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Methanotrophy inside-out: metals and metabolism

EMSL Project ID


Methylotrophs are among the key players in the environmental cycling of C1-compounds, such as methanol and/or methane. The oxidation of C1-compounds requires a number of minerals including Cu, Fe, Ca, W, and rare earth elements (REEs) for C1-carbon oxidation. This research is centered on uncovering the fundamental mechanisms that enable biological systems to scavenge metals from the environment and switch biochemical pathways in response to metal availability to advance the understanding of the intricacy of metabolic solutions in Methylotuvimicrobium alcaliphilum sp. 20ZR, a prominent microbial chassis for the utilization of single carbon (C1) substrates, such as methane and methanol.
The metabolic networks of methanotrophs are highly redundant, enabling growth across a wide range of metal availability. The first step of methane oxidation and respiration machinery in methanotrophs can be modulated by copper, with particulate enzymes expressed under copper supplementation, while soluble enzymes take over in Cu-limited environments. In the second step, methanol to formaldehyde is also redundant and can be performed by at least one of two methanol dehydrogenases (MDH), XoxF or MxaFI. These enzymes use different metal cofactors for methanol assimilation: MxaFI uses calcium as its metal cofactor while XoxF relies on REEs for its activity. Finally, formate utilization is controlled by Mo to W availability. While some other minerals, such as Ni and Zn are essential for optimal growth, their exact functions in C1-metabolic network have yet to be uncovered. Changes in metal availability impact not only cell growth, but also cell structure and formation of extracellular vesicles. As part of previous and on-going DOE-funded research, we established a computational framework encompassing the metabolic network, acquired large sets of multi-omic data, and developed a set of genetic tools. Here we will focus on methanotrophic surface layer proteins (SLP) and EVs, investigating their role in metal uptake. Under previous DOE/EMSL-sponsored proteomics studies, we found that the trace minerals play a key role in controlling the cellular redox and energy state. Our data suggest that metals can trigger significant changes in cell structure by inducing formation of ICMs and altering electron transport chain composition, which increases ATP generation. In addition to dramatic changes in the redox status, copper alters amino-acid biosynthesis pathways and modulates the activity of tRNA modification enzymes. These changes feedback into protein expression, alter the translation of key enzymes, and thus change the direction of carbon flow via central metabolic pathway enzymes. Furthermore, new biochemical and inhibition data suggest that methylotrophs possess a unique energy conservation system associated with ICMs. Metal starvation can also induce the formation of EVs.
Overall, this project seeks a better understanding of the intricate relationships between environmental conditions (i.e., metal availability), cell morphology (with/without ICMs, EVs), and cellular outputs (growth rate, carbon utilization efficiency, biomass composition) to quantitatively describe these relationships via constraint-based modeling for reliable engineering of a promising methylotrophic chassis for production of biofuels and biochemicals as well as metal sequestration.

Project Details

Project type
Exploratory Research
Start Date
End Date


Principal Investigator

Marina Kalyuzhnaya
San Diego State University

Team Members

Richard Hamilton
San Diego State University