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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 163 records · Page 9
All sourcesNASA NTRSDOE OSTI
DOE OSTI2025

Sub‐5 Ångstrom Porosity Tuning in Calixarene‐Derived Porous Liquids via Supramolecular Complexation Construction

Abstract Sub‐Ångstrom‐level porosity engineering, which is appealing in gas separations, has been demonstrated in solid carbon, polymer, and framework materials but rarely achieved in the liquid phase. In this work, a gas molecular sieving effect in the liquid phase at sub‐5 Ångstrom scale is created via sophisticated porosity tuning in calixarene‐derived porous liquids (PLs). Type II PLs are constructed via supramolecular complexation between the sodium salts of calixarene derivatives and crown ether solvents. The chemical structure variation and assembly behavior of the porous host upon PL construction are monitored by spectroscopy‐, X‐ray‐, and neutron‐scattering techniques. The presence of permanent porosity in calixarene‐derived PLs is verified by pressure swing gas uptake, altered CO 2 physisorption behavior, and molecular simulations. Sub‐5 Ångstrom porosity tuning within the PL phase is achieved by introducing bulky substituted groups on the benzene ring of the calixarene host, which then greatly affects the dynamic motion and transport behavior of CO 2 molecules and the Xe uptake performance. The approach being demonstrated in this work represents a promising pathway to tune and leverage the porosity effect for enhanced gas uptake capacity and selectivity in liquid sorbents.

Li, Errui [Department of Chemistry University of T↗
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 OSTI2018

Building a genome engineering toolbox in nonmodel prokaryotic microbes

The realization of a sustainable bioeconomy requires our ability to understand and engineer complex design principles for the development of platform organisms capable of efficient conversion of cheap and sustainable feedstocks (e.g. sunlight, CO 2 , non-food biomass) to biofuels and bioproducts at sufficient titers and costs. For model microbes such as E. coli , advances in DNA reading and writing technologies are driving adoption of new paradigms for engineering biological systems. Unfortunately, microbes with properties of interest for the utilization of cheap and renewable feedstocks such as photosynthesis, autotrophic growth, and cellulose degradation have very few, if any, genetic tools for metabolic engineering. Therefore, it is important to begin to develop 'design rules' for building a genetic toolbox for novel microbes. Furthermore, we present an overview of our current understanding of these rules for the genetic manipulation of prokaryotic microbes and available genetic tools to expand our ability to genetically engineer non-model systems.

09 BIOMASS FUELS↗
DOE OSTI2020

An Integrated Computer-Aided Design and Manufacturing Workflow for Synthetic Biology

Biological computer-aided design and manufacturing (bioCAD/CAM) tools facilitate the design and build processes of engineering biological systems using iterative design-build-test-learn (DBTL) cycles. In this book chapter, we highlight some of the bioCAD/CAM tools developed and used at the US Department of Energy (DOE) Joint Genome Institute (JGI), Joint BioEnergy Institute (JBEI), and Agile BioFoundry (ABF). We demonstrate the use of these bioCAD/CAM tools on a common workflow for designing and building a multigene pathway in a hierarchical fashion. Additionally, each tool presented in this book chapter is specifically tailored to support one or more specific steps in a workflow, can be integrated with the others into design and build workflows, and can be deployed at academic, government, or commercial entities.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

Biocatalytic gateway to convert glycerol into 3-hydroxypropionic acid in waste-based biorefineries: Fundamentals, limitations, and potential research strategies

Microbial conversion of bioenergy-derived waste glycerol into value-added chemicals has emerged as an important bioprocessing technology due to its eco-friendliness, feasible technoeconomics, and potential to provide sustainability in biodiesel and bioethanol production. Glycerol is an abundant liquid waste from bioenergy plants with a projected volume of 6 million tons by 2025, accounting for about 10% of biodiesel and 2.5% of bioethanol yields. 3-Hydroxypropionic acid (3-HP) is a major product of glycerol bioconversion, which is the third largest biobased platform compound with expected market size and value of 3.6 million tons/year and USD 10 billion/year, respectively. Despite these biorefinery values, 3-HP biosynthesis from glycerol is still at an immature stage of commercial exploitation. The main challenges behind this immaturity are the toxic effects of 3-HPA on cells, the distribution of carbon flux to undesirable pathways, low tolerance of cells to glycerol and 3-HP, co-factor dependence of enzymes, low enzyme activity and stability, and the problems of substrate inhibition and specificity of enzymes. To address these challenges, it is necessary to understand the fundamentals of glycerol bioconversion and 3-HP production in terms of metabolic pathways, related enzymes, cell factories, midstream process configurations, and downstream 3-HP recovery, as discussed in this review critically and comprehensively. It is equally important to know the current challenges and limitations in 3-HP production, which are discussed in detail along with recent research efforts and remaining gaps. Finally, possible research strategies are outlined considering the recent technological advances in microbial biosynthesis, aiming to attract further research efforts to achieve a sustainable and industrially exploitable 3-HP production technology. Here, by discussing the use of advanced tools and strategies to overcome the existing challenges in 3-HP biosynthesis, this review will attract researchers from many other similar biosynthesis technologies and provide a common gateway for their further development.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2020

Engineered bacterial microcompartments: apps for programming metabolism

Bacterial Microcompartments (BMCs) are used by diverse bacteria to compartmentalize enzymatic reactions, functioning analogously to the organelles of eukaryotes. The bounding membrane and encapsulated components are composed entirely of protein, which makes them ideal targets for modification by genetic engineering. In contrast to viruses, in which generally only one protein forms the capsid, the shells of BMCs consist of a variety of shell proteins, each a potential unit of selection. Despite their differences in permeability, the shell proteins are surprisingly interchangeable. Recent developments have shown that they are also highly amenable to engineered modifications which poise them for a variety of biotechnological applications. Additionally, given their modular structure, with a module defined as a semi-autonomous functional unit, BMCs can be considered apps for programming metabolism that can be de-bugged by adaptive evolution.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2019

Modification of RSF1010-Based Broad-Host-Range Plasmids for Improved Conjugation and Cyanobacterial Bioprospecting

To facilitate the genetic engineering of diverse cyanobacterial strains, we have modified broad-hostrange RSF1010-based plasmids to improve transmissibility, increase copy number, and facilitate cloning. RSF1010-based plasmids replicate in diverse bacterial strains but produce lowamounts of useable DNA for cloning. We previously engineered a mobAY25F mutation in RSF1010-based plasmids that improved cloning but decreased conjugation efficiency. Here, we engineered RSF1010-based plasmids to restore conjugation efficiency, which was demonstrated in three diverse laboratory strains of cyanobacteria. We then used an improved RSF1010-based plasmid in mating experiments with cultured samples of wild cyanobacteria. This plasmid, which confers antibiotic resistance and carries a yfp reporter gene, allowed selection of exconjugant cyanobacteria and facilitated the isolation of genetically tractable strains from mixed wild cultures. Improved RSF1010 vectors can be used for bioprospecting genetically tractable strains and are compatible with the CYANO-VECTOR cloning system, a versatile toolbox for constructing plasmids for cyanobacterial genetic engineering.

09 BIOMASS FUELS↗
DOE OSTI2022

Computational design and engineering of an Escherichia coli strain producing the nonstandard amino acid para -aminophenylalanine

Introducing heterologous pathways into host cells constitutes a promising strategy for synthesizing nonstandard amino acids (nsAAs) to enable the production of proteins with expanded chemistries. However, this strategy has proven challenging, as the expression of heterologous pathways can disrupt cellular homeostasis of the host cell. Here, we sought to optimize the heterologous production of the nsAA para -aminophenylalanine (pAF) in Escherichia coli . First, we incorporated a heterologous pAF biosynthesis pathway into a genome-scale model of E. coli metabolism and computationally identified metabolic interventions in the host’s native metabolism to improve pAF production. Next, we explored different approaches of imposing these flux interventions experimentally and found that the upregulation of flux in the chorismate biosynthesis pathway through the elimination of feedback inhibition mechanisms could significantly raise pAF titers (~20-fold) while maintaining a reasonable pAF production-growth rate trade-off. Overall, this study provides a promising strategy for the biosynthesis of nsAAs in engineered cells.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

Scripting a new dialogue between diazotrophs and crops

Diazotrophs are bacteria and archaea that can reduce atmospheric dinitrogen (N 2 ) into ammonium. Plant–diazotroph interactions have been explored for over a century as a nitrogen (N) source for crops to improve agricultural productivity and sustainability. This scientific quest has generated much information about the molecular mechanisms underlying the function, assembly, and regulation of nitrogenase, ammonium assimilation, and plant–diazotroph interactions. This review presents various approaches to manipulating N fixation activity, ammonium release by diazotrophs, and plant–diazotroph interactions. Here, we discuss the research avenues explored in this area, propose potential future routes, emphasizing engineering at the metabolic level via biorthogonal signaling, and conclude by highlighting the importance of biocontrol measures and public acceptance.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

Chromosomal integration of complex DNA constructs using CRAGE and CRAGE-Duet systems

Our recent development of the CRAGE (chassis-independent recombinase-assisted genome engineering) system enables single-step integration of large, complex DNA constructs directly into bacteria genomes across multiple phyla. This protocol describes the details of the experimental design and procedures of CRAGE and extended CRAGE-Duet systems. It also describes a strategy that combines CRISPR with CRAGE, which allows implementation of CRISPR-Cas9, CRISPRa, and CRISPRi in diverse bacteria, overcoming major limitations to broaden the application of CRISPR in non-model bacterial genome engineering. For complete details on the use and execution of this protocol, please refer to Wang et al. (2019), Wang et al. (2020), and Liu et al. (2020).

79 ASTRONOMY AND ASTROPHYSICS↗
DOE OSTI2025

Protocol for engineering poly(ethylene terephthalate) hydrolases via directed evolution using a high-throughput screening assay

Poly(ethylene terephthalate) (PET) hydrolases, which depolymerize PET to its monomers, have gained attention for their potential to facilitate bio-industrial recycling of this waste plastic. Here, we present a protocol for screening large, random mutagenesis enzyme libraries simultaneously for enhanced activity, solubility, and stability. We outline steps for library construction, screening using plate-based split GFP and model substrate assays, and determination of enzyme thermostability. We then detail procedures for validation assays on PET substrates and characterization of final variants.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

In Vitro Encapsulation of Functionally Active Abiotic Photosensitizers Inside a Bacterial Microcompartment Shell

Bacterial microcompartments (BMCs) are self-assembling, selectively permeable protein shells that encapsulate enzymes to enhance catalytic efficiency of segments of metabolic pathways through means of confinement. The modular nature of BMC shells' structure and assembly enables programming of shell permeability and underscores their promise in biotechnology engineering efforts for applications in industry, medicine, and clean energy. Realizing this potential requires methods for encapsulation of abiotic molecules, which have been developed here for the first time. We report in vitro cargo loading of BMC shells with ruthenium photosensitizers (RuPS) by two approaches-one involving site-specific covalent labeling and the other driven by diffusion, requiring no specific interactions between cargo molecules and shell proteins. The highly stable shells retain encapsulated cargo over 1 week without egress and preserve RuPS photophysical activity. Finally, this study is an important foundation for further work that will converge biological BMC architecture with synthetic chemistry to facilitate biohybrid photocatalysis.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2019

Visualizing in Vivo Dynamics of Designer Nanoscaffolds

Enzymes of natural biochemical pathways are routinely subcellularly organized in space and time in order to improve pathway efficacy and control. Designer scaffolding platforms are under development to confer similar benefits upon engineered pathways. Herein, we assess bacterial microcompartment shell (pfam0936-domain) proteins as modules for constructing well-defined nanometer scale scaffolds in vivo. We use a suite of visualization techniques to evaluate scaffold assembly and dynamics. We demonstrate recruitment of target cargo molecules onto assembled scaffolds by appending reciprocally interacting adaptor domains. These interactions can be refined by fine-tuning the scaffold expression level. Real-time observation of this system reveals a nucleation-limited step where multiple scaffolds initially form within a cell. Over time, nucleated scaffolds reorganize into a single intracellular assembly, likely due to interscaffold competition for protein subunits. Our findings suggest design considerations for using self-assembling proteins as building blocks to construct nanoscaffolds, while also providing a platform to visualize scaffold-cargo dynamics in vivo.

59 BASIC BIOLOGICAL SCIENCES↗