@article{LukanMachensColletal.2018, author = {Lukan, Tjaša and Machens, Fabian and Coll, Anna and Baebler, Špela and Messerschmidt, Katrin and Gruden, Kristina}, title = {Plant X-tender}, series = {PLOS ONE}, volume = {13}, journal = {PLOS ONE}, number = {1}, publisher = {Public Library of Science}, address = {San Fransisco}, issn = {1932-6203}, doi = {10.1371/journal.pone.0190526}, pages = {19}, year = {2018}, abstract = {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.}, language = {en} } @misc{MesserschmidtMachensHochreinetal.2018, author = {Messerschmidt, Katrin and Machens, Fabian and Hochrein, Lena and Naseri, Gita}, title = {Orthogonal, light-inducible protein expression platform in yeast Sacchararomyces cerevisiae}, series = {New biotechnology}, volume = {44}, journal = {New biotechnology}, publisher = {Elsevier}, address = {Amsterdam}, issn = {1871-6784}, doi = {10.1016/j.nbt.2018.05.153}, pages = {S19 -- S19}, year = {2018}, language = {en} } @article{delaCruzMachensMesserschmidtetal.2019, author = {de la Cruz, Jorge Gonzalez and Machens, Fabian and Messerschmidt, Katrin and Bar-Even, Arren}, title = {Core Catalysis of the Reductive Glycine Pathway Demonstrated in Yeast}, series = {ACS synthetic biology}, volume = {8}, journal = {ACS synthetic biology}, number = {5}, publisher = {American Chemical Society}, address = {Washington}, issn = {2161-5063}, doi = {10.1021/acssynbio.8b00464}, pages = {911 -- 917}, year = {2019}, abstract = {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.}, language = {en} } @misc{DasGuptaRoeschHochreinetal.2019, author = {Das Gupta, Mainak and Roesch, Florian and Hochrein, Lena and Machens, Fabian and M{\"u}ller-R{\"o}ber, Bernd}, title = {Facilitating Genome Engineering Through RNP-mediated Precise Gene Targeting}, series = {In Vitro Cellular \& Developmental Biology - Plant}, volume = {55}, journal = {In Vitro Cellular \& Developmental Biology - Plant}, number = {4}, publisher = {Springer}, address = {New York}, issn = {1054-5476}, pages = {481 -- 481}, year = {2019}, language = {en} } @misc{LukanMachensColletal.2018, author = {Lukan, Tjaša and Machens, Fabian and Coll, Anna and Baebler, Špela and Messerschmidt, Katrin and Gruden, Kristina}, title = {Plant X-tender}, series = {Postprints der Universit{\"a}t Potsdam : Mathematisch-Naturwissenschaftliche Reihe}, journal = {Postprints der Universit{\"a}t Potsdam : Mathematisch-Naturwissenschaftliche Reihe}, number = {990}, issn = {1866-8372}, doi = {10.25932/publishup-44628}, url = {http://nbn-resolving.de/urn:nbn:de:kobv:517-opus4-446281}, pages = {21}, year = {2018}, abstract = {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.}, language = {en} } @article{HochreinMachensGremmelsetal.2017, author = {Hochrein, Lena and Machens, Fabian and Gremmels, Juergen and Schulz, Karina and Messerschmidt, Katrin and Mueller-Roeber, Bernd}, title = {AssemblX: a user-friendly toolkit for rapid and reliable multi-gene assemblies}, series = {Nucleic acids research}, volume = {45}, journal = {Nucleic acids research}, publisher = {Oxford Univ. Press}, address = {Oxford}, issn = {0305-1048}, doi = {10.1093/nar/gkx034}, pages = {12}, year = {2017}, abstract = {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.}, language = {en} } @article{NaseriBalazadehMachensetal.2017, author = {Naseri, Gita and Balazadeh, Salma and Machens, Fabian and Kamranfar, Iman and Messerschmidt, Katrin and M{\"u}ller-R{\"o}ber, Bernd}, title = {Plant-Derived Transcription Factors for Orthologous Regulation of Gene Expression in the Yeast Saccharomyces cerevisiae}, series = {ACS synthetic biology}, volume = {6}, journal = {ACS synthetic biology}, publisher = {American Chemical Society}, address = {Washington}, issn = {2161-5063}, doi = {10.1021/acssynbio.7b00094}, pages = {1742 -- 1756}, year = {2017}, abstract = {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.}, language = {en} } @article{HochreinMachensMesserschmidtetal.2017, author = {Hochrein, Lena and Machens, Fabian and Messerschmidt, Katrin and M{\"u}ller-R{\"o}ber, Bernd}, title = {PhiReX: a programmable and red light-regulated protein expression switch for yeast}, series = {Nucleic acids research}, volume = {45}, journal = {Nucleic acids research}, publisher = {Oxford Univ. Press}, address = {Oxford}, issn = {0305-1048}, doi = {10.1093/nar/gkx610}, pages = {9193 -- 9205}, year = {2017}, abstract = {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.}, language = {en} } @article{LehmeyerKanofskyHankoetal.2016, author = {Lehmeyer, Mona and Kanofsky, Konstantin and Hanko, Erik K. R. and Ahrendt, Sarah and Wehrs, Maren and Machens, Fabian and Hehl, Reinhard}, title = {Functional dissection of a strong and specific microbe-associated molecular pattern-responsive synthetic promoter}, series = {Plant Biotechnology Journal}, volume = {14}, journal = {Plant Biotechnology Journal}, publisher = {Wiley}, address = {Hoboken}, issn = {1467-7644}, doi = {10.1111/pbi.12357}, pages = {61 -- 71}, year = {2016}, abstract = {Synthetic promoters are important for temporal and spatial gene expression in transgenic plants. To identify novel microbe-associated molecular pattern (MAMP)-responsive cis-regulatory sequences for synthetic promoter design, a combination of bioinformatics and experimental approaches was employed. One cis-sequence was identified which confers strong MAMP-responsive reporter gene activity with low background activity. The 35-bp-long cis-sequence was identified in the promoter of the Arabidopsis thaliana DJ1E gene, a homologue of the human oncogene DJ1. In this study, this cis-sequence is shown to be a tripartite cis-regulatory module (CRM). A synthetic promoter with four copies of the CRM linked to a minimal promoter increases MAMP-responsive reporter gene expression compared to the wild-type DJ1E promoter. The CRM consists of two WT-boxes (GGACTTTT and GGACTTTG) and a variant of the GCC-box (GCCACC), all required for MAMP and salicylic acid (SA) responsivity. Yeast one-hybrid screenings using a transcription factor (TF)-only prey library identified two AP2/ERFs, ORA59 and ERF10, interacting antagonistically with the CRM. ORA59 activates reporter gene activity and requires the consensus core sequence GCCNCC for gene expression activation. ERF10 down-regulates MAMP-responsive gene expression. No TFs interacting with the WT-boxes GGACTTTT and GGACTTTG were selected in yeast onehybrid screenings with the TF-only prey library. In transgenic Arabidopsis, the synthetic promoter confers strong and specific reporter gene activity in response to biotrophs and necrotrophs as well as SA.}, language = {en} } @misc{MachensBalazadehMuellerRoeberetal.2017, author = {Machens, Fabian and Balazadeh, Salma and M{\"u}ller-R{\"o}ber, Bernd and Messerschmidt, Katrin}, title = {Synthetic Promoters and Transcription Factors for Heterologous Protein Expression in Saccharomyces cerevisiae}, url = {http://nbn-resolving.de/urn:nbn:de:kobv:517-opus4-403804}, pages = {11}, year = {2017}, abstract = {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.}, language = {en} }