COMPASS for rapid combinatorial optimization of biochemical pathways based on artificial transcription factors

Gita Naseri, Jessica Behrend, Lisa Rieper and Bernd Mueller-Roeber ()
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Gita Naseri: University of Potsdam, Cell2Fab Research Unit
Jessica Behrend: University of Potsdam, Cell2Fab Research Unit
Lisa Rieper: University of Potsdam, Cell2Fab Research Unit
Bernd Mueller-Roeber: University of Potsdam, Department Molecular Biology

Nature Communications, 2019, vol. 10, issue 1, 1-18

Abstract: 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 β-carotene and co-producing β-ionone and biosensor-responsive naringenin. COMPASS will have many applications in synthetic biology projects that require gene expression balancing.

Date: 2019
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DOI: 10.1038/s41467-019-10224-x

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