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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 127 records · Page 7
All sourcesNASA NTRSDOE OSTI
DOE OSTI2023

Development of Scalable Reactive Transport Framework for PFAS

Research on PFAS chemicals is extensive and covers all aspects, including analytical quantification, determination of properties, toxicology, ex situ treatment, and in situ remediation. PFAS chemicals are very stable and thus persistent/recalcitrant in the environment. Although there are many unknowns about PFAS chemicals, degradation pathways, reaction rates, etc., many different sorption, oxidation, reduction, biological, and innovative treatment approaches are being developed. Sorption with activated carbon is currently the only fully available in situ treatment technology for PFAS-impacted groundwater. Given the wide array of PFAS chemicals and transformation products, remediation may need multi-step treatment trains to fully address the PFAS contamination. The work here provides kinetic reaction modules that represent an initial cut at functionality representing PFAS migration and reaction in groundwater aquifer flow and transport models. One reaction kinetics module provides a method to model kinetically limited adsorption using a mass transfer model. The second reaction module represents biological transformation of 8:2 FTOH and daughter species, illustrating how a complex reaction pathway network can be represented. Both reaction modules allow for spatially variable parameter values so that a variety of remediation approaches (e.g., a PRB or volumetric treatment or variations in geochemical conditions) can be simulated. The intent with these PFAS reaction modules is to provide tools for practitioners to aid in the selection, design, and assessment of potential in situ PFAS remediation strategies. It is anticipated that, as PFAS remediation technologies and scientific understanding advances, these modules would be refined or replaced to match new knowledge.

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

Enhanced production of taxadiene in Saccharomyces cerevisiae

Cost-effective production of the highly effective anti-cancer drug, paclitaxel (Taxol ® ), remains limited despite growing global demands. Low yields of the critical taxadiene precursor remains a key bottleneck in microbial production. In this study, the key challenge of poor taxadiene synthase (TASY) solubility in S. cerevisiae was revealed, and the strains were strategically engineered to relieve this bottleneck. Multi-copy chromosomal integration of TASY harbouring a selection of fusion solubility tags improved taxadiene titres 22-fold, up to 57 ± 3 mg/L at 30 °C at microscale, compared to expressing a single episomal copy of TASY. The scalability of the process was highlighted through achieving similar titres during scale up to 25 mL and 250 mL in shake flask and bioreactor cultivations, respectively at 20 and 30 °C. Maximum taxadiene titres of 129 ± 15 mg/L and 127 mg/L were achieved through shake flask and bioreactor cultivations, respectively, of the optimal strain at a reduced temperature of 20 °C. The results of this study highlight the benefit of employing a combination of molecular biology and bioprocess tools during synthetic pathway development, with which TASY activity was successfully improved by 6.5-fold compared to the highest literature titre in S. cerevisiae cell factories.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2020

A Hybrid Pathway to Biojet Fuel via 2,3-Butanediol

Production of biomass-derived sustainable alternative jet fuels (SAJF) has been considered as an important approach to decarbonize the aviation industry but still possesses various challenges in technology advancement, particularly in achieving high carbon efficiency. Here we report a hybrid pathway to SAJF from 2,3-butanediol (2,3-BDO), integrating biologically converting biomass to 2,3-BDO with catalytically upgrading of 2,3-BDO to jet-range hydrocarbons. This pathway is demonstrated to have a high carbon recovery to liquid hydrocarbons from corn stover (25–28%) (74–82% of the theoretical maximum efficiency). The catalytic conversion steps involve 2,3-BDO to C3+ olefins, oligomerization, and hydrogenation where the first two steps are the focus of this study. Under optimum reaction conditions (523 K, 115 kPa, 1.0 h-1 weight hourly space velocity), 2,3-BDO conversion and C3+ olefin selectivity are >97% and 94–98% during 40 h time on stream, respectively. To demonstrate the adaptability of this technology with bio-derived 2,3-BDO, we also investigated the impact of water and other organic coproducts (acetoin and acetic acid) inherited from the fermentation broth on the catalyst performance and product selectivities. We have shown that the catalyst can handle a significant amount of water in the liquid feed (40 wt% water/60 wt% 2,3-BDO) and maintain catalyst stability for ~40 h. Acetoin can be converted to similar C3+ olefins as 2,3-BDO with complete conversion of acetoin. Co-feeding 10 wt% acetoin with 2,3-BDO is found to have no impact on 2,3-BDO conversion, C3+ olefin selectivity, and catalyst stability. The utilization of organic coproduct like acetoin can help to improve overall carbon conversion efficiency when using real biomass-derived 2,3-BDO. On the other hand, the presence of 10 wt% acetic acid is shown to drastically inhibit methyl ethyl ketone (MEK) hydrogenation and butene oligomerization as revealed by the increased MEK and butene selectivities, implying the importance of separating organic acids when feeding bio-derived 2,3-BDO. The formed C3–C6 olefins from 2,3-BDO are further oligomerized over Amberlyst-36 catalyst to longer-chain hydrocarbons with >70 wt% jet-range hydrocarbons including predominantly iso-olefins/iso-paraffins. The overall carbon efficiency for the jet-range hydrocarbons is 19–22%, exceeding most of the reported biojet pathways, which makes it a promising approach for SAJF production.

29 ENERGY PLANNING, POLICY, AND ECONOMY↗
DOE OSTI2021

Maize tissue culture, transformation, and genome editing

The importance of maize (Zea mays L.) to global agriculture, world economy, and food security is widely known and increasing. Current maize breeding programs are deeply integrated with recent and rapid technological advances in genome sequencing, computational biology, and new genotyping and phenotyping technologies. Transformation and genome editing capabilities are a central hub to an array of advanced molecular and breeding approaches to crop improvement. Tissue culture and somatic embryogenesis play essential and central roles in maize transformation biology. Synergistic applications of maize transformation, advanced genomics, and genome editing provide a potent interdependent triad for functional genomics research and advanced molecular breeding. Implementation of advanced capabilities to transform maize and conduct genome editing will profoundly influence the dynamics of global agriculture ushering in a new era of varietal development and molecular breeding. With over 60.9 Mha planted in 2019 alone, biotech maize accounts for 31% of the world’s maize production. Up to 10% higher yields are achieved using new varieties generated using genetic modification technologies compared to similar conventional varieties. By extension, the impact new varietal releases developed through genome editing will likely be more significant. Further, advances in transformation and genome editing technologies will facilitate an even wider applicability for the development of new varieties with increasingly complex traits. The introduction of biochemical pathways and the use of synthetic biology have become increasingly more attainable. The future is of genotype independent maize transformation and genome editing, as the working platform will impact world agriculture, global food security, and plant science well into the future.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

Light-Dependence of Formate (C1) and Acetate (C2) Transport and Oxidation in Poplar Trees

Although apparent light inhibition of leaf day respiration is a widespread reported phenomenon, the mechanisms involved, including utilization of alternate respiratory pathways and substrates and light inhibition of TCA cycle enzymes are under active investigation. Recently, acetate fermentation was highlighted as a key drought survival strategy mediated through protein acetylation and jasmonate signaling. Here, we evaluate the light-dependence of acetate transport and assimilation in Populus trichocarpa trees using the dynamic xylem solution injection (DXSI) method developed here for continuous studies of C1 and C2 organic acid transport and light-dependent metabolism. Over 7 days, 1.0 L of [13C]formate and [13C2]acetate solutions were delivered to the stem base of 2-year old potted poplar trees, while continuous diurnal observations were made in the canopy of CO2, H2O, and isoprene gas exchange together with δ13CO2. Stem base injection of 10 mM [13C2]acetate induced an overall pattern of canopy branch headspace 13CO2 enrichment (δ13CO2 +27‰) with a diurnal structure in δ13CO2 reaching a mid-day minimum followed by a maximum shortly after darkening where δ13CO2 values rapidly increased up to +12‰. In contrast, 50 mM injections of [13C]formate were required to reach similar δ13CO2 enrichment levels in the canopy with δ13CO2 following diurnal patterns of transpiration. Illuminated leaves of detached poplar branches pretreated with 10 mM [13C2]acetate showed lower δ13CO2 (+20‰) compared to leaves treated with 10 mM [13C]formate (+320‰), the opposite pattern observed at the whole plant scale. Following dark/light cycles at the leaf-scale, rapid, strong, and reversible enhancements in headspace δ13CO2 by up to +60‰ were observed in [13C2]acetate-treated leaves which showed enhanced dihydrojasmonic acid and TCA cycle intermediate concentrations. The results are consistent with acetate in the transpiration stream as an effective activator of the jasmonate signaling pathway and respiratory substrate. The shorter lifetime of formate relative to acetate in the transpiration stream suggests rapid formate oxidation to CO2 during transport to the canopy. In contrast, acetate is efficiently transported to the canopy where an increased allocation towards mitochondrial dark respiration occurs at night. The results highlight the potential for an effective integration of acetate into glyoxylate and TCA cycles and the light-inhibition of citrate synthase as a potential regulatory mechanism controlling the diurnal allocation of acetate between anabolic and catabolic processes.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2025

Comparative Performance Evaluation of Large Language Models for Extracting Molecular Interactions and Pathway Knowledge

Understanding the interactions and regulatory relationships among biomolecules is essential for deciphering complex biological systems and elucidating the mechanisms behind diverse biological functions. Traditionally, the collection of such molecular interaction data has relied on expert curation, a process that is both time-consuming and labor-intensive. To address these limitations, this study explores the use of large language models (LLMs) to automate the genome-scale extraction of molecular interaction knowledge. Here, we evaluate the performance of various LLMs on key biological tasks, including the identification of protein-protein interactions, detection of genes associated with pathways influenced by low-dose radiation, and inference of gene regulatory relationships. Our findings demonstrate that larger LLMs tend to perform better, particularly in extracting intricate gene and protein interactions. Despite their strengths, these models face challenges in recognizing functionally diverse gene groups and highly correlated regulatory relationships. Through a comprehensive analysis using established molecular interaction and pathway databases, we show that LLMs possess the potential to identify relevant biomolecules and predict their interactions, offering valuable insights and marking a significant step toward AI-driven biological knowledge discovery.

63 RADIATION, THERMAL, AND OTHER ENVIRON. POLLUTAN↗
DOE OSTI2020

Mathematical Model of ATM Activation and Chromatin Relaxation by Ionizing Radiation

We propose a comprehensive mathematical model to study the dynamics of ionizing radiation induced Ataxia-telangiectasia mutated (ATM) activation that consists of ATM activation through dual mechanisms: the initiative activation pathway triggered by the DNA damage-induced local chromatin relaxation and the primary activation pathway consisting of a self-activation loop by interplay with chromatin relaxation. The model is expressed as a series of biochemical reactions, governed by a system of differential equations and analyzed by dynamical systems techniques. Radiation induced double strand breaks (DSBs) cause rapid local chromatin relaxation, which is independent of ATM but initiates ATM activation at damage sites. Key to the model description is how chromatin relaxation follows when active ATM phosphorylates KAP-1, which subsequently spreads throughout the chromatin and induces global chromatin relaxation. Additionally, the model describes how oxidative stress activation of ATM triggers a self-activation loop in which PP2A and ATF2 are released so that ATM can undergo autophosphorylation and acetylation for full activation in relaxed chromatin. In contrast, oxidative stress alone can partially activate ATM because phosphorylated ATM remains as a dimer. The model leads to predictions on ATM mediated responses to DSBs, oxidative stress, or both that can be tested by experiments.

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

Machine learning enables identification of an alternative yeast galactose utilization pathway

How genomic differences contribute to phenotypic differences is a major question in biology. The recently characterized genomes, isolation environments, and qualitative patterns of growth on 122 sources and conditions of 1,154 strains from 1,049 fungal species (nearly all known) in the yeast subphylum Saccharomycotina provide a powerful, yet complex, dataset for addressing this question. We used a random forest algorithm trained on these genomic, metabolic, and environmental data to predict growth on several carbon sources with high accuracy. Known structural genes involved in assimilation of these sources and presence/absence patterns of growth in other sources were important features contributing to prediction accuracy. By further examining growth on galactose, we found that it can be predicted with high accuracy from either genomic (92.2%) or growth data (82.6%) but not from isolation environment data (65.6%). Prediction accuracy was even higher (93.3%) when we combined genomic and growth data. After the GALactose utilization genes, the most important feature for predicting growth on galactose was growth on galactitol, raising the hypothesis that several species in two orders, Serinales and Pichiales (containing the emerging pathogen Candida auris and the genus Ogataea, respectively), have an alternative galactose utilization pathway because they lack the GAL genes. Growth and biochemical assays confirmed that several of these species utilize galactose through an alternative oxidoreductive D-galactose pathway, rather than the canonical GAL pathway. Machine learning approaches are powerful for investigating the evolution of the yeast genotype–phenotype map, and their application will uncover novel biology, even in well-studied traits.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2026

Abiotic manganese oxide formation induced by light and copper-driven Fenton chemistry

Redox-active transition metals such as manganese (Mn) and copper (Cu) frequently coexist in surface water systems, where their interactions can significantly influence contaminant fate and water quality. It is widely known that the oxidation of Mn 2+ (aq) to Mn(IV) oxide solids is driven mainly by biotic processes in which Cu 2+ serves as an inhibitor of Mn oxidation. However, Cu 2+ can also trigger Fenton reactions to produce reactive oxygen species (ROS) capable of promoting the abiotic Mn oxidation (i.e., without the involvement of biological organisms), through an alternative natural pathway that remains overlooked in water. In this study, we found that the abiotic oxidation of Mn 2+ can be facilitated by the co-existence of sunlight and Cu 2+ . By contrasting Cu 2+ with other divalent cations, such as Ca 2+ , Mg 2+ , and Zn 2+ , this investigation highlights the distinct role of Cu 2+ in promoting Mn oxidation. Moreover, the study elucidated how light induces ROS to trigger Cu-driven photo-Fenton reactions and identified the roles of ROS in Mn oxidation. Chemical-Fenton reactions (i.e., the interaction between Cu and H 2 O 2 in the absence of light) were also examined for their contribution to the Cu-driven Mn photo-oxidation, supporting the involvement of Fenton chemistry in Mn oxidation. By elucidating the novel photochemical mechanism of Mn oxide formation, our findings advance the understanding of heavy metal interactions in aqueous environments and highlight the intricate roles of Cu 2+ in affecting elemental cycling and the evaluation of heavy metal contamination in water resources. This discovery also underscores the critical contribution of co-existing Fenton-active metals in forming Mn oxide solids.

Abiotic Mn oxidation↗
DOE OSTI2021

Rickettsia increases its infection and spread in whitefly populations by manipulating the defense patterns of the host plant

ABSTRACT The whitefly Bemisia tabaci is a destructive agricultural pest that frequently harbors various species of secondary symbionts including Rickettsia. Previous studies have revealed that the infection of Rickettsia can improve whitefly performance on food plants; however, to date, no evidence has shown, if, and how, Rickettsia manipulates the plant-insect interactions. In the current study, the effects of Rickettsia persistence on the induced plant defenses and the consequent performance of whitefly B. tabaci were investigated. Results revealed that Rickettsia can be transmitted into plants via whitefly feeding and remain alive within the cotton plants for at least 2 weeks. The different expression genes of cotton plants were mostly concentrated in the phytohormone signaling pathways, the marker genes of jasmonic-acid signaling pathway (AOC, AOS, LOX, MYC2) were significantly downregulated, while the marker genes of the salicylic-acid signaling pathway (WRKY70, PR-1) were upregulated. Biological experiments revealed that the fecundity of Rickettsia negative B. tabaci significantly increased when they fed on Rickettsia-persistent cotton plants. Taken together, we provide experimental evidence that the persistence of Rickettsia and its induced defense responses in cotton plants can increase the fitness of whitefly and, by this, Rickettsia may increase its infection and spread within its whitefly host.

Shi, Pei-Qiong↗
DOE OSTI2022

Rerouting of the lignin biosynthetic pathway by inhibition of cytosolic shikimate recycling in transgenic hybrid aspen

Lignin is a phenolic polymer deposited in the plant cell wall and is mainly polymerized from three canonical monomers (monolignols), i.e., p-coumaryl, coniferyl, and sinapyl alcohols. After polymerization, these alcohols form different lignin substructures. In dicotyledons, monolignols are biosynthesized from phenylalanine, an aromatic amino acid. Shikimate acts at two positions in the route to the lignin building blocks. It is part of the shikimate pathway which provides the precursor for the biosynthesis of phenylalanine, and is involved in the transesterification of pcoumaroyl- CoA to p-coumaroyl shikimate, one of key steps in the biosynthesis of coniferyl and sinapyl alcohols. The shikimate residue in p-coumaroyl shikimate is released in later steps, and the resulting shikimate becomes available again for the biosynthesis of new p-coumaroyl shikimate molecules. In this study, we inhibited cytosolic shikimate recycling in transgenic hybrid aspen by accelerated phosphorylation of shikimate in the cytosol through expression of a bacterial shikimate kinase. Here, this expression elicited an increase in p-hydroxyphenyl units of lignin and, by contrast, a decrease in guaiacyl and syringyl units. Transgenic plants with high shikimate kinase activity produced a lignin content comparable to that in wild-type plants and had an increased processability via enzymatic saccharification. Although expression of many genes was altered in the transgenic plants, elevated shikimate kinase activity did not exert a significant effect on the expression of the majority of genes responsible for lignin biosynthesis. The present results indicate that cytosolic shikimate recycling is crucial to the monomeric composition of lignin rather than for lignin content.

shikimate pathway↗
DOE OSTI2023

Characterization of a novel aromatic substrate-processing microcompartment in Actinobacteria

ABSTRACT We have discovered a new cluster of genes that is found exclusively in the Actinobacteria phylum. This locus includes genes for the 2-aminophenol meta -cleavage pathway and the shell proteins of a bacterial microcompartment (BMC) and has been named aromatics (ARO) for its putative role in the breakdown of aromatic compounds. In this study, we provide details about the distribution and composition of the ARO BMC locus and conduct phylogenetic, structural, and functional analyses of the first two enzymes in the catabolic pathway: a unique 2-aminophenol dioxygenase, which is exclusively found alongside BMC shell genes in Actinobacteria, and a semialdehyde dehydrogenase, which works downstream of the dioxygenase. Genomic analysis reveals variations in the complexity of the ARO loci across different orders. Some loci are simple, containing shell proteins and enzymes for the initial steps of the catabolic pathway, while others are extensive, encompassing all the necessary genes for the complete breakdown of 2-aminophenol into pyruvate and acetyl-CoA. Furthermore, our analysis uncovers two subtypes of ARO BMC that likely degrade either 2-aminophenol or catechol, depending on the presence of a pathway-specific gene within the ARO locus. The precise precursor of 2-aminophenol, which serves as the initial substrate and/or inducer for the ARO pathway, remains unknown, as our model organism Micromonospora rosaria cannot utilize 2-aminophenol as its sole energy source. However, using enzymatic assays, we demonstrate the dioxygenase’s ability to cleave both 2-aminophenol and catechol in vitro , in collaboration with the aldehyde dehydrogenase, to facilitate the rapid conversion of these unstable and toxic intermediates. IMPORTANCE Bacterial microcompartments (BMCs) are proteinaceous organelles that are widespread among bacteria and provide a competitive advantage in specific environmental niches. Studies have shown that the genetic information necessary to form functional BMCs is encoded in loci that contain genes encoding shell proteins and the enzymatic core. This allows the bioinformatic discovery of BMCs with novel functions and expands our understanding of the metabolic diversity of BMCs. ARO loci, found only in Actinobacteria, contain genes encoding for phylogenetically remote shell proteins and homologs of the meta -cleavage degradation pathway enzymes that were shown to convert central aromatic intermediates into pyruvate and acetyl-CoA in gamma Proteobacteria. By analyzing the gene composition of ARO BMC loci and characterizing two core enzymes phylogenetically, structurally, and functionally, we provide an initial functional characterization of the ARO BMC, the most unusual BMC identified to date, distinctive among the repertoire of studied BMCs.

2-AP 1,6-dioxygenase↗
DOE OSTI2021

Isotope ratio-based quantification of carbon assimilation highlights the role of plastidial isoprenoid precursor availability in photosynthesis

Abstract Background We report a method to estimate carbon assimilation based on isotope ratio-mass spectrometry (IRMS) of 13 CO 2 labeled plant tissue. Photosynthetic carbon assimilation is the principal experimental observable which integrates important aspects of primary plant metabolism. It is traditionally measured through gas exchange. Despite its centrality in plant research, gas exchange performs poorly with rosette growth habits typical of Arabidopsis thaliana , mutant lines with limited biomass, and accounts poorly for leaf shading. Results IRMS-based carbon assimilation values from plants labeled at different light intensities were compared to those obtained by gas exchange, and the two methods yielded similar values. Using this method, we observed a strong correlation between 13 C content and labeling time (R 2 = 0.999) for 158 wild-type plants labeled for 6 to 42 min. Plants cultivated under different light regimes showed a linear response with respect to carbon assimilation, varying from 7.38 nmol 13 C mg −1 leaf tissue min −1 at 80 PAR to 19.27 nmol 13 C mg −1 leaf tissue min −1 at 500 PAR. We applied this method to examine the link between inhibition of the 2 C -methyl- d -erythritol-4-phosphate (MEP) pathway and suppression of photosynthesis. A significant decrease in carbon assimilation was observed when metabolic activity in the MEP pathway was compromised by mutation or herbicides targeting the MEP pathway. Mutants affected in MEP pathway genes 1-DEOXY- d -XYLULOSE 5-PHOSPHATE SYNTHASE (DXS) or 1-HYDROXY-2-METHYL-2-(E)-BUTENYL 4-DIPHOSPHATE SYNTHASE (HDS) showed assimilation rates 36% and 61% lower than wild type. Similarly, wild type plants treated with the MEP pathway inhibitors clomazone or fosmidomycin showed reductions of 52% and 43%, respectively, while inhibition of the analogous mevalonic acid pathway, which supplies the same isoprenoid intermediates in the cytosol, did not, suggesting inhibition of photosynthesis was specific to disruption of the MEP pathway. Conclusions This method provides an alternative to gas exchange that offers several advantages: resilience to differences in leaf overlap, measurements based on tissue mass rather than leaf surface area, and compatibility with mutant Arabidopsis lines which are not amenable to gas exchange measurements due to low biomass and limited leaf surface area. It is suitable for screening large numbers of replicates simultaneously as well as post-hoc analysis of previously labeled plant tissue and is complementary to downstream detection of isotopic label in targeted metabolite pools.

2C‑methyl‑d‑erythritol 4‑phosphate pathway↗
DOE OSTI2021

A rich and bountiful harvest: Key discoveries in plant cell biology

Abstract The field of plant cell biology has a rich history of discovery, going back to Robert Hooke’s discovery of cells themselves. The development of microscopes and preparation techniques has allowed for the visualization of subcellular structures, and the use of protein biochemistry, genetics, and molecular biology has enabled the identification of proteins and mechanisms that regulate key cellular processes. In this review, seven senior plant cell biologists reflect on the development of this research field in the past decades, including the foundational contributions that their teams have made to our rich, current insights into cell biology. Topics covered include signaling and cell morphogenesis, membrane trafficking, cytokinesis, cytoskeletal regulation, and cell wall biology. In addition, these scientists illustrate the pathways to discovery in this exciting research field.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2020

Biophysical and Biochemical Characterization of TP0037, a D-Lactate Dehydrogenase, Supports an Acetogenic Energy Conservation Pathway in Treponema pallidum

A longstanding conundrum in Treponema pallidum biology concerns how the spirochete generates sufficient energy to fulfill its complex pathogenesis processes during human syphilitic infection. For decades, it has been assumed that the bacterium relies solely on glucose catabolism (via glycolysis) for generation of its ATP. However, the organism’s robust motility, believed to be essential for human tissue invasion and dissemination, would require abundant ATP likely not provided by the parsimony of glycolysis. As such, additional ATP generation, either via a chemiosmotic gradient, substrate-level phosphorylation, or both, likely exists in T. pallidum. Along these lines, we have hypothesized that T. pallidum exploits an acetogenic energy conservation pathway that relies on the redox chemistry of flavins. Central to this hypothesis is the apparent existence in T. pallidum of an acetogenic pathway for the conversion of d-lactate to acetate. Herein we have characterized the structural, biophysical, and biochemical properties of the first enzyme (d-lactate dehydrogenase [d-LDH]; TP0037) predicted in this pathway. Binding and enzymatic studies showed that recombinant TP0037 consumed d-lactate and NAD+ to produce pyruvate and NADH. The crystal structure of TP0037 revealed a fold similar to that of other d-acid dehydrogenases; residues in the cofactor-binding and active sites were homologous to those of other known d-LDHs. The crystal structure and solution biophysical experiments revealed the protein’s propensity to dimerize, akin to other d-LDHs. This study is the first to elucidate the enzymatic properties of T. pallidum’s d-LDH, thereby providing new compelling evidence for a flavin-dependent acetogenic energy conservation (ATP-generating) pathway in T. pallidum.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

Generating Protein Structures for Pathway Discovery Using Deep Learning

Resolving the intricate details of biological phenomena at the molecular level is fundamentally limited by both length- and time scales that can be probed experimentally. Molecular dynamics (MD) simulations at various scales are powerful tools frequently employed to offer valuable biological insights beyond experimental resolution. However, while it is relatively simple to observe long-lived, stable configurations of, for example, proteins, at the required spatial resolution, simulating the more interesting rare transitions between such states often takes orders of magnitude longer than what is feasible even on the largest supercomputers available today. One common aspect of this challenge is pathway discovery, where the start and end states of a scientific phenomenon are known or can be approximated, but the mechanistic details in between are unknown. Here, we propose a representation-learning-based solution that uses interpolation and extrapolation in an abstract representation space to synthesize potential transition states, which are automatically validated using MD simulations. The new simulations of the synthesized transition states are subsequently incorporated into the representation learning, leading to an iterative framework for targeted path sampling. Our approach is demonstrated by recovering the transition of a RAS-RAF protein domain (CRD) from membrane-free to interacting with the membrane using coarse-grain MD simulations.

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

Evolution_and_engineering_of_pathways_for_aromatic_O-demethylation_in_Pseudomonas_putida_KT2440

Biological conversion of lignin from biomass offers a promising strategy for sustainable production of fuels and chemicals. However,aromatic compoundsderived from lignin commonly contain methoxy groups, andO-demethylation of these substrates is often a rate-limiting reaction that influences catabolic efficiency. Severalenzymefamilies catalyze aromaticO-demethylation, but they are rarely comparedin vivoto determine an optimal biocatalytic strategy. Here, two pathways for aromaticO-demethylation were compared inPseudomonas putidaKT2440. The native Rieske non-heme ironmonooxygenase(VanAB) and, separately, a heterologous tetrahydrofolate-dependentdemethylase(LigM) were constitutively expressed inP. putida, and the strains were optimized via adaptive laboratory evolution (ALE) withvanillateas a model substrate. All evolved strains displayed improved growth phenotypes, with the evolved strains harboring the native VanAB pathway exhibiting growth rates ∼1.8x faster than those harboring the heterologous LigM pathway.Enzyme kineticsandtranscriptomicsstudies investigated the contribution of selected mutations toward enhanced utilization of vanillate. The VanAB-overexpressing strains contained the most impactful mutations, including those in VanB, thereductasefor vanillateO-demethylase, PP_3494, a global regulator of vanillate catabolism, andfghA, involved in formaldehydedetoxification. These three mutations were combined into a single strain, which exhibited approximately 5x faster vanillate consumption than the wild-type strain in the first 8 h of cultivation. Overall, this study illuminates the details of vanillate catabolism in the context of two distinct enzymatic mechanisms,yielding a platform strain for efficientO-demethylation of lignin-related aromatic compounds to value-added products. This DOI contains supplementary material associated with the published manuscript.

CBI↗