aromatic prenyltransferase NphB, first isolated from Streptomyces sp. strain CL190, was investigated as an alternative. Beside the desired product CBGA, NphB forms a major side-product, 2-O-geranyl olivetolic acid. Therefore, NphB variants were tested in a low-throughput screening system in order to improve the product specificity towards CBGA and to enable higher specific activities. Additionally, the simultaneous functional expression of nphB and the final enzyme of the cannabinoid pathway, tetrahydrocannabinolic acid synthase (THCAS), in S. cerevisiae is presented. This study presented for the first time that the soluble aromatic prenyltransferase NphB produces CBGA from precursors OA and GPP. The obtained results therefore promote the generation of a cannabinoid producing yeast for a biotechnological approach in order to face the increasing demand of the phytocannabinoids CBD and THC in the medical sector.Item Emerging Trends in Natural Product Biotechnology(2018-09-20)Item Recombinant expression and functional characterization of cannabinoid producing enzymes in komagataella phaffii(2018) Zirpel, Bastian; Kayser, Oliver; Kley, Ida van derCannabinoids are secondary natural products predominantly found in the oil compartments of trichomes of the plant Cannabis sativa L. Up to now more than 100 cannabinoids have been isolated, the most prominent being the psychoactive Δ9-tetrahydrocannabinol (THC) and cannabidiol (CBD). As THC-derived products are already available for medical use and CBD is currently investigated for its application in several disease treatments, the demand of cannabinoid-based products is expected to further rise in the future. A biotechnological production presents a promising alternative to meet the demand, while also offering environmentally friendly, highly controllable and reproducible, GMP compliant processes as well as the reduction of agricultural area. In this regard, the cannabinoid producing enzymes from C. sativa, Δ9-tetrahydrocannabinolic acid synthase (THCAS) and cannabidiolic acid synthase (CBDAS), were recombinantly produced in the yeast Komagataella phaffii. The expression of thcas was enhanced in K. phaffii by optimization of cultivation conditions as well as identification and co-expression of helper protein genes. Insights were gained into structure-function relationships of the THCAS and CBDAS. The influence on the enzyme activities of glycosylation sites, of amino acid residues in the berberine-bridge-domain as well as in the active site were investigated. Important amino acid residues for enzyme activity were identified yielding enzyme variants with improved catalytic and stability properties. Additionally, a first approach to establish the late cannabinoid biosynthesis pathway in yeasts is presented. Production of the soluble prenyltransferase NphB from Streptomyces sp. strain CL190, which enables the replacement of the native transmembrane prenyltransferase cannabigerolic acid synthase from C. sativa, was simultaneously produced with THCAS, thereby enabling a production of Δ9-tetrahydrocannabinolic acid from precursors olivetolic acid and geranyl diphosphate. The studies presented here contribute to increased fundamental and industrially relevant knowledge and will help promoting a biotechnological cannabinoid production in yeasts.Item Identification, isolation and functional characterization of prenyltransferases in Cannabis sativa L.(2016) Pamplaniyil, Kathleen; Kayser, Oliver; Wessjohann, LudgerPrenylierte Substanzen tragen zur Vielfalt der Sekundärmetabolite bei. Prenylierte Metabolite besitzen entzündungshemmende und antibakterielle Eigenschaften. Die Enzyme, die die Prenylierung durch Bildung einer neuen C-C oder C-O Bindung katalysieren sind Prenyltransferasen. Eines dieser Enzyme ist an der Biosynthese des psychoaktiven Wirkstoffs THC aus Cannabis beteiligt. Olivetolsäure wird mit Geranyldiphosphat bei dieser Reaktion zu Cannabigerolsäure prenyliert, dem Ausgangsstoff von THCA. Das Ziel dieser Arbeit war die Identifikation von Enzymen die potentiell diese Reaktion katalysieren können. Eine in silico Analyse war der erste Schritt, um neue Mitglieder der Familie der aromatischen, membrangebundenen Prenyltransferasen zu finden. Die Suche beinhaltete die Charakterisierung unterschiedlicher Gruppen von Prenyltransferasen bezüglich Unterschiede in ihrer genetischen Sequenz mithilfe von webbasierten Software-Programmen. Die Suche nach neuen Prenyltransferasen in DNA-Bibliotheken wurde durch einen spezifisch dafür entwickelten mathematischen Algorithmus verwirklicht, welcher die verschiedenen Gruppen von Prenyltransferasen anhand des Aufbaus ihrer genetischen Sequenz unterschied. Die Berechnung ergab fünf mögliche neue aromatische Prenyltransferasen. Die evolutionäre Verwandtschaft anhand der genetischen Sequenzen ermöglichte eine Einteilung in verschiedene Gruppen. Anhand der in silico berechneten Ergebnisse wurden die cDNA Sequenzen für in vivo Experimente bestimmt. Wirtsorganismen für in vivo Experimente der möglichen Enzyme waren die Pflanze Nicotiana benthamiana und die Hefe Saccharomyces cerevisiae Y05416. Für die transiente Expression in Tabakpflanzen wurde das innovative Verfahren magnICON® verwendet. Für die heterologe Expression in Hefe wurden zwei Strategien verfolgt: (i) die Klonierung der cDNA Sequenzen mit dem nativen (pflanzeneigenen) Signalpeptid, (ii) die Klonierung der cDNA Sequenzen mit dem Signalpeptid des F1F0 ATP Synthase Enzymkomplexes aus Hefezellen. Eines der möglichen Pflanzenenzyme wurde aktiv in Saccharomyces cerevisiae Y05416 exprimiert. Dieses Ergebnis zeigt, dass das verwendete Expressionssystem für die Expression von aromatischen Prenyltransferasen aus Pflanzen geeignet ist und Hefezellen als Plattformorganismus zur Expression von Pflanzengenen unter bestimmten Bedingungen genutzt werden kann. Die gesamte Arbeit leistet einen Beitrag zur erfolgreichen Expression von Pflanzengenen in Saccharomyces cerevisiae und erweitert die Kenntnisse über die Familie der membran-gebundene Prenyltransferasen. Die Arbeit zeigt ebenfalls die Vielfalt an Klonierungsmethoden und Wirtsorganismen auf.