Anthropogenic climate change necessitates a societal shift toward sustainable manufacturing practices. The chemical and polymer industry, which accounts for a fifth of the global CO2 emissions, has been slow to adapt due to factors such as economic pressures and risk aversion. Biomanufacturing, which usually relies on microorganisms such as bacteria, offers a promising alternative by leveraging native biological pathways that can perform complex chemistry without the requirement for extremes of temperature and pressure, often needed in traditional chemical processes. Beyond the model bacterium E. coli, the diverse metabolisms of environmental bacteria can be harnessed to produce a wide range of chemicals, with some bacteria capable of directly fixing atmospheric CO2 (autotrophs) and N2 (diazotrophs) when provided with a source of reducing power. Biomolecular engineering and ‘domestication’ of these non-model bacteria is required to make sustainable biomanufacturing a reality. However, these efforts are hindered by a lack of genetic tools. Here, I present efforts to (i) address the challenges of introducing recombinant DNA into environmental Mycobacteriales, an industrially relevant yet largely genetically intractable clade of bacteria, (ii) investigate the metabolism of these bacteria under conditions of extreme nutrient limitation and isolate novel CO2 fixing species, and (iii) develop a modular DNA assembly framework for the model diazotroph Azotobacter vinelandii. From this work, we have developed a suite of new genetic tools which function in several Mycobacteriales genera, characterized a novel Gordonia species with potential autotrophic metabolism, and identified the presence of multiple restriction-modification systems as a dominant barrier to genetic manipulation and future biomolecular engineering efforts in these species.