A supernumerary designer chromosome for modularin vivopathway assembly inSaccharomyces cerevisiae

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 transcriptionalunit-sized fragments in a single transformation. These assemblies were remarkably efficient and faithful to their in silico design. 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 in S. cerevisiae and paves the way for de novo designer chromosomes as modular genome engineering platforms in S. cerevisiae.

Rapid 40 kb genome construction from 52 parts

“…In most cases these constructs are too long to be reliably generated by continuous chemical DNA synthesis methodologies or PCR, and thus are typically assembled from constituent DNA fragments over multiple rounds of assembly [1,2]. Several commonly used methodologies to manufacture large DNA constructs rely on in vivo recombination, and these high capacity systems allow researchers to join up to 40-50 DNA fragments in a single assembly round [3,4].…”