Optimized Tools and Methods for Methanotroph Genome Editing

Microbes with the capacity to use methane (CH₄) as a carbon source (methanotrophs) have significant potential for the bioconversion of CH₄-containing natural gas and anaerobic digestion-derived biogas to high value products. These organisms also play a vital role in the biogeochemical cycling of atmospheric CH₄ by serving as the only known biological sink of this gas in terrestrial and aquatic ecosystems. Much is known regarding the enzymes and central metabolic pathways mediating CH₄ utilization in these bacteria. However, large fundamental knowledge gaps exist regarding methanotroph physiology and responses to environmental stimuli, primarily due to a lack of efficient molecular tools to probe gene-function relationships. In this chapter, we describe several recently developed genetic tools and optimized genome editing methods that can be used for methanotroph metabolic engineering and to probe metabolic and physiological governing mechanisms in these unique bacteria.