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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 OSTI2022

Crystal structure of an RNA/DNA strand exchange junction

Short segments of RNA displace one strand of a DNA duplex during diverse processes including transcription and CRISPR-mediated immunity and genome editing. These strand exchange events involve the intersection of two geometrically distinct helix types—an RNA:DNA hybrid (A-form) and a DNA:DNA homoduplex (B-form). Although previous evidence suggests that these two helices can stack on each other, it is unknown what local geometric adjustments could enable A-on-B stacking. Here we report the X-ray crystal structure of an RNA-5′/DNA-3′ strand exchange junction at an anisotropic resolution of 1.6 to 2.2 Å. The structure reveals that the A-to-B helical transition involves a combination of helical axis misalignment, helical axis tilting and compression of the DNA strand within the RNA:DNA helix, where nucleotides exhibit a mixture of A- and B-form geometry. These structural principles explain previous observations of conformational stability in RNA/DNA exchange junctions, enabling a nucleic acid architecture that is repeatedly populated during biological strand exchange events.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

TCF High Efficiency Anaerobic Electroporation (CRADA Final Report)

The Joint BioEnergy Institute (JBEI) researchers were co-inventors of the technology that will be used on this project and have developed a more current version of the chip and controller. JBEI will also assist with the design of the pathways and implementation of pathways on the chip. LanzaTech has developed novel gas fermentation technology that captures and utilizes greenhouse gases for production of fuels and chemicals. In contrast to traditional fermentation that uses sugars as substrate (and releases CO2 as a byproduct), gas fermentation utilizes C1 substrates carbon monoxide (CO) or CO2. This enables a diverse range of feedstock options including waste gases from industrial sources (e.g., steel mills and processing plants) or syngas generated from any biomass resource (e.g., agricultural waste, municipal solid waste, or organic industrial waste). Biomass is then gasified, allowing for maximum yields and complete carbon utilization including the recalcitrant lignocellulosic fraction that cannot be utilized in traditional sugar fermentation. To maximize the value that can be added to the array of gas resources that the LanzaTech process can use as an input, LanzaTech has pioneered genetic modification of acetogens and developed a comprehensive set of genetic tools to perform routine strain modification, including genome editing tools as CRISPR/Cas9 and libraries of validated genetic parts as promoters and terminators. Using this platform, production of over 50 new molecules have been demonstrated directly from gas. For a few selected molecules production rates and yields have been optimized and surpass production of native producers and engineered E. coli or yeast strains, but a higher throughput approach for combinatorial optimization of pathways is required to further advance synthesis of additional products in parallel.

09 BIOMASS FUELS↗
DOE OSTI2024

Rational Optimization of Microbial Processing for High Yield CO 2 -to-Isopropanol Conversion: Cooperative Research and Development Final Report, CRADA Number CRD-20-17114

This project focuses on the production of the fuel blendstock isopropanol using a CO 2 -fixing Clostridium by metabolic engineering and process optimizations. The project will initiate from a baseline isopropanol producer and pursue isopropanol production at high carbon-conversion efficiency. We will lead engineering work by in-depth pathway analyses including thermodynamics optimization, enzyme expense analysis, metabolic robustness analysis, and -omics analysis. The isopropanol production will be optimized via genome editing followed by fermentation optimizations. This project will deliver a novel microbial process that efficiently converts waste CO 2 to isopropanol at ~g/L titer level within 18-months. This project will layout a solid knowledge basis and technology platform for renewable CO 2 valorization to bio-blendstock that help achieve Co-Optima and Shell's goals.

09 BIOMASS FUELS↗
DOE OSTI2024

Establishing a clostridia foundry for biosystems design by integrating computational modeling, systems-level analyses, and cell-free engineering technologies (Final Report)

Rapid population growth, a rise in global living standards, and economic competitiveness have intensified the need for sustainable, low-cost biofuels and bioproducts production. Industrial biotechnology using microbial cell factories – and waste and/or renewable feedstocks – is one of the most attractive approaches for addressing this need, particularly when large-scale chemical synthesis is untenable. Unfortunately, designing, building, and optimizing biosynthetic pathways in cells remains a complex challenge. With support from the Department of Energy, we worked to address this challenge in a new interdisciplinary venture that established the world’s first clostridial Foundry for Biosystems Design (cBioFAB). Working both in vitro (cell-free) and in vivo, the goal of this project was to interweave and advance state-of-the-art computational modeling, genome editing, omics measurements, systems-biology analyses, and cell-free technologies to expand the set of platform organisms that meet DOE bioenergy goals. Specifically, we manufactured fuel and chemical intermediates via existing and de novo pathways. This report covers the outcomes of our research project.

09 BIOMASS FUELS↗
DOE OSTI2026

Engineering Synthetic Anaerobic Consortia Inspired by the Rumen for Biomass Breakdown and Conversion

Lignocellulosic plant biomass is a widely-abundant renewable resource that can be harnessed for value-added production of fuels & chemicals. While microbes have been engineered to breakdown lignocellulose and turn released sugars into products, this remains an energy-intensive process that requires expensive pre-treatment and separation steps. Furthermore, it is difficult to engineer all desirable traits for breakdown and conversion into one organism. This project developed a new strategy that relies on microbial partnerships formed in the herbivore rumen to liberate sugars from crude plant biomass and convert that sugar to value-added chemicals. Microbial consortia consisting of fungi, bacteria, and archaea form tight associations in the herbivore rumen, which divide-and-conquer the difficult tasks of biomass breakdown. This project leveraged a “synthetic rumen” consortium composed of anaerobic fungi and chain-elongating bacteria to study which metabolites are shared and exchanged between microbes and identify strategies to bolster lignocellulose conversion to value-added products. Our approach developed high-throughput systems and synthetic biology approaches to realize stable synthetic consortia that route lignocellulosic carbon into short and medium chain fatty acids (SCFAs/MCFAs) rather than methane. Key research objectives were to (1) design and predict anaerobic fungal and bacterial consortia that efficiently convert lignocellulosic biomass into medium-chain fatty acids (MCFAs), (2) understand how fermentation parameters and microbe-microbe interactions regulate and drive microbiome metabolic fluxes, and (3) use genomic editing to alter the fermentation byproducts of anaerobic fungi and bolster MCFA titers and yields.

09 BIOMASS FUELS↗
DOE OSTI2024

Cas9 deletion of lutein biosynthesis in the marine alga Picochlorum celeri reduces photosynthetic pigments while sustaining high biomass productivity

Domestication of algae for food and renewable biofuels remains limited by the low photosynthetic efficiencies of processes that have evolved to be competitive for optimal light capture, incentivizing the development of large antennas in light-limiting conditions, thus decreasing efficient light utilization in cultivated ponds or photobioreactors. Reducing the pigment content to improve biomass productivity has been a strategy discussed for several decades and the ability to reduce pigment significantly is now fully at hand thanks to the widespread use of genome editing tools. Picochlorum celeri is one of the fastest growing marine algae identified and holds particular promise for outdoor cultivation, especially in saline water and warm climates. We show that while chlorophyll b is essential to sustain high biomass productivities under dense cultivation, removing Picochlorum celeri’s main carotenoid, lutein, leads to a decreased total chlorophyll content, higher a/ b ratio, reduced functional LHCII cross section and higher maximum quantum efficiencies at lower light intensities, resulting in an incremental increase in biomass productivity and increased PAR-to-biomass conversion efficiency. These findings further strengthen the existing strategies to improve photosynthetic efficiency and biomass production in algae.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

Comparison of genotyping assays for detection of targeted CRISPR/Cas mutagenesis in highly polyploid sugarcane

Sugarcane (Saccharum spp.) is an important biofuel feedstock and a leading source of global table sugar. Saccharum hybrid cultivars are highly polyploid (2n = 100–130), containing large numbers of functionally redundant hom(e)ologs in their genomes. Genome editing with sequence-specific nucleases holds tremendous promise for sugarcane breeding. However, identification of plants with the desired level of co-editing within a pool of primary transformants can be difficult. While DNA sequencing provides direct evidence of targeted mutagenesis, it is cost-prohibitive as a primary screening method in sugarcane and most other methods of identifying mutant lines have not been optimized for use in highly polyploid species. In this study, non-sequencing methods of mutant screening, including capillary electrophoresis (CE), Cas9 RNP assay, and high-resolution melt analysis (HRMA), were compared to assess their potential for CRISPR/Cas9-mediated mutant screening in sugarcane. These assays were used to analyze sugarcane lines containing mutations at one or more of six sgRNA target sites. All three methods distinguished edited lines from wild type, with co-mutation frequencies ranging from 2% to 100%. Cas9 RNP assays were able to identify mutant sugarcane lines with as low as 3.2% co-mutation frequency, and samples could be scored based on undigested band intensity. CE was highlighted as the most comprehensive assay, delivering precise information on both mutagenesis frequency and indel size to a 1 bp resolution across all six targets. This represents an economical and comprehensive alternative to sequencing-based genotyping methods which could be applied in other polyploid species.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2025

Leveraging CRISPR Cas9 RNPs and Cre- loxP in Picochlorum celeri for generation of field deployable strains and selection marker recycling

As new highly productive strains of algae are discovered and developed to meet the energy, chemical, and food requirements of the future, genetic engineering of those strains in a manner that yields deployable transformants is paramount. This study introduces the novel CRoxP ($\underline{\textrm{C}}$$\textrm{as9}$ $\underline{\textrm{R}}$$\textrm{NPs}$ coupled with an inducible $\underline{\textrm{CR}}$$\textrm{e}$-$\textrm{l}\underline{\textrm{oxP}}$) system for rapid generation of marker- and transgene-free strains of Picochlorum celeri. The CRoxP system allows reuse of selection markers without Cas9 expression in vivo, eliminating many of the bottlenecks associated with conventional CRISPR Cas9 use for precise genome editing. In P. celeri, transformants were generated with a turnaround time as short as 21 days between transformation and being ready for another round of transformation with the same selection marker by using the CRoxP system. As a use-case for CRoxP, depigmented strains of P. celeri were generated by multiplexed Cas9 disruption of major LHCII genes followed by either a second round of LHCII targeting, or knockout of an LHCI gene. One transformant tested in flask culture (R6) exhibited similar biomass production to the wild type with 46% less Chl a + b on a biomass basis. In photobioreactors and under diel light simulating a solar day, a transformant (LhcBM31) exhibited 34 g AFDW m –2 d –1 with 54% less Chl a + b on a biomass basis vs. wild type.

09 BIOMASS FUELS↗
DOE OSTI2021

Transcriptional Regulation of Drought Response in Arabidopsis and Woody Plants

Within the context of global warming, long-living plants such as perennial woody species endure adverse conditions. Among all of the abiotic stresses, drought stress is one of the most detrimental stresses that inhibit plant growth and productivity. Plants have evolved multiple mechanisms to respond to drought stress, among which transcriptional regulation is one of the key mechanisms. In this review, we summarize recent progress on the regulation of drought response by transcription factor (TF) families, which include abscisic acid (ABA)-dependent ABA-responsive element/ABRE-binding factors (ABRE/ABF), WRKY, and Nuclear Factor Y families, as well as ABA-independent AP2/ERF and NAC families, in the model plant Arabidopsis. We also review what is known in woody species, particularly Populus, due to its importance and relevance in economic and ecological processes. We discuss opportunities for a deeper understanding of drought response in woody plants with the development of high-throughput omics analyses and advanced genome editing techniques.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

Novel Mutant Alleles Reveal a Role of the Extra-Large G Protein in Rice Grain Filling, Panicle Architecture, Plant Growth, and Disease Resistance

Plant growth and grain filling are the key agronomical traits for grain weight and yield of rice. The continuous improvement in rice yield is required for a future sustainable global economy and food security. The heterotrimeric G protein complex containing a canonical α subunit (RGA1) couples extracellular signals perceived by receptors to modulate cell function including plant development and grain weight. We hypothesized that, besides RGA1, three atypical, extra-large GTP-binding protein (XLG) subunits also regulate panicle architecture, plant growth, development, grain weight, and disease resistance. Here, we identified a role of XLGs in agronomic traits and stress tolerance by genetically ablating all three rice XLGs individually and in combination using the CRISPR/Cas9 genome editing in rice. For this study, eight (three single, two double, and three triple) null mutants were selected. Three XLG proteins combinatorically regulate seed filling, because loss confers a decrease in grain weight from 14% with loss of one XLG and loss of three to 32% decrease in grain weight. Null mutations in XLG2 and XLG4 increase grain size. The mutants showed significantly reduced panicle length and number per plant including lesser number of grains per panicle compared to the controls. Loss-of-function of all individual XLGs contributed to 9% more aerial biomass compared to wild type (WT). The double mutant showed improved salinity tolerance. Moreover, loss of the XLG gene family confers hypersensitivity to pathogens. Our findings suggest that the non-canonical XLGs play important roles in regulating rice plant growth, grain filling, panicle phenotype, stress tolerance, and disease resistance. Genetic manipulation of XLG s has the potential to improve agronomic properties in rice.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2026

Decreases in polyunsaturated fatty acid content improve heat stress tolerance during flowering and silicle development in pennycress (Thlaspi arvense L.)

Introduction: Pennycress (Thlaspi arvense L.) is an emerging intermediate oilseed crop grown in the offseason between primary summer crops to produce three cash crops in two years. Previous efforts to improve seed oil quality produced Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) genome-edited lines with decreased polyunsaturated fatty acids (PUFAs) levels, through loss of function of the FATTY ACID DESATURASE 2 (FAD2), REDUCED OLEATE DESATURATION1 (ROD1), and FATTY ACID ELONGASE1 (FAE1) genes. While seed oil compositions were previously characterized, it remains unknown how vegetative and reproductive tissue compositions might differ and affect tolerance to high temperature (HT) conditions.Methods: In four growth chamber experiments, we explored HT tolerance during flowering and silicle development. Plants were subjected to a 34 °C day/28 °C night regime and compared to control plants maintained at 20 °C. Pollen grain viability at a range of temperatures, lipid peroxidation and proline content in leaves and silicles following HT, and seed yield were measured.Results: Both fad2 and rod1 mutant lines had relatively higher pollen viability (71% and 54% respectively) under moderately elevated temperature (28 °C) compared to wild-type controls (37%). They also showed smaller decreases in seed yield (0% and 40% for fad2 and rod1 respectively, compared to 61% for wild type), following HT exposure during late flowering and early silicle development. Silicles of fad2 plants experienced 65% less lipid peroxidation under HT and 55% less buildup of proline, signifying less stress.Discussion: The differential results of fad2 and rod1 are likely due to the role of FAD2 in membranes in all tissues, whereas ROD1 predominantly affects triacylglycerol (TAG) composition in oil-accumulating tissues including pollen. Our results indicate that decreasing PUFAs, through gene editing, can increase heat tolerance in reproductive tissues as an auxiliary benefit accompanying improved seed oil quality.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2020

Photosynthetic Co-production of Succinate and Ethylene in a Fast-Growing Cyanobacterium, Synechococcus elongatus PCC 11801

Cyanobacteria are emerging as hosts for photoautotrophic production of chemicals. Recent studies have attempted to stretch the limits of photosynthetic production, typically focusing on one product at a time, possibly to minimise the additional burden of product separation. Here, we explore the simultaneous production of two products that can be easily separated: ethylene, a gaseous product, and succinate, an organic acid that accumulates in the culture medium. This was achieved by expressing a single copy of the ethylene forming enzyme (efe) under the control of P cpcB , the inducer-free super-strong promoter of phycocyanin β subunit. We chose the recently reported, fast-growing and robust cyanobacterium, Synechococcus elongatus PCC 11801, as the host strain. A stable recombinant strain was constructed using CRISPR-Cpf1 in a first report of markerless genome editing of this cyanobacterium. Under photoautotrophic conditions, the recombinant strain shows specific productivities of 338.26 and 1044.18 μmole/g dry cell weight/h for ethylene and succinate, respectively. These results compare favourably with the reported productivities for individual products in cyanobacteria that are highly engineered. Metabolome profiling and 13 C labelling studies indicate carbon flux redistribution and suggest avenues for further improvement. Our results show that S. elongatus PCC 11801 is a promising candidate for metabolic engineering.

13C isotopic labelling↗
DOE OSTI2022

Is Genetic Engineering a Route to Enhance Microalgae-Mediated Bioremediation of Heavy Metal-Containing Effluents?

Contamination of the biosphere by heavy metals has been rising, due to accelerated anthropogenic activities, and is nowadays, a matter of serious global concern. Removal of such inorganic pollutants from aquatic environments via biological processes has earned great popularity, for its cost-effectiveness and high efficiency, compared to conventional physicochemical methods. Among candidate organisms, microalgae offer several competitive advantages; phycoremediation has even been claimed as the next generation of wastewater treatment technologies. Furthermore, integration of microalgae-mediated wastewater treatment and bioenergy production adds favorably to the economic feasibility of the former process—with energy security coming along with environmental sustainability. However, poor biomass productivity under abiotic stress conditions has hindered the large-scale deployment of microalgae. Recent advances encompassing molecular tools for genome editing, together with the advent of multiomics technologies and computational approaches, have permitted the design of tailor-made microalgal cell factories, which encompass multiple beneficial traits, while circumventing those associated with the bioaccumulation of unfavorable chemicals. Previous studies unfolded several routes through which genetic engineering-mediated improvements appear feasible (encompassing sequestration/uptake capacity and specificity for heavy metals); they can be categorized as metal transportation, chelation, or biotransformation, with regulation of metal- and oxidative stress response, as well as cell surface engineering playing a crucial role therein. This review covers the state-of-the-art metal stress mitigation mechanisms prevalent in microalgae, and discusses putative and tested metabolic engineering approaches, aimed at further improvement of those biological processes. Finally, current research gaps and future prospects arising from use of transgenic microalgae for heavy metal phycoremediation are reviewed.

Ranjbar, Saeed (ORCID:0000000256518605)↗
DOE OSTI2025

Woody Plant Transformation: Current Status, Challenges, and Future Perspectives

Woody plants, comprising forest and fruit tree species, provide essential ecological and economic benefits to society. Their genetic improvement is challenging due to long generation intervals and high heterozygosity. Genetic transformation, which combines targeted DNA delivery with plant regeneration from transformed cells, offers a powerful alternative to accelerating their domestication and improvement. Agrobacterium tumefaciens, Rhizobium rhizogenes, and particle bombardment have been widely used for DNA delivery into a wide variety of explants, including leaves, stems, hypocotyls, roots, and embryos, with regeneration occurring via direct organogenesis, callus-mediated organogenesis, somatic embryogenesis, or hairy root formation. Despite successes, conventional approaches are hampered by low efficiency, genotype dependency, and a reliance on challenging tissue culture. This review provides a critical analysis of the current landscape in woody plant transformation, moving beyond a simple summary of techniques to evaluate the co-evolution of established platforms with disruptive technologies. Key advances among these include the use of developmental regulators to engineer regeneration, the rise in in planta systems to bypass tissue culture, and the imperative for DNA-free genome editing to meet regulatory and public expectations. By examining species-specific breakthroughs in key genera, including Populus, Malus, Citrus, and Pinus, this review highlights a paradigm shift from empirical optimization towards rational, predictable engineering of woody plants for a sustainable future.

Agrobacterium tumefaciens↗
DOE OSTI2020

Biosystems Design to Accelerate C 3 -to-CAM Progression

Global demand for food and bioenergy production has increased rapidly, while the area of arable land has been declining for decades due to damage caused by erosion, pollution, sea level rise, urban development, soil salinization, and water scarcity driven by global climate change. In order to overcome this conflict, there is an urgent need to adapt conventional agriculture to water-limited and hotter conditions with plant crop systems that display higher water-use efficiency (WUE). Crassulacean acid metabolism (CAM) species have substantially higher WUE than species performing C 3 or C 4 photosynthesis. CAM plants are derived from C 3 photosynthesis ancestors. However, it is extremely unlikely that the C 3 or C 4 crop plants would evolve rapidly into CAM photosynthesis without human intervention. Currently, there is growing interest in improving WUE through transferring CAM into C 3 crops. However, engineering a major metabolic plant pathway, like CAM, is challenging and requires a comprehensive deep understanding of the enzymatic reactions and regulatory networks in both C 3 and CAM photosynthesis, as well as overcoming physiometabolic limitations such as diurnal stomatal regulation. Recent advances in CAM evolutionary genomics research, genome editing, and synthetic biology have increased the likelihood of successful acceleration of C 3 -to-CAM progression. Here, we first summarize the systems biology-level understanding of the molecular processes in the CAM pathway. Then, we review the principles of CAM engineering in an evolutionary context. Lastly, we discuss the technical approaches to accelerate the C 3 -to-CAM transition in plants using synthetic biology toolboxes.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2025

Supplementary table, figures and DNA sequences of sorghum gene models SbiRTx430.01G455400 and SbiRTx.02G006600 that feature primers, gRNAs and indels created

In-context promoter bashing via genome editing is a route to identify and characterize critical regulatory regions that govern expression of genes of interest. The outcomes of in-context promoter bashing can be used to inform editing strategies to modulate the expression of selected gene models in a desired fashion. Here we employed in-context promoter bashing to characterize the proximal upstream regulatory regions of sorghum genes encoding phosphoenolpyruvate carboxykinase (Sb.PEPCK.BS, SbiTx430.01G455400) and alanine aminotransferase (SbiTx430.02G006600, SbAlaAT.BS), two proteins involved in the PCK C4 pathway. Characterized germinal edits within the targeted regions upstream of these two genes ranged in size from 138 bp up to 1790 bp. A 138 bp within the Sb.PEPCK.BS upstream region and a 1643 bp element within the Sb.AlaAT.BS upstream region were determined to be important for maintenance of transcription levels. No change in development or various physiological parameters was observed in characterized lineages carrying promoter edits. However, significant changes in seed reserves and a reduction in 100 seed weight were consistently observed, under both greenhouse and field environments, in plants carrying an edit in the promoter of Sb.PEPCK.BS gene were significantly reduced in transcript accumulation for this gene.

Quach, Truyen [Center for Plant Science Innovation↗
DOE OSTI2020

Generation of heritable chimeric plant traits

The present invention provides methods and compositions for targeting enzymes involved in lignin or xylan biosynthesis using genome editing nucleases to specifically reduce content in a desired plant cell type(s).

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

Methods of identifying and modulating pathogen resistance in plants

Pathogenic fungi from the genus Sphaerulina cause damage to a diverse array of economically important plant species. The present disclosure provides methods of determining whether a plant is susceptible to pathogenic fungi infections. The disclosure further provides methods of engineering pathogenic fungi-resistant plants from susceptible plants using targeted genome editing techniques.

Muchero, Wellington↗