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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 55 records · Page 3
All sourcesNASA NTRSDOE OSTI
DOE OSTI2023

Rare ribosomal RNA sequences from archaea stabilize the bacterial ribosome

The ribosome serves as the universally conserved translator of the genetic code into proteins and supports life across diverse temperatures ranging from below freezing to above 120°C. Ribosomes are capable of functioning across this wide range of temperatures even though the catalytic site for peptide bond formation, the peptidyl transferase center, is nearly universally conserved. Here we find that Thermoproteota, a phylum of thermophilic Archaea, substitute cytidine for uridine at large subunit rRNA positions 2554 and 2555 (Escherichia coli numbering) in the A loop, immediately adjacent to the binding site for the 3'-end of A-site tRNA. We show by cryo-EM that E. coli ribosomes with uridine to cytidine mutations at these positions retain the proper fold and post-transcriptional modification of the A loop. Additionally, these mutations do not affect cellular growth, protect the large ribosomal subunit from thermal denaturation, and increase the mutational robustness of nucleotides in the peptidyl transferase center. This work identifies sequence variation across archaeal ribosomes in the peptidyl transferase center that likely confers stabilization of the ribosome at high temperatures and develops a stable mutant bacterial ribosome that can act as a scaffold for future ribosome engineering efforts.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

Manipulation of Lignin Monomer Composition Combined with the Introduction of Monolignol Conjugate Biosynthesis Leads to Synergistic Changes in Lignin Structure

Abstract The complexity of lignin structure impedes efficient cell wall digestibility. Native lignin is composed of a mixture of three dominant monomers, coupled together through a variety of linkages. Work over the past few decades has demonstrated that lignin composition can be altered through a variety of mutational and transgenic approaches such that the polymer is derived almost entirely from a single monomer. In this study, we investigated changes to lignin structure and digestibility in Arabidopsis thaliana in near-single-monolignol transgenics and mutants and determined whether novel monolignol conjugates, produced by a FERULOYL-CoA MONOLIGNOL TRANSFERASE (FMT) or a p-COUMAROYL-CoA MONOLIGNOL TRANSFERASE (PMT), could be integrated into these novel polymers to further improve saccharification efficiency. Monolignol conjugates, including a new conjugate of interest, p-coumaryl p-coumarate, were successfully integrated into high-H, high-G and high-S lignins in A. thaliana. Regardless of lignin composition, FMT- and PMT-expressing plants produced monolignol ferulates and monolignol p-coumarates, respectively, and incorporated them into their lignin. Through the production and incorporation of monolignol conjugates into near-single-monolignol lignins, we demonstrated that substrate availability, rather than monolignol transferase substrate preference, is the most important determining factor in the production of monolignol conjugates, and lignin composition helps dictate cell wall digestibility.

2D-HSQC-NMR↗
DOE OSTI2022

Enzymatic Nitrogen Insertion into Unactivated C–H Bonds

Selective functionalization of aliphatic C–H bonds, ubiquitous in molecular structures, could allow ready access to diverse chemical products. While enzymatic oxygenation of C–H bonds is well established, the analogous enzymatic nitrogen functionalization is still unknown; nature is reliant on pre-oxidized compounds for nitrogen incorporation. Likewise, synthetic methods for selective nitrogen derivatization of unbiased C–H bonds remain elusive. In this work, new-to-nature heme-containing nitrene transferases were used as starting points for the directed evolution of enzymes to selectively aminate and amidate unactivated C(sp3)–H sites. The desymmetrization of methyl- and ethylcyclohexane with divergent site selectivity is offered as demonstration. The evolved enzymes in these lineages are highly promiscuous and show activity towards a wide array of substrates, providing a foundation for further evolution of nitrene transferase function. Computational studies and kinetic isotope effects (KIEs) are consistent with a stepwise radical pathway involving an irreversible, enantiodetermining hydrogen atom transfer (HAT), followed by a lower-barrier diastereoselectivity determining radical rebound step. In-enzyme molecular dynamics (MD) simulations reveal a predominantly hydrophobic pocket with favorable dispersion interactions with the substrate. Here, by offering a direct path from saturated precursors, these enzymes present a new biochemical logic for accessing nitrogen-containing compounds.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2023

Structural basis for heparan sulfate co-polymerase action by the EXT1–2 complex

Heparan sulfate (HS) proteoglycans are extended (-GlcAβ1,4GlcNAcα1,4-)n co-polymers containing decorations of sulfation and epimerization that are linked to cell surface and extracellular matrix proteins. In mammals, HS repeat units are extended by an obligate heterocomplex of two exostosin family members, EXT1 and EXT2, where each protein monomer contains distinct GT47 (GT-B fold) and GT64 (GT-A fold) glycosyltransferase domains. In this study, we generated human EXT1–EXT2 (EXT1–2) as a functional heterocomplex and determined its structure in the presence of bound donor and acceptor substrates. Structural data and enzyme activity of catalytic site mutants demonstrate that only two of the four glycosyltransferase domains are major contributors to co-polymer syntheses: the EXT1 GT-B fold β1,4GlcA transferase domain and the EXT2 GT-A fold α1,4GlcNAc transferase domain. Furthermore, the two catalytic sites are over 90 Å apart, indicating that HS is synthesized by a dissociative process that involves a single catalytic site on each monomer.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2020

Chemical structure and genetic organization of the E. coli O6:K15 capsular polysaccharide

Capsular polysaccharides are important virulence factors in pathogenic bacteria. Characterizing the structural components and biosynthetic pathways for these polysaccharides is key to our ability to design vaccines and other preventative therapies that target encapsulated pathogens. Many gramnegative pathogens such as Neisseria meningitidis and Escherichia coli express acidic capsules. The E. coli K15 serotype has been identifed as both an enterotoxigenic and uropathogenic pathogen. Despite its relevance as a disease-causing serotype, the associated capsular polysaccharide remains poorly characterized. We describe in this report the chemical structure of the K15 polysaccharide, based on chemical analysis and nuclear magnetic resonance (NMR) data. The repeating structure of the K15 polysaccharide consists of 4)-α-GlcpNAc-(1→5)-α-KDOp-(2→partially O-acetylated at 3-hydroxyl of GlcNAc. We also report, the organization of the gene cluster responsible for capsule biosynthesis. We identify genes in this cluster that potentially encode an O-acetyltransferase, an N-acetylglucosamine transferase, and a KDO transferase consistent with the structure we report.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

Stacking AsFMT overexpression with BdPMT loss of function enhances monolignol ferulate production in Brachypodium distachyon

To what degree can the lignin subunits in a monocot be derived from monolignol ferulate (ML-FA) conjugates? This simple question comes with a complex set of variables. Three potential requirements for optimizing ML-FA production are as follows: (1) The presence of an active FERULOYL-CoA MONOLIGNOL TRANSFERASE (FMT) enzyme throughout monolignol production; (2) Suppression or elimination of enzymatic pathways competing for monolignols and intermediates during lignin biosynthesis; and (3) Exclusion of alternative phenolic compounds that participate in lignification. A 16-fold increase in lignin-bound ML-FA incorporation was observed by introducing an AsFMT gene into Brachypodium distachyon. On its own, knocking out the native p-COUMAROYL-CoA MONOLIGNOL TRANSFERASE (BdPMT) pathway that competes for monolignols and the p-coumaroyl-CoA intermediate did not change ML-FA incorporation, nor did partial loss of CINNAMOYL-CoA REDUCTASE1 (CCR1) function, which reduced metabolic flux to monolignols. However, stacking AsFMT into the Bdpmt-1 mutant resulted in a 32-fold increase in ML-FA incorporation into lignin over the wild-type level.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2020

Deletion of a Peptidylprolyl Isomerase Gene Results in the Inability of Caldicellulosiruptor bescii To Grow on Crystalline Cellulose without Affecting Protein Glycosylation or Growth on Soluble Substrates

Caldicellulosiruptor bescii secretes a large number of complementary multifunctional enzymes with unique activities for biomass deconstruction. The most abundant enzymes in the C. bescii secretome are found in a unique gene cluster containing a glycosyl transferase (GT39) and a putative peptidyl prolyl cis-trans isomerase. Deletion of the glycosyl transferase in this cluster resulted in loss of detectable protein glycosylation in C. bescii, and its activity has been shown to be responsible for the glycosylation of the proline-threonine rich linkers found in many of the multifunctional cellulases. The presence of a putative peptidyl prolyl cis-trans isomerase within this gene cluster suggested that it might also play a role in cellulase modification. Here, we identify this gene as a putative prsA prolyl cis-trans isomerase. Deletion of prsA2 leads to the inability of C. bescii to grow on insoluble substrates such as Avicel, the model cellulose substrate, while exhibiting no differences in phenotype with the wild-type strain on soluble substrates. Finally, we provide evidence that the prsA2 gene is likely needed to increase solubility of multifunctional cellulases and that this unique gene cluster was likely acquired by members of the Caldicellulosiruptor genus with a group of genes to optimize the production and activity of multifunctional cellulases.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2026

Data for A Generalized Platform for Artificial Intelligence-powered Autonomous Protein Engineering

Proteins are the molecular machines of life with numerous applications in energy, health, and sustainability. However, engineering proteins with desired functions for practical applications remains slow, expensive, and specialist-dependent. Here we report a generally applicable platform for autonomous enzyme engineering that integrates machine learning and large language models with biofoundry automation to eliminate the need for human intervention, judgement, and domain expertise. Requiring only an input protein sequence and a quantifiable way to measure fitness, this automated platform can be applied to engineer a wide array of proteins. As a proof of concept, we engineer Arabidopsis thaliana halide methyltransferase (AtHMT) for a 90-foldimprovement in substrate preference and 16-fold improvement in ethyl-transferase activity, along with developing a Yersinia mollaretii phytase (YmPhytase) variant with 26-fold improvement in activity at neutral pH. This is accomplished in four rounds over 4 weeks, while requiring construction and characterization of fewer than 500 variants for each enzyme. This platform for autonomous experimentation paves the way for rapid advancements across diverse industries, from medicine and biotechnology to renewable energy and sustainable chemistry.

AI/ML↗
DOE OSTI2026

Data for "Metabolic Engineering Strategies to Produce Medium-Chain Oleochemicals via Acyl-ACP:CoA Transacylase Activity"

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.

Bioproducts↗
DOE OSTI2026

Data for High Yield Production of 3-Hydroxypropionic Acid Using Issatchenkia orientalis

Biomanufacturing provides a more sustainable alternative to fossil-based chemical manufacturing. 3-Hydroxypropionic acid (3HP) is a top Department of Energy value-added chemical and precursor to bioplastics, yet cost-effective microbial production remains elusive. Here, we establish the acid-tolerant yeast Issatchenkia orientalis as a robust host for low-pH 3HP biosynthesis. Genome-scale modeling identifies the β-alanine pathway as optimal, offering the highest theoretical yield and lowest oxygen requirement. Thermodynamic analysis confirms its favorability under acidic conditions. Using sequence similarity network analysis, we discover highly active aspartate 1-decarboxylase (PAND), β-alanine-pyruvate aminotransferase (BAPAT), and 3HP dehydrogenase (YDFG), which significantly improve the pathway efficiency. Next, to further elevate the production, pathway optimization through multi-copy PAND integration, byproduct elimination (knockouts of pyruvate decarboxylase and glycerol-3-phosphate dehydrogenase), and reinforcement of aspartate flux by overexpression of pyruvate carboxylase and aspartate amino transferase improves the titer to 29 g/L in shake flasks. Fed-batch fermentation at pH 4 with low-cost corn steep liquor medium further increases the production to 92 g/L with 0.7 g/g yield and 0.55 g/L/h productivity. Techno-economic analysis indicates that such performance could potentially enable a financially viable process for sustainable acrylic acid production. This work establishes I. orientalis as a next-generation platform for cost-effective 3HP production and paves the way toward industrial commercialization.

Bioproducts↗
DOE OSTI2024

Enhancing monolignol ferulate conjugate levels in poplar lignin via OsFMT1

The phenolic polymer lignin is one of the primary chemical constituents of the plant secondary cell wall. Due to the inherent plasticity of lignin biosynthesis, several phenolic monomers have been shown to be incorporated into the polymer, as long as the monomer can undergo radicalization so it can participate in coupling reactions. In this study, we significantly enhance the level of incorporation of monolignol ferulate conjugates into the lignin polymer to improve the digestibility of lignocellulosic biomass. Overexpression of a rice Feruloyl-CoA Monolignol Transferase (FMT), OsFMT1, in hybrid poplar (Populus alba x grandidentata) produced transgenic trees clearly displaying increased cell wall-bound ester-linked ferulate, p-hydroxybenzoate, and p-coumarate, all of which are in the lignin cell wall fraction, as shown by NMR and DFRC. We also demonstrate the use of a novel UV–Vis spectroscopic technique to rapidly screen plants for the presence of both ferulate and p-hydroxybenzoate esters. Lastly we show, via saccharification assays, that the OsFMT1 transgenic poplars have significantly improved processing efficiency compared to wild-type and Angelica sinensis-FMT-expressing poplars. The findings demonstrate that OsFMT1 has a broad substrate specificity and a higher catalytic efficiency compared to the previously published FMT from Angelica sinensis (AsFMT). Importantly, enhanced wood processability makes OsFMT1 a promising gene to optimize the composition of lignocellulosic biomass.

09 BIOMASS FUELS↗
DOE OSTI2022

Design and evaluation of novel analogs of 2-amino-4-boronobutanoic acid (ABBA) as inhibitors of human gamma-glutamyl transpeptidase

Inhibitors of gamma-glutamyl transpeptidase (GGT1, aka gamma-glutamyl transferase) are needed for the treatment of cancer, cardiovascular illness and other diseases. Compounds that inhibit GGT1 have been evaluated in the clinic, but no inhibitor has successfully demonstrated specific and systemic GGT1 inhibition. All have severe side effects. L-2-amino-4-boronobutanoic acid (l-ABBA), a glutamate analog, is the most potent GGT1 inhibitor in vitro. In this study, we have solved the crystal structure of human GGT1 (hGGT1) with ABBA bound in the active site. The structure was interrogated to identify interactions between the enzyme and the inhibitor. Based on these data, a series of novel ABBA analogs were designed and synthesized. Their inhibitory activity against the hydrolysis and transpeptidation activities of hGGT1 were determined. The lead compounds were crystalized with hGGT1 and the structures solved. The kinetic data and structures of the complexes provide new insights into the critical role of protein structure dynamics in developing compounds for inhibition of hGGT1.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2023

Iron-sulfur clusters are involved in post-translational arginylation

Eukaryotic arginylation is an essential post-translational modification that modulates protein stability and regulates protein half-life. Arginylation is catalyzed by a family of enzymes known as the arginyl-tRNA transferases (ATE1s), which are conserved across the eukaryotic domain. Despite their conservation and importance, little is known regarding the structure, mechanism, and regulation of ATE1s. In this work, we show that ATE1s bind a previously undiscovered [Fe-S] cluster that is conserved across evolution. We characterize the nature of this [Fe-S] cluster and find that the presence of the [Fe-S] cluster in ATE1 is linked to its arginylation activity, both in vitro and in vivo, and the initiation of the yeast stress response. Importantly, the ATE1 [Fe-S] cluster is oxygen-sensitive, which could be a molecular mechanism of the N-degron pathway to sense oxidative stress. Taken together, our data provide the framework of a cluster-based paradigm of ATE1 regulatory control.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2025

Understanding and Harnessing the Robustness of Undomesticated Yarrowia lipolytica Strains for Biosynthesis of Designer Bioesters (Final Report)

This project seeks to elucidate and harness the exceptional robustness of novel and undomesticated Y. lipolytica isolates, which were identified from a genetic diversity screening for compatibility with bioenergy development. Bioenergy-relevant isolates were further developed as microbial platforms for efficient conversion of undetoxified biomass hydrolysates into designer bio-esters continuously recovered by solvent extraction. The project has three major goals. Goal 1. Elucidate and enhance the endogenous metabolism of Y. lipolytica for superior growth, sugar utilization, and lipid accumulation in undetoxified biomass hydrolysates under hypoxic conditions. Goal 2. Understand and enhance the underlying mechanism of exceptional tolerance of Y. lipolytica to organic solvents. Goal 3. Elucidate and rewire endogenous metabolism of the most robust Y. lipolytica strains for effective conversion of accumulating lipids to designer bio-esters. Significant progress has been made toward completing all research goals. We elucidated and optimized the robustness of Y. lipolytica by utilizing mixed C5 and C6 sugars in switchgrass hydrolysates (SGH) for lipid production (Aim 1). We conducted extensive omics analysis to investigate how genetic diversity among Yarrowia strains, derived from natural isolates or developed through adaptive laboratory evolution, influences lipid production when utilizing SGH. In Aim 2, novel mechanisms and underlying genetics were discovered that enabled Yarrowia strains to thrive in cultures containing high ionic liquid (IL) concentrations. Amongst other novel findings, it was found that sterols strengthened cell membranes to confer IL toxicity resistance, specifically via increased ergosterol content upon exposure to IL. In Aim 3, mechanistic studies elucidated how Y. lipolytica utilized intracellular lipids and alkanes/alkenes, leading to our discovery of novel enzymes and pathways for making short-chain esters. Most notably, thermostable chloramphenicol transferases were repurposed to function as alcohol acetyltransferases in Y. lipolytica, as well as the Gram-negative and Gram-positive bacteria Escherichia coli and thermophile Clostridium thermocellum, respectively.

09 BIOMASS FUELS↗
DOE OSTI2022

The Impact of Cell Wall Feruloylation on Plant Growth, Responses to Environmental Stress, Plant Pathogens and Cell Wall Degradability

This article summarizes evolving concepts and scientific findings on cell wall feruloylation and ferulate oxidative coupling processes in grasses, and the effects these have on the wide range of cell wall properties and consequent plant responses to biotic and abiotic stress and tissue degradability. Updates of the different strategies that have been applied to genetically modifying cell wall feruloylation are presented. Special emphasis is given to the modification of cell wall feruloylation by heterologous expression of cell wall ferulic acid esterase, as this strategy has provided insights into the impact of feruloylation on the changes in the physicochemical properties of the cell wall with consequent effects on different plant processes. Emerging feruloyl transferase candidate genes codifying enzymes accounting for ferulate incorporation into grass arabinoxylans are also highlighted.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

Programmable Site‐Specific Functionalization of DNA Origami with Polynucleotide Brushes

Abstract Combining surface‐initiated, TdT (terminal deoxynucleotidyl transferase) catalyzed enzymatic polymerization (SI‐TcEP) with precisely engineered DNA origami nanostructures (DONs) presents an innovative pathway for the generation of stable, polynucleotide brush‐functionalized DNA nanostructures. We demonstrate that SI‐TcEP can site‐specifically pattern DONs with brushes containing both natural and non‐natural nucleotides. The brush functionalization can be precisely controlled in terms of the location of initiation sites on the origami core and the brush height and composition. Coarse‐grained simulations predict the conformation of the brush‐functionalized DONs that agree well with the experimentally observed morphologies. We find that polynucleotide brush‐functionalization increases the nuclease resistance of DONs significantly, and that this stability can be spatially programmed through the site‐specific growth of polynucleotide brushes. The ability to site‐specifically decorate DONs with brushes of natural and non‐natural nucleotides provides access to a large range of functionalized DON architectures that would allow for further supramolecular assembly, and for potential applications in smart nanoscale delivery systems.

Yang, Yunqi↗
DOE OSTI2021

Programmable Site-Specific Functionalization of DNA Origami with Polynucleotide Brushes

Combining surface-initiated, TdT (terminal deoxynucleotidyl transferase) catalyzed enzymatic polymerization (SI-TcEP) with precisely engineered DNA origami nanostructures (DONs) presents an innovative pathway for the generation of stable, polynucleotide brush-functionalized DNA nanostructures. Here we demonstrate that SI-TcEP can site-specifically pattern DONs with brushes containing both natural and non-natural nucleotides. The brush functionalization can be precisely controlled in terms of the location of initiation sites on the origami core and the brush height and composition. Coarse-grained simulations predict the conformation of the brush-functionalized DONs that agree well with the experimentally observed morphologies. We find that polynucleotide brush-functionalization increases the nuclease resistance of DONs significantly, and that this stability can be spatially programmed through the site-specific growth of polynucleotide brushes. The ability to site-specifically decorate DONs with brushes of natural and non-natural nucleotides provides access to a large range of functionalized DON architectures that would allow for further supramolecular assembly, and for potential applications in smart nanoscale delivery systems.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2024

Development of inducible promoters for regulating gene expression in Clostridium tyrobutyricum for biobutanol production

Abstract Clostridium tyrobutyricumis an anaerobe known for its ability to produce short‐chain fatty acids, alcohols, and esters. We aimed to develop inducible promoters for fine‐tuning gene expression inC. tyrobutyricum. Synthetic inducible promoters were created by employing anEscherichia coli lacoperator to regulate the thiolase promoter (PCathl) fromClostridium acetobutylicum, with the best one (LacI‐Pto4s) showing a 5.86‐fold dynamic range with isopropyl β‐d‐thiogalactoside (IPTG) induction. A LT‐Pt7 system with a dynamic range of 11.6‐fold was then created by combining LacI‐Pto4s with a T7 expression system composing of RNA polymerase (T7RNAP) and Pt7lacpromoter. Furthermore, two inducible expression systems BgaR‐PbgaLA and BgaR‐PbgaLB with a dynamic range of ~40‐fold were developed by optimizing a lactose‐inducible expression system fromClostridium perfringenswith modified 5′ untranslated region (5′ UTR) and ribosome‐binding site (RBS). BgaR‐PbgaLB was then used to regulate the expressions of a bifunctional aldehyde/alcohol dehydrogenase encoded byadhE2 and butyryl‐CoA/acetate Co‐A transferase encoded bycat1 inC. tyrobutyricumwild type and Δcat1::adhE2, respectively, demonstrating its efficient inducible gene regulation. The regulatedcat1 expression also confirmed that the Cat1‐catalyzed reaction was responsible for acetate assimilation inC. tyrobutyricum. The inducible promoters offer new tools for tuning gene expression inC. tyrobutyricumfor industrial applications.

Biotechnology & Applied Microbiology↗