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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 181 records · Page 10
All sourcesNASA NTRSDOE OSTI
DOE OSTI2022

Perspective: Biochemical and Physical Constraints Associated With Preparing Thin Specimens for Single-Particle Cryo-EM

While many aspects of single-particle electron cryo-microscopy (cryo-EM) of biological macromolecules have reached a sophisticated level of development, this is not yet the case when it comes to preparing thin samples on specimen grids. As a result, there currently is considerable interest in achieving better control of both the sample thickness and the amount of area that is useful, but this is only one aspect in which improvement is needed. This Perspective addresses the further need to prevent the macromolecular particles from making contact with the air-water interface, something that can result in preferential orientation and even structural disruption of macromolecular particles. This unwanted contact can occur either as the result of free diffusion of particles during the interval between application, thinning and vitrification of the remaining buffer, or—when particles have been immobilized—by the film of buffer becoming too thin prior to vitrification. An opportunity now exists to apply theoretical and practical insights from the fields of thin-film physical chemistry and interfacial science, in an effort to bring cryo-EM sample preparation to a level of sophistication that is comparable to that of current data collection and analysis.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

Genetic and Biochemical Investigation of Seed Fatty Acid Accumulation in Arabidopsis

As a vegetable oil, consisting principally of triacylglycerols, is the major storage form of photosynthetically-fixed carbon in oilseeds which are of significant agricultural and industrial value. Photosynthesis in chlorophyll-containing green seeds, along with photosynthesis in leaves and other green organs, generates ATP and reductant (NADPH and NADH) needed for seed fatty acid production. However, contribution of seed photosynthesis to fatty acid accumulation in seeds have not been well-defined. Here, we report the contribution of seed-photosynthesis to fatty acid production by probing segregating green (photosynthetically-competent) and non-green or yellow (photosynthetically-non-competent) seeds in siliques of an Arabidopsis chlorophyll synthase mutant. Using this mutant, we found that yellow seeds lacking photosynthetic capacity reached 80% of amounts of oil in green seeds at maturity. Combining this with studies using shaded siliques, we determined that seed-photosynthesis accounts for 20% and silique and leaf/stem photosynthesis each account for ~40% of the ATP and reductant for seed oil production. Transmission electron microscopy (TEM) and pyridine nucleotides and ATP analyses revealed that seed photosynthesis provides ATP and reductant for oil production mostly during early development, as evidenced by delayed oil accumulation in non-green seeds. Transcriptomic analyses suggests that the oxidative pentose phosphate pathway could be the source of carbon, energy and reductants required for fatty acid synthesis beyond the early stages of seed development.

Nwafor, Chinedu Charles↗
DOE OSTI2021

Identification and Biochemical Characterization of a Novel Hormone-Sensitive Lipase Family Esterase Est19 from the Antarctic Bacterium Pseudomonas sp. E2-15

Esterases represent an important class of enzymes with a wide variety of industrial applications. A novel hormone-sensitive lipase (HSL) family esterase, Est19, from the Antarctic bacterium Pseudomonas sp. E2-15 is identified, cloned, and expressed. The enzyme possesses a GESAG motif containing an active serine (S) located within a highly conserved catalytic triad of Ser 155 , Asp 253 , and His 282 residues. The catalytic efficiency (k cat /K m ) of Est19 for the pNPC6 substrate is 148.68 s –1 mM –1 at 40 °C. Replacing Glu 154 juxtaposed to the critical catalytic serine with Asp (E154→D substitution) reduced the activity and catalytic efficiency of the enzyme two-fold, with little change in the substrate affinity. The wild-type enzyme retained near complete activity over a temperature range of 10–60 °C, while ~50% of its activity was retained at 0 °C. A phylogenetic analysis suggested that Est19 and its homologs may represent a new subfamily of HSL. The thermal stability and stereo-specificity suggest that the Est19 esterase may be useful for cold and chiral catalyses.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2020

Biochemical upgrading of high-protein biomass and grain products

The present invention relates to methods of upgrading biomass to provide useful chemical intermediates, fuels, amino acids, nutrients, etc. In particular examples, the biomass is a by-product of ethanol production and is mainly used as high-protein feed. Described herein are methods for upgrading such biomass, such as by implementing pre-treatment conditions and by employing fermentation conditions including modified organisms.

09 BIOMASS FUELS↗
DOE OSTI2021

Host cells and methods for producing toluene biochemically

The present invention provides for a genetically modified host cell comprising a first polypeptide comprising a sequence having at least 70% amino acid sequence identity with a phenylacetate decarboxylase, and having an enzymatic activity to decarboxylate a phenylacetic acid into a toluene and a carbon dioxide, and a second polypeptide comprising a sequence having at least 70% amino acid sequence identity with a phenylacetate decarboxylase activating enzyme, and having an enzymatic activity to cleave a S-adenosylmethionine (SAM) to form a methionine and a 5′-deoxyadenosyl radical.

Beller, Harry R.↗
DOE OSTI2022

Biochemical upgrading of high-protein biomass and grain products

The present invention relates to methods of upgrading biomass to provide useful chemical intermediates, fuels, amino acids, nutrients, etc. In particular examples, the biomass is a by-product of ethanol production and is mainly used as high-protein feed. Described herein are methods for upgrading such biomass, such as by implementing pre-treatment conditions and by employing fermentation conditions including modified organisms.

Davis, Ryan Wesley↗
DOE OSTI2020

Structural conserved moiety splitting of a stoichiometric matrix

Characterising biochemical reaction network structure in mathematical terms enables the inference of functional biochemical consequences from network structure with existing mathematical techniques and spurs the development of new mathematics that exploits the peculiarities of biochemical network structure. The structure of a biochemical network may be specified by reaction stoichiometry, that is, the relative quantities of each molecule produced and consumed in each reaction of the network. A biochemical network may also be specified at a higher level of resolution in terms of the internal structure of each molecule and how molecular structures are transformed by each reaction in a network. The stoichiometry for a set of reactions can be compiled into a stoichiometric matrix N ϵ Z mxn where each row corresponds to a molecule and each column corresponds to a reaction. We demonstrate that a stoichiometric matrix may be split into the sum of m - rank(N) moiety transition matrices, each of which corresponds to a subnetwork accessible to a structurally identifiable conserved moiety. The existence of this moiety matrix splitting is a property that distinguishes a stoichiometric matrix from an arbitrary rectangular matrix.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

Cell Free and Immobilization Technologies (CFIT)

Today, several key factors negatively impact the production of fuels and chemicals from renewable sources. Common hindrances in the biological production of biochemicals are: (1) end-product or intermediate toxicity to the microbial biocatalyst, (2) the diversion of carbon to biomass formation, and (3) co-production of undesired byproducts. A particularly attractive alternative is to eliminate the biocatalyst entirely and instead operate the desired metabolic pathways in isolation, thus circumventing the roadblocks of biological toxicity, lower yields, and lack of specificity. However, cell-free enzyme systems still suffer from low productivities owing in part to the effects of free diffusion of intermediates, lack of long term enzyme stability, cofactor cost or inefficient recycling rates, and finally, the cost of enzyme production/purification. This project represents a new effort to propose innovative and cost competitive routes to producing biochemicals from a variety of feedstocks using cell free approaches. These routes will help reduce the current risk and cost associated with classical cell free production. Cell free technologies show promise for application to the production of toxic/inhibitory products or products difficult to separate from microbial growth media and can help reduce the production barriers in multiple areas of biological conversion of feedstocks to biochemicals. More specifically, we are developing new technologies and routes that could be used to produce high value biochemicals such as 2,3 BDO and terpenes (among many others) from biomass derived C5/C6 sugars or lignin but also from waste byproducts such as glycerol. This project will lead to significant innovation and also lead to new concepts and rational design of pathways and enzymes. Within this project, we will develop new metabolic enzyme cascades that will represent natural or artificial combinations of enzymes to produce the desired biochemicals from a variety of feedstocks. We are also developing basic design principals for constructing synthetic metabolons, using fusion proteins and synthetic protein scaffolds, to promote substrate channeling and stability while conserving peak activity. Additionally, our efforts include a techno economic analysis (TEA) of cell free approaches to provide the sensitivities of the process to enzyme loading, activity, pH, reactor volumes, cofactor recycling rates. Finally, we are focusing on further increasing stability, operating lifetime and efficiency of the pathway enzymes by immobilization on support surfaces. We are also focus on immobilizing pathway enzymes or combinations of enzymes on several different conducting polymers and evaluate the effect on stability and operating lifetime. As more combinations become available we will conduct a more systematic study of the means of immobilizing these enzymes. This preliminary work will enable the in-depth study of cofactor recycling at these interfaces using mediators for electron transfer. Taken together, these approaches will enable process intensification, continuous operation, lower capital and separations costs and end-product flexibility, and thus has the potential to contribute significantly to BETO's goals of cost competitive biofuels and bioproducts. To date we have demonstrated 1) the conversion of pyruvate to 2,3 BDO (4 enzymes with cofactor recycling, no additional cofactors needed) at >100g/L (>3g/L/h) using our enzyme tethering approach, 2) Generated mutants of a key redox enzyme with >180- fold improvement in NADH utilization over WT to >85% of NADPH utilization. 3) Produced mevalonate at >10g/L from glucose, limonene at >10g/L from mevalonate and more than >5g/L from glucose with crude enzyme preparations and complete cofactor recycling. 4) successfully generated fully active cross-linked aggregates (CLEAs) of NOX, a cofactor-regenerating water-forming oxidase that is key to our process with increased stability 5) Engineered and identified enzymes able to use the biomimetic cofactor NMN and synthesized new synthetic cofactors.

biocatalysis↗
DOE OSTI2023

Water chemistry in flume channel and hyporheic zone (i.e., porewater) associated with: “Rethinking Aerobic Respiration in the Hyporheic Zone Under Variation in Carbon and Nitrogen Stoichiometry”

Dissolved oxygen (DO), total organic carbon (TOC), total nitrogen (TN), molecular data for organic matter, and biochemical reactions for surface water and porewater (i.e., hyporheic zone) collected from a water recirculating flume located at the University of Texas, Austin. The flume contained real river water from Lower Colorado River(Austin, TX) and clean sand. Hyporheic exchange in the flume was induced through The study aims to understand relationships between aerobic metabolism of organic matter and molecular characteristics of organic matter, such as thermodynamic signature and nitrogen content, through the extent of the hyporheic zone at 10 cm- resolution, and through time. During the experiment, organic matter (dry leaves) was added to the flume and removed after 24 hours. The water samples were collected before the addition of leaves, at the time of removal of leaves, and at hour 72. The water samples were analyzed using ultrahigh resolution Fourier transform ion cyclotron resonance mass spectrometry (FTICR-MS) and total organic carbon (TOC) and total nitrogen (TN) analysis. Dissolved oxygen content throughout the surface water and the hyporheic zone of the flume was measured with a large planar optode. This data package is associated with the publication ’ Rethinking Aerobic Respiration in the Hyporheic Zone Under Variation in Carbon and Nitrogen Stoichiometry’ published in Environmental Science and Technology (Turețcaia et al., 2023 https://doi.org/10.1021/acs.est.3c04765). The dataset is comprised of five folders (1) Diss_O2_pic, (2) input_files (3) output_files; (4) python_code; and (5) R_code . Diss_O2_pic contains siximages of dissolved oxygen distribution in a bedform at hours 0, 24, and 72 of the experiment conducted in a large recirculation flume. Images are in separate R and G channels (i.e., RGB). The input_files contains (1) a csv file with FTICR peaks identified within each sample, (2) a csv file with molecular information pertinent to FTICR data with Gibbs free energy calculations adjusted for environmental temperature, (3) a csv file containing concentrations of non-purgeable organic carbon measured throughout the experiment , (4) a csv file containing concentrations of total nitrogen measured throughout the experiment, (5) a csv file containing total biochemical reactions (i.e., transformations) identified in the dataset, (6) a csv containing transformation profiles, and (7) a csv file containing transformations with formulas, and (8) a jpg file with schematic representation of locations for sample collection. The output_files contains (1) and xlsx file containing percent biochemical reactions containing nitrogen identified across all 39 sample, (2) a csv file of merged FTICR data and molecular information files, (3) a csv files containing average Gibbs free energy within sampling domains and at each sampling location, (4) a csv file with average concentrations of dissolved oxygen across sampling locations at hour 0, (5) a csv file with average concentrations of dissolved oxygen across sampling locations at hour 24, (6) a csv file with average concentrations of dissolved oxygen across sampling locations at hour 72, (7) a csv file with percent chemical classes identified across sampling locations at hour 0, (8) a csv file with percent chemical classes identified across sampling locations at hour 24, (9) a csv file with percent chemical classes identified across sampling locations at hour 72, and (10) a csv file containing percent nitrogen containing biochemical reactions identified across sampling locations at hours 0, 24, and 72. The python_code contains seven ipynb files which are Jupyter Notebooks used for data analysis and figures generation. The R_code contains 3 R files with R code used for data analysis and figures generation. This data package contains the processed data used in the associated manuscript. This data has not been previously published.

54 ENVIRONMENTAL SCIENCES↗
DOE OSTI2026

Catabolism of lignin-related methoxylated compounds in white-rot fungi utilizes non-canonical oxidoreductases

White-rot fungi (WRF) are the most effective lignin-degrading organisms in nature, making them essential to Earth’s carbon cycle. Lignin is a highly methoxylated, heterogeneous biopolymer derived from plants. However, the pathways WRF use to metabolize methoxylated aromatic monomericcompounds as carbon sources remain unidentified. Here, we employ a systems biology approach to elucidate the intracellular catabolism of vanillate – a monomethoxylated aromatic compound – in two white-rot fungi (WRF), Gelatoporia subvermispora and Trametes versicolor. We identified and biochemically validated a four-enzyme pathway that converts vanillate into ß-ketoadipate – a metabolite that enters central carbon metabolism. This pathway deviates from typical bacterial pathways, where vanillate is initially demethylated and ring-cleaved by intradiol dioxygenases; instead, oxidative decarboxylation occurs prior to ring cleavage by extradiol dioxygenases. Thus, we conducted an in-depth investigation of ring cleavage and further downstream catabolism by the identified fungal enzymes using biochemical and structural approaches. This revealed non-canonical enzymes, including a highly substrate-specific extradiol dioxygenase and a metal-free, promiscuous reductase, the latter capable of acting on catabolic intermediates derived from both methoxylated and non-methoxylated aromatic compounds. This work emphasizes the potential of WRF and their enzymes to advance lignin valorization and enhance our understanding of their role during wood decay.

dioxygenase↗