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Results for “anaerobic catabolic pathways”

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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9 records
All sourcesNASA NTRSDOE OSTI
DOE OSTI2023

Methyl-Based Methanogenesis: an Ecological and Genomic Review

Methyl-based methanogenesis is one of three broad categories of archaeal anaerobic methanogenesis, including both the methyl dismutation (methylotrophic) pathway and the methyl-reducing (also known as hydrogen-dependent methylotrophic) pathway. Methyl-based methanogenesis is increasingly recognized as an important source of methane in a variety of environments. Here, we provide an overview of methyl-based methanogenesis research, including the conditions under which methyl-based methanogenesis can be a dominant source of methane emissions, experimental methods for distinguishing different pathways of methane production, molecular details of the biochemical pathways involved, and the genes and organisms involved in these processes. In this study, we also identify the current gaps in knowledge and present a genomic and metagenomic survey of methyl-based methanogenesis genes, highlighting the diversity of methyl-based methanogens at multiple taxonomic levels and the widespread distribution of known methyl-based methanogenesis genes and families across different environments.

methanogens↗
DOE OSTI2024

Phenotypic and genomic characterization of Methanothermobacter wolfeii strain BSEL, a CO 2 -capturing archaeon with minimal nutrient requirements

A new variant of Methanothermobacter wolfeii was isolated from an anaerobic digester using enrichment cultivation in anaerobic conditions. Here, the new isolate was taxonomically identified via 16S rRNA gene sequencing and tagged as M. wolfeii BSEL. The whole genome of the new variant was sequenced and de novo assembled. Genomic variations between the BSEL strain and the type strain were discovered, suggesting evolutionary adaptations of the BSEL strain that conferred advantages while growing under a low concentration of nutrients. M. wolfeii BSEL displayed the highest specific growth rate ever reported for the wolfeii species (0.27 ± 0.03 h –1 ) using carbon dioxide (CO 2 ) as unique carbon source and hydrogen (H 2 ) as electron donor. M. wolfeii BSEL grew at this rate in an environment with ammonium (NH 4 + ) as sole nitrogen source. The minerals content required to cultivate the BSEL strain was relatively low and resembled the ionic background of tap water without mineral supplements. Optimum growth rate for the new isolate was observed at 64°C and pH 8.3. In this work, it was shown that wastewater from a wastewater treatment facility can be used as a low-cost alternative medium to cultivate M. wolfeii BSEL. Continuous gas fermentation fed with a synthetic biogas mimic along with H 2 in a bubble column bioreactor using M. wolfeii BSEL as biocatalyst resulted in a CO 2 conversion efficiency of 97% and a final methane (CH 4 ) titer of 98.5%v, demonstrating the ability of the new strain for upgrading biogas to renewable natural gas.

09 BIOMASS FUELS↗
DOE OSTI2021

Anaerobic Microbial Metabolism of Dichloroacetate

Dichloroacetate (DCA) commonly occurs in the environment due to natural production and anthropogenic releases, but its fate under anoxic conditions is uncertain. Mixed culture RM comprising “Candidatus Dichloromethanomonas elyunquensis” strain RM utilizes DCA as an energy source, and the transient formation of formate, H 2 , and carbon monoxide (CO) was observed during growth. Only about half of the DCA was recovered as acetate, suggesting a fermentative catabolic route rather than a reductive dechlorination pathway. Sequencing of 16S rRNA gene amplicons and 16S rRNA gene-targeted quantitative real-time PCR (qPCR) implicated “Candidatus Dichloromethanomonas elyunquensis” strain RM in DCA degradation. An (S)-2-haloacid dehalogenase (HAD) encoded on the genome of strain RM was heterologously expressed, and the purified HAD demonstrated the cofactor-independent stoichiometric conversion of DCA to glyoxylate at a rate of 90 ± 4.6 nkat mg -1 protein. Differential protein expression analysis identified enzymes catalyzing the conversion of DCA to acetyl coenzyme A (acetyl-CoA) via glyoxylate as well as enzymes of the Wood-Ljungdahl pathway. Glyoxylate carboligase, which catalyzes the condensation of two molecules of glyoxylate to form tartronate semialdehyde, was highly abundant in DCA-grown cells. The physiological, biochemical, and proteogenomic data demonstrate the involvement of an HAD and the Wood-Ljungdahl pathway in the anaerobic fermentation of DCA, which has implications for DCA turnover in natural and engineered environments, as well as the metabolism of the cancer drug DCA by gut microbiota.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2020

Molecular basis for catabolism of the abundant metabolite trans-4-hydroxy-L-proline by a microbial glycyl radical enzyme

The glycyl radical enzyme (GRE) superfamily utilizes a glycyl radical cofactor to catalyze difficult chemical reactions in a variety of anaerobic microbial metabolic pathways. Recently, a GRE, trans-4-hydroxy-L-proline (Hyp) dehydratase (HypD), was discovered that catalyzes the dehydration of Hyp to (S)-Δ 1 -pyrroline-5-carboxylic acid (P5C). This enzyme is abundant in the human gut microbiome and also present in prominent bacterial pathogens. However, we lack an understanding of how HypD performs its unusual chemistry. Here, we have solved the crystal structure of HypD from the pathogen Clostridioides difficile with Hyp bound in the active site. Biochemical studies have led to the identification of key catalytic residues and have provided insight into the radical mechanism of Hyp dehydration.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2026

Engineering Escherichia coli to produce medium chain oleochemicals from C 2 substrates

Many strategies to create a circular bioeconomy have been proposed. To be successful, CO 2 must be reduced with renewable energy into chemical building blocks, from which the chemical industry can be supported. Circular strategies include leveraging photosynthesis to produce sugar and lipid intermediates or renewable electricity to produce hydrogen or other electron carriers to support CO 2 reduction. Acetogens can anaerobically reduce CO 2 with H 2 to produce mixtures of small organic molecules in gas fermentations. We previously demonstrated that acetate, a common product of gas fermentation, can be converted to the model oleochemical dodecanol in engineered Escherichia coli. Here, we explored the conversion of ethanol and mixtures of ethanol and acetate to the same model oleochemicals. Co-feeding ethanol can supply both carbon and additional reducing power relative to acetate alone. In this work, we engineered E. coli to catabolize ethanol and expressed two distinct ethanol metabolism pathways in different operons and combined them with improved engineered acetate activation. We evaluated the performance of these operons in dodecanol-producing strains when fed ethanol or acetate and found ethanol to be a better carbon source when judged by product titers. The engineered strains fed ethanol produced about two-fold more dodecanol than the strains fed acetate. This increase was in part, due to change in product distribution. Cells fed ethanol produced predominantly dodecanol, whereas cells fed acetate generated a mixture of dodecanol and dodecanoic acid. Dodecanol titers were further improved by employing feeding strategies in controlled bioreactors.

Escherichia coli↗
DOE OSTI2022

Combinatorial library design for improving isobutanol production in Saccharomyces cerevisiae

Saccharomyces cerevisiae is the dominant fermentative producer of ethanol in industry and a preferred host for production of other biofuels. That said, rewiring the metabolism of S. cerevisiae to produce other fermentation products, such as isobutanol, remains an academic challenge. Many studies report aerobic production of isobutanol, but ethanol remains a substantial by-product under these conditions due to the Crabtree effect. These studies indicate that the native isobutanol pathway is incapable of carrying sufficient flux to displace ethanol. In this report, we screened a combinatorial library of pathway enzymes to identify an isobutanol pathway cassette capable of supporting the growth of a non-ethanol producing S. cerevisiae . We began by identifying a diverse set of isobutanol pathway enzyme homologs and combined each open reading frame with varied-strength promoters in a combinatorial, pooled fashion. We applied a growth-coupled screen where a functional isobutanol pathway restored NAD + regeneration during glucose catabolism that is otherwise repressed via the Crabtree effect. Using this screen, we isolated a cassette consisting of a mosaic of bacterial and cytosol-localized fungal enzymes that conferred under aerobic conditions the ability to produce 364 mg/L isobutanol (8.8% of the theoretical maximum yield). We next shifted the cofactor usage of the isolated ketol-acid reductoisomerase enzyme in the cassette from NADPH to NADH-preferring to improve redox balance. The approach used herein isolated isobutanol producing strains that approach the best in the literature without producing substantial ethanol titers. Still, the best isolated cassette was insufficient to support anaerobic growth in the absence of ethanol fermentation - indicating the presence of further fundamental gaps in our understanding of yeast fermentation.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2025

Xylanolytic metabolism is regulated by coordination of transcription factors XynR and XylR in extremely thermophilic Caldicellulosiruptorales

ABSTRACT Global transcription factors (TFs) control metabolic processes in bacteria to efficiently utilize available carbon. The orderCaldicellulosiruptoraleshas drawn interest due to the ability of its members to degrade components of lignocellulosic biomass. Regulatory reconstruction ofAnaerocellum (f. Caldicellulosiruptor) besciiidentified two major global transcription factors for xylan utilization, XynR and XylR, and the corresponding putative transcription factor binding sites. Recombinant versions of XynR (LacI family) and XylR (ROK family) were subjected to fluorescence polarization (FP) and biolayer interferometry (BLI) analysis to confirm the predicted binding sites. Four XynR sites and two XylR sites were validated, accounting for 20 of 26 genes regulated by XynR and six of seven genes regulated by XylR. Bioinformatic analysis of the individual genes controlled by the two regulators showed an inter-dependent scheme for xylan conversion; the transport of xylooligosaccharides (XOS) is dependent on XylR, while enzymes responsible for hydrolysis are controlled by both regulators. For xylose catabolism by the xylose isomerase-xylulose kinase pathway, regulation is also split, with XylR controlling xylose isomerase and XynR controlling xylokinase. The XynR/XylR regulator pair withinA. besciiis conserved in all sequenced species ofCaldicellulosiruptorales, suggesting similarities in regulating linear xylan conversion. In other xylanolytic thermophiles, XylR homologs control xylan degradation, compared to just 6 out of 26 genes forA. bescii. These results show that two separate regulatory schemes (dual repression) are coordinated byA. besciito effectively regulate the hemicellulose inventory and xylan catabolism. IMPORTANCE To take full advantage of extreme thermophiles as platform metabolic engineering microorganisms, the tools for genetic manipulation must be further developed, and strategies that exploit a better understanding of metabolic regulation need to be discerned.Anaerocellum bescii, the most studied of the extremely thermophilic fermentative anaerobic bacteria that can utilize microcrystalline cellulose, can degrade microcrystalline cellulose and hemicellulose and has been metabolically engineered to convert the resulting sugars to products such as ethanol and acetone. For xylan, in particular, two major global transcription factors (TFs), XynR and XylR, play a role in sugar metabolism, although their predicted regulatory interdependence from bioinformatics analysis has not been elucidated experimentally. Here, fluorescence polarization (FP) and biolayer interferometry (BLI) were used to explore this issue to support metabolic engineering efforts aimed at improving carbohydrate processing to industrial chemicals.

Biotechnology & Applied Microbiology↗
DOE OSTI2025

Sulfoquinovose is exclusively metabolized by the gut microbiota and degraded differently in mice and humans

Abstract Background Sulfoquinovose (SQ) is a green-diet-derived sulfonated glucose and a selective substrate for a limited number of human gut bacteria. Complete anaerobic SQ degradation via interspecies metabolite transfer to sulfonate-respiring bacteria produces hydrogen sulfide, which has dose- and context-dependent health effects. Here, we studied potential SQ degradation by the mammalian host and the impact of SQ supplementation on human and murine gut microbiota diversity and metabolism. Results 13 CO 2 breath tests with germ-free C57BL/6 mice gavaged with 13 C-SQ were negative. Also, SQ was not degraded by human intestinal cells in vitro, indicating that SQ is not directly metabolized by mice and humans. Addition of increasing SQ concentrations to human fecal microcosms revealed dose-dependent responses of the microbiota and corroborated the relevance ofAgathobacter rectalisandBilophila wadsworthiain cooperative degradation of SQ to hydrogen sulfide via interspecies transfer of 2,3-dihydroxy-1-propanesulfonate (DHPS). Similar to the human gut microbiome, the genetic capacity for SQ or DHPS degradation is sparsely distributed among bacterial species in the gut of conventional laboratory mice.Escherichia coliandEnterocloster clostridioformiswere identified as primary SQ degraders in the mouse gut. SQ and DHPS supplementation experiments with conventional laboratory mice and their intestinal contents showed that SQ was incompletely catabolized to DHPS. Although someE. clostridioformisgenomes encode an extended sulfoglycolytic pathway for both SQ and DHPS fermentation, SQ was only degraded to DHPS by a mouse-derivedE. clostridioformisstrain. Conclusions Our findings suggest that SQ is solely a nutrient for the gut microbiota and not for mice and humans, emphasizing its potential as a prebiotic. SQ degradation by the microbiota of conventional laboratory mice differs from the human gut microbiota by absence of DHPS degradation activity. Hence, the microbiota of conventional laboratory mice does not fully represent the SQ metabolism in humans, indicating the need for alternative model systems to assess the impact of SQ on human health. This study advances our understanding of how individual dietary compounds shape the microbial community structure and metabolism in the gut and thereby potentially influence host health.

Microbiology↗