Creating a Genetic\nToolbox for the Carbon-Fixing,\nNitrogen-Fixing and Dehalogenating Bacterium Xanthobacter\nautotrophicus

Xanthobacter\nautotrophicus is a\nmetabolically flexible microorganism with two key features: (1) The\norganism has adapted to grow on a wide variety of carbon sources including\nCO<sub>2</sub>, methanol, formate, propylene, haloalkanes and haloacids;\nand (2) X. autotrophicus was the first\nchemoautotroph identified that could also simultaneously fix N<sub>2</sub>, meaning the organism can utilize CO<sub>2</sub>, N<sub>2</sub>, and H<sub>2</sub> for growth. This metabolic flexibility has enabled\nuse of X. autotrophicus for gas fixation,\nthe creation of fertilizers and foods from gases, and the dehalogenation\nof environmental contaminants. Despite the wide variety of applications\nthat have already been demonstrated for this organism, there are few\ngenetic tools available to explore and exploit its metabolism. Here,\nwe report a genetic toolbox for use in X. autotrophicus. We first identified suitable origins of replication and quantified\ntheir copy number, and identified antibiotic resistance cassettes\nthat could be used as selectable markers. We then tested several constitutive\nand inducible promoters and terminators and quantified their promoter\nstrengths and termination efficiencies. Finally, we demonstrated that\ngene expression tools remain effective under both autotrophic and\ndehalogenative metabolic conditions to show that these tools can be\nused in the environments that make X. autotrophicus unique. Our extensive characterization of these tools in X. autotrophicus will enable genetic and metabolic\nengineering to optimize production of fertilizers and foods from gases,\nand enable bioremediation of halogenated environmental contaminants.