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.…
Author details: | 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 |
Title of parent work (English): | Nature Communications |
Publisher: | Nature Publ. Group |
Place of publishing: | London |
Publication type: | Article |
Language: | English |
Date of first publication: | 2019/06/13 |
Publication year: | 2019 |
Release date: | 2021/01/21 |
Volume: | 10 |
Number of pages: | 18 |
Funding 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 |
Organizational units: | Mathematisch-Naturwissenschaftliche Fakultät / Institut für Biochemie und Biologie |
DDC classification: | 5 Naturwissenschaften und Mathematik / 57 Biowissenschaften; Biologie / 570 Biowissenschaften; Biologie |
Peer review: | Referiert |
Publishing method: | Open Access / Gold Open-Access |
DOAJ gelistet | |
License (German): | CC-BY - Namensnennung 4.0 International |