TY - GEN A1 - Das Gupta, Mainak A1 - Roesch, Florian A1 - Hochrein, Lena A1 - Machens, Fabian A1 - Müller-Röber, Bernd T1 - Facilitating Genome Engineering Through RNP-mediated Precise Gene Targeting T2 - In Vitro Cellular & Developmental Biology - Plant Y1 - 2019 SN - 1054-5476 SN - 1475-2689 VL - 55 IS - 4 SP - 481 EP - 481 PB - Springer CY - New York 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 - TY - JOUR A1 - Hochrein, Lena A1 - Machens, Fabian A1 - Gremmels, Juergen A1 - Schulz, Karina A1 - Messerschmidt, Katrin A1 - Mueller-Roeber, Bernd T1 - AssemblX: a user-friendly toolkit for rapid and reliable multi-gene assemblies JF - Nucleic acids research N2 - The assembly of large DNA constructs coding for entire pathways poses a major challenge in the field of synthetic biology. Here, we present AssemblX, a novel, user-friendly and highly efficient multi-gene assembly strategy. The software-assisted AssemblX process allows even unexperienced users to rapidly design, build and test DNA constructs with currently up to 25 functional units, from 75 or more subunits. At the gene level, AssemblX uses scar-free, overlap-based and sequence-independent methods, allowing the unrestricted design of transcriptional units without laborious parts domestication. The assembly into multi-gene modules is enabled via a standardized, highly efficient, polymerase chain reaction-free and virtually sequence-independent scheme, which relies on rare cutting restriction enzymes and optimized adapter sequences. Selection and marker switching strategies render the whole process reliable, rapid and very effective. The assembly product can be easily transferred to any desired expression host, making AssemblX useful for researchers from various fields. Y1 - 2017 U6 - https://doi.org/10.1093/nar/gkx034 SN - 0305-1048 SN - 1362-4962 VL - 45 PB - Oxford Univ. Press CY - Oxford ER - TY - JOUR A1 - Hochrein, Lena A1 - Machens, Fabian A1 - Messerschmidt, Katrin A1 - Müller-Röber, Bernd T1 - PhiReX: a programmable and red light-regulated protein expression switch for yeast JF - Nucleic acids research N2 - Highly regulated induction systems enabling dose-dependent and reversible fine-tuning of protein expression output are beneficial for engineering complex biosynthetic pathways. To address this, we developed PhiReX, a novel red/far-red light-regulated protein expression system for use in Saccharomyces cerevisiae. PhiReX is based on the combination of a customizable synTALE DNA-binding domain, the VP64 activation domain and the light-sensitive dimerization of the photoreceptor PhyB and its interacting partner PIF3 from Arabidopsis thaliana. Robust gene expression and high protein levels are achieved by combining genome integrated red light-sensing components with an episomal high-copy reporter construct. The gene of interest as well as the synTALE DNA-binding domain can be easily exchanged, allowing the flexible regulation of any desired gene by targeting endogenous or heterologous promoter regions. To allow low-cost induction of gene expression for industrial fermentation processes, we engineered yeast to endogenously produce the chromophore required for the effective dimerization of PhyB and PIF3. Time course experiments demonstrate high-level induction over a period of at least 48 h. Y1 - 2017 U6 - https://doi.org/10.1093/nar/gkx610 SN - 0305-1048 SN - 1362-4962 VL - 45 SP - 9193 EP - 9205 PB - Oxford Univ. Press CY - Oxford ER - TY - JOUR A1 - Hochrein, Lena A1 - Mitchell, Leslie A. A1 - Schulz, Karina A1 - Messerschmidt, Katrin A1 - Müller-Röber, Bernd T1 - L-SCRaMbLE as a tool for light-controlled Cre-mediated recombination in yeast JF - Nature Communications N2 - The synthetic yeast genome constructed by the International Synthetic Yeast Sc2.0 consortium adds thousands of loxPsym recombination sites to all 16 redesigned chromosomes, allowing the shuffling of Sc2.0 chromosome parts by the Cre-loxP recombination system thereby enabling genome evolution experiments. Here, we present L-SCRaMbLE, a lightcontrolled Cre recombinase for use in the yeast Saccharomyces cerevisiae. L-SCRaMbLE allows tight regulation of recombinase activity with up to 179-fold induction upon exposure to red light. The extent of recombination depends on induction time and concentration of the chromophore phycocyanobilin (PCB), which can be easily adjusted. The tool presented here provides improved recombination control over the previously reported estradiol-dependent SCRaMbLE induction system, mediating a larger variety of possible recombination events in SCRaMbLE-ing a reporter plasmid. Thereby, L-SCRaMbLE boosts the potential for further customization and provides a facile application for use in the S. cerevisiae genome reengineering project Sc2.0 or in other recombination-based systems. Y1 - 2018 U6 - https://doi.org/10.1038/s41467-017-02208-6 SN - 2041-1723 VL - 9 PB - Nature Publ. Group CY - London ER - TY - JOUR A1 - Messerschmidt, Katrin A1 - Hochrein, Lena A1 - Dehm, Daniel A1 - Schulz, Karina A1 - Mueller-Roeber, Bernd T1 - Characterizing seamless ligation cloning extract for synthetic biological applications JF - Analytical biochemistry : methods in the biological sciences N2 - Synthetic biology aims at designing and engineering organisms. The engineering process typically requires the establishment of suitable DNA constructs generated through fusion of multiple protein coding and regulatory sequences. Conventional cloning techniques, including those involving restriction enzymes and ligases, are often of limited scope, in particular when many DNA fragments must be joined or scar-free fusions are mandatory. Overlap-based-cloning methods have the potential to overcome such limitations. One such method uses seamless ligation cloning extract (SLiCE) prepared from Escherichia coli cells for straightforward and efficient in vitro fusion of DNA fragments. Here, we systematically characterized extracts prepared from the unmodified E. coli strain DH10B for SLiCE-mediated cloning and determined DNA sequence-associated parameters that affect cloning efficiency. Our data revealed the virtual absence of length restrictions for vector backbone (up to 13.5 kbp) and insert (90 bp to 1.6 kbp). Furthermore, differences in GC content in homology regions are easily tolerated and the deletion of unwanted vector sequences concomitant with targeted fragment insertion is straightforward. Thus, SLiCE represents a highly versatile DNA fusion method suitable for cloning projects in virtually all molecular. and synthetic biology projects. (C) 2016 Elsevier Inc. All rights reserved. KW - SLiCE KW - Seamless ligation cloning KW - Homologous recombination KW - Synthetic biology Y1 - 2016 U6 - https://doi.org/10.1016/j.ab.2016.05.029 SN - 0003-2697 SN - 1096-0309 VL - 509 SP - 24 EP - 32 PB - Elsevier CY - San Diego ER - TY - GEN A1 - Messerschmidt, Katrin A1 - Machens, Fabian A1 - Hochrein, Lena A1 - Naseri, Gita T1 - Orthogonal, light-inducible protein expression platform in yeast Sacchararomyces cerevisiae T2 - New biotechnology Y1 - 2018 U6 - https://doi.org/10.1016/j.nbt.2018.05.153 SN - 1871-6784 SN - 1876-4347 VL - 44 SP - S19 EP - S19 PB - Elsevier CY - Amsterdam ER -