bioRxiv preprint doi: https://doi.org/10.1101/2020.02.18.954131; this version posted February 19, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. A supernumerary designer chromosome for modular in vivo pathway assembly in Saccharomyces cerevisiae Eline D. Postma1, Sofia Dashko1, Lars van Breemen1, Shannara K. Taylor Parkins1, Marcel van den Broek1, Jean-Marc Daran1, Pascale Daran-Lapujade1* 1 Department of Biotechnology, Delft University of Technology, van der Maasweg 9, 2627HZ Delft, The Netherlands * Corresponding author: Tel: +31 15 278 9965 Email:
[email protected] 1 bioRxiv preprint doi: https://doi.org/10.1101/2020.02.18.954131; this version posted February 19, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. ABSTRACT The construction of microbial cell factories for sustainable production of chemicals and pharmaceuticals requires extensive genome engineering. Using Saccharomyces cerevisiae, this study proposes Synthetic Chromosomes (SynChs) as orthogonal expression platforms for rewiring native cellular processes and implementing new functionalities. Capitalizing the powerful homologous recombination capability of S. cerevisiae, modular SynChs of 50 and 100 Kb were fully assembled de novo from up to 44 transcriptional- unit-sized fragments in a single transformation.