XanthoMoClo─A Robust Modular Cloning Genetic Toolkit for the Genera and .

Interest in species is increasing due to their unique metabolic capabilities. They can grow in both heterotrophic and fully autotrophic environments, including carbon dioxide, dinitrogen gas, and hydrogen as the sole carbon, nitrogen, and energy sources, respectively. Academic and industrial groups looking to leverage these metabolic properties are already using strains for the sustainable production of food and commodities. However, only a handful of genetic parts and protocols exist in scattered genetic backgrounds, and there is an unmet need for reliable genetic engineering tools to manipulate species. Here, we developed XanthoMoClo, a robust modular cloning genetic toolkit for and species and strains, providing extensive tools to transform them, manipulate their metabolism, and express genes of interest. The toolkit contains plasmid parts, such as replication origins, antibiotic selection markers, fluorescent proteins, constitutive and inducible promoters, a standardized framework to incorporate novel components into the toolkit, and a conjugation donor to transform and strains easily with no or minimal optimization. We validated these plasmid components in depth in three of the most commonly studied strains: Py2, GZ29, and GJ10, as well as in VTT E-85241. Finally, we demonstrate robust toolkit functionality across 21 different species of and , comprising 23 strains in total. The XanthoMoClo genetic toolkit is available to the research community (through AddGene) and will help accelerate the genetic engineering of to further their applications in sustainability and bioremediation efforts.