@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{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{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} } @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} } @article{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}, series = {Frontiers in Bioengineering and Biotechnology}, volume = {5}, journal = {Frontiers in Bioengineering and Biotechnology}, publisher = {Frontiers}, address = {Lausanne}, issn = {2296-4185}, doi = {10.3389/fbioe.2017.00063}, pages = {1 -- 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} }