A supernumerary designer chromosome for modular in vivo pathway assembly in Saccharomyces cerevisiae

The construction of microbial cell factories for sustainable production of chemicals and pharmaceuticals requires extensive genome engineering. UsingSaccharomyces 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 ofS. cerevisiae, modular SynChs of 50 and 100 Kb were fully assembledde novofrom up to 44 transcriptional-unit-sized fragments in a single transformation. These assemblies were remarkably efficient and faithful to theirin silicodesign. SynChs made of non-coding DNA were stably replicated and segregated irrespective of their size without affecting the physiology of their host. These non-coding SynChs were successfully used as landing pad and as exclusive expression platform for the essential glycolytic pathway. This work pushes the limit of DNA assembly inS. cerevisiaeand paves the way forde novodesigner chromosomes as modular genome engineering platforms inS. cerevisiae.