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Microviridins are a prominent family of ribosomally synthesized and posttranslationally modified peptides (RiPPs) featuring characteristic lactone and lactam rings. Their unusual cage-like architecture renders them highly potent serine protease inhibitors of which individual variants specifically inhibit different types of proteases of pharmacological interest.
While posttranslational modifications are key for the stability and bioactivity of RiPPs, additional attractive properties can be introduced by functional tags.
To date - although highly desirable - no method has been reported to incorporate functional tags in microviridin scaffolds or the overarching class of graspetides.
In this study, a chemoenzymatic in vitro platform is used to introduce functional tags in various microviridin variants yielding biotinylated, dansylated or propargylated congeners.
This straightforward approach paves the way for customized protease inhibitors with built-in functionalities that can help to unravel the still elusive ecological roles and targets of this remarkable class of compounds and to foster applications based on protease inhibition.
Overcoming natural biomass limitations in gram-negative bacteria through synthetic carbon fixation
(2024)
The carbon demands of an ever-increasing human population and the concomitant rise in net carbon emissions requires CO2 sequestering approaches for production of carbon-containing molecules. Microbial production of carbon-containing products from plant-based sugars could replace current fossil-based production. However, this form of sugar-based microbial production directly competes with human food supply and natural ecosystems. Instead, one-carbon feedstocks derived from CO2 and renewable energy were proposed as an alternative. The one carbon molecule formate is a stable, readily soluble and safe-to-store energetic mediator that can be electrochemically generated from CO2 and (excess off-peak) renewable electricity. Formate-based microbial production could represent a promising approach for a circular carbon economy. However, easy-to-engineer and efficient formate-utilizing microbes are lacking. Multiple synthetic metabolic pathways were designed for better-than-nature carbon fixation. Among them, the reductive glycine pathway was proposed as the most efficient pathway for aerobic formate assimilation. While some of these pathways have been successfully engineered in microbial hosts, these synthetic strains did so far not exceed the performance of natural strains. In this work, I engineered and optimized two different synthetic formate assimilation pathways in gram-negative bacteria to exceed the limits of a natural carbon fixation pathway, the Calvin cycle.
The first chapter solidified Cupriavidus necator as a promising formatotrophic host to produce value-added chemicals. The formate tolerance of C. necator was assessed and a production pathway for crotonate established in a modularized fashion. Last, bioprocess optimization was leveraged to produce crotonate from formate at a titer of 148 mg/L.
In the second chapter, I chromosomally integrated and optimized the synthetic reductive glycine pathway in C. necator using a transposon-mediated selection approach. The insertion methodology allowed selection for condition-specific tailored pathway expression as improved pathway performance led to better growth. I then showed my engineered strains to exceed the biomass yields of the Calvin cycle utilizing wildtype C. necator on formate. This demonstrated for the first time the superiority of a synthetic formate assimilation pathway and by extension of synthetic carbon fixation efforts as a whole.
In chapter 3, I engineered a segment of a synthetic carbon fixation cycle in Escherichia coli. The GED cycle was proposed as a Calvin cycle alternative that does not perform a wasteful oxygenation reaction and is more energy efficient. The pathways simple architecture and reasonable driving force made it a promising candidate for enhanced carbon fixation. I created a deletion strain that coupled growth to carboxylation via the GED pathway segment. The CO2 dependence of the engineered strain and 13C-tracer analysis confirmed operation of the pathway in vivo.
In the final chapter, I present my efforts of implementing the GED cycle also in C. necator, which might be a better-suited host, as it is accustomed to formatotrophic and hydrogenotrophic growth. To provide the carboxylation substrate in vivo, I engineered C. necator to utilize xylose as carbon source and created a selection strain for carboxylase activity. I verify activity of the key enzyme, the carboxylase, in the decarboxylative direction. Although CO2-dependent growth of the strain was not obtained, I showed that all enzymes required for operation of the GED cycle are active in vivo in C. necator.
I then evaluate my success with engineering a linear and cyclical one-carbon fixation pathway in two different microbial hosts. The linear reductive glycine pathway presents itself as a much simpler metabolic solution for formate dependent growth over the sophisticated establishment of hard-to-balance carbon fixation cycles. Last, I highlight advantages and disadvantages of C. necator as an upcoming microbial benchmark organism for synthetic metabolism efforts and give and outlook on its potential for the future of C1-based manufacturing.
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.
Synthetische Transkriptionsfaktoren bestehen wie natürliche Transkriptionsfaktoren aus einer DNA-Bindedomäne, die sich spezifisch an die Bindestellensequenz vor dem Ziel-Gen anlagert, und einer Aktivierungsdomäne, die die Transkriptionsmaschinerie rekrutiert, sodass das Zielgen exprimiert wird. Der Unterschied zu den natürlichen Transkriptionsfaktoren ist, sowohl dass die DNA-Bindedomäne als auch die Aktivierungsdomäne wirtsfremd sein können und dadurch künstliche Stoffwechselwege im Wirt, größtenteils chemisch, induziert werden können. Optogenetische synthetische Transkriptionsfaktoren, die hier entwickelt wurden, gehen einen Schritt weiter. Dabei ist die DNA-Bindedomäne nicht mehr an die Aktivierungsdomäne, sondern mit dem Blaulicht-Photorezeptor CRY2 gekoppelt. Die Aktivierungsdomäne wurde mit dem Interaktionspartner CIB1 fusioniert. Unter Blaulichtbestrahlung dimerisieren CRY2 und CIB1 und damit einhergehend die beiden Domänen, sodass ein funktionsfähiger Transkriptionsfaktor entsteht. Dieses System wurde in die Saccharomyces cerevisiae genomisch integriert. Verifiziert wurde das konstruierte System mit Hilfe des Reporters yEGFP, welcher durchflusszytometrisch detektiert werden konnte. Es konnte gezeigt werden, dass die yEGFP Expression variabel gestaltet werden kann, indem unterschiedlich lange Blaulichtimpulse ausgesendet wurden, die DNA-Bindedomäne, die Aktivierungsdomäne oder die Anzahl der Bindestellen, an dem sich die DNA-Bindedomäne anlagert, verändert wurden. Um das System für industrielle Anwendungen attraktiv zu gestalten, wurde das System vom Deepwell-Maßstab auf Photobioreaktor-Maßstab hochskaliert. Außerdem erwies sich das Blaulichtsystem sowohl im Laborstamm YPH500 als auch im industriell oft verwendeten Hefestamm CEN.PK als funktional. Des Weiteren konnte ein industrierelevante Protein ebenso mit Hilfe des verifizierten Systems exprimiert werden. Schlussendlich konnte in dieser Arbeit das etablierte Blaulicht-System erfolgreich mit einem Rotlichtsystem kombiniert werden, was zuvor noch nicht beschrieben wurde.
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.
Membrane contact sites are of particular interest in the field of synthetic biology and biophysics. They are involved in a great variety of cellular functions. They form in between two cellular organelles or an organelle and the plasma membrane in order to establish a communication path for molecule transport or signal transmission.
The development of an artificial membrane system which can mimic membrane contact sites using bottom up synthetic biology was the goal of this research study. For this, a multi - compartmentalised giant unilamellar vesicle (GUV) system was created with the membrane of the outer vesicle mimicking the plasma membrane and the inner GUVs posing as cellular organelles.
In the following steps, three different strategies were used to achieve an internal membrane - membrane adhesion.
We have developed a genetic circuit in Escherichia coli that can be used to select for protein-protein interactions of different strengths by changing antibiotic concentrations in the media. The genetic circuit links protein-protein interaction strength to beta-lactamase activity while simultaneously imposing tuneable positive and negative selection pressure for beta-lactamase activity. Cells only survive if they express interacting proteins with affinities that fall within set high- and low-pass thresholds; i.e. the circuit therefore acts as a bandpass filter for protein-protein interactions. We show that the circuit can be used to recover protein-protein interactions of desired affinity from a mixed population with a range of affinities. The circuit can also be used to select for inhibitors of protein-protein interactions of defined strength. (C) 2018 Elsevier Ltd. All rights reserved.
One-carbon (C1) compounds are attractive microbial feedstocks as they can be efficiently produced from widely available resources. Formate, in particular, represents a promising growth substrate, as it can be generated from electrochemical reduction of CO2 and fed to microorganisms in a soluble form. We previously identified the synthetic reductive glycine pathway as the most efficient route for aerobic growth on formate. We further demonstrated pathway activity in Escherichia coli after expression of both native and foreign genes. Here, we explore whether the reductive glycine pathway could be established in a model microorganism using only native enzymes. We used the yeast Saccharomyces cerevisiae as host and show that overexpression of only endogenous enzymes enables glycine biosynthesis from formate and CO2 in a strain that is otherwise auxotrophic for glycine. We find the pathway to be highly active in this host, where 0.125 mM formate is sufficient to support growth. Notably, the formate-dependent growth rate of the engineered S. cerevisiae strain remained roughly constant over a very wide range of formate concentrations, 1-500 mM, indicating both high affinity for formate use and high tolerance toward elevated concentration of this C1 feedstock. Our results, as well the availability of endogenous NAD-dependent formate dehydrogenase, indicate that yeast might be an especially suitable host for engineering growth on formate.
Plant X-tender
(2018)
Cloning multiple DNA fragments for delivery of several genes of interest into the plant genome is one of the main technological challenges in plant synthetic biology. Despite several modular assembly methods developed in recent years, the plant biotechnology community has not widely adopted them yet, probably due to the lack of appropriate vectors and software tools. Here we present Plant X-tender, an extension of the highly efficient, scarfree and sequence-independent multigene assembly strategy AssemblX,based on overlapdepended cloning methods and rare-cutting restriction enzymes. Plant X-tender consists of a set of plant expression vectors and the protocols for most efficient cloning into the novel vector set needed for plant expression and thus introduces advantages of AssemblX into plant synthetic biology. The novel vector set covers different backbones and selection markers to allow full design flexibility. We have included ccdB counterselection, thereby allowing the transfer of multigene constructs into the novel vector set in a straightforward and highly efficient way. Vectors are available as empty backbones and are fully flexible regarding the orientation of expression cassettes and addition of linkers between them, if required. We optimised the assembly and subcloning protocol by testing different scar-less assembly approaches: the noncommercial SLiCE and TAR methods and the commercial Gibson assembly and NEBuilder HiFi DNA assembly kits. Plant X-tender was applicable even in combination with low efficient homemade chemically competent or electrocompetent Escherichia coli. We have further validated the developed procedure for plant protein expression by cloning two cassettes into the newly developed vectors and subsequently transferred them to Nicotiana benthamiana in a transient expression setup. Thereby we show that multigene constructs can be delivered into plant cells in a streamlined and highly efficient way. Our results will support faster introduction of synthetic biology into plant science.
Control of gene expression by transcription factors (TFs) is central in many synthetic biology projects for which a 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 harboring 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.