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COMPASS for rapid combinatorial optimization of biochemical pathways based on artificial transcription factors
- Balanced expression of multiple genes is central for establishing new biosynthetic pathways or multiprotein cellular complexes. Methods for efficient combinatorial assembly of regulatory sequences (promoters) and protein coding sequences are therefore highly wanted. Here, we report a high-throughput cloning method, called COMPASS for COMbinatorial Pathway ASSembly, for the balanced expression of multiple genes in Saccharomyces cerevisiae. COMPASS employs orthogonal, plant-derived artificial transcription factors (ATFs) and homologous recombination-based cloning for the generation of thousands of individual DNA constructs in parallel. The method relies on a positive selection of correctly assembled pathway variants from both, in vivo and in vitro cloning procedures. To decrease the turnaround time in genomic engineering, COMPASS is equipped with multi-locus CRISPR/Cas9-mediated modification capacity. We demonstrate the application of COMPASS by generating cell libraries producing n-carotene and co-producing p-ionone andBalanced expression of multiple genes is central for establishing new biosynthetic pathways or multiprotein cellular complexes. Methods for efficient combinatorial assembly of regulatory sequences (promoters) and protein coding sequences are therefore highly wanted. Here, we report a high-throughput cloning method, called COMPASS for COMbinatorial Pathway ASSembly, for the balanced expression of multiple genes in Saccharomyces cerevisiae. COMPASS employs orthogonal, plant-derived artificial transcription factors (ATFs) and homologous recombination-based cloning for the generation of thousands of individual DNA constructs in parallel. The method relies on a positive selection of correctly assembled pathway variants from both, in vivo and in vitro cloning procedures. To decrease the turnaround time in genomic engineering, COMPASS is equipped with multi-locus CRISPR/Cas9-mediated modification capacity. We demonstrate the application of COMPASS by generating cell libraries producing n-carotene and co-producing p-ionone and biosensor-responsive naringenin. COMPASS will have many applications in synthetic biology projects that require gene expression balancing.…
Verfasserangaben: | Gita NaseriORCiDGND, Jessica Behrend, Lisa Rieper, Bernd Müller-RöberORCiDGND |
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DOI: | https://doi.org/10.1038/s41467-019-10224-x |
ISSN: | 2041-1723 |
Pubmed ID: | https://pubmed.ncbi.nlm.nih.gov/31197154 |
Titel des übergeordneten Werks (Englisch): | Nature Communications |
Verlag: | Nature Publ. Group |
Verlagsort: | London |
Publikationstyp: | Wissenschaftlicher Artikel |
Sprache: | Englisch |
Datum der Erstveröffentlichung: | 13.06.2019 |
Erscheinungsjahr: | 2019 |
Datum der Freischaltung: | 21.01.2021 |
Band: | 10 |
Seitenanzahl: | 18 |
Fördernde Institution: | Federal Ministry of Education and Research of Germany (BMBF)Federal Ministry of Education & Research (BMBF) [031A172, 031B0223]; European Union H2020 project PlantaSYST (SGA-CSA under FPA) [739582, 664620]; fellowship of the Potsdam Graduate School, University of Potsdam |
Organisationseinheiten: | Mathematisch-Naturwissenschaftliche Fakultät / Institut für Biochemie und Biologie |
DDC-Klassifikation: | 5 Naturwissenschaften und Mathematik / 57 Biowissenschaften; Biologie / 570 Biowissenschaften; Biologie |
Peer Review: | Referiert |
Publikationsweg: | Open Access / Gold Open-Access |
DOAJ gelistet | |
Lizenz (Deutsch): | CC-BY - Namensnennung 4.0 International |