Design and control of extrachromosomal elements in Methylorubrum extorquens AM1.

Genetic tools are a prerequisite to engineer cellular factories for synthetic biology and biotechnology. AM1 is an important platform organism of a future C1-bioeconomy. However, its application is currently limited by the availability of genetic tools. Here we systematically tested regions to maintain extrachromosal DNA in . We used three elements to construct mini-chromosomes that are stably inherited at single copy number and can be shuttled between and . These mini-chromosomes are compatible among each other and with high copy number plasmids of . We also developed a set of inducible promoters of wide expression range reaching levels exceeding those currently available, notably the P-promoter. In summary, we provide a set of tools to control the dynamic expression and copy number of genetic elements in , which opens new ways to unleash the metabolic and biotechnological potential of this organism for future applications.