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COMPASS for rapid combinatorial optimization of biochemical pathways based on artificial transcription factors.
Nat Commun ; 10(1): 2615, 2019 06 13.
Artigo em Inglês | MEDLINE | ID: mdl-31197154
ABSTRACT
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 ß-carotene and co-producing ß-ionone and biosensor-responsive naringenin. COMPASS will have many applications in synthetic biology projects that require gene expression balancing.
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Texto completo: Disponível Coleções: Bases de dados internacionais Base de dados: MEDLINE Assunto principal: Saccharomyces cerevisiae / Fatores de Transcrição / Proteínas de Saccharomyces cerevisiae / Vias Biossintéticas / Engenharia Metabólica Idioma: Inglês Revista: Nat Commun Assunto da revista: Biologia / Ciência Ano de publicação: 2019 Tipo de documento: Artigo País de afiliação: Alemanha