UC San Diego

Microbial methane utilization (also known as methanotrophy) stands out as an exceptional form of chemoorganotrophy, which requires a set of unique cellular machineries (membrane compartments and metabolic pathways) fully dedicated for energy recovery from the simplest organic compounds (methane or methanol) and reconstruction of all cellular parts from a single carbon unit. Despite significant progress in understanding fundamentals of methanotrophy, several critical elements remain unresolved. The most critical knowledge gaps include the lack of mechanistic understanding of methane oxidation, such as the source of electrons for the copper dependent methane monooxygenase (pMMO), the plasticity of metabolic pathways, and their dynamic interplay modulated by metal availability. These elements need to be thoroughly understood before they can be established as a chassis organism for biomanufacturing. To solve these mysteries, the research in this thesis is presented in three chapters, each dedicated to different cellular and molecular aspects of the methane metabolism using Methylotuvimicrobium alcaliphilum 20ZR as a model methanotrophic organism. The first chapter of my thesis is focused on characterization of methane oxidation machinery in the strain 20ZR at a cellular level. Exploration into this machinery was performed by a series of biochemical studies with strains that lack different elements of the methane oxidation network, such as methanol dehydrogenase (MDH), formate dehydrogenase (FDH), and downregulation/ expression of particulate methane monooxygenase (pMMO) proteins. This is followed by the second chapter of the thesis, which investigates cellular function of the surface layer proteins (SLPs). I performed comparative genetic analysis, knockout studies and a series of phenotyping tests complemented by -omics studies to gain insights into the SLP function. The data revealed that the surface layer protein matrix not only stabilizes the cell envelope at suboptimal conditions, but also contributes to metal scavenging. The last chapter of my thesis is dedicated to evaluating the feasibility of utilizing SLPs from methanotrophic bacteria for biomanufacturing. I developed an array of genetic tools to functionalize the surface layer proteins and use them as a platform to display heterologous proteins and peptides. The identification and utilization of surface layer proteins in methanotrophic bacteria introduces a novel approach for biomining.