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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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All sourcesNASA NTRSDOE OSTI
DOE OSTI2021

Editorial: Bioconversion and Biorefinery of C1 Compounds

The past decade has seen significant progress in the field of metabolic engineering and synthetic biology. The exponentially growing multi-omics data and technological advances in the development of efficient genetic manipulation tools and techniques have allowed scientists to explore and expand their understanding of microbial metabolisms and further develop sophisticated engineering strategies to realize the use of industrial "workhorses" and non-conventional microorganisms for sustainable bioconversion and biorefinery. There is of great interest for the research community in using C1 compounds (i.e., CO 2 , CO/syngas, methane, methanol) as the next generation feedstocks for microbial cell factories and biocatalysts to promote the sustainable development of a green economy (Figure 1). Considering lowering input costs is also a main consideration for successful business ventures, the use of inexpensive, abundant, and widely accessible C1 compounds is envisioned as a promising route for the sustainable production of fine chemicals, fuels, and other high-value products. Many C1 compounds are waste gases from industrial activities and may have detrimental effects on climate change upon emission into the atmosphere. Therefore, promoting the use of C1 compounds as renewable carbon feedstocks can greatly contribute to the reduction of anthropogenic emission of air pollutants.

09 BIOMASS FUELS↗
DOE OSTI2021

Rationally Programming Nanomaterials with DNA for Biomedical Applications

Abstract DNA is not only a carrier of genetic information, but also a versatile structural tool for the engineering and self‐assembling of nanostructures. In this regard, the DNA template has dramatically enhanced the scalability, programmability, and functionality of the self‐assembled DNA nanostructures. These capabilities provide opportunities for a wide range of biomedical applications in biosensing, bioimaging, drug delivery, and disease therapy. In this review, the importance and advantages of DNA for programming and fabricating of DNA nanostructures are first highlighted. The recent progress in design and construction of DNA nanostructures are then summarized, including DNA conjugated nanoparticle systems, DNA‐based clusters and extended organizations, and DNA origami‐templated assemblies. An overview on biomedical applications of the self‐assembled DNA nanostructures is provided. Finally, the conclusion and perspectives on the self‐assembled DNA nanostructures are presented.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2025

Direct sensitivity analysis on the parameterization of crystal plasticity models

Various methods for calibrating crystal plasticity finite element (CPFE) models lead to non-unique input parameter values, which subsequently introduce uncertainty in the predicted mechanical response. Sensitivity analysis (SA) conducted on crystal plasticity models is used to identify how variability in these parameters contribute to output uncertainty. Traditional SA on CPFE parameters uses simplified surrogate models to save computational time. However, the accuracy of the surrogate models depends on the quantity of training data used, and any modeling error can propagate into the SA results, potentially affecting their reliability. In this work, the elementary effects test (EET) method, a global SA technique using direct CPFE simulations was employed, and the results obtained were compared with the First Order Second Moment (FOSM) method. ExaConstit, an open-source GPU-enabled CPFE code, was used to perform the simulations and direct SA. The EET method was accurately able to capture the non-linear effects of all the input parameters on the output and is a valuable approach for reliably attributing parameter sensitivities in CPFE models. Based on the results, efficient strategies to perform future parameter calibration and SA are discussed. Additionally, the SA trends observed in different single crystal orientations closely mirrored the activity of the slip systems.

Elementary Effects Test↗
DOE OSTI2024

Design and Characterization of a Transcriptional Repression Toolkit for Plants

Regulation of gene expression is essential for all life. Tools to manipulate the gene expression level have therefore proven to be very valuable in efforts to engineer biological systems. However, there are few well-characterized genetic parts that reduce gene expression in plants, commonly known as transcriptional repressors. We characterized the repression activity of a library consisting of repression motifs from approximately 25% of the members of the largest known family of repressors. Combining sequence information with our trans-regulatory function data, we next generated a library of synthetic transcriptional repression motifs with function predicted in advance. After characterizing our synthetic library, we demonstrated not only that many of our synthetic constructs were functional as repressors but also that our advance predictions of repression strength were better than random guesses. Finally, we assessed the functionality of known transcriptional repression motifs from a wide range of eukaryotes. Our study represents the largest plant repressor motif library experimentally characterized to date, providing unique opportunities for tuning transcription in plants.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

teemi: An open-source literate programming approach for iterative design-build-test-learn cycles in bioengineering

Synthetic biology dictates the data-driven engineering of biocatalysis, cellular functions, and organism behavior. Integral to synthetic biology is the aspiration to efficiently find, access, interoperate, and reuse high-quality data on genotype-phenotype relationships of native and engineered biosystems under FAIR principles, and from this facilitate forward-engineering strategies. However, biology is complex at the regulatory level, and noisy at the operational level, thus necessitating systematic and diligent data handling at all levels of the design, build, and test phases in order to maximize learning in the iterative design-build-test-learn engineering cycle. To enable user-friendly simulation, organization, and guidance for the engineering of biosystems, we have developed an open-source python-based computer-aided design and analysis platform operating under a literate programming user-interface hosted on Github. The platform is called teemi and is fully compliant with FAIR principles. In this study we apply teemi for i) designing and simulating bioengineering, ii) integrating and analyzing multivariate datasets, and iii) machine-learning for predictive engineering of metabolic pathway designs for production of a key precursor to medicinal alkaloids in yeast. The teemi platform is publicly available at PyPi and GitHub.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2025

Which Plant Traits Increase Soil Carbon Sequestration? Empirical Evidence From a Long‐Term Poplar Genetic Diversity Trial

Plants play a key role in mediating soil response to global change, and breeding or engineering crops to increase soil organic carbon (SOC) storage is a potential route to land-based carbon dioxide removal in agricultural systems. However, due to limited observational datasets plus shifting paradigms of SOC stabilization, it is unclear which plant traits are most important for enhancing different types of soil organic matter. Existing long-term common gardens of genetically diverse plant populations may provide an opportunity to evaluate biological controls on SOC, separate from environmental or management variability. Here we report on soil and root chemical data collected for 24 genotypes within a 13-year-old common garden in northwestern Oregon planted with a large natural variant population of Populus trichocarpa. Fractionating surface soil (0–15 cm) revealed substantial variation in stocks of mineral-associated organic matter (MAOM; 18–67 t C/ha) and particulate organic matter (POM; 2–22 t C/ha). Tree genotype explained 24% and 26% of the MAOM and POM stock variability, respectively, after controlling for background variability. We found minimal association between SOC concentration and either aboveground tree productivity or root biomass recalcitrance (C/N ratios and lignin content). In contrast, root elemental content appeared influential for MAOM-C concentration, which showed a strong positive association with root aluminum (Al) and a strong negative association with root boron (B) and magnesium (Mg). Furthermore, root concentrations of these elements were highly heritable (57%–78%) and not simply a reflection of background variation in soil elemental concentrations. We estimate that surface SOC stocks under these 24 genotypes have diverged at rates of up to 1.2–4.3 t C/ha/year. These results suggest that long-term genetic diversity trials have value for elucidating biological controls on soil organic matter dynamics, and that traits associated with root elemental content may be a useful target for enhancing biosequestration.

biomass recalcitrance↗
DOE OSTI2022

Plant-based engineering for production of high-valued natural products

Plants are a unique source of complex specialized metabolites, many of which play significant roles in human society. In many cases, however, the availability of these metabolites from naturally occurring sources fails to meet current demands. Thus, there is much interest in expanding the production capacity of target plant molecules. Traditionally, plant breeding, chemical synthesis, and microbial fermentation are considered the primary routes towards large scale production of natural products. Here, in this paper, we explore the advances, challenges, and future of plant engineering as a complementary path. Although plants are an integral part of our food and agricultural systems and sustain an extensive array of chemical constituents, their complex genetics and physiology have prevented the optimal exploitation of plants as a production chassis. We highlight emerging engineering tools and scientific advances developed in recent years that have improved the prospects of using plants as a sustainable and scalable production platform. We also discuss technological limitations and overall economic outlook of plant-based production of natural products.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2020

Engineering the cellulolytic extreme thermophile Caldicellulosiruptor bescii to reduce carboxylic acids to alcohols using plant biomass as the energy source

Abstract Caldicellulosiruptor bescii is the most thermophilic cellulolytic organism yet identified (Topt 78 °C). It grows on untreated plant biomass and has an established genetic system thereby making it a promising microbial platform for lignocellulose conversion to bio-products. Here, we investigated the ability of engineered C. bescii to generate alcohols from carboxylic acids. Expression of aldehyde ferredoxin oxidoreductase (aor from Pyrococcus furiosus) and alcohol dehydrogenase (adhA from Thermoanaerobacter sp. X514) enabled C. bescii to generate ethanol from crystalline cellulose and from biomass by reducing the acetate produced by fermentation. Deletion of lactate dehydrogenase in a strain expressing the AOR–Adh pathway increased ethanol production. Engineered strains also converted exogenously supplied organic acids (isobutyrate and n-caproate) to the corresponding alcohol (isobutanol and hexanol) using both crystalline cellulose and switchgrass as sources of reductant for alcohol production. This is the first instance of an acid to alcohol conversion pathway in a cellulolytic microbe.

Biotechnology & Applied Microbiology↗
DOE OSTI2025

An Innovative High Throughput Genome Releaser for Rapid and Efficient PCR Screening

High-throughput PCR screening is vital in synthetic biology and metabolic engineering as it allows researchers to rapidly analyze and detect numerous targeted genetic mutation in the genome. Current challenges for high-throughput PCR screening in synthetic biology include efficiently preparing genomic DNA, optimizing protocols for diverse sample types, managing contamination risks, and effectively analyzing the large volumes of data generated while ensuring consistent and accurate results. In this study, we present the development of a High Throughput Genome Releaser (HTGR), an innovative device addressing common challenges in screening PCR. This genome DNA releaser is designed based on a squash method for rapid, cost-effective, and efficient DNA release, optimized for subsequent PCR reactions. After experimenting with various synthetic materials, we selected a plastic that closely replicates the smooth surface and compression properties of microscope slides, ensuring reliable performance. We engineered a device featuring a 96-Well Plate and a shear applicator, operable both manually and automatically, and compatible with standard liquid-handling robot platform. This compatibility enhances ease of use in high-throughput PCR workflows. Additionally, we developed software to support its automatic functions. Our results demonstrated that the specially engineered 96-Well Plate and HTGR can effectively squash fungal spores , which release enough genome DNA for PCR screening. The genome releaser facilitates the preparation of PCR-amplifiable genomic DNA substrate from 96 samples within minutes, eliminates the need for extraction buffers, and is adaptable to a wide range of microorganisms and cells, which could significantly advance biomanufacturing processes.

Yuan, Guoliang [BATTELLE (PACIFIC NW LAB)]↗
DOE OSTI2022

Rapid Targeted Quantitation of Protein Overexpression with Direct Infusion Shotgun Proteome Analysis (DISPA-PRM)

While much effort has been placed on comprehensive quantitative proteome analysis, certain applications demand measurement of only a few target proteins from complex systems. Traditional approaches to targeted proteomics rely on nano-liquid chromatography (nLC) and targeted mass spectrometry (MS) methods, e.g., parallel reaction monitoring (PRM). However, the time requirement for nLC can limit throughput of targeted proteomics. To achieve rapid and high-throughput targeted methods, here we show that nLC separations can be eliminated and replaced with direct infusion shotgun proteome analysis (DISPA) using high-field asymmetric waveform ion mobility spectrometry (FAIMS) with PRM. We demonstrate application of DISPA-PRM for rapid targeted quantification of bacterial enzymes utilized in the production of biofuels by monitoring temporal expression in 72 metabolically engineered bacterial cultures in under 2.5 hours, with a measured dynamic range >1200-fold. Here, we conclude that DISPA-PRM presents a valuable innovative tool with results comparable to nLC-MS/MS, enabling fast and rapid detection of targeted proteins in complex mixtures.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2022

Synthetic genetic circuits as a means of reprogramming plant roots

We report the shape of a plant’s root system influences its ability to reach essential nutrients in the soil and to acquire water during drought. Progress in engineering plant roots to optimize water and nutrient acquisition has been limited by our capacity to design and build genetic programs that alter root growth in a predictable manner. We developed a collection of synthetic transcriptional regulators for plants that can be compiled to create genetic circuits. These circuits control gene expression by performing Boolean logic operations and can be used to predictably alter root structure. This work demonstrates the potential of synthetic genetic circuits to control gene expression across tissues and reprogram plant growth.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

Light‐Driven Metabolic Pathways in Non‐Photosynthetic Biohybrid Bacteria

Biomanufacturing via microorganisms relies on carbon substrates for molecular feedstocks and a source of energy to carry out enzymatic reactions. This creates metabolic bottlenecks and lowers the efficiency for substrate conversion. Nanoparticle biohybridization with proteins and whole cell surfaces can bypass the need for redox cofactor regeneration for improved secondary metabolite production in a non-specific manner. Here we propose using nanobiohybrid organisms (Nanorgs), intracellular protein-nanoparticle hybrids formed through the spontaneous coupling of core-shell quantum dots (QDs) with histidine-tagged enzymes in non-photosynthetic bacteria, for light-mediated control of bacterial metabolism. Furthermore, this proved to eliminate metabolic constrictions and replace glucose with light as the source of energy in Escherichia coli, with an increase in growth by 1.7-fold in 75 % reduced nutrient media. Metabolomic tracking through carbon isotope labeling confirmed flux shunting through targeted pathways, with accumulation of metabolites downstream of respective targets. Finally, application of Nanorgs with the Ehrlich pathway improved isobutanol titers/yield by 3.9-fold in 75 % less sugar from E. coli strains with no genetic alterations. These results demonstrate the promise of Nanorgs for metabolic engineering and low-cost biomanufacturing.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2022

In Situ Optical Quantification of Extracellular Electron Transfer Using Plasmonic Metal Oxide Nanocrystals**

Extracellular electron transfer (EET) is a critical form of microbial metabolism that enables respiration on a variety of inorganic substrates, including metal oxides. However, quantifying current generated by electroactive bacteria has been predominately limited to biofilms formed on electrodes. To address this, we developed a platform for quantifying EET flux from cell suspensions using aqueous dispersions of infrared plasmonic tin-doped indium oxide nanocrystals. Furthermore, tracking the change in optical extinction during electron transfer enabled quantification of current generated by planktonic Shewanella oneidensis cultures. Using this method, we differentiated between starved and actively respiring cells, cells of varying genotype, and cells engineered to differentially express a key EET gene using an inducible genetic circuit. Overall, our results validate the utility of colloidally stable plasmonic metal oxide nanocrystals as quantitative biosensors in aqueous environments and contribute to a fundamental understanding of planktonic S. oneidensis electrophysiology using simple in situ spectroscopy.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2021

An integrated in vivo/in vitro framework to enhance cell-free biosynthesis with metabolically rewired yeast extracts

Cell-free systems using crude cell extracts present appealing opportunities for designing biosynthetic pathways and enabling sustainable chemical synthesis. However, the lack of tools to effectively manipulate the underlying host metabolism in vitro limits the potential of these systems. Here, we create an integrated framework to address this gap that leverages cell extracts from host strains genetically rewired by multiplexed CRISPR-dCas9 modulation and other metabolic engineering techniques. As a model, we explore conversion of glucose to 2,3-butanediol in extracts from flux-enhanced Saccharomyces cerevisiae strains. We show that cellular flux rewiring in several strains of S. cerevisiae combined with systematic optimization of the cell-free reaction environment significantly increases 2,3-butanediol titers and volumetric productivities, reaching productivities greater than 0.9 g/L-h. We then show the generalizability of the framework by improving cell-free itaconic acid and glycerol biosynthesis. Our coupled in vivo/in vitro metabolic engineering approach opens opportunities for synthetic biology prototyping efforts and cell-free biomanufacturing.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2020

Development of genetic tools for the thermophilic filamentous fungus Thermoascus aurantiacus

Abstract Background Fungal enzymes are vital for industrial biotechnology, including the conversion of plant biomass to biofuels and bio-based chemicals. In recent years, there is increasing interest in using enzymes from thermophilic fungi, which often have higher reaction rates and thermal tolerance compared to currently used fungal enzymes. The thermophilic filamentous fungus Thermoascus aurantiacus produces large amounts of highly thermostable plant cell wall-degrading enzymes. However, no genetic tools have yet been developed for this fungus, which prevents strain engineering efforts. The goal of this study was to develop strain engineering tools such as a transformation system, a CRISPR/Cas9 gene editing system and a sexual crossing protocol to improve the enzyme production. Results Here, we report Agrobacterium tumefaciens -mediated transformation (ATMT) of T. aurantiacus using the hph marker gene, conferring resistance to hygromycin B. The newly developed transformation protocol was optimized and used to integrate an expression cassette of the transcriptional xylanase regulator xlnR , which led to up to 500% increased xylanase activity. Furthermore, a CRISPR/Cas9 gene editing system was established in this fungus, and two different gRNAs were tested to delete the pyrG orthologue with 10% and 35% deletion efficiency, respectively. Lastly, a sexual crossing protocol was established using a hygromycin B- and a 5-fluoroorotic acid-resistant parent strain. Crossing and isolation of progeny on selective media were completed in a week. Conclusion The genetic tools developed for T. aurantiacus can now be used individually or in combination to further improve thermostable enzyme production by this fungus.

09 BIOMASS FUELS↗
DOE OSTI2022

Cas9-Based Metabolic Engineering of Issatchenkia orientalis for Enhanced Utilization of Cellulosic Hydrolysates

Issatchenkia orientalis, exhibiting high tolerance against harsh environmental conditions, is a promising metabolic engineering host for producing fuels and chemicals from cellulosic hydrolysates containing fermentation inhibitors under acidic conditions. Although genetic tools for I. orientalis exist, they require auxotrophic mutants so that the selection of a host strain is limited. We developed a drug resistance gene (cloNAT)-based genome-editing method for engineering any I. orientalis strains and engineered I. orientalis strains isolated from various sources for xylose fermentation. Specifically, xylose reductase, xylitol dehydrogenase, and xylulokinase from Scheffersomyces stipitis were integrated into an intended chromosomal locus in four I. orientalis strains (SD108, IO21, IO45, and IO46) through Cas9-based genome editing. Furthermore, the resulting strains (SD108X, IO21X, IO45X, and IO46X) efficiently produced ethanol from cellulosic and hemicellulosic hydrolysates even though the pH adjustment and nitrogen source were not provided. As they presented different fermenting capacities, selection of a host I. orientalis strain was crucial for producing fuels and chemicals using cellulosic hydrolysates.

09 BIOMASS FUELS↗
DOE OSTI2024

Systems-Level Modeling for CRISPR-Based Metabolic Engineering

The CRISPR-Cas system has enabled the development of sophisticated, multigene metabolic engineering programs through the use of guide RNA-directed activation or repression of target genes. To optimize biosynthetic pathways in microbial systems, we need improved models to inform design and implementation of transcriptional programs. Recent progress has resulted in new modeling approaches for identifying gene targets and predicting the efficacy of guide RNA targeting. Genome-scale and flux balance models have successfully been applied to identify targets for improving biosynthetic production yields using combinatorial CRISPR-interference (CRISPRi) programs. Here, the advent of new approaches for tunable and dynamic CRISPR activation (CRISPRa) promises to further advance these engineering capabilities. Once appropriate targets are identified, guide RNA prediction models can lead to increased efficacy in gene targeting. Developing improved models and incorporating approaches from machine learning may be able to overcome current limitations and greatly expand the capabilities of CRISPR-Cas9 tools for metabolic engineering.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

Comparison of Shape Optimization Methods for Heat Exchanger Fins Using Computational Fluid Dynamics

Inverse design techniques are one way to leverage advances in 3D printing, artificial intelligence, and computational resources to achieve increased performance of heat exchangers. Two optimization techniques (genetic algorithm and particle swarm) and three geometry representations (binary level set, composite Bézier, and free form deformation) are used to increase the heat transfer and reduce the pressure drop of a heat exchanger fin. After running 210,810 OpenFOAM simulations, results indicate that a significant performance increase of the fin can be realized in less than 48 hrs, allowing for such a process to be integrated in traditional design processes. The best design increased the performance of the objective function, compared to the baseline rectangular geometry by 75%. A custom distributed infrastructure was built, allowing for all methods to reach 95% of the final objective values in a little over 4 hrs, handling 1674 OpenFoam simulations per hour.

42 ENGINEERING↗