Replacement of petrochemistry by bio-based processes is key to sustainable development and requires microbes equipped with outstanding, novel-to-nature capabilities. Constructing such advanced ‘cell factories’ requires large-scale remodelling of their core machinery, and therefore of their genome. However, microbes often harbour mosaic genomes in which thousands of genes are scattered over dozens of chromosomes. This absence of a modular organization tremendously restricts genetic accessibility and defies current technologies. To tackle this limitation we develop microbial platforms with specialized, interchangeable synthetic chromosomes that will enable the core machinery to be remodeled at will. Access to a sheer endless variety of configurations of core metabolism offers unique, new possibilities for fundamental understanding and rational engineering of microbes. Our research paves the way for a modular approach to engineering of microbial cells.