TY - JOUR A1 - de la Cruz, Jorge Gonzalez A1 - Machens, Fabian A1 - Messerschmidt, Katrin A1 - Bar-Even, Arren T1 - Core Catalysis of the Reductive Glycine Pathway Demonstrated in Yeast JF - ACS synthetic biology N2 - 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. KW - metabolic engineering KW - synthetic biology KW - one-carbon metabolism KW - carbon labeling KW - tetrahydrofolate KW - glycine cleavage system Y1 - 2019 U6 - https://doi.org/10.1021/acssynbio.8b00464 SN - 2161-5063 VL - 8 IS - 5 SP - 911 EP - 917 PB - American Chemical Society CY - Washington ER - TY - GEN A1 - Göthel, Markus A1 - Listek, Martin A1 - Messerschmidt, Katrin A1 - Schlör, Anja A1 - Hönow, Anja A1 - Hanack, Katja T1 - A New Workflow to Generate Monoclonal Antibodies against Microorganisms T2 - Mathematisch-Naturwissenschaftliche Reihe N2 - Monoclonal antibodies are used worldwide as highly potent and efficient detection reagents for research and diagnostic applications. Nevertheless, the specific targeting of complex antigens such as whole microorganisms remains a challenge. To provide a comprehensive workflow, we combined bioinformatic analyses with novel immunization and selection tools to design monoclonal antibodies for the detection of whole microorganisms. In our initial study, we used the human pathogenic strain E. coli O157:H7 as a model target and identified 53 potential protein candidates by using reverse vaccinology methodology. Five different peptide epitopes were selected for immunization using epitope-engineered viral proteins. The identification of antibody-producing hybridomas was performed by using a novel screening technology based on transgenic fusion cell lines. Using an artificial cell surface receptor expressed by all hybridomas, the desired antigen-specific cells can be sorted fast and efficiently out of the fusion cell pool. Selected antibody candidates were characterized and showed strong binding to the target strain E. coli O157:H7 with minor or no cross-reactivity to other relevant microorganisms such as Legionella pneumophila and Bacillus ssp. This approach could be useful as a highly efficient workflow for the generation of antibodies against microorganisms. T3 - Zweitveröffentlichungen der Universität Potsdam : Mathematisch-Naturwissenschaftliche Reihe - 1174 KW - monoclonal antibody KW - antibody producing cell selection KW - hybridoma KW - epitope prediction Y1 - 2021 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:kobv:517-opus4-523341 SN - 1866-8372 IS - 20 ER - TY - JOUR A1 - Göthel, Markus A1 - Listek, Martin A1 - Messerschmidt, Katrin A1 - Schlör, Anja A1 - Hönow, Anja A1 - Hanack, Katja T1 - A New Workflow to Generate Monoclonal Antibodies against Microorganisms JF - Applied Sciences N2 - Monoclonal antibodies are used worldwide as highly potent and efficient detection reagents for research and diagnostic applications. Nevertheless, the specific targeting of complex antigens such as whole microorganisms remains a challenge. To provide a comprehensive workflow, we combined bioinformatic analyses with novel immunization and selection tools to design monoclonal antibodies for the detection of whole microorganisms. In our initial study, we used the human pathogenic strain E. coli O157:H7 as a model target and identified 53 potential protein candidates by using reverse vaccinology methodology. Five different peptide epitopes were selected for immunization using epitope-engineered viral proteins. The identification of antibody-producing hybridomas was performed by using a novel screening technology based on transgenic fusion cell lines. Using an artificial cell surface receptor expressed by all hybridomas, the desired antigen-specific cells can be sorted fast and efficiently out of the fusion cell pool. Selected antibody candidates were characterized and showed strong binding to the target strain E. coli O157:H7 with minor or no cross-reactivity to other relevant microorganisms such as Legionella pneumophila and Bacillus ssp. This approach could be useful as a highly efficient workflow for the generation of antibodies against microorganisms. KW - monoclonal antibody KW - antibody producing cell selection KW - hybridoma KW - epitope prediction Y1 - 2021 U6 - https://doi.org/10.3390/app11209359 SN - 1454-5101 VL - 11 IS - 20 PB - MDPI CY - Basel ER - TY - JOUR A1 - Hess, Anne-Katrin A1 - Bartel, Manuela A1 - Roth, Karina A1 - Messerschmidt, Katrin A1 - Heilmann, Katja A1 - Kenchington, Ellen A1 - Micheel, Burkhard A1 - Stuckas, Heiko T1 - Expression of M6 and M7 lysin in Mytilus edulis is not restricted to sperm, but occurs also in oocytes and somatic tissue of males and females JF - Molecular reproduction and development N2 - Sperm proteins of marine sessile invertebrates have been extensively studied to understand the molecular basis of reproductive isolation. Apart from molecules such as bindin of sea urchins or lysin of abalone species, the acrosomal protein M7 lysin of Mytilus edulis has been analyzed. M7 lysin was found to be under positive selection, but mechanisms driving the evolution of this protein are not fully understood. To explore functional aspects, this study investigated the protein expression pattern of M7 and M6 lysin in gametes and somatic tissue of male and female M. edulis. The study employs a previously published monoclonal antibody (G26-AG8) to investigate M6 and M7 lysin protein expression, and explores expression of both genes. It is shown that these proteins and their encoding genes are expressed in gametes and somatic tissue of both sexes. This is in contrast to sea urchin bindin and abalone lysin, in which gene expression is strictly limited to males. Although future studies need to clarify the functional importance of both acrosomal proteins in male and female somatic tissue, new insights into the evolution of sperm proteins in marine sessile invertebrates are possible. This is because proteins with male-specific expression (bindin, lysin) might evolve differently than proteins with expression in both sexes (M6/M7 lysin), and the putative function of both proteins in females opens the possibility that the evolution of M6/M7 lysin is under sexual antagonistic selection, for example, mutations beneficial to the acrosomal function that are less beneficial the function in somatic tissue of females.Mol. Reprod. Dev. 79: 517-524, 2012. Y1 - 2012 U6 - https://doi.org/10.1002/mrd.22056 SN - 1040-452X VL - 79 IS - 8 SP - 517 EP - 524 PB - Wiley-Blackwell CY - Hoboken 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 - GEN A1 - Lukan, Tjaša A1 - Machens, Fabian A1 - Coll, Anna A1 - Baebler, Špela A1 - Messerschmidt, Katrin A1 - Gruden, Kristina T1 - Plant X-tender BT - an extension of the AssemblX system for the assembly and expression of multigene constructs in plants T2 - Postprints der Universität Potsdam : Mathematisch-Naturwissenschaftliche Reihe N2 - 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. T3 - Zweitveröffentlichungen der Universität Potsdam : Mathematisch-Naturwissenschaftliche Reihe - 990 KW - ligation cloning extract KW - DNA cloning KW - synthetic biology KW - multiple genes KW - vector system KW - transformation KW - recombination KW - protein KW - RNA KW - Methylation Y1 - 2020 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:kobv:517-opus4-446281 SN - 1866-8372 IS - 990 ER - TY - JOUR A1 - Lukan, Tjaša A1 - Machens, Fabian A1 - Coll, Anna A1 - Baebler, Špela A1 - Messerschmidt, Katrin A1 - Gruden, Kristina T1 - Plant X-tender BT - an extension of the AssemblX system for the assembly and expression of multigene constructs in plants JF - PLOS ONE N2 - 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, scar-free and sequence-independent multigene assembly strategy AssemblX, based on overlap-depended 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. Y1 - 2018 U6 - https://doi.org/10.1371/journal.pone.0190526 SN - 1932-6203 VL - 13 IS - 1 PB - Public Library of Science CY - San Fransisco ER - TY - GEN A1 - Machens, Fabian A1 - Balazadeh, Salma A1 - Müller-Röber, Bernd A1 - Messerschmidt, Katrin T1 - Synthetic Promoters and Transcription Factors for Heterologous Protein Expression in Saccharomyces cerevisiae N2 - Orthogonal systems for heterologous protein expression as well as for the engineering of synthetic gene regulatory circuits in hosts like Saccharomyces cerevisiae depend on synthetic transcription factors (synTFs) and corresponding cis-regulatory binding sites. We have constructed and characterized a set of synTFs based on either transcription activator-like effectors or CRISPR/Cas9, and corresponding small synthetic promoters (synPs) with minimal sequence identity to the host’s endogenous promoters. The resulting collection of functional synTF/synP pairs confers very low background expression under uninduced conditions, while expression output upon induction of the various synTFs covers a wide range and reaches induction factors of up to 400. The broad spectrum of expression strengths that is achieved will be useful for various experimental setups, e.g., the transcriptional balancing of expression levels within heterologous pathways or the construction of artificial regulatory networks. Furthermore, our analyses reveal simple rules that enable the tuning of synTF expression output, thereby allowing easy modification of a given synTF/synP pair. This will make it easier for researchers to construct tailored transcriptional control systems. T3 - Zweitveröffentlichungen der Universität Potsdam : Mathematisch-Naturwissenschaftliche Reihe - 393 KW - JUB1 KW - chimeric transcription factors KW - dead Cas9 KW - gene expression KW - synthetic biology KW - synthetic circuits KW - transcriptional regulation Y1 - 2017 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:kobv:517-opus4-403804 ER -