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Search indexed NASA NTRS and DOE OSTI research on propulsion, heat transfer, battery materials and energy systems. Follow report and document links to the original sources.

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At least 19 records
All sourcesNASA NTRSDOE OSTI
DOE OSTI2021

Improving the Methanol Tolerance of an Escherichia coli Methylotroph via Adaptive Laboratory Evolution Enhances Synthetic Methanol Utilization

There is great interest in developing synthetic methylotrophs that harbor methane and methanol utilization pathways in heterologous hosts such as Escherichia coli for industrial bioconversion of one-carbon compounds. While there are recent reports that describe the successful engineering of synthetic methylotrophs, additional efforts are required to achieve the robust methylotrophic phenotypes required for industrial realization. Here, we address an important issue of synthetic methylotrophy in E. coli : methanol toxicity. Both methanol, and its oxidation product, formaldehyde, are cytotoxic to cells. Methanol alters the fluidity and biological properties of cellular membranes while formaldehyde reacts readily with proteins and nucleic acids. Thus, efforts to enhance the methanol tolerance of synthetic methylotrophs are important. Here, adaptive laboratory evolution was performed to improve the methanol tolerance of several E. coli strains, both methylotrophic and non-methylotrophic. Serial batch passaging in rich medium containing toxic methanol concentrations yielded clones exhibiting improved methanol tolerance. In several cases, these evolved clones exhibited a > 50% improvement in growth rate and biomass yield in the presence of high methanol concentrations compared to the respective parental strains. Importantly, one evolved clone exhibited a two to threefold improvement in the methanol utilization phenotype, as determined via 13 C-labeling, at non-toxic, industrially relevant methanol concentrations compared to the respective parental strain. Whole genome sequencing was performed to identify causative mutations contributing to methanol tolerance. Common mutations were identified in 30S ribosomal subunit proteins, which increased translational accuracy and provided insight into a novel methanol tolerance mechanism. This study addresses an important issue of synthetic methylotrophy in E. coli and provides insight as to how methanol toxicity can be alleviated via enhancing methanol tolerance. Coupled improvement of methanol tolerance and synthetic methanol utilization is an important advancement for the field of synthetic methylotrophy.

Bennett, R. Kyle↗
DOE OSTI2020

Adaptive laboratory evolution of methylotrophic Escherichia coli enables synthesis of all amino acids from methanol-derived carbon

Recent attempts to create synthetic Escherichia coli methylotrophs identified that de novo biosynthesis of amino acids, in the presence of methanol, presents significant challenges in achieving autonomous methylotrophic growth. Previously engineered methanol-dependent strains required co-utilization of stoichiometric amounts of co-substrates and methanol. As such, these strains could not be evolved to grow on methanol alone. In this work, we have explored an alternative approach to enable biosynthesis of all amino acids from methanol-derived carbon in minimal media without stoichiometric coupling. First, we identified that biosynthesis of threonine was limiting the growth of our methylotrophic E. coli. To address this, we performed adaptive laboratory evolution to generate a strain that grew efficiently in minimal medium with methanol and threonine. Methanol assimilation and growth of the evolved strain were analyzed and, interestingly, we found that the evolved strain synthesized all amino acids, including threonine, from methanol-derived carbon. Here, the evolved strain was then further engineered through overexpression of an optimized threonine biosynthetic pathway. We show that the resulting methylotrophic E. coli strain has a methanol-dependent growth phenotype with homoserine as co-substrate. In contrast to previous methanol-dependent strains, co-utilization of homoserine is not stoichiometrically linked to methanol assimilation. As such, future engineering of this strain and successive adaptive evolution could enable autonomous growth on methanol as the sole carbon source.

13C-labeling analysis↗
DOE OSTI2025

Spatial evidence of cryptic methane cycling and methylotrophic metabolisms along a land–ocean transect in salt marsh sediment

Methylotrophic methanogenesis in the sulfate-rich zone of coastal and marine sediments couples with anaerobic oxidation of methane (AOM), forming the cryptic methane cycle. This study provides evidence of cryptic methane cycling in the sulfate-rich zone across a land–ocean transect of four stations–two brackish, one marine, and one hypersaline–within the Carpinteria Salt Marsh Reserve (CSMR), southern California, USA. Samples from the top 20 cm of sediment from the transect were analyzed through geochemical and molecular (16S rRNA) techniques, in-vitro methanogenesis incubations, and radiotracer incubations utilizing 35 S-SO 4 , 14 C-mono-methylamine, and 14 C-CH 4 . Sediment methane concentrations were consistently low (3 to 28 µM) at all stations, except for the marine station, where methane increased with depth reaching 665 µM. Methanogenesis from mono-methylamine was detected throughout the sediment at all stations with estimated CH 4 production rates in the sub-nanomolar to nanomolar range per cm 3 sediment and day. 16S rRNA analysis identified methanogenic archaea (Methanosarcinaceae, Methanomassiliicoccales, and Methanonatronarchaeacea) capable of producing methane from methylamines in sediment where methylotrophic methanogenesis was found to be active. Metabolomic analysis of porewater showed mono-methylamine was mostly undetectable (<3 µM) or present in trace amounts (<10 µM) suggesting rapid metabolic turnover. In-vitro methanogenesis incubations of natural sediment showed no linear methane buildup, suggesting a process limiting methane emissions. AOM activity, measured with 14 C-CH 4 , overlapped with methanogenesis from mono-methylamine activity at all stations, with rates ranging from 0.03 to 19.4 nmol cm −3 d −1 . Geochemical porewater analysis showed the CSMR sediments are rich in sulfate and iron. Porewater sulfate concentrations (9–91 mM) were non-limiting across the transect, supporting sulfate reduction activity (1.5–2,506 nmol cm −3 d −1 ). Porewater sulfide and iron (II) profiles indicated that the sediment transitioned from a predominantly iron-reducing environment at the two brackish stations to a predominantly sulfate-reducing environment at the marine and hypersaline stations, which coincided with the presence of phyla (Desulfobacterota) involved in these processes. AOM activity overlapped with sulfate reduction and porewater iron (II) concentrations suggesting that AOM is likely coupled to sulfate and possibly iron reduction at all stations. However, 16S rRNA analysis identified anaerobic methanotrophs (ANME-2) only at the marine and hypersaline stations while putative methanogens were found in sediment across all stations. In one sediment horizon at the marine station, methanogen families (Methanosarcinaceae, Methanosaetaceae, Methanomassiliicoccales, and Methanoregulaceae) and ANME 2a,2b, and 2c groups were found together. Collectively, our data suggest that at the brackish stations methanogens alone may be involved in cryptic methane cycling, while at the marine and hypersaline stations both groups may be involved in the process. Differences in rate constants from incubations with 14 C-labeled methane and mono-methylamine suggest a non-methanogenic process oxidizing mono-methylamine to inorganic carbon, likely mediated by sulfate-reducing bacteria. Understanding the potential competition of sulfate reducers with methanogens for mono-methylamine needs further investigation as it might be another important process responsible for low methane emissions in salt marshes.

Anaerobic oxidation of methane↗
DOE OSTI2025

Expanding on the ability of trivalent actinides to support microbial alcohol metabolism in evolved methylotrophic bacterium

The 4 f elements, known as the lanthanides, have only recently been recognized as biologically essential metals. These elements can be found in the active site of certain alcohol dehydrogenase (ADH) enzymes, which are used by methylotrophic bacteria to metabolize simple alcohols such as methanol and ethanol. The 5 f elements, known as the actinides, often show similar chemical behaviour to the lanthanides and recent reports demonstrated that americium (Am) and curium (Cm) can replace lanthanides in certain ADHs to yield a catalytically competent enzyme and support bacterial life. In this work, we expand on these results to show the trivalent ions actinium (Ac3+), americium (Am3+), curium (Cm3+), berkelium (Bk3+), and californium (Cf3+) all support growth of an evolved strain of the model methylotroph Methylobacterium extorquens AM1. Further in vitro experiments using a reconstituted ethanol dehydrogenase enzyme confirm the ability of this enzyme to utilize trivalent actinide ions.

Woods, Joshua J↗
DOE OSTI2024

Diverse and unconventional methanogens, methanotrophs, and methylotrophs in metagenome-assembled genomes from subsurface sediments of the Slate River floodplain, Crested Butte, CO, USA

We use metagenome-assembled genomes (MAGs) to understand single-carbon (C1) compound-cycling—particularly methane-cycling—microorganisms in montane riparian floodplain sediments. We generated 1,233 MAGs (>50% completeness and <10% contamination) from 50- to 150-cm depth below the sediment surface capturing the transition between oxic, unsaturated sediments and anoxic, saturated sediments in the Slate River (SR) floodplain (Crested Butte, CO, USA). We recovered genomes of putative methanogens, methanotrophs, and methylotrophs (n = 57). Methanogens, found only in deep, anoxic depths at SR, originate from three different clades (Methanoregulaceae, Methanotrichaceae, and Methanomassiliicoccales), each with a different methanogenesis pathway; putative methanotrophic MAGs originate from within the Archaea (Candidatus Methanoperedens) in anoxic depths and uncultured bacteria (Ca. Binatia) in oxic depths. Genomes for canonical aerobic methanotrophs were not recovered. Ca. Methanoperedens were exceptionally abundant (~1,400× coverage, >50% abundance in the MAG library) in one sample that also contained aceticlastic methanogens, indicating a potential C1/methane-cycling hotspot. Ca. Methylomirabilis MAGs from SR encode pathways for methylotrophy but do not harbor methane monooxygenase or nitrogen reduction genes. Comparative genomic analysis supports that one clade within the Ca. Methylomirabilis genus is not methanotrophic. The genetic potential for methylotrophy was widespread, with over 10% and 19% of SR MAGs encoding a methanol dehydrogenase or substrate-specific methyltransferase, respectively. MAGs from uncultured Thermoplasmata archaea in the Ca. Gimiplasmatales (UBA10834) contain pathways that may allow for anaerobic methylotrophic acetogenesis. Overall, MAGs from SR floodplain sediments reveal a potential for methane production and consumption in the system and a robust potential for methylotrophy.

58 GEOSCIENCES↗
DOE OSTI2022

Employing methylotrophs for a green economy: one-carbon to fuel them all and through metabolism redesign them

Microbial platforms are currently being optimized to revolutionize industrial energy production while mitigating shortages of global resources and food supplies. Here, in this work, we address recent advances to develop bacterial methylotrophic platforms as promising platforms enabling the reuse of products and materials (at their highest value) while reducing waste and pollution.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

Transcriptomic Network Analysis of Cyanobacterial-Methylotroph Interactions in Coculture and Axenic Conditions

A previous study demonstrated the potential for Cyanobacterial-Methylotroph cocultures to facilitate biogas processing as well as to be used in other biotechnological applications. To advance this technology, we investigated potential interactions between Cyanobacterium stanieri HL-69 (HL69) and Methylotuvimicrobium alkaliphilum 20Z (20Z) by inferring and analyzing gene co-expression networks under co-culture and axenic conditions. Five different co-expression networks were examined. These networks were inferred using gene expression profiles for 20Z axenic condition, HL-69 axenic, HL-69, 20Z coculture, HL-69 coculture, and cross-species HL-69-20Z coculture. Through the analysis of node (gene) betweenness and node normalized degree values in all five network cases, we compared adjustments in gene expression between growth conditions (axenic vs co-culture) as well as identify biological functions relevant to interspecies interactions. This analysis was done to distinguish between gene interactions within an organism and gene interactions between two organisms. Moreover, for all five cases we investigated two different network cutoff levels of 3,000 and 10,000. By shedding light on inter- and intra- species interactions, we hope to gain a better understanding of how these two organisms interact. This research will allow the investigation of further biotechnological applications of coculture systems and optimization of such applications for biotechnological purposes.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

Methods and systems for methylotrophic production of organic compounds

The present disclosure identifies pathways, mechanisms, systems and methods to confer production of carbon-based products of interest, such as sugars, alcohols, chemicals, amino acids, polymers, fatty acids and their derivatives, hydrocarbons, isoprenoids, and intermediates thereof, in engineered and/or evolved methylotrophs such that these organisms efficiently convert C1 compounds, such as formate, formic acid, formaldehyde or methanol, to organic carbon-based products of interest, and in particular the use of organisms for the commercial production of various carbon-based products of interest.

Shetty, Reshma P.↗
DOE OSTI2026

Bioelectrocatalytic conversion of CO₂ to PHA bioplastics using engineered methylotrophs

The sustainable generation of biodegradable plastics represents an opportunity to capture atmospheric CO 2 while reducing plastic waste accumulation in the environment. This study implements an integrated platform for bioelectrocatalytic CO 2 conversion to medium-chain-length polyhydroxyalkanoates (mcl-PHAs). Immobilizing cobalt phthalocyanine electrocatalysts on a covalent-organic framework in a gas recirculation electrolyzer enabled CO 2 -to-methanol conversion with a carbon conversion efficiency of 98%. Integration of polymer biosynthesis pathways enabled Methylotuvimicrobium alcaliphilum 20Z R to produce ~20% mcl-PHA of the dry cell weight with a CO 2 -to-bioproducts carbon conversion efficiency of 50%. This cell line was adapted to high sodium bicarbonate media, eliminating costly intermediate separation steps while improving economic potential. Transcriptomic analysis revealed sulfate transporters and peptidoglycan biosynthesis as key pathways involved in sodium bicarbonate halotolerance. Altogether, this research presents a foundation for integrating divergent chemical and biological processes into a transformative electrobiomanufacturing platform, addressing the need for alternative pipelines for generating valuable plastics and chemicals.

CO2 utilization↗
DOE OSTI2021

Genomic Analysis of the Yet-Uncultured Binatota Reveals Broad Methylotrophic, Alkane-Degradation, and Pigment Production Capacities

A wide range of microbial lineages remain uncultured, yet little is known regarding their metabolic capacities, physiological preferences, and ecological roles in various ecosystems. We conducted a thorough comparative genomic analysis of 108 genomes belonging to the Binatota (UBP10), a globally distributed, yet-uncharacterized bacterial phylum.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2020

Synthetic methylotrophs

The present invention provides a non-naturally occurring microbe capable of growing in a medium comprising methanol, comprising a heterologous polynucleotide encoding a heterologous methanol dehydrogenase (MDH) derived from a Corynebacterium organism (Cor), wherein the MDH is expressed in the microbe, and wherein the MDH exhibits a Km of no more than 3 mM for methanol. Also provided are uses of the non-naturally occurring microbe for oxidizing methanol and producing a metabolite as well as the preparation of the non-naturally occurring microbe.

09 BIOMASS FUELS↗
DOE OSTI2024

Engineering Terpene Production Pathways in Methylobacterium extorquens AM1

Terpenes are diverse specialized metabolites naturally found within plants and have important roles in inter-species communication, adaptation and interaction with the environment. Their industrial applications span a broad range, including fragrances, flavors, cosmetics, natural colorants to agrochemicals and therapeutics, yet formal chemical synthesis is economically challenging due to structural complexities. Engineering terpene biosynthesis could represent an alternative in microbial biotechnological workhorses, such as Saccharomyces cerevisiae or Escherichi coli, utilizing sugars or complex media as feedstocks. Host species that metabolize renewable and affordable carbon sources may offer unique sustainable biotechnological alternatives. Methylotrophs are bacteria with the capacity to utilize one-carbon feedstocks, such as methanol or formate. They colonize the phyllosphere (above-ground area) of plants, and many accumulate abundant carotenoid pigments. Methylotrophs have the capacity to take up and use a subset of the rare earth elements known as lanthanides. These metals can enhance one-carbon (methylotrophic) metabolism. Here, we investigated whether manipulating the metabolism enables and enhances terpene production. A carotenoid-deficient mutant potentially liberates carbon, which may contribute to bioproduct accumulation. To test this hypothesis, terpene-producing bacterial strains regulated by two distinct promoters were generated. Wildtype Methylobacterium extorquens, ∆Meta1_3665, a methylotrophic mutant lacking the carotenoid pathway, and an E. coli strain were transformed with an exogenous terpene pathway and grown both in the presence and absence of lanthanides. The extraction, and the comparison of analytical profiles, provided evidence that engineered cultured M. extorquens under control of a native, inducible methylotrophic promoter can yield the sesquiterpene patchoulol when supplemented with lanthanide. In contrast, using a moderate-strength constitutive promoter failed to give production. We demonstrated colonization of the phyllosphere with the engineered strains, supporting the future engineering of selected species of the plant microbiome and with promising implications for the synthetic biology of small molecules.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

Methylotrophy in the Mire: direct and indirect routes for methane production in thawing permafrost

While wetlands are major sources of biogenic methane (CH 4 ), our understanding of resident microbial metabolism is incomplete, which compromises the prediction of CH 4 emissions under ongoing climate change. Here, we employed genome-resolved multi-omics to expand our understanding of methanogenesis in the thawing permafrost peatland of Stordalen Mire in Arctic Sweden. In quadrupling the genomic representation of the site’s methanogens and examining their encoded metabolism, we revealed that nearly 20% of the metagenome-assembled genomes (MAGs) encoded the potential for methylotrophic methanogenesis. Further, 27% of the transcriptionally active methanogens expressed methylotrophic genes; for Methanosarcinales and Methanobacteriales MAGs, these data indicated the use of methylated oxygen compounds (e.g., methanol), while for Methanomassiliicoccales, they primarily implicated methyl sulfides and methylamines. In addition to methanogenic methylotrophy, >1,700 bacterial MAGs across 19 phyla encoded anaerobic methylotrophic potential, with expression across 12 phyla. Metabolomic analyses revealed the presence of diverse methylated compounds in the Mire, including some known methylotrophic substrates. Active methylotrophy was observed across all stages of a permafrost thaw gradient in Stordalen, with the most frozen non-methanogenic palsa found to host bacterial methylotrophy and the partially thawed bog and fully thawed fen seen to house both methanogenic and bacterial methylotrophic activities. Methanogenesis across increasing permafrost thaw is thus revised from the sole dominance of hydrogenotrophic production and the appearance of acetoclastic at full thaw to consider the co-occurrence of methylotrophy throughout. Collectively, these findings indicate that methanogenic and bacterial methylotrophy may be an important and previously underappreciated component of carbon cycling and emissions in these rapidly changing wetland habitats.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2020

Regulatory interventions improve the biosynthesis of limiting amino acids from methanol carbon to improve synthetic methylotrophy in Escherichia coli

Synthetic methylotrophy aims to engineer methane and methanol utilization pathways in platform hosts like Escherichia coli for industrial bioprocessing of natural gas and biogas. While recent attempts to engineer synthetic methylotrophs have proved successful, autonomous methylotrophy, that is, the ability to utilize methane or methanol as sole carbon and energy substrates, has not yet been realized. Here, we address an important limitation of autonomous methylotrophy in E. coli: the inability of the organism to synthesize several amino acids when grown on methanol. We targeted global and local amino acid regulatory networks. Those include removal of amino acid allosteric feedback inhibition (argA H15Y , ilvA L447F , hisG E271K , leuA G462D , proB D107N , thrA S345F, trpE S40F ), knockouts of transcriptional repressors (ihfA, metJ); and overexpression of amino acid biosynthetic operons (hisGDCBHAFI, leuABCD, thrABC, trpEDCBA) and transcriptional regulators (crp, purR). Compared to the parent methylotrophic E. coli strain that was unable to synthesize these amino acids from methanol carbon, these strategies resulted in improved biosynthesis of limiting proteinogenic amino acids (histidine, leucine, lysine, methionine, phenylalanine, threonine, tyrosine) from methanol carbon. In several cases, improved amino acid biosynthesis from methanol carbon led to improvements in methylotrophic growth in methanol minimal medium supplemented with a small amount of yeast extract. Here, this study addresses a key limitation currently preventing autonomous methylotrophy in E. coli and possibly other synthetic methylotrophs and provides insight as to how this limitation can be alleviated via global and local regulatory modifications.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2025

Sulfide stress tolerance as a controller of methane production in temperate wetlands

Abstract Wetlands are a major source of methane emissions and contribute to the observed increase in atmospheric methane over the last 20 years. Methane production in wetlands is the final step of carbon decomposition performed by anaerobic archaea. Although hydrogen/carbon dioxide and acetate are the substrates most often attributed to methanogenesis, other substrates—such as methylated compounds—may additionally play important roles in driving methane production in wetland systems. Here we conducted mesocosm experiments combined with genome-resolved metatranscriptomics to investigate the impact of diverse methanogenic substrate amendment on methanogenesis in two high methane-emitting wetlands with distinct geochemistry, termed P7 and P8. Methanol amendment resulted in high methane production at both sites, whereas acetate and formate amendment only stimulated methanogenesis in P7 mesocosms, where aqueous sulfide concentrations were lower. In P7 sediments, formate amendment fueled acetogenic microbes that produced acetate, which was subsequently utilized by acetoclastic methanogens. In contrast to expression profiles in P7 mesocosms, active methylotrophic methanogen genomes from P8 showed increased expression of genes related to membrane remodeling and DNA damage repair, indicative of stress tolerance mechanisms to counter sulfide toxicity. Methylotrophic methanogenesis generates higher free energy yields than acetoclastic methanogenesis, which likely enables allocation of more energy toward stress responses. These findings contribute to the growing body of literature highlighting methylotrophic methanogenesis as an important methane production pathway in wetlands. By using less competitive substrates like methanol that provide greater energy yields, methylotrophic methanogens may invest in physiological strategies that provide competitive advantages across a range of environmental stresses.

Environmental Sciences & Ecology↗
DOE OSTI2020

Triggering the stringent response enhances synthetic methanol utilization in Escherichia coli

Synthetic methylotrophy aims to engineer methane and methanol utilization pathways in platform hosts like Escherichia coli for industrial bioprocessing of natural gas and biogas. While recent attempts to engineer synthetic methylotrophs have proved successful, autonomous methylotrophy, i.e. the ability to utilize methane or methanol as sole carbon and energy substrates, has not yet been realized. Here in this paper, we address an important limitation of autonomous methylotrophy in E. coli: the inability of the organism to synthesize several amino acids when grown on methanol. By activating the stringent/stress response via ppGpp overproduction, or DksA and RpoS overexpression, we demonstrate improved biosynthesis of proteinogenic amino acids via endogenous upregulation of amino acid synthesis pathway genes. Thus, we were able to achieve biosynthesis of several limiting amino acids from methanol-derived carbon, in contrast to the control methylotrophic E. coli strain. This study addresses a key limitation currently preventing autonomous methylotrophy in E. coli and possibly other synthetic methylotrophs and provides insight as to how this limitation can be alleviated via stringent/stress response activation.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2021

EfgA is a conserved formaldehyde sensor that leads to bacterial growth arrest in response to elevated formaldehyde

Normal cellular processes give rise to toxic metabolites that cells must mitigate. Formaldehyde is a universal stressor and potent metabolic toxin that is generated in organisms from bacteria to humans. Methylotrophic bacteria such as Methylorubrum extorquens face an acute challenge due to their production of formaldehyde as an obligate central intermediate of single-carbon metabolism. Mechanisms to sense and respond to formaldehyde were speculated to exist in methylotrophs for decades but had never been discovered. Here, we identify a member of the DUF336 domain family, named efgA for enhanced formaldehyde growth, that plays an important role in endogenous formaldehyde stress response in M . extorquens PA1 and is found almost exclusively in methylotrophic taxa. Our experimental analyses reveal that EfgA is a formaldehyde sensor that rapidly arrests growth in response to elevated levels of formaldehyde. Heterologous expression of EfgA in Escherichia coli increases formaldehyde resistance, indicating that its interaction partners are widespread and conserved. EfgA represents the first example of a formaldehyde stress response system that does not involve enzymatic detoxification. Thus, EfgA comprises a unique stress response mechanism in bacteria, whereby a single protein directly senses elevated levels of a toxic intracellular metabolite and safeguards cells from potential damage.

59 BASIC BIOLOGICAL SCIENCES↗