TY  - THES
A1  - Pramanik, Shreya
T1  - Protein reconstitution in giant vesicles
N2  - Das Leben auf der Erde ist vielfältig und reicht von einzelligen Organismen bis hin zu mehrzelligen Lebewesen wie dem Menschen. Obwohl es Theorien darüber gibt, wie sich diese Organismen entwickelt haben könnten, verstehen wir nur wenig darüber, wie "Leben" aus Molekülen entstanden ist. Die synthetische Bottom-up-Biologie zielt darauf ab, minimale Zellen zu schaffen, indem sie verschiedene Module wie Kompartimentierung, Wachstum, Teilung und zelluläre Kommunikation kombiniert.
Alle lebenden Zellen haben eine Membran, die sie von dem sie umgebenden wässrigen Medium trennt und sie schützt. Darüber hinaus haben alle eukaryotischen Zellen Organellen, die von intrazellulären Membranen umschlossen sind. Jede Zellmembran besteht hauptsächlich aus einer Lipiddoppelschicht mit Membranproteinen. Lipide sind amphiphile Moleküle, die molekulare Doppelschichten aus zwei Lipid-Monoschichten oder Blättchen bilden. Die hydrophoben Ketten der Lipide sind einander zugewandt, während ihre hydrophilen Kopfgruppen die Grenzflächen zur wässrigen Umgebung bilden. Riesenvesikel sind Modellmembransysteme, die Kompartimente mit einer Größe von mehreren Mikrometern bilden und von einer einzigen Lipiddoppelschicht umgeben sind. Die Größe der Riesenvesikel ist mit der Größe von Zellen vergleichbar und macht sie zu guten Membranmodellen, die mit einem Lichtmikroskop untersucht werden können. Allerdings fehlen den Riesenvesikelmembranen nach der ersten Präparation Membranproteine, die in weiteren Präparationsschritten in diese Membranen eingebaut werden müssen. Je nach Protein kann es entweder über Ankerlipide an eines der Membranblättchen gebunden oder über seine Transmembrandomänen in die Lipiddoppelschicht eingebaut werden.
Diese Arbeit befasst sich mit der Herstellung von Riesenvesikeln und der Rekonstitution von Proteinen in diesen Vesikeln. Außerdem wird ein mikrofluidischer Chip entworfen, der in verschiedenen Experimenten verwendet werden kann. Die Ergebnisse dieser Arbeit werden anderen Forschern helfen, die Protokolle für die Herstellung von GUVs zu verstehen, Proteine in GUVs zu rekonstituieren und Experimente mit dem mikrofluidischen Chip durchzuführen. Auf diese Weise wird die vorliegende Arbeit für das langfristige Ziel von Nutzen sein, die verschiedenen Module der synthetischen Biologie zu kombinieren, um eine Minimalzelle zu schaffen.
N2  - Life on Earth is diverse and ranges from unicellular organisms to multicellular creatures like humans. Although there are theories about how these organisms might have evolved, we understand little about how ‘life’ started from molecules. Bottom-up synthetic biology aims to create minimal cells by combining different modules, such as compartmentalization, growth, division, and cellular communication.
All living cells have a membrane that separates them from the surrounding aqueous medium and helps to protect them. In addition, all eukaryotic cells have organelles that are enclosed by intracellular membranes. Each cellular membrane is primarily made of a lipid bilayer with membrane proteins. Lipids are amphiphilic molecules that assemble into molecular bilayers consisting of two leaflets. The hydrophobic chains of the lipids in the two leaflets face each other, and their hydrophilic headgroups face the aqueous surroundings. Giant unilamellar vesicles (GUVs) are model membrane systems that form large compartments with a size of many micrometers and enclosed by a single lipid bilayer. The size of GUVs is comparable to the size of cells, making them good membrane models which can be studied using an optical microscope. However, after the initial preparation, GUV membranes lack membrane proteins which have to be reconstituted into these membranes by subsequent preparation steps. Depending on the protein, it can be either attached via anchor lipids to one of the membrane leaflets or inserted into the lipid bilayer via its transmembrane domains.
The first step is to prepare the GUVs and then expose them to an exterior solution with proteins. Various protocols have been developed for the initial preparation of GUVs. For the second step, the GUVs can be exposed to a bulk solution of protein or can be trapped in a microfluidic device and then supplied with the protein solution. To minimize the amount of solution and for more precise measurements, I have designed a microfluidic device that has a main channel, and several dead-end side channels that are perpendicular to the main channel. The GUVs are trapped in the dead-end channels. This design exchanges the solution around the GUVs via diffusion from the main channel, thus shielding the GUVs from the flow within the main channel. This device has a small volume of just 2.5 μL, can be used without a pump and can be combined with a confocal microscope, enabling uninterrupted imaging of the GUVs during the experiments. I used this device for most of the experiments on GUVs that are discussed in this thesis.
In the first project of the thesis, a lipid mixture doped with an anchor lipid was used that can bind to a histidine chain (referred to as His-tag(ged) or 6H) via the metal cation Ni2+. This method is widely used for the biofunctionalization of GUVs by attaching proteins without a transmembrane domain. Fluorescently labeled His-tags which are bound to a membrane can be observed in a confocal microscope. Using the same lipid mixture, I prepared the GUVs with different protocols and investigated the membrane composition of the resulting GUVs by evaluating the amount of fluorescently labeled His-tagged molecules bound to their membranes. I used the microfluidic device described above to expose the outer leaflet of the vesicle to a constant concentration of the His-tagged molecules. Two fluorescent molecules with a His-tag were studied and compared: green fluorescent protein (6H-GFP) and fluorescein isothiocyanate (6H-FITC). Although the quantum yield in solution is similar for both molecules, the brightness of the membrane-bound 6H-GFP is higher than the brightness of the membrane-bound 6H-FITC. The observed difference in the brightness reveals that the fluorescence of the 6H-FITC is quenched by the anchor lipid via the Ni2+ ion. Furthermore, my measurements also showed that the fluorescence intensity of the membranebound His-tagged molecules depends on microenvironmental factors such as pH. For both 6H-GFP and 6H-FITC, the interaction with the membrane is quantified by evaluating the equilibrium dissociation constant. The membrane fluorescence is measured as a function of the fluorophores’ molar concentration. Theoretical analysis of these data leads to the equilibrium dissociation constants of (37.5 ± 7.5) nM for 6H-GFP and (18.5 ± 3.7) nM for 6H-FITC.
The anchor lipid mentioned previously used the metal cation Ni2+ to mediate the bond between the anchor lipid and the His-tag. The Ni2+ ion can be replaced by other transition metal ions. Studies have shown that Co3+ forms the strongest bonds with the His-tags attached to proteins. In these studies, strong oxidizing agents were used to oxidize the Co2+ mediated complex with the His-tagged protein to a Co3+ mediated complex. This procedure puts the proteins at risk of being oxidized as well. In this thesis, the vesicles were first prepared with anchor lipids without any metal cation. The Co3+ was added to these anchor lipids and finally the His-tagged protein was added to the GUVs to form the Co3+ mediated bond. This system was also established using the microfluidic device.
The different preparation procedures of GUVs usually lead to vesicles with a spherical morphology. On the other hand, many cell organelles have a more complex architecture with a non spherical topology. One fascinating example is provided by the endoplasmic reticulum (ER) which is made of a continuous membrane and extends throughout the cell in the form of tubes and sheets. The tubes are connected by three-way junctions and form a tubular network of irregular polygons. The formation and maintenance of these reticular networks requires membrane proteins that hydrolyize guanosine triphosphate (GTP). One of these membrane proteins is atlastin. In this thesis, I reconstituted the atlastin protein in GUV membranes using detergent-assisted reconstitution protocols to insert the proteins directly into lipid bilayers.
This thesis focuses on protein reconstitution by binding His-tagged proteins to anchor lipids and by detergent-assisted insertion of proteins with transmembrane domains. It also provides the design of a microfluidic device that can be used in various experiments, one example is the evaluation of the equilibrium dissociation constant for membrane-protein interactions. The results of this thesis will help other researchers to understand the protocols for preparing GUVs, to reconstitute proteins in GUVs, and to perform experiments using the microfluidic device. This knowledge should be beneficial for the long-term goal of combining the different modules of synthetic biology to make a minimal cell.
KW  - protein reconstitution
KW  - giant vesicles
KW  - microfluidics
KW  - synthetic biology
KW  - Riesenvesikel
KW  - Mikrofluidik
KW  - Proteinrekonstitution
KW  - synthetische Biologie
Y1  - 2023
U6  - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:kobv:517-opus4-612781
ER  - 
TY  - THES
A1  - Chandrakanth Shetty, Sunidhi
T1  - Directed chemical communication in artificial eukaryotic cells
T1  - Gezielte chemische Kommunikation in künstlichen eukaryotischen Zellen
N2  - Eukaryotic cells can be regarded as complex microreactors capable of performing various biochemical reactions in parallel which are necessary to sustain life. An essential prerequisite for these complex metabolic reactions to occur is the evolution of lipid membrane-bound organelles enabling compartmental- ization of reactions and biomolecules. This allows for a spatiotemporal control over the metabolic reactions within the cellular system. Intracellular organi- zation arising due to compartmentalization is a key feature of all living cells and has inspired synthetic biologists to engineer such systems with bottom-up approaches.
Artificial cells provide an ideal platform to isolate and study specific re- actions without the interference from the complex network of biomolecules present in biological cells. To mimic the hierarchical architecture of eukaryotic cells, multi-compartment assemblies with nested liposomal structures also re- ferred to as multi-vesicular vesicles (MVVs) have been widely adopted. Most of the previously reported multi-compartment systems adopt bulk method- ologies which suffer from low yield and poor control over size. Microfluidic strategies help circumvent these issues and facilitate a high-throughput and robust technique to assemble MVVs of uniform size distribution.
In this thesis, firstly, the bulk methodologies are explored to build MVVs and implement a synthetic signalling cascade. Next, a polydimethylsiloxane (PDMS)-based microfluidic platform is introduced to build MVVs and the significance of PEGylated lipids for the successful encapsulation of inner com- partments to generate stable multi-compartment systems is highlighted.
Next, a novel two-inlet channel PDMS-based microfluidic device to create MVVs encompassing a three-step enzymatic reaction cascade is presented. A directed reaction pathway comprising of the enzymes α-glucosidase (α-Glc), glucose oxidase (GOx), and horseradish peroxidase (HRP) spanning across three compartments via reconstitution of size-selective membrane proteins is described. Furthermore, owing to the monodispersity of our MVVs due to microfluidic strategies, this platform is employed to study the effect of com- partmentalization on reaction kinetics.
Further integration of cell-free expression module into the MVVs would allow for gene-mediated signal transduction within artificial eukaryotic cells. Therefore, the chemically inducible cell-free expression of a membrane protein alpha-hemolysin and its further reconstitution into liposomes is carried out.
In conclusion, the present thesis aims to build artificial eukaryotic cells to achieve size-selective chemical communication that also show potential for applications as micro reactors and as vehicles for drug delivery.
N2  - Eukaryontische Zellen können als komplexe Mikroreaktoren betrachtet werden, die in der Lage sind, verschiedene biochemische Reaktionen parallel durchzuführen, die für die Aufrechterhaltung des Lebens notwendig sind. Eine wesentliche Voraussetzung für die Durchführung dieser komplexen Stoffwechselreaktionen ist die Entwicklung von Organellen mit Lipidmembranen, die eine Kompartimentierung von Reaktionen und Biomolekülen ermöglichen. Dies ermöglicht eine räumlich-zeitliche Kontrolle über die Stoffwechselreaktionen innerhalb des zellulären Systems. Die durch die Kompartimentierung entstehende intrazelluläre Organisation ist ein Schlüsselmerkmal aller lebenden Zellen und hat synthetische Biologen dazu inspiriert, solche Systeme mit Bottom-up-Ansätzen zu entwickeln.

Künstliche Zellen bieten eine ideale Plattform, um spezifische Reaktionen zu isolieren und zu untersuchen, ohne dass das komplexe Netzwerk von Biomolekülen, das in biologischen Zellen vorhanden ist, stört. Um die hierarchische Architektur eukaryontischer Zellen zu imitieren, haben sich Multikompartiment-Anordnungen mit verschachtelten liposomalen Strukturen, die auch als multivesikuläre Vesikel (MVV) bezeichnet werden, durchgesetzt. Die meisten der bisher vorgestellten Multikompartiment-Systeme basieren auf Bulk-Methoden, die eine geringe Ausbeute und eine schlechte Kontrolle über die Größe aufweisen. Mikrofluidische Strategien helfen, diese Probleme zu umgehen und ermöglichen eine robuste Technik mit hohem Durchsatz, um MVVs mit einheitlicher Größenverteilung herzustellen.

In dieser Dissertation werden zunächst die Bulk-Methoden zum Aufbau von MVVs und zur Implementierung einer synthetischen Signalkaskade untersucht. Anschließend wird eine auf Polydimethylsiloxan (PDMS) basierende mikrofluidische Plattform zur Herstellung von MVVs vorgestellt und die Bedeutung von PEGylierten Lipiden für die erfolgreiche Verkapselung der inneren Kompartimente zur Erzeugung stabiler Multikompartiment-Systeme hervorgehoben.

Es wird ein neuartiges mikrofluidisches Gerät mit zwei Einlasskanälen auf PDMS-Basis zur Herstellung von MVVs vorgestellt, das eine dreistufige enzymatische Reaktionskaskade umfasst. Es wird ein gerichteter Reaktionsweg beschrieben, der die Enzyme α-Glucosidase (α-Glc), Glucoseoxidase (GOx) und Meerrettichperoxidase (HRP) umfasst und sich über drei Kompartimente erstreckt, die durch die Rekonstitution von größenselektiven Membranproteinen entstehen. Aufgrund der Monodispersität unserer MVVs durch mikrofluidische Strategien nutze ich diese Plattform außerdem, um die Auswirkungen der Kompartimentierung auf die Reaktionskinetik zu untersuchen.
Eine weitere Integration von zellfreien Expressionsmodulen in MVVs würde eine genvermittelte Signaltransduktion in künstlichen eukaryotischen Zellen ermöglichen. Daher wird die chemisch induzierbare zellfreie Expression eines Membranproteins alpha-Hämolysin und seine weitere Rekonstitution in Liposomen durchgeführt.

Zusammenfassend lässt sich sagen, dass die vorliegende Arbeit darauf abzielt, künstliche eukaryotische Zellen zu bauen, um eine größenselektive chemische Kommunikation zu erreichen, und das Potenzial für Anwendungen als Mikroreaktoren und als Vehikel für die Verabreichung von Medikamenten aufweisen.
KW  - microfluidics
KW  - synthetic biology
KW  - Mikrofluidik
KW  - synthetische Biologie
Y1  - 2021
U6  - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:kobv:517-opus4-533642
ER  - 
TY  - THES
A1  - Naseri, Gita
T1  - Plant-derived transcription factors and their application for synthetic biology approaches in Saccharomyces cerevisiae
T1  - Pflanzenbasierte Transkriptionsfaktoren und ihre Anwendungen in der synthetischen Biologie in Saccharomyces cerevisiae
N2  - Bereits seit 9000 Jahren verwendet die Menschheit die Bäckerhefe Saccharomyces cerevisiae für das Brauen von Bier, aber erst seit 150 Jahren wissen wir, dass es sich bei diesem unermüdlichen Helfer im Brauprozess um einzellige, lebende Organismen handelt. Und die Bäckerhefe kann noch viel mehr. Im Rahmen des Forschungsgebietes der Synthetischen Biologie soll unter anderem die Bäckerhefe als innovatives Werkzeug für die biobasierte Herstellung verschiedenster Substanzen etabliert werden. Zu diesen Substanzen zählen unter anderem Feinchemikalien, Biokraftstoffe und Biopolymere sowie pharmakologisch und medizinisch interessante Pflanzenstoffe.
Damit diese verschiedensten Substanzen in der Bäckerhefe hergestellt werden können, müssen große Mengen an Produktionsinformationen zum Beispiel aus Pflanzen in die Hefezellen übertragen werden. Darüber hinaus müssen die neu eingebrachten Biosynthesewege reguliert und kontrolliert in den Zellen ablaufen. Auch Optimierungsprozesse zur Erhöhung der Produktivität sind notwendig. Für alle diese Arbeitsschritte mangelt es bis heute an anwendungsbereiten Technologien und umfassenden Plattformen. Daher wurden im Rahmen dieser Doktorarbeit verschiedene Technologien und Plattformen zur Informationsübertragung, Regulation und Prozessoptimierung geplant und erzeugt.
Für die Konstruktion von Biosynthesewegen in der Bäckerhefe wurde als erstes eine Plattform aus neuartigen Regulatoren und Kontrollelementen auf der Basis pflanzlicher Kontrollelemente generiert und charakterisiert. Im zweiten Schritt erfolgte die Entwicklung einer Technologie zur kombinatorischen Verwendung der Regulatoren in der Planung und Optimierung von Biosynthesewegen (COMPASS). Abschließend wurde eine Technologie für die Prozessoptimierung der veränderten Hefezellen entwickelt (CapRedit). Die Leistungsfähigkeit der entwickelten Plattformen und Technologien wurde durch eine Optimierung der Produktion von Carotenoiden (Beta-Carotin und Beta-Ionon) und Flavonoiden (Naringenin) in Hefezellen nachgewiesen.
Die im Rahmen der Arbeit etablierten neuartigen Plattformen und innovativen Technologien sind ein wertvoller Grundbaustein für die Erweiterung der Nutzbarkeit der Bäckerhefe. Sie ermöglichen den Einsatz der Hefezellen in kosteneffizienten Produktionswegen und alternativen chemischen Wertschöpfungsketten. Dadurch können zum Beispiel Biokraftstoffe und pharmakologisch interessante Pflanzenstoffe unter Verwendung von nachwachsenden Rohstoffen, Reststoffen und Nebenprodukten hergestellt werden. Darüber hinaus ergeben sich Anwendungsmöglichkeiten zur Bodensanierung und Wasseraufbereitung.
N2  - Plant-derived Transcription Factors for Orthologous Regulation of Gene Expression in the Yeast Saccharomyces cerevisiae
Control of gene expression by transcription factors (TFs) is central in many synthetic biology projects where tailored expression of one or multiple genes is often needed. As TFs from evolutionary distant organisms are unlikely to affect gene expression in a host of choice, they represent excellent candidates for establishing orthogonal control systems. To establish orthogonal regulators for use in yeast (Saccharomyces cerevisiae), we chose TFs from the plant Arabidopsis thaliana. We established a library of 106 different combinations of chromosomally integrated TFs, activation domains (yeast GAL4 AD, herpes simplex virus VP64, and plant EDLL) and synthetic promoters harbouring cognate cis-regulatory motifs driving a yEGFP reporter. Transcriptional output of the different driver / reporter combinations varied over a wide spectrum, with EDLL being a considerably stronger transcription activation domain in yeast, than the GAL4 activation domain, in particular when fused to Arabidopsis NAC TFs. Notably, the strength of several NAC - EDLL fusions exceeded that of the strong yeast TDH3 promoter by 6- to 10-fold. We furthermore show that plant TFs can be used to build regulatory systems encoded by centromeric or episomal plasmids. Our library of TF – DNA-binding site combinations offers an excellent tool for diverse synthetic biology applications in yeast.

COMPASS: Rapid combinatorial optimization of biochemical pathways based on artificial transcription factors

We established a high-throughput cloning method, called COMPASS for COMbinatorial Pathway ASSembly, for the balanced expression of multiple genes in Saccharomyces cerevisiae. COMPASS employs orthogonal, plant-derived artificial transcription factors (ATFs) for controlling the expression of pathway genes, and homologous recombination-based cloning for the generation of thousands of individual DNA constructs in parallel. The method relies on a positive selection of correctly assembled pathway variants from both, in vivo and in vitro cloning procedures. To decrease the turnaround time in genomic engineering, we equipped COMPASS with multi-locus CRISPR/Cas9-mediated modification capacity. In its current realization, COMPASS allows combinatorial optimization of up to ten pathway genes, each transcriptionally controlled by nine different ATFs spanning a 10-fold difference in expression strength. The application of COMPASS was demonstrated by generating cell libraries producing beta-carotene and co-producing beta-ionone and biosensor-responsive naringenin. COMPASS will have many applications in other synthetic biology projects that require gene expression balancing. 

CaPRedit: Genome editing using CRISPR-Cas9 and plant-derived transcriptional regulators for the redirection of flux through the FPP branch-point in yeast. Technologies developed over the past decade have made Saccharomyces cerevisiae a promising platform for production of different natural products. We developed CRISPR/Ca9- and plant derived regulator-mediated genome editing approach (CaPRedit) to greatly accelerate strain modification and to facilitate very low to very high expression of key enzymes using inducible regulators. CaPRedit can be implemented to enhance the production of yeast endogenous or heterologous metabolites in the yeast S. cerevisiae. The CaPRedit system aims to faciltiate modification of multiple targets within a complex metabolic pathway through providing new tools for increased expression of genes encoding rate-limiting enzymes, decreased expression of essential genes, and removed expression of competing pathways. This approach is based on CRISPR/Cas9-mediated one-step double-strand breaks to integrate modules containing IPTG-inducible plant-derived artificial transcription factor and promoter pair(s) in a desired locus or loci. Here, we used CaPRedit to redirect the yeast endogenous metabolic flux toward production of farnesyl diphosphate (FPP), a central precursor of nearly all yeast isoprenoid products, by overexpression of the enzymes lead to produce FPP from glutamate. We found significantly higher beta-carotene accumulation in the CaPRedit-mediated modified strain than in the wild type (WT) strain. More specifically, CaPRedit_FPP 1.0 strain was generated, in which three genes involved in FPP synthesis, tHMG1, ERG20, and GDH2, were inducibly overexpressed under the control of strong plant-derived ATFPs. The beta–carotene accumulated in CaPRedit_FPP 1.0 strain to a level 1.3-fold higher than the previously reported optimized strain that carries the same overexpressed genes (as well as additional genetic modifications to redirect yeast endogenous metabolism toward FPP production). Furthermore, the genetic modifications implemented in CaPRedit_FPP 1.0 strain resulted in only a very small growth defect (growth rate relative to the WT is ~ -0.03).
KW  - synthetic biology
KW  - Saccharomyces cerevisiae
KW  - artificial transcription factor
KW  - combinatorial optimization
KW  - biosensor
KW  - DNA assembly
KW  - pathway engineering
KW  - artifizielle Transkriptionsfaktoren
KW  - Biosensor
KW  - kombinatorische Optimierung
KW  - DNA assembly
KW  - Saccharomyces cerevisiae
KW  - synthetische Biologie
KW  - pathway engineering
Y1  - 2018
U6  - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:kobv:517-opus4-421514
ER  - 
TY  - THES
A1  - Hochrein, Lena
T1  - Development of a new DNA-assembly method and its application for the establishment of a red light-sensing regulation system
T1  - Entwicklung einer neuartigen DNS-Assemblierungsmethode und ihre Anwendung für die Etablierung eines Rotlicht-responsiven Regulierungssystems
N2  - In der hier vorgelegten Doktorarbeit wurde eine Strategie zur schnellen, einfachen und zuverlässigen Assemblierung von DNS-Fragmenten, genannt AssemblX, entwickelt. Diese kann genutzt werden, um komplexe DNS-Konstrukte, wie beispielsweise komplette Biosynthesewege, aufzubauen. Dies dient der Produktion von technisch oder medizinisch relevanten Produkten in biotechnologisch nutzbaren Organismen. Die Vorteile der Klonierungsstrategie liegen in der Schnelligkeit der Klonierung, der Flexibilität bezüglich des Wirtsorganismus, sowie der hohen Effektivität, die durch gezielte Optimierung erreicht wurde. Die entwickelte Technik erlaubt die nahtlose Assemblierung von Genfragmenten und bietet eine Komplettlösung von der Software-gestützten Planung bis zur Fertigstellung von DNS-Konstrukten, welche die Größe von Mini-Chromosomen erreichen können. 
Mit Hilfe der oben beschriebenen AssemblX Strategie wurde eine optogenetische Plattform für die Bäckerhefe Saccharomyces cerevisiae etabliert. Diese besteht aus einem Rotlicht-sensitiven Photorezeptor und seinem interagierenden Partner aus Arabidopsis thaliana, welche in lichtabhängiger Weise miteinander agieren. Diese Interaktion wurde genutzt, um zwei Rotlicht-aktivierbare Proteine zu erstellen: Einen Transkriptionsfaktor, der nach Applikation eines Lichtpulses die Produktion eines frei wählbaren Proteins stimuliert, sowie eine Cre Rekombinase, die ebenfalls nach Bestrahlung mit einer bestimmten Wellenlänge die zufallsbasierte Reorganisation bestimmter DNS-Konstrukte ermöglicht. Zusammenfassend wurden damit drei Werkzeuge für die synthetische Biologie etabliert. Diese ermöglichen den Aufbau von komplexen Biosynthesewegen, deren Licht-abhängige Regulation, sowie die zufallsbasierte Rekombination zu Optimierungszwecken.
N2  - With Saccharomyces cerevisiae being a commonly used host organism for synthetic biology and biotechnology approaches, the work presented here aims at the development of novel tools to improve and facilitate pathway engineering and heterologous protein production in yeast. Initially, the multi-part assembly strategy AssemblX was established, which allows the fast, user-friendly and highly efficient construction of up to 25 units, e.g. genes, into a single DNA construct. To speed up complex assembly projects, starting from sub-gene fragments and resulting in mini-chromosome sized constructs, AssemblX follows a level-based approach: Level 0 stands for the assembly of genes from multiple sub-gene fragments; Level 1 for the combination of up to five Level 0 units into one Level 1 module; Level 2 for linkages of up to five Level 1 modules into one Level 2 module. This way, all Level 0 and subsequently all Level 1 assemblies can be carried out simultaneously. Individually planned, overlap-based Level 0 assemblies enable scar-free and sequence-independent assemblies of transcriptional units, without limitations in fragment number, size or content. Level 1 and Level 2 assemblies, which are carried out via predefined, computationally optimized homology regions, follow a standardized, highly efficient and PCR-free scheme. AssemblX follows a virtually sequence-independent scheme with no need for time-consuming domestication of assembly parts. To minimize the risk of human error and to facilitate the planning of assembly projects, especially for individually designed Level 0 constructs, the whole AssemblX process is accompanied by a user-friendly webtool. This webtool provides the user with an easy-to-use operating surface and returns a bench-protocol including all cloning steps. The efficiency of the assembly process is further boosted through the implementation of different features, e.g. ccdB counter selection and marker switching/reconstitution. Due to the design of homology regions and vector backbones the user can flexibly choose between various overlap-based cloning methods, enabling cost-efficient assemblies which can be carried out either in E. coli or yeast. Protein production in yeast is additionally supported by a characterized library of 40 constitutive promoters, fully integrated into the AssemblX toolbox. This provides the user with a starting point for protein balancing and pathway engineering. Furthermore, the final assembly cassette can be subcloned into any vector, giving the user the flexibility to transfer the individual construct into any host organism different from yeast.
As successful production of heterologous compounds generally requires a precise adjustment of protein levels or even manipulation of the host genome to e.g. inhibit unwanted feedback regulations, the optogenetic transcriptional regulation tool PhiReX was designed. In recent years, light induction was reported to enable easy, reversible, fast, non-toxic and nearly gratuitous regulation, thereby providing manifold advantages compared to conventional chemical inducers. The optogenetic interface established in this study is based on the photoreceptor PhyB and its interacting protein PIF3. Both proteins, derived from Arabidopsis thaliana, dimerize in a red/far-red light-responsive manner. This interaction depends on a chromophore, naturally not available in yeast. By fusing split proteins to both components of the optical dimerizer, active enzymes can be reconstituted in a light-dependent manner. For the construction of the red/far-red light sensing gene expression system PhiReX, a customizable synTALE-DNA binding domain was fused to PhyB, and a VP64 activation domain to PIF3. The synTALE-based transcription factor allows programmable targeting of any desired promoter region. The first, plasmid-based PhiReX version mediates chromophore- and light-dependent expression of the reporter gene, but required further optimization regarding its robustness, basal expression and maximum output. This was achieved by genome-integration of the optical regulator pair, by cloning the reporter cassette on a high-copy plasmid and by additional molecular modifications of the fusion proteins regarding their cellular localization. In combination, this results in a robust and efficient activation of cells over an incubation time of at least 48 h. Finally, to boost the potential of PhiReX for biotechnological applications, yeast was engineered to produce the chromophore. This overcomes the need to supply the expensive and photo-labile compound exogenously. The expression output mediated through PhiReX is comparable to the strong constitutive yeast TDH3 promoter and - in the experiments described here - clearly exceeds the commonly used galactose inducible GAL1 promoter. 
The fast-developing field of synthetic biology enables the construction of complete synthetic genomes. The upcoming Synthetic Yeast Sc2.0 Project is currently underway to redesign and synthesize the S. cerevisiae genome. As a prerequisite for the so-called “SCRaMbLE” system, all Sc2.0 chromosomes incorporate symmetrical target sites for Cre recombinase (loxPsym sites), enabling rearrangement of the yeast genome after induction of Cre with the toxic hormonal substance beta-estradiol. To overcome the safety concern linked to the use of beta-estradiol, a red light-inducible Cre recombinase, dubbed L-SCRaMbLE, was established in this study. L-SCRaMbLE was demonstrated to allow a time- and chromophore-dependent recombination with reliable off-states when applied to a plasmid containing four genes of the beta-carotene pathway, each flanked with loxPsym sites. When directly compared to the original induction system, L-SCRaMbLE generates a larger variety of recombination events and lower basal activity. In conclusion, L-SCRaMbLE provides a promising and powerful tool for genome rearrangement.
The three tools developed in this study provide so far unmatched possibilities to tackle complex synthetic biology projects in yeast by addressing three different stages: fast and reliable biosynthetic pathway assembly; highly specific, orthogonal gene regulation; and tightly controlled synthetic evolution of loxPsym-containing DNA constructs.
KW  - synthetic biology
KW  - pathway engineering
KW  - DNA assembly
KW  - transcription factor
KW  - Cre recombinase
KW  - optogenetics
KW  - synthetische Biologie
KW  - Optimierung von Biosynthesewegen
KW  - DNS Assemblierung
KW  - Transkriptionsfaktor
KW  - Cre Rekombinase
KW  - Optogenetik
Y1  - 2018
U6  - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:kobv:517-opus4-404441
ER  -