Microbial physiology represents an intricate network of processes related to microbial growth, function, and regulation as a response to their environment. Therefore, understanding microbial physiology is the first step in understanding far-ranging phenomena from global biogeochemical cycles to agriculture to human health and disease. Manipulations of microbial physiologies have formed the basis of modern society, allowing for the development of antibiotics and therapies, increased agricultural and food output, and synthetic biology and bioproduct production, to name just a few examples.Methylotrophy, defined as the ability of microorganisms to utilize reduced one-carbon compounds lacking carbon-carbon bonds (e.g. methanol, methane, methylated amines), is a unique microbial physiology by which to investigate the relationship between microbial metabolism and the environment for several reasons. First, the use of ubiquitous C1 substrates such as methanol and methane position methylotrophic bacteria as key players in global carbon cycles. As such, methylotrophic bacteria can be found in a variety of ecosystems including air, marine, soil, and plant environments. Second, methylotrophy necessarily proceeds via the obligate toxic intermediate, formaldehyde; the evolution of pathways that allow methylotrophs to not only routinely encounter and detoxify, but also assimilate, formaldehyde has led to a metabolism that is highly plastic and robust. Third, the recent discovery of the role of lanthanide metals as cofactors in methanol dehydrogenases has expanded our understanding of trace metal utilization in biology. Taken together, these core facets of methylotrophic physiology have massive implications beyond just their roles in metabolism: growth on renewable C1 substrates can be coupled to production of various bioproducts for biotechnologies; understanding the symbiotic interaction between methylotrophic bacteria and plants can lead to enhanced crop production; the resilience against toxic intermediates allows for development of biotechnologies using substrates that would be otherwise excluded from microbial substrate repertoires; the dependence on lanthanides for growth can be manipulated for biorecovery of critical metals. The relationship between methylotrophic physiology and the environment, as well as potential applications for these processes, serve as the basis for this dissertation.In this dissertation, I provide a comprehensive overview of methoxylated aromatic acid metabolism in methylotrophic bacteria. Methoxylated aromatic acids (e.g., vanillic acid) are monomers that comprise lignin, the main constituent of woody plant cell walls and one of the most abundant and renewable carbon sources on Earth. Degradation of lignin releases toxic C1 byproducts such as formaldehyde, and while methylotrophic strains capable of growth on methoxylated aromatic acids have been identified, the details surrounding their metabolism – and especially, the fate of formaldehyde—remained unknown.In Chapter 1, I describe the current state of methylotrophic metabolisms in biotechnology, highlighting recent breakthroughs in metabolic engineering that have allowed for use of diverse feedstocks for production of various commodities. I emphasize the challenges associated with lignin conversion, such as accumulation of formaldehyde, and propose that methylotrophic bacteria capable of methoxylated aromatic acid metabolism could efficiently overcome these challenges while coupling growth to production of value-added products. In Chapter 2, I describe the isolation of a community of methylotrophic bacteria from the leaves of soybean plants, including dozens of strains capable of robust growth on methoxylated aromatic acids. It is from this isolate community that the model organism for methoxylated aromatic acid metabolism in methylotrophs, Methylobacterium extorquens SLI 505, originates. In Chapter 3, I dive into genetic and metabolic characterizations of aromatic acid metabolism to identify the fate of formaldehyde and how methylotrophs cope with seemingly overlapping methylotrophic and heterotrophic pathways during growth on aromatic acids. I demonstrate the importance of formaldehyde dissimilation during growth of M. extorquens SLI 505 on methoxylated aromatic acids and expand on this idea in Chapter 4 where I compare various formaldehyde oxidation modules in M. extorquens SLI 505 and their bioenergetic consequences during growth on vanillic acid. Taken together, this dissertation establishes aromatic acids as a model by which to understand how M. extorquens SLI 505 balances methylotrophic and heterotrophic pathways during growth to provide strategies for growth optimization when using complex substrates in both ecological and industrial fermentation applications
