2022 Molecular Basis of Microbial One-Carbon Metabolism Gordon Research Conference and Seminar
Final Conference Program and Registration List
Marx, Christopher J↗
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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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Novel microbial based enzymatic CO2 fixation mechanisms: Conformational control of enzymatic reactivity
The broad, long-term goal of the research is to provide insight into the mechanism of novel carboxylation and electron bifurcation reactions as paradigms to study how enzyme utilized protein conformational change to protect reactive intermediates and/or control the pathway in a catalytic cycle. catalyzed by enzymes involved in bacterial alkene, ketone, and epoxide metabolism. We are examining two paradigms in which conformational change is a key mechanistic feature of 1) protecting reaction intermediates that are sensitive to breakdown in the presence of water and 2) directing electron flow in reactions that split electron pairs along two pathways in reactions that fine tune metabolic efficiency in microbial metabolism. Both paradigms are of key importance in defining the molecular determinants of reactions relevant to CO 2 capture and fuel upcycling. In the first paradigm the compounds acetone and bicarbonate are activated by the addition of phosphate creating reaction intermediates (phosphoenolacetone and carboxyphosphate) that are very sensitive to breakdown in the presence of water. We are studying the mechanism of how the enzyme protects these reactive intermediates from water by encapsulating them upon their formation within a hydrophobic protein channel leading to an enzyme active site approximately 40 Å away. In the second paradigm, we are examining how the direction of electron transfer reactions can be modulated through protein conformational change that alters either the difference driving force or distance or both between electron transfer pathways in enzymes that catalyze electron bifurcation. We are using a combination of enzyme kinetics, mass spectrometry, spectroscopy, and structural analysis through X-ray diffraction and scattering methods to characterize the mechanism of conformational changes along the catalytic path. Within the context of the mission of the Department of Energy and the core activities of the Energy Biosciences, the results obtained in the study will reveal new insights into the fundamentals of novel carboxylation / CO 2 capture chemical transformations and electron transfer reactions that equate to biological fuel upcycling.
37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
Metabolic Potential and Activity in Fluids of the Coast Range Ophiolite Microbial Observatory, California, USA
Metabolic potential and activity associated with hydrogen and carbon monoxide were characterized in fluids sampled from the the Coast Range Ophiolite Microbial Observatory (CROMO). CROMO consists of two clusters of science-dedicated wells drilled to varying depths up to 35m in the actively serpentinizing, Jurassic-age Coast Range Ophiolite of Northern California, along with a suite of pre-existing monitoring wells at the same site. Consistent with the fluid chemistry observed in other serpentinizing systems, CROMO fluids are highly alkaline, with pH up to 12.5, high in methane, with concentrations up 1600 micromolar, and low in dissolved inorganic carbon (DIC), with concentrations of 10's to 100's of micromolar. CROMO is conspicuous for fluid H2 concentrations that are consistently sub-micromolar, orders of magnitude lower than is typical of other systems. However, higher H2 concentrations (10's -100's of micromolar) at an earlier stage of fluid chemical evolution are predicted by, or consistent with: thermodynamic models for fluid chemistry based on parent rock composition equivalent to local peridotite and with water:rock ratio constrained by observed pH; the presence of magnetite at several wt% in CROMO drill cores; and concentrations of formate and carbon monoxide that would require elevated H2 if formed in equilibrium with H2 and DIC. Calculated Gibbs energy changes for reaction of H2 and CO in each of several metabolisms, across the range of fluid composition encompassed by the CROMO wells, range from bioenergetically feasible (capable of driving ATP synthesis) to thermodynamically unfavorable. Active consumption relative to killed controls was observed for both CO and H2 during incubation of fluids from the pre-existing monitoring wells; in incubations of freshly cored solids, consumption was only observed in one sample set (corresponding to the lowest pH) out of three. The specific metabolisms by which H2 and CO are consumed remain to be determined.
Hydrogen↗
Biogeochemical Processes in Microbial Ecosystems
The hierarchical organization of microbial ecosystems determines process rates that shape Earth's environment, create the biomarker sedimentary and atmospheric signatures of life and define the stage upon which major evolutionary events occurred. In order to understand how microorganisms have shaped the global environment of Earth and potentially, other worlds, we must develop an experimental paradigm that links biogeochemical processes with ever-changing temporal and spatial distributions of microbial population, and their metabolic properties. Photosynthetic microbial mats offer an opportunity to define holistic functionality at the millimeter scale. At the same time, their Biogeochemistry contributes to environmental processes on a planetary scale. These mats are possibly direct descendents of the most ancient biological communities; communities in which oxygenic photosynthesis might have been invented. Mats provide one of the best natural systems to study how microbial populations associate to control dynamic biogeochemical gradients. These are self-sustaining, complete ecosystems in which light energy absorbed over a diel (24 hour) cycle drives the synthesis of spatially-organized, diverse biomass. Tightly-coupled microorganisms in the mat have specialized metabolisms that catalyze transformations of carbon, nitrogen. sulfur, and a host of other elements.
DesMarais, David J.↗
Metabolic engineering strategies to produce medium-chain oleochemicals via acyl-ACP:CoA transacylase activity
Abstract Microbial lipid metabolism is an attractive route for producing oleochemicals. The predominant strategy centers on heterologous thioesterases to synthesize desired chain-length fatty acids. To convert acids to oleochemicals (e.g., fatty alcohols, ketones), the narrowed fatty acid pool needs to be reactivated as coenzyme A thioesters at cost of one ATP per reactivation - an expense that could be saved if the acyl-chain was directly transferred from ACP- to CoA-thioester. Here, we demonstrate such an alternative acyl-transferase strategy by heterologous expression of PhaG, an enzyme first identified in Pseudomonads , that transfers 3-hydroxy acyl-chains between acyl-carrier protein and coenzyme A thioester forms for creating polyhydroxyalkanoate monomers. We use it to create a pool of acyl-CoA’s that can be redirected to oleochemical products. Through bioprospecting, mutagenesis, and metabolic engineering, we develop three strains of Escherichia coli capable of producing over 1 g/L of medium-chain free fatty acids, fatty alcohols, and methyl ketones.
37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
Non-canonical d-xylose and l-arabinose metabolism via d-arabitol in the oleaginous yeast Rhodosporidium toruloides
Abstract R. toruloides is an oleaginous yeast, with diverse metabolic capacities and high tolerance for inhibitory compounds abundant in plant biomass hydrolysates. While R. toruloides grows on several pentose sugars and alcohols, further engineering of the native pathway is required for efficient conversion of biomass-derived sugars to higher value bioproducts. A previous high-throughput study inferred that R. toruloides possesses a non-canonical l -arabinose and d -xylose metabolism proceeding through d -arabitol and d -ribulose. In this study, we present a combination of genetic and metabolite data that refine and extend that model. Chiral separations definitively illustrate that d -arabitol is the enantiomer that accumulates under pentose metabolism. Deletion of putative d -arabitol-2-dehydrogenase (RTO4_9990) results in > 75% conversion of d -xylose to d -arabitol, and is growth-complemented on pentoses by heterologous xylulose kinase expression. Deletion of putative d -ribulose kinase (RTO4_14368) arrests all growth on any pentose tested. Analysis of several pentose dehydrogenase mutants elucidates a complex pathway with multiple enzymes mediating multiple different reactions in differing combinations, from which we also inferred a putative l -ribulose utilization pathway. Our results suggest that we have identified enzymes responsible for the majority of pathway flux, with additional unknown enzymes providing accessory activity at multiple steps. Further biochemical characterization of the enzymes described here will enable a more complete and quantitative understanding of R. toruloides pentose metabolism. These findings add to a growing understanding of the diversity and complexity of microbial pentose metabolism.
59 BASIC BIOLOGICAL SCIENCES↗
Kinetic, metabolic, and statistical analytics: addressing metabolic transport limitations among organelles and microbial communities
Microbial organisms engage in a variety of metabolic interactions. A crucial part of these interactions is the exchange of molecules between different organelles, cells, and the environment. The main forces mediating this metabolic exchange are transporters. This transport can be difficult to measure experimentally because several transport mechanisms remain opaque. However, theoretical calculations about the inputs and outputs of cells via metabolic exchanges have enabled the successful inference of the workings of intra-organismal and inter-organismal systems. Kinetic, metabolic, and statistical modeling approaches in combination with omics data are enhancing our knowledge and understanding about metabolic exchange and mass resource allocation. Furthermore, this model-driven analytics approach can guide effective experimental design and yield new insights into biological function and control.
59 BASIC BIOLOGICAL SCIENCES↗
Modification and analysis of context-specific genome-scale metabolic models: methane-utilizing microbial chassis as a case study
ABSTRACT Context-specific genome-scale model (CS-GSM) reconstruction is becoming an efficient strategy for integrating and cross-comparing experimental multi-scale data to explore the relationship between cellular genotypes, facilitating fundamental or applied research discoveries. However, the application of CS modeling for non-conventional microbes is still challenging. Here, we present a graphical user interface that integrates COBRApy, EscherPy, and RIPTiDe, Python-based tools within the BioUML platform, and streamlines the reconstruction and interrogation of the CS genome-scale metabolic frameworks via Jupyter Notebook. The approach was tested using -omics data collected for Methylotuvimicrobium alcaliphilum 20Z R , a prominent microbial chassis for methane capturing and valorization. We optimized the previously reconstructed whole genome-scale metabolic network by adjusting the flux distribution using gene expression data. The outputs of the automatically reconstructed CS metabolic network were comparable to manually optimized i IA409 models for Ca-growth conditions. However, the CS model questions the reversibility of the phosphoketolase pathway and suggests higher flux via primary oxidation pathways. The model also highlighted unresolved carbon partitioning between assimilatory and catabolic pathways at the formaldehyde-formate node. Only a very few genes and only one enzyme with a predicted function in C1 metabolism, a homolog of the formaldehyde oxidation enzyme ( fae1-2 ), showed a significant change in expression in La-growth conditions. The CS-GSM predictions agreed with the experimental measurements under the assumption that the Fae1-2 is a part of the tetrahydrofolate-linked pathway. The cellular roles of the tungsten (W)-dependent formate dehydrogenase ( fdhAB ) and fae homologs ( fae1-2 and fae3 ) were investigated via mutagenesis. The phenotype of the f dhAB mutant followed the model prediction. Furthermore, a more significant reduction of the biomass yield was observed during growth in La-supplemented media, confirming a higher flux through formate. M. alcaliphilum 20Z R mutants lacking fae1-2 did not display any significant defects in methane or methanol-dependent growth. However, contrary to fae1, the fae1-2 homolog failed to restore the formaldehyde-activating enzyme function in complementation tests. Overall, the presented data suggest that the developed computational workflow supports the reconstruction and validation of CS-GSM networks of non-model microbes. IMPORTANCE The interrogation of various types of data is a routine strategy to explore the relationship between genotype and phenotype. An efficient approach for integrating and cross-comparing experimental multi-scale data in the context of whole-genome-based metabolic network reconstruction becomes a powerful tool that facilitates fundamental and applied research discoveries. The present study describes the reconstruction of a context-specific (CS) model for the methane-utilizing bacterium, Methylotuvimicrobium alcaliphilum 20Z R . M. alcaliphilum 20Z R is becoming an attractive microbial platform for the production of biofuels, chemicals, pharmaceuticals, and bio-sorbents for capturing atmospheric methane. We demonstrate that this pipeline can help reconstruct metabolic models that are similar to manually curated networks. Furthermore, the model is able to highlight previously overlooked pathways, thus advancing fundamental knowledge of non-model microbial systems or promoting their development toward biotechnological or environmental implementations.
Kulyashov, M. A.↗
Distinct Depth-Discrete Profiles of Microbial Communities and Geochemical Insights in the Subsurface Critical Zone
Microbial assembly and metabolic potential in the subsurface critical zone (SCZ) are substantially impacted by subsurface geochemistry and hydrogeology, selecting for microbes distinct from those in surficial soils. In this study, we integrated metagenomics and geochemistry to elucidate how microbial composition and metabolic potential are shaped and impacted by vertical variations in geochemistry and hydrogeology in terrestrial subsurface sediment. A sediment core from an uncontaminated, pristine well at Oak Ridge Field Research Center in Oak Ridge, Tennessee, including the shallow subsurface, vadose zone, capillary fringe, and saturated zone, was used in this study. Our results showed that subsurface microbes were highly localized and that communities were rarely interconnected. Microbial community composition as well as metabolic potential in carbon and nitrogen cycling varied even over short vertical distances. Further analyses indicated a strong depth-related covariation of community composition with a subset of 12 environmental variables. An analysis of dissolved organic carbon (DOC) quality via ultrahigh resolution mass spectrometry suggested that the SCZ was generally a low-carbon environment, with the relative portion of labile DOC decreasing and that of recalcitrant DOC increasing along the depth, selecting microbes from copiotrophs to oligotrophs and also impacting the microbial metabolic potential in the carbon cycle. Our study demonstrates that sediment geochemistry and hydrogeology are vital in the selection of distinct microbial populations and metabolism in the SCZ.
59 BASIC BIOLOGICAL SCIENCES↗
Transitory Microbial Habitat in the Hyperarid Atacama Desert
Traces of life are nearly ubiquitous on Earth. However, a central unresolved question is whether these traces always indicate an active microbial community or whether, in extreme environments, such as hyperarid deserts, they instead reflect just dormant or dead cells. Although microbial biomass and diversity decrease with increasing aridity in the Atacama Desert, we provide multiple lines of evidence for the presence of an at times metabolically active, microbial community in one of the driest places on Earth. We base this observation on four major lines of evidence: (i) a physico-chemical characterization of the soil habitability after an exceptional rain event, (ii) identified biomolecules indicative of potentially active cells [e.g., presence of ATP, phospholipid fatty acids (PLFAs), metabolites, and enzymatic activity], (iii) measurements of in situ replication rates of genomes of uncultivated bacteria reconstructed from selected samples, and (iv) microbial community patterns specific to soil parameters and depths. We infer that the microbial populations have undergone selection and adaptation in response to their specific soil microenvironment and in particular to the degree of aridity. Collectively, our results highlight that even the hyperarid Atacama Desert can provide a habitable environment for microorganisms that allows them to become metabolically active following an episodic increase in moisture and that once it decreases, so does the activity of the microbiota. These results have implications for the prospect of life on other planets such as Mars, which has transitioned from an earlier wetter environment to today’s extreme hyperaridity.
Schulze-Makuch, Dirk↗
The anaerobic fungus Caecomyces churrovis produces H 2 via a non-bifurcating NADH-dependent enzyme complex
ABSTRACT Hydrogenosomes are mitochondria-derived organelles that produce ATP and H 2 to support energy metabolism in anaerobic eukaryotes. H 2 production allows reoxidation of reduced cofactors generated during fermentative metabolism; however, the metabolic mechanisms for H 2 production in anaerobic eukaryotes remains incompletely understood. In particular, it remains unclear whether anaerobic fungi (AF) hydrogenosomes use a ferredoxin-dependent pathway or a distinct mechanism to regenerate NAD(P) + and link electron transfer to H 2 formation. Here, by combining genomic search, proteomic analysis, and enzymology, we reveal the molecular mechanism for H 2 production in the AF strain Caecomyces churrovis . Our enzyme assays on the organelle fraction of C. churrovis revealed the activity of H 2 :NAD + oxidoreductase but not pyruvate:ferredoxin oxidoreductase, which is usually linked to H 2 formation. We identified genes encoding [FeFe] hydrogenase (Hyd) and NADH dehydrogenase subunits E and F (NuoE, NuoF) in C. churrovis , and confirmed their expression in the isolated hydrogenosomal fractions by proteomic analysis. Combining the individually purified enzymes, we found Hyd and NuoEF proteins formed H 2 directly from NADH independently of ferredoxin, functioning as a non-bifurcating NADH-dependent enzyme rather than an electron-bifurcating enzyme. We identified homologs of hydrogenosomal NuoE, NuoF, and Hyd in many other AF, indicating this pathway is commonly shared among the AF. This work demonstrates the existence of a non-bifurcating NADH-dependent enzyme complex in eukaryotes. Moreover, this complex could potentially be exploited as a target for controlling AF H 2 production and altering fungal metabolism. IMPORTANCE H 2 production is a prominent feature of anaerobic energy metabolism, yet our understanding of eukaryotic mechanisms remains limited. Anaerobic fungi (AF) are key decomposers of lignocellulose and contribute to hydrogen flux in anaerobic environments. Although it has been more than 40 years since the H 2 production from AF was first reported, the molecular mechanism for hydrogenosomal H 2 production and redox balance remains unclear. We demonstrate that AF produce H 2 from NADH utilizing a non-bifurcating NADH-dependent enzyme complex rather than an electron-bifurcating, ferredoxin-dependent variant. We show that this enzyme complex is conserved across multiple AF lineages and thus demonstrate the occurrence of a non-bifurcating NADH-dependent enzyme in eukaryotes. This discovery expands our understanding of eukaryotic hydrogenosomal metabolism, reveals a previously unknown strategy for redox balancing, and highlights potential targets for manipulating H 2 production. These insights have broad implications for microbial energy metabolism, anaerobic ecosystems, and bioengineering of H 2 -producing systems.
Zhang, Bo [Department of Chemical Engineering, Uni↗
Engineering Methanogenic Microbiomes to Redirect Flux to Biomass
In this study, we present a method for acquiring and characterizing novel microbial consortia that regulates methanogens and methanotrophs through selective cultivation and metagenomic analysis of indigenous microorganisms in the environment. In addition, we present the work performed as part of this project to model the pathways that act as limiting factors in microbial methane metabolism based on a carbon cycle model. In this report, we describe the methods for selective cultivation of methane-metabolism-related microorganisms from environmental samples, the method for monitoring their methane consumption performance, and the method and results for verifying their functions using quantitative PCR and metagenomics techniques. The microbial consortia containing methanotrophs were obtained through selective cultivation and molecular biological verification, and their methane consumption performance was evaluated. In addition, the potential of the existence of bacteriophages interacting with methane metabolism-related microorganisms was identified through metagenomic sequencing.
09 BIOMASS FUELS↗
Distinct Assembly Processes and Microbial Communities Constrain Soil Organic Carbon Formation
Soil stores more carbon (C) than all vegetation and the atmosphere combined. Soil C stocks are broadly shaped by temperature, moisture, soil physical characteristics, vegetation, and microbial-mediated metabolic processes. The efficiency with which microorganisms use soil C regulates the balance between C storage in soil and the atmosphere. In this review, we discuss how microbial physiology and community assembly processes determine microbial growth rate and efficiency and, in turn, soil organic C cycling through the lens of community ecology. We introduce a conceptual framework cataloging life history (i.e., growth rate, resource acquisition, and stress tolerance) and assembly traits (i.e., competition, facilitation, and dispersal) that correspond with different growth efficiencies. We also compare how dominant mycorrhizal fungal type affects growth efficiency. We propose that further development and inclusion of specific community parameters in microbial-explicit Earth system models are needed for accurately predicting soil organic C responses to global change.
carbon sequestration↗
Arsenic Accumulation in Microbial Biomass and the Interpretation of Signals of Early Arsenic‐Based Metabolisms
Carbonaceous particles that concentrate arsenic in microbialites as old as ~3.5 Ga are similar to As-rich organic globules in modern microbialites. The former particles have been interpreted as tracers of As cycling by early microbial metabolisms. However, it is unclear if arsenic accumulation is a consequence of biological activity or passive postmortem binding of arsenic by organic matter during diagenesis in volcanically influenced, As-rich environments. Here, we address this uncertainty by evaluating the concentrations, speciation, and detectability of As in active or heat-killed biofilms formed by cyanobacteria or anoxygenic photosynthetic microbes exposed to environmentally relevant concentrations of As(III) or As(V) (50 μM to 3 mM). The genomes or metagenomes of these biofilms contain genes involved in detoxifying or energy-yielding As metabolisms. Biomass accumulates As from the solution in a concentration-dependent manner and with a preference for oxidized As(V) over As(III). Autoclaved biomass accumulates As even more strongly than active biomass, likely because living biofilms actively detoxify As. Active biofilms oxidize and reduce As and accumulate both As(III) and As(V), whereas a small fraction of As(V) can be reduced in inactive biofilms that bind As during diagenesis. Arsenic enrichments in the biomass are detectable by X-ray based spectroscopy techniques (XRF, EPMA-WDS) that are commonly used to analyze geological materials. These findings enable the reconstruction of past active and passive interactions of microbial biomass with arsenic in fossilized microbial biofilms and microbialites from the early Earth.
Madrigal‐Trejo, David↗
A multi-organ maize metabolic model connects temperature stress with energy production and reducing power generation
Not Available
59 BASIC BIOLOGICAL SCIENCES↗
Bayesian Inference for Integrating Yarrowia lipolytica Multiomics Datasets with Metabolic Modeling
Optimizing the metabolism of microbial cell factories for yields and titers is a critical step for economically viable production of bioproducts and biofuels. In this process, tuning the expression of individual enzymes to obtain the desired pathway flux is a challenging step, in which data from separate multiomics techniques must be integrated with existing biological knowledge to determine where changes should be made. Following a design-build-test-learn strategy, building on recent advances in Bayesian metabolic control analysis, we identify in this work key enzymes in the oleaginous yeast Yarrowia lipolytica that correlate with the production of itaconate by integrating a metabolic model with multiomics measurements. To this extent, we quantify the uncertainty for a variety of key parameters, known as flux control coefficients (FCCs), needed to improve the bioproduction of target metabolites and statistically obtain key correlations between the measured enzymes and boundary flux. Based on the top five significant FCCs and five correlated enzymes, our results show phosphoglycerate mutase, acetyl-CoA synthetase (ACSm), carbonic anhydrase (HCO3E), pyrophosphatase (PPAm), and homoserine dehydrogenase (HSDxi) enzymes in rate-limiting reactions that can lead to increased itaconic acid production.
59 BASIC BIOLOGICAL SCIENCES↗
Coarse-Grained Modeling Elucidates Differential Metabolism of Saccharomyces cerevisiae under Varied Nutrient Limitations
Microorganisms such as Saccharomyces cerevisiae have a native ability to adapt their metabolism to varying nutrient conditions. Understanding their responses to nutrient limitations is critical for decoding cellular physiology and designing strategies for metabolic engineering. While the influence of carbon availability on yeast metabolism has been extensively studied, the role of nitrogen availability remains relatively underexplored. In this study, we utilized a coarse-grained kinetic model to systematically analyze and compare the effects of carbon and nitrogen limitations on yeast metabolism. Our model successfully revealed the differential metabolic characteristics of S. cerevisiae under carbon- and nitrogen-limited chemostat conditions. It also highlighted the significance of protein activity regulation at varying carbon-to-nitrogen ratios, and elucidated distinct strategies employed to maintain ATP homeostasis. Furthermore, this study provides a computational tool for investigating yeast physiology under nutrient limitations and offers quantitative and mechanistic insights into yeast metabolism.
59 BASIC BIOLOGICAL SCIENCES↗
Gut enterotype-dependent modulation of gut microbiota and their metabolism in response to xanthohumol supplementation in healthy adults
Xanthohumol (XN), a polyphenol found in the hop plant (Humulus lupulus), has antioxidant, anti-inflammatory, prebiotic, and anti-hyperlipidemic activity. Preclinical evidence suggests the gut microbiome is essential in mediating these bioactivities; however, relatively little is known about XN’s impact on human gut microbiota in vivo. We conducted a randomized, triple-blinded, placebo-controlled clinical trial (ClinicalTrials.gov NCT03735420) to determine safety and tolerability of XN in healthy adults. Thirty healthy participants were randomized to 24 mg/day XN or placebo for 8 weeks. As secondary outcomes, quantification of bacterial metabolites and 16S rRNA gene sequencing were utilized to explore the relationships between XN supplementation, gut microbiota, and biomarkers of gut health. Although XN did not significantly change gut microbiota composition, it did re-shape individual taxa in an enterotype-dependent manner. High levels of inter-individual variation in metabolic profiles and bioavailability of XN metabolites were observed. Moreover, reductions in microbiota-derived bile acid metabolism were observed, which were enterotype-dependent. These results suggest interactions between XN and gut microbiota in healthy adults are highly inter-individualized and potentially indicate that XN elicits effects on gut health in an enterotype-dependent manner.
59 BASIC BIOLOGICAL SCIENCES↗