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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 325 records · Page 18
All sourcesNASA NTRSDOE OSTI
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 OSTI2024

Predicting Anaerobic Membrane Bioreactor Performance Using Flow-Cytometry-Derived High and Low Nucleic Acid Content Cells

Having a tool to monitor the microbial abundances rapidly and to utilize the data to predict the reactor performance would facilitate the operation of an anaerobic membrane bioreactor (AnMBR). This study aims to achieve the aforementioned scenario by developing a linear regression model that incorporates a time-lagging mode. The model uses low nucleic acid (LNA) cell numbers and the ratio of high nucleic acid (HNA) to LNA cells as an input data set. First, the model was trained using data sets obtained from a 35 L pilot-scale AnMBR. The model was able to predict the chemical oxygen demand (COD) removal efficiency and methane production 3.5 days in advance. Subsequent validation of the model using flow cytometry (FCM)-derived data (at time t – 3.5 days) obtained from another biologically independent reactor did not exhibit any substantial difference between predicted and actual measurements of reactor performance at time t. Further cell sorting, 16S rRNA gene sequencing, and correlation analysis partly attributed this accurate prediction to HNA genera (e.g., Anaerovibrio and unclassified Bacteroidales) and LNA genera (e.g., Achromobacter, Ochrobactrum, and unclassified Anaerolineae). In summary, our findings suggest that HNA and LNA cell routine enumeration, along with the trained model, can derive a fast approach to predict the AnMBR performance.

42 ENGINEERING↗
DOE OSTI2024

Emergence of Orchestrated and Dynamic Metabolism of Saccharomyces cerevisiae

Microbial metabolism is a fundamental cellular process that involves many biochemical events and is distinguished by its emergent properties. While the molecular details of individual reactions have been increasingly elucidated, it is not well understood how these reactions are quantitatively orchestrated to produce collective cellular behaviors. Here we developed a coarse-grained, systems, and dynamic mathematical framework, which integrates metabolic reactions with signal transduction and gene regulation to dissect the emergent metabolic traits of Saccharomyces cerevisiae. Our framework mechanistically captures a set of characteristic cellular behaviors, including the Crabtree effect, diauxic shift, diauxic lag time, and differential growth under nutrient-altered environments. It also allows modular expansion for zooming in on specific pathways for detailed metabolic profiles. Furthermore, this study provides a systems mathematical framework for yeast metabolic behaviors, providing insights into yeast physiology and metabolic engineering.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

Genome engineering allows selective conversions of terephthalaldehyde to multiple valorized products in bacterial cells

Deconstruction of polyethylene terephthalate (PET) plastic waste generates opportunities for valorization to alternative products. We recently designed an enzymatic cascade that could produce terephthalaldehyde (TPAL) from terephthalic acid. Here, we showed that the addition of TPAL to growing cultures of Escherichia coli wild-type strain MG1655 and an engineered strain for reduced aromatic aldehyde reduction (RARE) strain resulted in substantial reduction. We then investigated if we could mitigate this reduction using multiplex automatable genome engineering (MAGE) to create an E. coli strain with 10 additional knockouts in RARE. Encouragingly, we found this newly engineered strain enabled a 2.5-fold higher retention of TPAL over RARE after 24 h. We applied this new strain for the production of para-xylylenediamine (pXYL) and observed a 6.8-fold increase in pXYL titer compared with RARE. Altogether, our study demonstrates the potential of TPAL as a versatile intermediate in microbial biosynthesis of chemicals that derived from waste PET.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2025

Decompartmentalization of the yeast mitochondrial metabolism to improve chemical production in Issatchenkia orientalis

Microbial production of chemicals may suffer from inadequate cofactor provision, a challenge further exacerbated in yeasts due to compartmentalized cofactor metabolism. Here, we perform cofactor engineering through the decompartmentalization of mitochondrial metabolism to improve succinic acid (SA) production in Issatchenkia orientalis. We localize the reducing equivalents of mitochondrial NADH to the cytosol through cytosolic expression of its pyruvate dehydrogenase (PDH) complex and couple a reductive tricarboxylic acid pathway with a glyoxylate shunt, partially bypassing an NADH-dependent malate dehydrogenase to conserve NADH. Cytosolic SA production reaches a titer of 104 g/L and a yield of 0.85 g/g glucose, surpassing the yield of 0.66 g/g glucose constrained by cytosolic NADH availability. Additionally, expressing cytosolic PDH, we expand our I. orientalis platform to enhance acetyl-CoA-derived citramalic acid and triacetic acid lactone production by 1.22- and 4.35-fold, respectively. Our work establishes I. orientalis as a versatile platform to produce markedly reduced and acetyl-CoA-derived chemicals.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2020

Stress-tolerant non-conventional microbes enable next-generation chemical biosynthesis

Microbial chemical production is a rapidly growing industry, with much of the growth fueled by advances in synthetic biology. New approaches have enabled rapid strain engineering for the production of various compounds; however, translation to industry is often problematic because native phenotypes of model hosts prevent the design of new low-cost bioprocesses. Here, in this work, we argue for a new approach that leverages the native stress-tolerant phenotypes of non-conventional microbes that directly address design challenges from the outset. Growth at high temperature, high salt and solvent concentrations, and low pH can enable cost savings by reducing the energy required for product separation, bioreactor cooling, and maintaining sterile conditions. These phenotypes have the added benefit of allowing for the use of low-cost sugar and water resources. Non-conventional hosts are needed because these phenotypes are polygenic and thus far have proven difficult to recapitulate in the common hosts Escherichia coli and Saccharomyces cerevisiae.

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

Data for "Decompartmentalization of the yeast mitochondrial metabolism to improve chemical production in Issatchenkia orientalis "

Microbial production of chemicals may suffer from inadequate cofactor provision, a challenge further exacerbated in yeasts due to compartmentalized cofactor metabolism. Here, we perform cofactor engineering through the decompartmentalization of mitochondrial metabolism to improve succinic acid (SA) production in Issatchenkia orientalis . We localize the reducing equivalents of mitochondrial NADH to the cytosol through cytosolic expression of its pyruvate dehydrogenase (PDH) complex and couple a reductive tricarboxylic acid pathway with a glyoxylate shunt, partially bypassing an NADH-dependent malate dehydrogenase to conserve NADH. Cytosolic SA production reaches a titer of 104 g/L and a yield of 0.85 g/g glucose, surpassing the yield of 0.66 g/g glucose constrained by cytosolic NADH availability. Additionally, expressing cytosolic PDH, we expand our I. orientalis platform to enhance acetyl-CoA-derived citramalic acid and triacetic acid lactone production by 1.22- and 4.35-fold, respectively. Our work establishes I. orientalis as a versatile platform to produce markedly reduced and acetyl-CoA-derived chemicals.

bioproducts↗
DOE OSTI2024

Species-specific ribosomal RNA-FISH identifies interspecies cellular-material exchange, active-cell population dynamics and cellular localization of translation machinery in clostridial cultures and co-cultures

ABSTRACT The development of synthetic microbial consortia in recent years has revealed that complex interspecies interactions, notably the exchange of cytoplasmic material, exist even among organisms that originate from different ecological niches. Although morphogenetic characteristics, viable RNA and protein dyes, and fluorescent reporter proteins have played an essential role in exploring such interactions, we hypothesized that ribosomal RNA-fluorescence in situ hybridization (rRNA-FISH) could be adapted and applied to further investigate interactions in synthetic or semisynthetic consortia. Despite its maturity, several challenges exist in using rRNA-FISH as a tool to quantify individual species population dynamics and interspecies interactions using high-throughput instrumentation such as flow cytometry. In this work, we resolve such challenges and apply rRNA-FISH to double and triple co-cultures of Clostridium acetobutylicum, Clostridium ljungdahlii, and Clostridium kluyveri . In pursuing our goal to capture each organism’s population dynamics, we demonstrate dynamic rRNA, and thus ribosome, exchange between the three species leading to the formation of hybrid cells. We also characterize the localization patterns of the translation machinery in the three species, identifying distinct, dynamic localization patterns among them. Our data also support the use of rRNA-FISH to assess the culture’s health and expansion potential, and, here again, our data find surprising differences among the three species examined. Taken together, our study argues for rRNA-FISH as a valuable and accessible tool for quantitative exploration of interspecies interactions, especially in organisms which cannot be genetically engineered or in consortia where selective pressures to maintain recombinant species cannot be used. IMPORTANCE Though dyes and fluorescent reporter proteins have played an essential role in identifying microbial species in co-cultures, we hypothesized that ribosomal RNA-fluorescence in situ hybridization (rRNA-FISH) could be adapted and applied to quantitatively probe complex interactions between organisms in synthetic consortia. Despite its maturity, several challenges existed before rRNA-FISH could be used to study Clostridium co-cultures of interest. First, species-specific probes for Clostridium acetobutylicum and Clostridium ljungdahlii had not been developed. Second, “state-of-the-art” labeling protocols were tedious and often resulted in sample loss. Third, it was unclear if FISH was compatible with existing fluorescent reporter proteins. We resolved these key challenges and applied the technique to co-cultures of C. acetobutylicum , C. ljungdahlii , and Clostridium kluyveri . We demonstrate that rRNA-FISH is capable of identifying rRNA/ribosome exchange between the three organisms and characterized rRNA localization patterns in each. In combination with flow cytometry, rRNA-FISH can capture sub-population dynamics in co-cultures.

Hill, John D.↗
DOE OSTI2026

The anaerobic fungus Caecomyces churrovis produces H 2 via a non-bifurcating NADH-dependent enzyme complex

ABSTRACT Hydrogenosomes are mitochondria-derived organelles that produce ATP and H 2 to support energy metabolism in anaerobic eukaryotes. H 2 production allows reoxidation of reduced cofactors generated during fermentative metabolism; however, the metabolic mechanisms for H 2 production in anaerobic eukaryotes remains incompletely understood. In particular, it remains unclear whether anaerobic fungi (AF) hydrogenosomes use a ferredoxin-dependent pathway or a distinct mechanism to regenerate NAD(P) + and link electron transfer to H 2 formation. Here, by combining genomic search, proteomic analysis, and enzymology, we reveal the molecular mechanism for H 2 production in the AF strain Caecomyces churrovis . Our enzyme assays on the organelle fraction of C. churrovis revealed the activity of H 2 :NAD + oxidoreductase but not pyruvate:ferredoxin oxidoreductase, which is usually linked to H 2 formation. We identified genes encoding [FeFe] hydrogenase (Hyd) and NADH dehydrogenase subunits E and F (NuoE, NuoF) in C. churrovis , and confirmed their expression in the isolated hydrogenosomal fractions by proteomic analysis. Combining the individually purified enzymes, we found Hyd and NuoEF proteins formed H 2 directly from NADH independently of ferredoxin, functioning as a non-bifurcating NADH-dependent enzyme rather than an electron-bifurcating enzyme. We identified homologs of hydrogenosomal NuoE, NuoF, and Hyd in many other AF, indicating this pathway is commonly shared among the AF. This work demonstrates the existence of a non-bifurcating NADH-dependent enzyme complex in eukaryotes. Moreover, this complex could potentially be exploited as a target for controlling AF H 2 production and altering fungal metabolism. IMPORTANCE H 2 production is a prominent feature of anaerobic energy metabolism, yet our understanding of eukaryotic mechanisms remains limited. Anaerobic fungi (AF) are key decomposers of lignocellulose and contribute to hydrogen flux in anaerobic environments. Although it has been more than 40 years since the H 2 production from AF was first reported, the molecular mechanism for hydrogenosomal H 2 production and redox balance remains unclear. We demonstrate that AF produce H 2 from NADH utilizing a non-bifurcating NADH-dependent enzyme complex rather than an electron-bifurcating, ferredoxin-dependent variant. We show that this enzyme complex is conserved across multiple AF lineages and thus demonstrate the occurrence of a non-bifurcating NADH-dependent enzyme in eukaryotes. This discovery expands our understanding of eukaryotic hydrogenosomal metabolism, reveals a previously unknown strategy for redox balancing, and highlights potential targets for manipulating H 2 production. These insights have broad implications for microbial energy metabolism, anaerobic ecosystems, and bioengineering of H 2 -producing systems.

Zhang, Bo [Department of Chemical Engineering, Uni↗
DOE OSTI2025

De Novo Design of High‐Affinity Miniprotein Binders Targeting Francisella Tularensis Virulence Factor

Abstract Francisella tularensis poses considerable public health risk due to its high infectivity and potential for bioterrorism. Francisella‐like lipoprotein (Flpp3), a key virulence factor unique to Francisella, plays critical roles in infection and immune evasion, making it a promising target for therapeutic development. However, the lack of well‐defined binding pockets and structural information on native interactions has hindered structure‐guided ligand discovery against Flpp3. Here, we used a combination of physics‐based and deep‐learning methods to design high‐affinity miniprotein binders targeting two distinct sites on Flpp3. We identified four binders for site I with binding affinities ranging between 24–110 nM. For the second site, an initial binder showed a dissociation constant ( K D ) of 81 nM, and subsequent site saturation mutagenesis yielded variants with sub‐nanomolar affinities. Circular dichroism confirmed the topology of designed miniproteins. The X‐ray crystal structure of Flpp3 in complex with a site I binder is nearly identical to the design model (Cα root‐mean‐square deviation (RMSD): 0.9 Å). These designed miniproteins provide research tools to explore the roles of Flpp3 in tularemia and should enable the development of new therapeutic candidates.

Gokce‐Alpkilic, Gizem [Molecular Engineering and S↗
DOE OSTI2021

The Anaerobic Fungi: Challenges and Opportunities for Industrial Lignocellulosic Biofuel Production

Lignocellulose is a promising feedstock for biofuel production as a renewable, carbohydrate-rich and globally abundant source of biomass. However, challenges faced include environmental and/or financial costs associated with typical lignocellulose pretreatments needed to overcome the natural recalcitrance of the material before conversion to biofuel. Anaerobic fungi are a group of underexplored microorganisms belonging to the early diverging phylum Neocallimastigomycota and are native to the intricately evolved digestive system of mammalian herbivores. Anaerobic fungi have promising potential for application in biofuel production processes due to the combination of their highly effective ability to hydrolyse lignocellulose and capability to convert this substrate to H2 and ethanol. Furthermore, they can produce volatile fatty acid precursors for subsequent biological conversion to H2 or CH4 by other microorganisms. The complex biological characteristics of their natural habitat are described, and these features are contextualised towards the development of suitable industrial systems for in vitro growth. Moreover, progress towards achieving that goal is reviewed in terms of process and genetic engineering. In addition, emerging opportunities are presented for the use of anaerobic fungi for lignocellulose pretreatment; dark fermentation; bioethanol production; and the potential for integration with methanogenesis, microbial electrolysis cells and photofermentation.

pretreatment↗
DOE OSTI2023

Electrons to Molecules by Engineering and Evolution: Biological Upgrading of Formate by Cupriavidus necator

Waste carbon from industrial point sources can be captured, stored, and/or transformed using electrochemical conversion or "electrons to molecules" technologies using low-cost renewable electricity. One such process involves electrocatalytic reduction of CO2 to generate formate/formic acid, a C1 carboxylic acid. Formate is a promising potential feedstock for microbial upgrading, as it is water soluble and can be consumed as the sole source of carbon and energy by some microbial species, such as the soil bacterium Cupriavidus necator. Here we will present progress toward improving C. necator as a host for biological conversion of formate to value-added products. Using the power of adaptive laboratory evolution, we were able to isolate mutants of C. necator with significantly faster growth rates on formate. We then sequenced the genomes of these strains, elucidated the metabolic role of the mutations we found, and then used these insights to build rationally engineered strains that outperform even the best evolved isolates. These results highlight the utility of "genome streamlining" as a route for generating platform strains with potential industrial applications.

adaptive laboratory evolution↗
DOE OSTI2026

Microbial valorization of lignin to malic acid by Aspergillus niger

Lignin is the largest renewable source of aromatic carbon, yet its heterogeneity and recalcitrance limit its use in higher-value bioconversion processes. In this study, Aspergillus niger was engineered to enable the bioconversion of lignin-derived aromatics and base-catalyzed depolymerized (BCD) lignin streams into malic acid, a value-added C4 dicarboxylic acid with broad industrial relevance. Overexpression of the C4 dicarboxylate transporter C4T318 from Aspergillus oryzae enhanced malic acid secretion, while medium optimization under buffered conditions further improved the production. The engineered strain efficiently assimilated representative lignin-derived aromatics, including 4-hydroxybenzoic acid and p-coumaric acid, producing up to 3.9 g/L malic acid. Conversion of BCD lignin liquors from poplar and sorghum demonstrated effective utilization of heterogeneous aromatic mixtures, generating up to 0.82 g/L malic acid. This work demonstrates direct fungal conversion of real lignin streams into malic acid and establishes A. niger as a promising platform for sustainable lignin valorization.

Aromatic bioconversion↗
DOE OSTI2026

Bioadaptive Ni single atoms unlock high rate microbial electrosynthesis of isopropanol from CO 2

Hybrid systems that integrate electrochemical CO 2 reduction with microbial upgrading offer a viable route to high value organic compounds from CO 2 at ambient conditions. However, electrocatalyst deactivation in microbial growth media remains a key barrier, limiting efficiency and increasing cost. Here we show that a bioadaptive single-atom nickel catalyst (Ni SAC), coupled with genetically engineered Clostridium ljungdahlii, enables robust electrosynthesis of isopropanol (IPA) from CO 2 via a CO-mediated pathway. Instead of relying on H 2 as an electron carrier, the system applies high-rate CO formation in complex growth media, maintaining a tunable CO Faradaic efficiency up to 92%, which is 9.4 to 52.7 times greater than conventional Ag catalysts. This performance supports stable IPA production at current density of 10.8 A/m 2 and production rate of 161.3 mg/L/day. In situ Raman and X-ray absorption spectroscopy, together with theoretical calculations, indicate that the Ni SAC can resist competing organic adsorption and retain its coordination structure during CO 2 reduction in bioelectrolytes, providing a mechanistic basis for the catalyst stability and integrated process performance.

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

Williston Basin Resource Study for Commercial-Scale Subsurface Hydrogen Storage

This closeout presentation summarizes a Department of Energy–funded study that evaluated whether large amounts of hydrogen can be safely stored underground in the North Dakota portion of the Williston Basin. The project combined lab testing, computer simulations, and basin‑wide analysis to assess saline formations, depleted oil and gas reservoirs, and salt formations for hydrogen storage capacity, recovery, and risks. Results show that underground hydrogen storage is technically feasible across multiple formation types, with depleted oil and gas reservoirs offering higher recovery and saline formations providing large long‑term storage potential. The study also identifies key challenges—such as wellbore material durability and gas purity management—and recommends pilot projects and further site‑specific studies to support future commercialization.

08 HYDROGEN↗