Navigating methane metabolism: Enzymes, compartments, and networks.

Microbial methane utilization-a key node in the global carbon cycle-controls and often eliminates emission of methane into the atmosphere. The diversity and distribution of microbes capable of methane oxidation is astounding. However, from a biochemical point of view, only a very narrow set of unique enzymes underlies their metabolic capabilities. Despite this restriction, the successful integration of the enzymes into nonmethanotrophs, if judged by the ability of the trait to grow on methane, remains to be achieved. Failures and small victories with heterologous expression have highlighted a set of challenges linked to structure, compartmentalization, and regulation of the methanotrophic metabolic network. A better understanding of how these challenges are handled by cells of native methane-consuming bacteria is required. In this chapter we focus on key experimental aspects of working with native methanotrophic bacteria, including routine cultivation strategies, lab-scale bioreactor setups, genetic alteration, imaging, and basic -omic-level approaches.