Methane, as natural gas or biogas, is the least expensive source of carbon for (bio)chemical synthesis. Scalable biological upgrading of this simple alkane to chemicals and fuels can bring new ...sustainable solutions to a number of industries with large environmental footprints, such as natural gas/petroleum production, landfills, wastewater treatment, and livestock. Microbial biocatalysis with methane as a feedstock has been pursued off and on for almost a half century, with little enduring success. Today, biological engineering and systems biology provide new opportunities for metabolic system modulation and give new optimism to the concept of a methane-based bio-industry. Here we present an overview of the most recent advances pertaining to metabolic engineering of microbial methane utilization. Some ideas concerning metabolic improvements for production of acetyl-CoA and pyruvate, two main precursors for bioconversion, are presented. We also discuss main gaps in the current knowledge of aerobic methane utilization, which must be solved in order to release the full potential of methane-based biosystems.
•Methanotrophs have the potential to play a role in our sustainable energy future.•A system-level understanding of methanotrophic metabolism will lay the groundwork for metabolic engineering to generate value-added products from methane.•A set of gaps in our fundamental knowledge of methanotrophy make predictive metabolic engineering more difficult, but approaches are available to accommodate the uncertainty.•A suite of available cultures and tools exist that will facilitate metabolic engineering efforts.•A set of approaches are suggested that have the potential to increase flux to key precursors for many value-added products.
In the past few years, the field of methylotrophy has undergone a significant transformation in terms of discovery of novel types of methylotrophs, novel modes of methylotrophy, and novel metabolic ...pathways. This time has also been marked by the resolution of long-standing questions regarding methylotrophy and the challenge of long-standing dogmas. This chapter is not intended to provide a comprehensive review of metabolism of methylotrophic bacteria. Instead we focus on significant recent discoveries that are both refining and transforming the current understanding of methylotrophy as a metabolic phenomenon. We also review new directions in methylotroph ecology that improve our understanding of the role of methylotrophy in global biogeochemical processes, along with an outlook for the future challenges in the field.
Methane is an essential component of the global carbon cycle and one of the most powerful greenhouse gases, yet it is also a promising alternative source of carbon for the biological production of ...value-added chemicals. Aerobic methane-consuming bacteria (methanotrophs) represent a potential biological platform for methane-based biocatalysis. Here we use a multi-pronged systems-level approach to reassess the metabolic functions for methane utilization in a promising bacterial biocatalyst. We demonstrate that methane assimilation is coupled with a highly efficient pyrophosphate-mediated glycolytic pathway, which under oxygen limitation participates in a novel form of fermentation-based methanotrophy. This surprising discovery suggests a novel mode of methane utilization in oxygen-limited environments, and opens new opportunities for a modular approach towards producing a variety of excreted chemical products using methane as a feedstock.
We describe five novel Methylophilaceae ecotypes from a single ecological niche in Lake Washington, USA, and compare them to three previously described ecotypes, in terms of their phenotype and ...genome sequence divergence. Two of the ecotypes appear to represent novel genera within the Methylophilaceae. Genome-based metabolic reconstruction highlights metabolic versatility of Methylophilaceae with respect to methylotrophy and nitrogen metabolism, different ecotypes possessing different combinations of primary substrate oxidation systems (MxaFI-type methanol dehydrogenase versus XoxF-type methanol dehydrogenase; methylamine dehydrogenase versus N-methylglutamate pathway) and different potentials for denitrification (assimilatory versus respiratory nitrate reduction). By comparing pairs of closely related genomes, we uncover that site-specific recombination is the main means of genomic evolution and strain divergence, including lateral transfers of genes from both closely- and distantly related taxa. The new ecotypes and the new genomes contribute significantly to our understanding of the extent of genomic and metabolic diversity among organisms of the same family inhabiting the same ecological niche. These organisms also provide novel experimental models for studying the complexity and the function of the microbial communities active in methylotrophy.
Aerobic methanotrophs oxidize methane at ambient temperatures and pressures and are therefore attractive systems for methane-based bioconversions. In this work, we developed and validated genetic ...tools for Methylomicrobium buryatense, a haloalkaliphilic gammaproteobacterial (type I) methanotroph. M. buryatense was isolated directly on natural gas and grows robustly in pure culture with a 3-h doubling time, enabling rapid genetic manipulation compared to many other methanotrophic species. As a proof of concept, we used a sucrose counterselection system to eliminate glycogen production in M. buryatense by constructing unmarked deletions in two redundant glycogen synthase genes. We also selected for a more genetically tractable variant strain that can be conjugated with small incompatibility group P (IncP)-based broad-host-range vectors and determined that this capability is due to loss of the native plasmid. These tools make M. buryatense a promising model system for studying aerobic methanotroph physiology and enable metabolic engineering in this bacterium for industrial biocatalysis of methane.
We investigated phenotypes of mutants of Methylotenera mobilis JLW8 with lesions in genes predicted to encode functions of the denitrification pathway, as well as mutants with mutations in methanol ...dehydrogenase-like structural genes xoxF1 and xoxF2, in order to obtain insights into denitrification and methanol metabolism by this bacterium. By monitoring the accumulation of nitrous oxide, we demonstrate that a periplasmic nitrate reductase, NAD(P)-linked and copper-containing nitrite reductases, and a nitric oxide reductase are involved in the denitrification pathway and that the pathway must be operational in aerobic conditions. However, only the assimilatory branch of the denitrification pathway was essential for growth on methanol in nitrate-supplemented medium. Mutants with mutations in each of the two xoxF genes maintained their ability to grow on methanol, but not the double XoxF mutant, suggesting that XoxF proteins act as methanol dehydrogenase enzymes in M. mobilis JLW8. Reduced levels of nitrous oxide accumulated by the XoxF mutants compared to the wild type suggest that these enzymes must be capable of donating electrons for denitrification.
We report observations on the dynamics of bacterial communities in response to methane stimulus in laboratory microcosm incubations prepared with lake sediment samples. We first measured taxonomic ...compositions of long-term enrichment cultures and determined that, although dominated by Methylococcaceae types, these cultures also contained accompanying types belonging to a limited number of bacterial taxa, methylotrophs and non-methylotrophs. We then followed the short-term community dynamics, in two oxygen tension regimens (150 μM and 15 μM), observing rapid loss of species diversity. In all microcosms, a single type of Methylobacter represented the major methane-oxidizing partner. The accompanying members of the communities revealed different trajectories in response to different oxygen tensions, with Methylotenera species being the early responders to methane stimulus under both conditions. The communities in both conditions were convergent in terms of their assemblage, suggesting selection for specific taxa. Our results support prior observations from metagenomics on distribution of carbon from methane among diverse bacterial populations and further suggest that communities are likely responsible for methane cycling, rather than a single type of microbe.
Fluorides are thought to be a major cause of osteocarcinogenesis, due to their widespread industrial use, ability to accumulate in bone tissue, and genotoxic and probable carcinogenic properties. In ...vitro experiments investigating the genotoxic potential of fluorides in bone tissue models can provide valuable indirect information on their involvement in osteocarcinogenesis. Here, we investigated whether sodium fluoride (NaF) has the ability to induce DNA damage and chromosomal abnormalities in human osteosarcoma cells after 48 and 72 h of exposure. The cell cultures were treated with NaF in concentrations of 0, 20, 100 and 200 μg/ml. The level of DNA damage was assessed by the comet assay, and the frequency of chromosomal abnormalities by a micronucleus test. A significant increase in DNA damage indicators was noted in the samples treated with fluoride concentrations of 100 and 200 µg/ml, after 48 and 72 h of exposure. The micronucleus test revealed a dose-dependent increase in cells with micronuclei, nucleoplasmic bridges and nuclear protrusions. Increasing the concentration of NaF led to an increase in the prevalence of cytogenetic indicators after both treatment durations. This demonstrated ability of fluorine to exert genotoxic effects on bone cells indirectly indicates the possible importance of fluoride in the aetiology of osteosarcoma.
Most microbes in the biosphere remain unculturable. Whole genome shotgun (WGS) sequencing of environmental DNA (metagenomics) can be used to study the genetic and metabolic properties of natural ...microbial communities. However, in communities of high complexity, metagenomics fails to link specific microbes to specific ecological functions. To overcome this limitation, we developed a method to target microbial subpopulations by labeling DNA through stable isotope probing (SIP), followed by WGS sequencing. Metagenome analysis of microbes from Lake Washington in Seattle that oxidize single-carbon (C1) compounds shows specific sequence enrichments in response to different C1 substrates, revealing the ecological roles of individual phylotypes. We also demonstrate the utility of our approach by extracting a nearly complete genome of a novel methylotroph, Methylotenera mobilis, reconstructing its metabolism and conducting genome-wide analyses. This high-resolution, targeted metagenomics approach may be applicable to a wide variety of ecosystems.
Three strains of methylotrophic Rhodocyclaceae (FAM1(T), RZ18-153 and RZ94) isolated from Lake Washington sediment samples were characterized. Based on phylogenetic analysis of 16S rRNA gene ...sequences the strains should be assigned to the genus Methyloversatilis. Similarly to other members of the family, the strains show broad metabolic capabilities and are able to utilize a number of organic acids, alcohols and aromatic compounds in addition to methanol and methylamine. The main fatty acids were 16:1ω7c (49-59%) and 16:0 (32-29%). Genomes of all isolates were sequenced, assembled and annotated in collaboration with the DOE Joint Genome Institute (JGI). Genome comparison revealed that the strains FAM1T, RZ18-153 and RZ94 are closely related to each other and almost equally distant from two previously described species of the genus Methyloversatilis, Methyloversatilis universalis and Methyloversatilis thermotolerans. Like other methylotrophic species of the genus Methyloversatilis, all three strains possess one-subunit PQQ-dependent ethanol/methanol dehydrogenase (Mdh-2), the N-methylglutamate pathway and the serine cycle (isocitrate lyase/malate synthase, Icl/ms(+) variant). Like M. universalis, strains FAM1(T), RZ18-153 and RZ94 have a quinohemoprotein amine dehydrogenase, a tungsten-containing formaldehyde ferredoxin oxidoreductase, phenol hydroxylase, and the complete Calvin cycle. Similarly to M. thermotolerans, the three strains possess two-subunit methanol dehydrogenase (MxaFI), monoamine oxidase (MAO) and nitrogenase. Based on the phenotypic and genomic data, the strains FAM1(T), RZ18-153 and RZ94 represent a novel species of the genus Methyloversatilis, for which the name Methyloversatilis discipulorum sp. nov. is proposed. The type strain is FAM1(T) ( = JCM 30542(T) = VKM = B-2888(T)).
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