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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 73 records · Page 4
All sourcesNASA NTRSDOE OSTI
DOE OSTI2026

Adaptive_Evolution_2026

Thermoanaerobacterium saccharolyticum, an anaerobic and thermophilic bacterium capable of metabolizing sugar monomers and soluble oligomers into ethanol, has been proposed for use in consolidated bioprocessing in coculture with compatible cellulolytic bacteria such asClostridium thermocellum. Although the mixed acid fermentation of both of these strains has been engineered to produce ethanol as the major fermentation product, the maximum titer produced thus far byC. thermocellum, about 3% (w/v) ethanol, is half that produced byT. saccharolyticum. There is thus motivation to understand the mechanistic basis of the robust ethanol pathway inT. saccharolyticumso that key features can be recapitulated inC. thermocellumand other organisms.Previously, we characterized theindividualrole of the main genes responsible for electron transfer in the ethanol production ofT. saccharolyticum. However, the consequences of thecombinedloss of function of all these genes have not been investigated, nor has the way in which fermentative metabolism adapts to such constraints. In this work, we combined knockouts of ferredoxin nicotinamide oxidoreductase (fnor) genes (nfnAandnfnB) and hydrogenase genes (hydAandhfsD) and studied their effects on fermentation and grow. We showed that these genetic modifications together impair growth and decrease electron transfer from reduced ferredoxin, thereby redirecting flux from the pyruvate ferredoxin oxidoreductase enzyme to the pyruvate formate lyase enzyme. We also performed adaptive evolution of these mutants to rescue their growth, and most notably, we observed a single nucleotide variation in the alcohol dehydrogenaseadhAgene. Through molecular dynamics simulations and enzymatic assays, we determined that this point mutation causes a structural change that impairs the AdhA specificity for the NADPH cofactor and increases NADH-linked activity to restore redox balance. These findings consolidate our understanding of the functioning of electron transfer pathways in this organism.

alcohol dehydrogenase↗
DOE OSTI2026

Catalytic Triad-Inspired Nanozyme Catalysts for Ester Hydrolysis in Organic Solvent Mixtures

We synthesized a series of molecular nanozyme catalysts containing functional groups (imidazole, carboxylic acid, and hydroxyl groups) inspired by the catalytic triad found in natural serine hydrolases. Different structural features were incorporated using two distinct synthesis routes to investigate the influence of interactions beyond the active site on catalytic activity in designing molecular nanozymes. Molecular dynamics simulations suggest that nanozyme activity is affected by structural features that influence nanozyme hydrophilicity and the organization of the local solvent environment. The most active nanozyme showed activity comparable to the enzyme α-chymotrypsin for the hydrolysis of a model ester (4-nitrophenyl 4- hydroxybenzoate) in a 95:5 (v/v%) water/acetonitrile mixture at ambient temperature. Increasing the temperature and organic solvent content decreases the activity of α-chymotrypsin while enhancing the activity of the nanozymes. The nanozymes can be immobilized on supported metal nanoparticles using a dithiol self-assembled monolayer, which facilitates their removal from the postreaction solution. These results demonstrate the potential in creating solvent-tolerant bioinspired catalysts, thereby combining the advantages of biocatalysts and chemical catalysts as next-generation industrial catalysts.

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

Thiol post-translational modifications modulate allosteric regulation of the OpcA–G6PDH complex through conformational gate control

In cyanobacteria, the redox-sensitive protein OpcA acts as a metabolic switch for G6PDH, enabling rapid adjustment of reducing power generation from glycogen catabolism and thereby precisely regulating carbon flux between anabolic and catabolic pathways. Although redox-sensitive cysteines in OpcA are known to regulate G6PDH, the mechanisms by which redox post-translational modifications (PTMs) on OpcA control G6PDH structure and activity remain unclear. Here, we combine computational modeling with experimental redox proteomics in Synechococcus elongatus PCC 7942 to dissect this mechanism. Experimentally, redox proteome analysis revealed differential redox PTM patterns, particularly on cysteines within the G6PDH-binding site of OpcA. These environmentally sensitive PTM changes at the interface suggest that thiol modifications in this region form a key regulatory node. More broadly, redox proteomics identified site-specific cysteine modifications under light/dark transitions and circadian cycling, linking distinct redox regimes to discrete PTM states. We employed PTM-Psi simulations to show that thiol PTMs near the OpcA–G6PDH interface are critical for allosteric regulation of G6PDH. The thiol PTMs on OpcA affect a putative gate region in G6PDH for substrate ingress and product egress as well as key hydrogen-bond networks within the active site. We infer that PTMs on OpcA tune the conformational landscapes of individual G6PDH subunits toward functionally relevant configurations according to environmental gradients, biasing the enzyme toward catalytically favorable states. Together, our results reveal a molecular mechanism in which thiol PTMs on OpcA modulate G6PDH structure and function through PTM-induced reorganization of conformational dynamics and allosteric communication. These findings demonstrate that PTM-level regulation provides a critical control layer from genotypes to phenotypes that enables cyanobacteria to rapidly adapt to environmental fluctuations through precise metabolic fine-tuning.

Allosteric regulation↗
DOE OSTI2025

Enzymatic carbon–fluorine bond cleavage by human gut microbes

Fluorinated compounds are used for agrochemical, pharmaceutical, and numerous industrial applications, resulting in global contamination. In many molecules, fluorine is incorporated to enhance the half-life and improve bioavailability. Fluorinated compounds enter the human body through food, water, and xenobiotics including pharmaceuticals, exposing gut microbes to these substances. The human gut microbiota is known for its xenobiotic biotransformation capabilities, but it was not previously known whether gut microbial enzymes could break carbon-fluorine bonds, potentially altering the toxicity of these compounds. Here, through the development of a rapid, miniaturized fluoride detection assay for whole-cell screening, we identified active gut microbial defluorinases. We biochemically characterized enzymes from diverse human gut microbial classes including Clostridia, Bacilli, and Coriobacteriia, with the capacity to hydrolyze (di)fluorinated organic acids and a fluorinated amino acid. Whole-protein alanine scanning, molecular dynamics simulations, and chimeric protein design enabled the identification of a disordered C-terminal protein segment involved in defluorination activity. Domain swapping exclusively of the C-terminus conferred defluorination activity to a nondefluorinating dehalogenase. To advance our understanding of the structural and sequence differences between defluorinating and nondefluorinating dehalogenases, we trained machine learning models which identified protein termini as important features. Models trained on 41-amino acid segments from protein C termini alone predicted defluorination activity with 83% accuracy (compared to 95% accuracy based on full-length protein features). This work is relevant for therapeutic interventions and environmental and human health by uncovering specificity-determining signatures of fluorine biochemistry from the gut microbiome.

Probst, Silke I↗
DOE OSTI2024

Library Screening, In Vivo Confirmation, and Structural and Bioinformatic Analysis of Pentapeptide Sequences as Substrates for Protein Farnesyltransferase

Protein farnesylation is a post-translational modification where a 15-carbon farnesyl isoprenoid is appended to the C-terminal end of a protein by farnesyltransferase (FTase). This process often causes proteins to associate with the membrane and participate in signal transduction pathways. The most common substrates of FTase are proteins that have C-terminal tetrapeptide CaaX box sequences where the cysteine is the site of modification. However, recent work has shown that five amino acid sequences can also be recognized, including the pentapeptides CMIIM and CSLMQ. In this work, peptide libraries were initially used to systematically vary the residues in those two parental sequences using an assay based on Matrix Assisted Laser Desorption Ionization–Mass Spectrometry (MALDI-MS). In addition, 192 pentapeptide sequences from the human proteome were screened using that assay to discover additional extended CaaaX-box motifs. Selected hits from that screening effort were rescreened using an in vivo yeast reporter protein assay. The X-ray crystal structure of CMIIM bound to FTase was also solved, showing that the C-terminal tripeptide of that sequence interacted with the enzyme in a similar manner as the C-terminal tripeptide of CVVM, suggesting that the tripeptide comprises a common structural element for substrate recognition in both tetrapeptide and pentapeptide sequences. Molecular dynamics simulation of CMIIM bound to FTase further shed light on the molecular interactions involved, showing that a putative catalytically competent Zn(II)-thiolate species was able to form. Bioinformatic predictions of tetrapeptide (CaaX-box) reactivity correlated well with the reactivity of pentapeptides obtained from in vivo analysis, reinforcing the importance of the C-terminal tripeptide motif. This analysis provides a structural framework for understanding the reactivity of extended CaaaX-box motifs and a method that may be useful for predicting the reactivity of additional FTase substrates bearing CaaaX-box sequences.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

Cu site differentiation in tetracopper(I) sulfide clusters enables biomimetic N 2 O reduction

Copper clusters feature prominently in both metalloenzymes and synthetic nanoclusters that mediate catalytic redox transformations of gaseous small molecules. Such reactions are critical to biological energy conversion and are expected to be crucial parts of renewable energy economies. However, the precise roles of individual metal atoms within clusters are difficult to elucidate, particularly for cluster systems that are dynamic under operating conditions. Here, we present a metal site-specific analysis of synthetic Cu 4 (μ 4 -S) clusters that mimic the Cu Z active site of the nitrous oxide reductase enzyme. Leveraging the ability to obtain structural snapshots of both inactive and active forms of the synthetic model system, we analyzed both states using resonant X-ray diffraction anomalous fine structure (DAFS), a technique that enables X-ray absorption profiles of individual metal sites within a cluster to be extracted independently. Using DAFS, we found that a change in cluster geometry between the inactive and active states is correlated to Cu site differentiation that is presumably required for efficient activation of N 2 O gas. More precisely, we hypothesize that the Cu δ+ ∙∙∙Cu δ- pairs produced upon site differentiation are poised for N 2 O activation, as supported by computational modeling. These results provide an unprecedented level of detail on the roles of individual metal sites within the synthetic cluster system and how those roles interplay with cluster geometry to impact the reactivity function. We expect this fundamental knowledge to inform understanding of metal clusters in settings ranging from (bio)molecular to nanocluster to extended solid systems involved in energy conversion.

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

Design, Synthesis, and Biological Activity of Novel Ornithine Decarboxylase (ODC) Inhibitors

We here describe the design, synthesis, and biological activity of novel ornithine decarboxylase (ODC) inhibitors that show significantly higher potency in vitro than α-difluoromethylornithine (DFMO), a U.S. Food and Drug Administration (FDA) approved drug. We report two X-ray structures of ODC complexed with new ODC inhibitors, computational docking, molecular dynamics, and binding free energy calculations to validate the experimental models. The X-ray structures reveal that covalent adducts with pyridoxal phosphate (PLP) are formed in the active site of the human ODC enzyme, as verified by their preparation and enzymatic testing. Finally, we verified that the cellular activity of endogenous ODC was inhibited, and polyamine levels were reduced. Given that ODC is a clinically validated target, combined with the fact that DFMO is currently the only ODC inhibitor in clinical use for several indications, the further development of more potent ODC inhibitors with superior activity and physical properties is warranted.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2024

Increasing Bacterial Tolerance and Metabolism of the Biofuel, N-Butanol Using Community-Level Evolution and Functional Genomics

Bioremediation capability should be developed along with biofuel technology to mitigate the potential damage of future spills. One biofuel that is being developed is biobutanol, since n-butanol is more energy dense and less volatile than ethanol. A bottleneck for industrial production of biobutanol is its toxicity; most microbes cannot survive about 1.5% v/v. Thus, microbial bioremediation of n-butanol would need microbes that can both tolerate and metabolize butanol. We used ecological and evolutionary biology approaches to find bacteria that could tolerate and metabolize butanol. We then tried to increase the metabolism of butanol by promising bacterial strains and communities. Ecological community-level assays and screenings were conducted followed by 16S amplicon sequencing to identify butanol-tolerant artificial bacterial communities. Promising communities were then tested for growth with butanol as the sole carbon source. Secondly, we looked for bacteria with alcohol dehydrogenase enzymes and looked to increase butanol metabolism. We found that the tolerance for n-butanol may be improved with repeated exposure, but it was difficult to switch from tolerance to metabolism. Bacterial community v dynamics may be influenced by n-butanol concentration, and there was putative butanol metabolism found with both research approaches.

09 BIOMASS FUELS↗
DOE OSTI2024

Seasonal Controls on Microbial Depolymerization and Oxidation of Organic Matter in Floodplain Soils

Floodplain soils are vast reservoirs of organic carbon often attributed to anaerobic conditions that impose metabolic constraints on organic matter degradation. What remains elusive is how such metabolic constraints respond to dynamic flooding and drainage cycles characteristic of floodplain soils. Here, in this work, we show that microbial depolymerization and respiration of organic compounds, two rate-limiting steps in decomposition, vary spatially and temporally with seasonal flooding of mountainous floodplain soils (Gothic, Colorado, USA). Combining metabolomics and -proteomics, we found a lower abundance of oxidative enzymes during flooding coincided with the accumulation of aromatic, high-molecular weight compounds, particularly in surface soils. In subsurface soils, we found that a lower oxidation state of carbon coincided with a greater abundance of chemically reduced, energetically less favorable low-molecular weight metabolites, irrespective of flooding condition. Our results suggest that seasonal flooding temporarily constrains oxidative depolymerization of larger, potentially plant-derived compounds in surface soils; in contrast, energetic constraints on microbial respiration persist in more reducing subsurface soils regardless of flooding. Our work underscores that the potential vulnerability of these distinct anaerobic carbon storage mechanisms to changing flooding dynamics should be considered, particularly as climate change shifts both the frequency and extent of flooding in floodplains globally.

54 ENVIRONMENTAL SCIENCES↗
DOE OSTI2024

Dual Enhancement of Thermostability and Activity of Xylanase through Computer-Aided Rational Design

In the realm of enzyme engineering, the dual enhancement of thermostability and activity remains a challenge. Herein, we employed a computer-aided approach integrating folding free energy calculations and evolutionary analysis to engineer Paecilomyces thermophila xylanase into a hyperthermophilic enzyme for application in the paper and pulp industry. Through the computational rational design, XynM9 with superior thermostability and enhanced activity was designed. Its optimal reaction temperature increases by 10 °C to 85 °C, its T m increases by 10 °C to 93 °C, and its half-life increases 11-fold to 5.8 h. Additionally, its catalytic efficiency improves by 57% to 3926 s –1 mM –1 . Molecular dynamics simulations revealed that XynM9 is stabilized by more hydrogen bonds and salt bridges than wild-type xylanase. The mutant’s narrower catalytic cleft enhances the substrate-binding affinity, thus improving the catalytic efficiency. In harsh conditions at 80 °C and pH 10, using XynM9 significantly reduced both hemicellulose and lignin, which makes it a good candidate for use in the paper and pulp process. Finally, our study presents an accurate and efficient strategy for the dual enhancement of enzyme properties, guiding further improvement of computational tools for protein stabilization.

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

Structural characterization of ligand binding and pH-specific enzymatic activity of mouse Acidic Mammalian Chitinase

Chitin is an abundant biopolymer and pathogen-associated molecular pattern that stimulates a host innate immune response. Mammals express chitin-binding and chitin-degrading proteins to remove chitin from the body. One of these proteins, Acidic Mammalian Chitinase (AMCase), is an enzyme known for its ability to function under acidic conditions in the stomach but is also active in tissues with more neutral pHs, such as the lung. Here, we used a combination of biochemical, structural, and computational modeling approaches to examine how the mouse homolog (mAMCase) can act in both acidic and neutral environments. We measured kinetic properties of mAMCase activity across a broad pH range, quantifying its unusual dual activity optima at pH 2 and 7. We also solved high-resolution crystal structures of mAMCase in complex with oligomeric GlcNAcn, the building block of chitin, where we identified extensive conformational ligand heterogeneity. Leveraging these data, we conducted molecular dynamics simulations that suggest how a key catalytic residue could be protonated via distinct mechanisms in each of the two environmental pH ranges. These results integrate structural, biochemical, and computational approaches to deliver a more complete understanding of the catalytic mechanism governing mAMCase activity at different pH. Engineering proteins with tunable pH optima may provide new opportunities to develop improved enzyme variants, including AMCase, for therapeutic purposes in chitin degradation.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2025

Mixed lipid bilayers enable enhanced stability and activity retention of Lipase A in low-pH environments

Lipase A (LipA) from Bacillus subtilis is a versatile and industrially relevant enzyme, but its activity is compromised under acidic conditions due to aggregation and deactivation. In this study, we investigated the use of mixed lipid bilayers to stabilize and improve activity retention of LipA at low pH. Attachment to lipid bilayers, particularly those containing cationic lipids, greatly enhanced the long-term stability of LipA in acidic conditions while also leading to improvements in activity. Tethering to cationic bilayers not only shifted the apparent pH activity profile toward more acidic conditions, but also significantly enhanced activity retention upon incubation at pH 6. Notably, this protective effect persisted even without direct tethering, indicating that reversible, non-covalent interactions with the bilayer surface are sufficient for long-term stability. Circular dichroism further revealed that the secondary structure of LipA was retained, while dynamic light scattering suggested that activity loss in solution was primarily due to aggregation of the native state. Together, these findings support a model in which lipid bilayers mitigate aggregation and stabilize LipA through transient interactions and electrostatic modulation of the local surface environment. Furthermore, this approach exemplifies a simple, tether-free strategy for enhancing enzyme performance in acidic or destabilizing conditions, with implications for biocatalysis, biosensing, and therapeutic delivery.

Biocatalysis↗
DOE OSTI2025

Exploring single-cell biosynthetic noise and dynamics for enhanced betaxanthin production in Escherichia coli

Cell-to-cell variability often limits the efficiency of microbial bioproduction, yet how individual cells fluctuate over time and how these fluctuations shape population-level output remain unclear. To address this issue, we tracked a heterologous betaxanthin pathway in Escherichia coli using microfluidics-assisted time-lapse microscopy, allowing simultaneous measurement of fluctuations in betaxanthin, its biosynthetic enzyme DOD and growth across generations. Here we show that over 50% of high betaxanthin producers become medium or low producers after two divisions. Betaxanthin variation primarily originates from DOD noise, with a smaller contribution from growth rate fluctuations. We further develop a stochastic model to explore various control circuits and find that pathway enzyme or metabolite-based growth selection strategies are most effective in enhancing production. We experimentally validate the model by coupling enzyme expression to nutrient availability, which enriches high producers and boosts titer by 4.4-fold. Our results highlight key sources of metabolic heterogeneity and provide a framework for designing robust microbial processes.

Bacterial systems biology↗
DOE OSTI2025

Assessing the fidelity of shallow-water carbonates as records of the Ni isotope composition of surface seawater

Nickel is a bioessential metal that is used in enzymes important to the C, N, and O cycles, and changes in its marine abundance and bioavailability may have affected the evolutionary trajectory of early life. Changes over time in the Ni isotope composition (δ 60 Ni) of surface seawater, which reflects biological demand for Ni, could allow for the reconstruction of the dynamics of Ni demand over Earth’s history, but this approach would require geologic records of surface seawater. Here, we investigate the fidelity of shallow-water carbonates as a record of the Ni isotope composition of surface seawater by determining how Ni is first partitioned into natural carbonates and then how post-depositional processes influence the Ni signal. Our samples come from the Great Bahama Bank, which is a well-studied, modern carbonate platform often used to study ancient platforms. We found that Ni is fractionated from seawater upon incorporation into carbonates capturing shallow (<18 cm), recent deposition (0.1 ‰–0.4 ‰ lighter than seawater). Variation among these [Ni] and δ 60 Ni values may be controlled by variation in mineral proportions. Meteoric diagenesis shifts δ 60 Ni to lower values, which we attribute to isotopically light meteoric fluids. In contrast, carbonates that experienced sediment-buffered marine diagenesis with respect to Ca isotopes and Sr/Ca ratios do not appear to differ in δ 60 Ni values from sediments generally representative of their initial deposition. The sensitivity of δ 60 Ni to diagenetic reset in these samples appears comparable to the sensitivities of Ca isotopes and Sr/Ca ratios, to first order. Thus, in general, carbonates that experienced sediment-buffered marine diagenesis with respect to these elements may hold the most promise as a record of the δ 60 Ni of coeval surface seawater. Additionally, we use our results to infer that the fraction of Ni removed from seawater into carbonates is less than 10 % of the total Ni output from the global oceans and incorporation of this Ni sink into global biogeochemical models will only have a minor impact on the modeled modern Ni budget.

Great Bahama Bank carbonates↗
DOE OSTI2025

A split luciferase system for studying coronavirus Mpro dimerization in vitro and in living cells

The main protease enzyme (Mpro) of coronaviruses cleaves the viral polyprotein into functional units essential for virus replication. Prior work has demonstrated that Mpro functions as a homodimer. However, studies on the mechanism of dimerization have been challenging because the purified protease is mostly dimeric, dimerization-defective mutants lack proteolytic activity, and robust cell-based assays have yet to be reported. To enable work on Mpro dimerization, we have developed a quantitative luciferase-based SARS-CoV-2 (SARS2) Mpro biosensor that accurately reports protein dimerization in living cells and, upon purification, also in vitro. Co-transfection of cells with a construct expressing Mpro fused to the 18 kDa LargeBiT of luciferase (LgBiT) and a second construct with Mpro fused to the 1 kDa SmallBiT of luciferase (SmBiT) results in a reconstitution of luciferase activity in a dose-dependent manner that requires conserved residues within the dimerization interface. Proteolytic activity is dispensable for dimerization and, uniquely, a C145A catalytically inactive mutant exhibits enhanced dimerization signal likely due to lower cytotoxicity. Mpro enzymes from multiple different coronaviruses also dimerize in this system, indicating mechanistic conservation. Interestingly, this dimerization biosensor also provides a quantitative read-out of inhibitor-facilitated dimerization. Covalent SARS2 Mpro inhibitors such as nirmatrelvir cause a 3- to 5-fold increase in luciferase activity. Together with corroborating structural, biophysical, and molecular dynamics experiments, our studies support a model in which covalent Mpro inhibitors such as nirmatrelvir simultaneously block catalytic activity and induce allosteric stabilization of the dimeric complex.

SARS-CoV-2 main protease (Mpro/3CLpro)↗
DOE OSTI2025

Harnessing the Power of Machine Learning and Omics to Identify Environmental Regulation on Microbial Functional Composition for Soil C, N, and P Cycling

Microbial enzyme-mediated soil organic matter (SOM) decomposition regulates many key ecosystem functions, such as elemental cycling, soil carbon sequestration, and soil fertility. However, representing microbial processes in Earth system models (ESMs) remains challenging due to a limited understanding of the spatial patterns of diverse microbial functions responsible for soil carbon (C), nitrogen (N), and phosphorus (P) cycling as well as the underlying mechanisms regulating their relative abundances across various environments. We collected published metagenomics data across the continental US (CONUS) to identify hundreds of microbial genes involved in soil C, N, and P cycling and grouped them into eight enzyme functional classes (EFCs). Each EFC represented a group of gene-encoded potential enzymes that decompose similar soil compounds. By integrating the abundances of omics-informed EFCs with the corresponding environmental information, we trained a machine learning (ML) model to identify key edaphic, climate, and vegetation factors regulating the abundances of each EFC. Quantitative analysis of effects of these factors revealed that the spatial distribution of eight EFCs for soil C, N, and P cycling across CONUS reflected potential resource optimization strategies of microbial communities under nutrient limitation, preferential organic-mineral associations, and climatological stresses. This insight, together with the interpreted ML tool and the CONUS-level benchmark for EFCs abundances, paves the way for parameterizing environmental-regulated microbial functional dynamics in biogeochemical models.

machine learning↗
DOE OSTI2025

Maximizing long-term biohydrogen production with Clostridium thermocellum for high solids conversion of lignocellulosic biomass

Biological hydrogen production from lignocellulosic biomass sustainably couples organic waste reduction with renewable energy generation. Efficient conversion is challenged by the structural complexity of lignocellulose and resulting recalcitrance to enzymatic degradation. Clostridium thermocellum natively breaks down biomass with highly effective hemi-/cellulases systems (i.e., cellulosomes) and generates hydrogen in anaerobic cultivation, creating a compelling platform for lignocellulosic biohydrogen production. Achieving commercially viable production rates requires balancing high biomass loading and throughput against uniform mixing conditions required for enzyme dispersion, pH and temperature control, and efficient hydrogen and metabolite removal in continuous operation. To address these barriers to process intensification, we implemented novel reactor and process designs for high-solids lignocellulosic biomass fermentations using the C. thermocellum KJC19-9 strain, genetically engineered for co-utilization of cellulose and hemicellulose sugars (i.e., xylose). Via computational fluid dynamics (CFD) modeling and experimental validation, we achieved a >50% improvement in biohydrogen production with an improved anchor-type impeller morphology, coupled to a threefold reduction in agitation rate. To further reduce rheological constraints and accumulation of toxic metabolites, we then transitioned the process to sequencing fed-batch operation. The resulting process generated 24.87 L H 2 L −1 from 160 g L −1 of deacetylated and mechanically refined (DMR)-pretreated corn stover biomass over 16 days while solubilizing >95% of influent cellulose and hemicellulose, setting a new performance benchmark for continuous production of biohydrogen from lignocellulose.

08 HYDROGEN↗
DOE OSTI2026

Determinants of Stereoselectivity in Monoterpene Synthases

Monoterpene synthases (MTSs) catalyze the conversion of an achiral prenyl diphosphate precursor, most commonly geranyl diphosphate (GPP), into structurally and stereochemically diverse products. However, remains poorly understood. Here, we combine enzymatic assays with six MTSs and selected variants, along with extensive molecular dynamics simulations to the mechanistic basis of stereoselectivity. We demonstrate that the preferred helical binding conformation of GPP, determined from free-energy calculations and selected crystal structures of MTSs, correlates well with the experimentally determined stereoselectivity of MTSs, whereas only a poor correlation is observed between the binding of enantiomers of linalyl diphosphate (LPP), a chiral intermediate of MTS catalysis, and the stereochemical reaction outcomes. Free energy maps indicate that enzyme-bound GPP conformers preferentially occupy regions that enable a direct and stereochemically faithful transition from GPP to subsequent carbocations. In contrast, LPP frequently populates regions of the energy surface where conformational scrambling can occur, thus leading to a lack of correlation between LPP enantiomer binding and product stereochemistry. Overall, these findings establish that the configuration of GPP is the primary determining factor for stereochemical outcomes in MTSs, offering a new framework for designing stereoselective terpene synthases.

Srividya, Narayanan (ORCID:0000000179347987)↗