About This Project
Remediating methane requires the uniquely potent enzyme methane monooxygenase (MMO). All attempts to express MMO heterologously (e.g., in transgenic plants or microbes) has failed, for unknown reasons. Here we will systematically profile the biophysical properties of MMO, including cofactors, pH, and lipid composition and order, using designed fluorescent biosensors. These results will aid efforts to express MMO for methane remediation and could power efforts to engineer improved MMO variants.
Ask the Scientists
Join The DiscussionMotivating Factor
Novel technology for methane removal (MR) at 1-100 MtCH4 scale is needed, particularly for oxidizing atmospheric concentrations (2 ppm) (NASEM, 2024, Abernethy, 2024) and emissions at 2-1000 ppm that are too dilute to be scalably oxidized with existing technology (Abernethy, 2023).
Several MR strategies have been proposed that would leverage biological CH4 oxidation in engineered contexts. One such strategy is engineered crops or managed trees expressing methane monooxygenase (MMO) in their leaves or roots, which could oxidize CH4 in soil or ambient air (Strand, 2022; Spatola Rossi, 2023).
Heterologous transduction of MMO subunits into plants has yielded expressed, but essentially nonfunctional, protein. The reasons for this failure are not understood. As such, more characterization, particularly the particulate MMO (pMMO), which is thought to have the highest affinity for CH4 (He, 2024; Reginato, 2024), is required.
Specific Bottleneck
Native pMMO is embedded in intracytoplasmic membranes (ICMs) in ordered arrays of multi-subunit complexes (Tucci, 2024). While non- or barely functional protein complexes have been assembled (Koo, 2022; Rossi, 2023; Gou, 2006), producing active pMMO heterologously has been impossible. Further, substantial open questions remain about how the native cellular environment provides the necessary biophysical conditions for pMMO to function, hindering efforts to engineer high-activity pMMO (Koo, 2021).
The membrane potential, concentrations of key ions, redox state, and pH in the native environment of pMMO substantially impact enzyme function, but are unknown. The active site contains critical copper ions, but it’s not known whether other transition metals are also required, nor if NADH or NADPH is needed. Determining these unknown biophysical parameters in the native context of pMMO would likely aid engineering and transplantation efforts involving pMMO.
Actionable Goals
To characterize biophysical factors in pMMO function, optical (fluorescent) biosensors for voltage, pH, reactive oxygen species, NAD(P)H, and ions will be adapted for use in model methanotrophs with protocols for quantitative measurement through microscopy. These systems will include development of specific ICM localization tags for structures inside methanotrophs. Dye-based studies of the same factors, where applicable, will also be pursued. Ideally, sensors would allow imaging of multiple factors simultaneously to understand interplay. Any constructs, expression vectors, and cell lines generated will be broadly distributed.
Budget
We will create a large set of expression plasmids for use in methanotrophs. This will require several months of molecular biology. Expression constructs will then be tested in target bacteria subjected to a range of conditions of methane/methanol level, pH, concentration of copper and other metals, etc. - with each condition tracked both by fluorescence microscopy and by a gold standard assay of methane level to determine MMO activity. This work will require a fair bit of lab supplies (dyes, microbiology supplies, microscope filters) - particularly for the delicate work on methane itself, which requires sophisticated gas-flow mechanisms for precise control of levels.
Meet the Team
Team Bio
Drs. Konopka and Looger are coming together to bring disparate skillsets - designing, optimizing, and deploying fluorescent biosensors for diverse analytes; combined with rigorous study of methanotrophs and the enzymology of particulate methane monooxygenase (pMMO). The team believes that such cross-disciplinary collaborations will be required to tackle the hardest problems facing our homeworld.
Michael Konopka
Michael Konopka is an Assistant Professor of Chemistry at the United States Naval Academy. He has >15 years of experience studying methanotrophic bacteria, starting from his postdoc. He has developed methods for both imaging membranes in methanotrophs, and for identifying and characterizing them in environmental samples. He also has extensive experience in quantitative image analysis, including developing code for high-throughput analysis programs.
Loren Looger
Loren Looger is a Professor of Neurosciences and HHMI Investigator at UC San Diego. He has >25 years experience in making fluorescent biosensors, and has made many of the sensors in wide use across neuroscience and other biological disciplines. Although his sensors are mostly used in animals and their cells, they work well in bacteria and indeed, his lab has imaged many of them in bacteria and other microbes, visualizing carbon metabolism and flux through specific transporters, for instance. His lab also has extensive expertise in scraping genomes for targeting peptides and similar features.
Project Backers
- 0Backers
- 100%Funded
- $135,285Total Donations
- $0Average Donation