Design and Control of Extrachromosomal Elements in Methylorubrum extorquens AM1

Genetic tools are a prerequisite to engineer cellular factories for synthetic biology and biotechnology. Methylorubrum extorquens AM1 is an important platform organism of a future C 1 -bioeconomy. However, its application is currently limited by the availability of genetic tools. Here we systematically tested repABC regions to maintain extrachromosomal DNA in M. extorquens . We used three elements to construct mini-chromosomes that are stably inherited at single copy number and can be shuttled between Escherichia coli and M. extorquens . These mini-chromosomes are compatible among each other and with high-copy number plasmids of M. extorquens . We also developed a set of inducible promoters of wide expression range, reaching levels exceeding those currently available, notably the P mxaF -promoter. In summary, we provide a set of tools to control the dynamic expression and copy number of genetic elements in M. extorquens , which opens new ways to unleash the metabolic and biotechnological potential of this organism for future applications.