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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 37 records · Page 2
All sourcesNASA NTRSDOE OSTI
NASA NTRS1979

Problems and potentialities of cultured plant cells in retrospect and prospect

The past, present and expected future accomplishments and limitations of plant cell and tissue culture are reviewed. Consideration is given to the pioneering insights of Haberlandt in 1902, the development of culture techniques, and past work on cell division, cell and tissue growth and development, somatic embryogenesis, and metabolism and respiration. Current activity in culture media and technique development for plant regions, organs, tissues, cells, protoplasts, organelles and embryos, totipotency, somatic embryogenesis and clonal propagation under normal and space conditions, biochemical potentialities, and genetic engineering is surveyed. Prospects for the investigation of the induced control of somatic cell division, the division of isolated protoplasts, the improvement of haploid cell cultures, liquid cultures for somatic embryogenesis, and the genetic control of development are outlined.

Steward, F. C.↗
DOE OSTI2025

Multigene engineering in plants: Technologies, applications, and future prospects

The emerging bioeconomy presents a promising solution to both economic and environmental challenges. Within the bioeconomy, plants serve as a renewable, sustainable, and cost-effective source of foods, fuels, chemicals, and materials. However, traditional breeding and single-gene engineering approaches fall short in addressing complex traits (e.g., drought tolerance, disease resistance, yield, nutrient use efficiency) which are controlled by multiple genes. The complexity of plant biology often necessitates the use of multigene engineering (MGE), which involves simultaneous ectopic expression, up/down-regulation, or editing of multiple genes, to enhance plant traits relevant to the bioeconomy. These genes may be associated with distinct traits or function as components of specific metabolic and regulatory pathways. This review summarizes current technologies for MGE within the synthetic biology-driven Design-Build-Test-Learn (DBTL) framework, detailing its four key stages: Design – gene construct development; Build – DNA assembly and plant transformation; Test – the molecular, biochemical, and physiological characterization of engineered plants; and Learn – computational modeling to refine, multiplex and iterate the process. Despite good progress in the applications of MGE in biofortification, metabolic engineering, and stress resilience, challenges remain in construct stability, coordinated gene expression, and regulatory predictability. We identified optimization paths and future directions to accelerate MGE deployment in sustainable agriculture, with possible societal benefits including reduced production costs, increased yield, and improved food and nutritional security.

AI-aided plant engineering↗
DOE OSTI2026

Prediction of non-intuitive metabolic targets with bayesian metabolic control analysis to improve 3-hydroxypropionic acid production in Aspergillus niger

Development of efficient bioconversion processes is limited by the ability to predictably improve metabolic flux. Here we deployed Bayesian Metabolic Control Analysis as a platform to integrate multi-omics data with metabolic modeling and evaluated its ability to predict genetic interventions that improve metabolic flux. Global Metabolomics and proteomics data was collected from 17 Aspergillus niger strains engineered to produce the platform biochemical 3-hydroxypropionic acid from which seven actional genetic interventions were predicted from significant flux control coefficients. Of the suggested genetic interventions, two were present within the intuitively designed strains used for training (malonic semialdehyde dehydrogenase and pyruvate carboxylase) while five predicted targets were present within non-intuitive areas of the metabolic network including 5-formyltetrahydrofolate deformylase and four mitochondrial enzymes, alcohol dehydrogenase, succinyl-CoA ligase, aspartate aminotransferase, and malate dehydrogenase. Six of the targets were validated in the highest performing 3-HP strain used for multi-omics data generation which contained a prior disruption of the highest scoring target malonic semialdehyde dehydrogenase. Predicted directional perturbation of five of the six tested targets significantly improved titer and rate of 3-HP production and two significantly improved yield. The greatest improvements were observed following disruption of the non-intuitive target succinyl-CoA ligase which increased titer by 39% and yield by 29% (to 20.4 g/L 3-HP and 0.31 g 3-HP/g glucose) over the strains used for training. This study demonstrates the utility of Bayesian Metabolic Control Analysis and highlights the ability to predict meaningful genetic targets in unexpected areas of metabolism to improve engineered strains for bioconversion.

3-hydroxypropionic acid↗
DOE OSTI2020

Nano on micro: tuning microbial metabolisms by nano-based artificial mediators to enhance and expand production of biochemicals

Advances in synthetic biology and metabolic engineering across the past few decades have enabled the successful production of many novel chemicals. However, bioproduction of such chemicals is often limited by low yield and titer due to disrupted metabolic homeostasis. Finely tuning cellular metabolism to restore robust metabolic functions entails various genetic modifications, which is often not practical. Alternatively, artificial mediators capable of tailoring microbial metabolisms open a new avenue for restoring physiological functions. In this context, nanoparticle-based artificial mediators have been pursued to tune cellular metabolisms. They can not only enhance production of molecules from endogenous metabolism, but also expand bioproducts spectrum. Here, we reviewed recent advances toward the employment of nano-based artificial mediators for the tuning of cellular metabolism, with a focus on their positive effects on electron transfer and pathway flux. Perspectives for potential applications of artificial mediators for mediating microbial metabolisms in the future were also provided.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2020

Recent developments in pretreatment technologies on lignocellulosic biomass: Effect of key parameters, technological improvements, and challenges

Lignocellulosic biomass is an inexpensive renewable source that can be used to produce biofuels and bioproducts. The recalcitrance nature of biomass hampers polysaccharide accessibility for enzymes and microbes. Several pretreatment methods have been developed for the conversion of lignocellulosic biomass into value-added products. Yet, these pretreatment methods also produce a wide range of secondary compounds, which are inhibitory to enzymes and microorganisms. The selection of an effective and efficient pretreatment method discussed in the review and its process optimization can significantly reduce the production of inhibitory compounds and may lead to enhanced production of fermentable sugars and biochemicals. Furthermore, evolutionary and genetic engineering approaches are being used for the improvement of microbial tolerance towards inhibitors. Advancements in pretreatment and detoxification technologies may help to increase the productivity of lignocellulose-based biorefinery. Here, we discuss the recent advancements in lignocellulosic biomass pretreatment technologies and strategies for the removal of inhibitors.

09 BIOMASS FUELS↗
NASA NTRS2017

Improving Satellite Compatible Microdevices to Study Biology in Space

The technology for biology in space lags far behind the gold standard for biological experiments on Earth. To remedy this disparity, the Rothschild lab works on proof of concept, prototyping, and developing of new sensors and devices to further the capabilities of biology research on satellites. One such device is the PowerCell Payload System. One goal for synthetic biology in aiding space travel and colonization is to genetically engineer living cells to produce biochemicals in space. However, such farming in space presupposes bacteria retain their functionality post-launch, bombarded by radiation, and without the 1G of Earth. Our questions is, does a co-culture of cyanobacteria and protein-synthesizing bacteria produce Earth-like yields of target proteins? Is the yield sensitive to variable gravitational forces? To answer these questions, a PowerCell Payload System will spend 1 year aboard the German Aerospace Center's Euglena and Combined Regenerative Organic-food Production In Space (Eu:CROPIS) mission satellite. The PowerCell system is a pair of two 48-well microfluidic cards, each well seeded with bacteria. The system integrates fluidic, thermal, optical, electronic, and control systems to germinate bacteria spores, then measure the protein synthesized for comparison to parallel experiments conducted on the Earth. In developing the PowerCell Payload, we gained insight into the shortcomings of biology experiments on satellites. To address these issues, we have started three new prototyping projects: 1) The development of an extremely stable and radiation resistant cell-free system, allowing for the construction of proteins utilizing only cell components instead of living cells. This can be lyophilized on a substrate, like paper. (2) Using paper as a microfluidic platform that is flexible, stable, cheap, and wicking. The capillary action eliminates the need for pumps, reducing volume, mass, and potential failing points. Electrodes can be printed on the paper to sense for biochemicals. (3) Developing a modular, semi-autonomous microfluidic device that can be easily adapted for a variety of common biological experiments. This versatility will allow for quicker and cheaper experimentation. These improvements to satellite experiment platforms have the potential to radically increase the return from NASA's biological and field studies with reduced development time, mass, and cost with increased robustness data and interpretation.

biology in spac↗
DOE OSTI2020

System-Level Optimization to Improve Biofuel Potential via Genetic Engineering and Hydrothermal Liquefaction

The economic success of biofuels and bioproducts depends on system-level optimization including biomass production and conversion. Hydrothermal liquefaction (HTL) can convert wet biomass such as microalgae into a biofuel intermediate (BFI) under elevated temperatures and pressure. An understanding of the impacts of biomass composition on BFI yield and quality can inform genetic engineering strategies in the improvement of biochemical composition for biofuel production. In this work, wild type cyanobacterium Synechocystis sp. PCC 6803 biomass was doped with various common cellular storage compounds in lab-scale HTL experiments. Doping with glycogen or polyhydroxybutyrate (PHB) significantly reduced BFI yields, while doping with triglycerides (TAG) or medium chain-length polyhydroxyalkanoate (mcl-PHA) increased BFI yield and quality. In light of these observations, a genetically engineered Synechocystis strain deficient in glycogen biosynthesis was cultivated to produce biomass for HTL, leading to a 17% increase in BFI yield. In addition, we built a multiphase component additivity (MCA) model that can predict BFI yield and quality with PHAs in the biomass. This work demonstrates an effective strategy to integrate strain development with downstream biomass conversion to maximize biofuel yield, with lessons applicable to microalgae as well as other biomass.

09 BIOMASS FUELS↗
DOE OSTI2020

Heterologous Expression and Engineering of the Nitrogenase Cofactor Biosynthesis Scaffold NifEN

Abstract NifEN plays a crucial role in the biosynthesis of nitrogenase, catalyzing the final step of cofactor maturation prior to delivering the cofactor to NifDK, the catalytic component of nitrogenase. The difficulty in expressing NifEN, a complex, heteromultimeric metalloprotein sharing structural/functional homology with NifDK, is a major challenge in the heterologous expression of nitrogenase. Herein, we report the expression and engineering of Azotobacter vinelandii NifEN in Escherichia coli . Biochemical and spectroscopic analyses demonstrate the integrity of the heterologously expressed NifEN in composition and functionality and, additionally, the ability of an engineered NifEN variant to mimic NifDK in retaining the matured cofactor at an analogous cofactor‐binding site. This is an important step toward piecing together a viable pathway for the heterologous expression of nitrogenase and identifying variants for the mechanistic investigation of this enzyme.

Solomon, Joseph B.↗
DOE OSTI2020

Heterologous Expression and Engineering of the Nitrogenase Cofactor Biosynthesis Scaffold NifEN

NifEN plays a crucial role in the biosynthesis of nitrogenase, catalyzing the final step of cofactor maturation prior to delivering the cofactor to NifDK, the catalytic component of nitrogenase. The difficulty in expressing NifEN, a complex, heteromultimeric metalloprotein sharing structural/functional homology with NifDK, is a major challenge in the heterologous expression of nitrogenase. Herein, we report the expression and engineering of Azotobacter vinelandii NifEN in Escherichia coli. Biochemical and spectroscopic analyses demonstrate the integrity of the heterologously expressed NifEN in composition and functionality and, additionally, the ability of an engineered NifEN variant to mimic NifDK in retaining the matured cofactor at an analogous cofactor-binding site. This is an important step toward piecing together a viable pathway for the heterologous expression of nitrogenase and identifying variants for the mechanistic investigation of this enzyme.

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

Adapting isotopic tracer and metabolic flux analysis approaches to study C1 metabolism

Single-carbon (C1, or one-carbon) substrates are promising feedstocks for sustainable biofuel and biochemical production. Crucial to the goal of engineering C1-utilizing strains for improved production is a quantitative understanding of the organization, regulation and rates of the reactions that underpin C1 metabolism. 13 C Metabolic flux analysis (MFA) is a well-established platform for interrogating these questions with multi-carbon substrates, and uses the differential labeling of metabolites that results from feeding a substrate with position-specific incorporation of 13 C in order to infer quantitative fluxes and pathway topology. Furthermore, adapting isotopic tracer approaches to C1 metabolism, where position-specific substrate labeling is impossible, requires additional experimental considerations. Here we review recent studies that have developed isotopic tracer approaches to overcome the challenge of uniform metabolite labeling and provide quantitative insight into C1 metabolism.

59 BASIC BIOLOGICAL SCIENCES↗
NASA NTRS2003

Use of fibroblast growth factor 2 for expansion of chondrocytes and tissue engineering

The present invention provides an improved method for expanding cells for use in tissue engineering. In particular the method provides specific biochemical factors to supplement cell culture medium during the expansion process in order to reproduce events occurring during embryonic development with the goal of regenerating tissue equivalents that resemble natural tissues both structurally and functionally. These specific biochemical factors improve proliferation of the cells and are capable of de-differentiation mature cells isolated from tissue so that the differentiation potential of the cells is preserved. The bioactive molecules also maintain the responsiveness of the cells to other bioactive molecules. Specifically, the invention provides methods for expanding chondrocytes in the presence of fibroblast growth factor 2 for use in regeneration of cartilage tissue.

Martin, Ivan↗
DOE OSTI2022

Genome‐wide CRISPR‐Cas9 screen reveals a persistent null‐hyphal phenotype that maintains high carotenoid production in Yarrowia lipolytica

Abstract Yarrowia lipolytica is a metabolic engineering host of growing industrial interest due to its ability to metabolize hydrocarbons, fatty acids, glycerol, and other renewable carbon sources. This dimorphic yeast undergoes a stress‐induced transition to a multicellular hyphal state, which can negatively impact biosynthetic activity, reduce oxygen and nutrient mass transfer in cell cultures, and increase culture viscosity. Identifying mutations that prevent the formation of hyphae would help alleviate the bioprocess challenges that they create. To this end, we conducted a genome‐wide CRISPR screen to identify genetic knockouts that prevent the transition to hyphal morphology. The screen identified five mutants with a null‐hyphal phenotype— ΔRAS2 , ΔRHO5 , ΔSFL1 , ΔSNF2 , and ΔPAXIP1 . Of these hits, only ΔRAS2 suppressed hyphal formation in an engineered lycopene production strain over a multiday culture. The RAS2 knockout was also the only genetic disruption characterized that did not affect lycopene production, producing more than 5 mg L −1 OD −1 from a heterologous pathway with enhanced carbon flux through the mevalonate pathway. These data suggest that a ΔRAS2 mutant of Y. lipolytica could prove useful in engineering a metabolic engineering host of the production of carotenoids and other biochemicals.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2020

Metabolic engineering of Zymomonas mobilis for anaerobic isobutanol production

Biofuels and value-added biochemicals derived from renewable biomass via biochemical conversion have attracted considerable attention to meet global sustainable energy and environmental goals. Isobutanol is a four-carbon alcohol with many advantages that make it attractive as a fossil-fuel alternative. Zymomonas mobilis is a highly efficient, anaerobic, ethanologenic bacterium making it a promising industrial platform for use in a biorefinery. In this study, the effect of isobutanol on Z. mobilis was investigated, and various isobutanol-producing recombinant strains were constructed. The results showed that the Z. mobilis parental strain was able to grow in the presence of isobutanol below 12 g/L while concentrations greater than 16 g/L inhibited cell growth. Integration of the heterologous gene encoding 2-ketoisovalerate decarboxylase such as kdcA from Lactococcus lactis is required for isobutanol production in Z. mobilis. Moreover, isobutanol production increased from nearly zero to 100-150 mg/L in recombinant strains containing the kdcA gene driven by the tetracycline-inducible promoter Ptet. In addition, we determined that overexpression of a heterologous als gene and two native genes (ilvC and ilvD) involved in valine metabolism in a recombinant Z. mobilis strain expressing kdcA can divert pyruvate from ethanol production to isobutanol biosynthesis. This engineering improved isobutanol production to above 1 g/L. Finally, recombinant strains containing both a synthetic operon, als-ilvC-ilvD, driven by Ptet and the kdcA gene driven by the constitutive strong promoter, Pgap, were determined to greatly enhance isobutanol production with a maximum titer about 4.0 g/L. Finally, isobutanol production was negatively affected by aeration with more isobutanol being produced in more poorly aerated flasks. This study demonstrated that overexpression of kdcA in combination with a synthetic heterologous operon, als-ilvC-ilvD, is crucial for diverting pyruvate from ethanol production for enhanced isobutanol biosynthesis. Moreover, this study also provides a strategy for harnessing the valine metabolic pathway for future production of other pyruvate-derived biochemicals in Z. mobilis.

09 BIOMASS FUELS↗
DOE OSTI2022

evSeq: Cost-Effective Amplicon Sequencing of Every Variant in a Protein Library

Widespread availability of protein sequence-fitness data would revolutionize both our biochemical understanding of proteins and our ability to engineer them. Unfortunately, even though thousands of protein variants are generated and evaluated for fitness during a typical protein engineering campaign, most are never sequenced, leaving a wealth of potential sequence-fitness information untapped. Primarily, this is because sequencing is unnecessary for many protein engineering strategies; the added cost and effort of sequencing is thus unjustified. It also results from the fact that, even though many lower cost sequencing strategies have been developed, they often require at least some sequencing or computational resources, both of which can be barriers to access. In this work, we present every variant sequencing (evSeq), a method and collection of tools/standardized components for sequencing a variable region within every variant gene produced during a protein engineering campaign at a cost of cents per variant. evSeq was designed to democratize low-cost sequencing for protein engineers and, indeed, anyone interested in engineering biological systems. Execution of its wet-lab component is simple, requires no sequencing experience to perform, relies only on resources and services typically available to biology labs, and slots neatly into existing protein engineering workflows. Analysis of evSeq data is likewise made simple by its accompanying software (found at github.com/fhalab/evSeq, documentation at fhalab.github.io/evSeq), which can be run on a personal laptop and was designed to be accessible to users with no computational experience. Here, low-cost and easy to use, evSeq makes collection of extensive protein variant sequence-fitness data practical.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

A new approach to zip–lignin: 3,4–dihydroxybenzoate is compatible with lignification

Renewed interests in the development of bioenergy, biochemicals, and biomaterials have elicited new strategies for engineering the lignin of biomass feedstock plants. Furthermore, this study shows, for the first time, that 3,4-dihydroxybenzoate (DHB) is compatible with the radical coupling reactions that assemble polymeric lignin in plants. We introduced a bacterial 3-dehydroshikimate dehydratase into hybrid poplar (Populus alba × grandidentata) to divert carbon flux away from the shikimate pathway, which lies upstream of lignin biosynthesis. Transgenic poplar wood had up to 33% less lignin with p-hydroxyphenyl units comprising as much as 10% of the lignin. Mild alkaline hydrolysis of transgenic wood released fewer ester-linked p-hydroxybenzoate groups than control trees, and revealed the novel incorporation of cell-wall-bound DHB, as well as glycosides of 3,4-dihydroxybenzoic acid (DHBA). Two-dimensional nuclear magnetic resonance (2D-NMR) analysis uncovered DHBA-derived benzodioxane structures suggesting that DHB moieties were integrated into the lignin polymer backbone. In addition, up to 40% more glucose was released from transgenic wood following ionic liquid pretreatment and enzymatic hydrolysis. This work highlights the potential of diverting carbon flux from the shikimate pathway for lignin engineering and describes a new type of ‘zip-lignin’ derived from the incorporation of DHB into poplar lignin.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2025

State of the Art in Thermal Catalytic Upgrading of Biomass and Biomass-Derived Intermediates

Biomass-derived energy sources represent a promising domestic route for fuel and chemical production, taking advantage of largely underutilized biological and waste resources. Heterogeneous catalysis plays a key role in these biomass conversion processes, as reflected by all American Society for Testing and Materials–approved pathways for producing sustainable aviation fuel proceeding through a catalytic step. This concise review seeks to establish the state of the art in thermal catalytic process development for various biomass-derived feedstocks and the current enabling capabilities that aid this development. Research needs are identified and described throughout the article, as further advancements in heterogeneous catalysis are required to improve the affordability and realize the full potential of biomass-derived products.

09 BIOMASS FUELS↗
DOE OSTI2025

Genetically pliable green algae for bioproduction of modified fatty acids, nutritional therapeutic oils, and biopharmaceuticals

Homologous recombination (HR) is an essential tool for complex metabolic engineering in yeast, but transgene integration into plant and green algal nuclear genomes predominantly occurs by non-homologous end-joining. Species of the closely related, oleaginous trebouxiophytes Auxenochlorella and Prototheca, are unusual among the green algae in that HR is the favored mechanism for DNA integration into the nuclear genome. This property enables locus-specific targeting of gene cassettes encoding multiple enzymes for manipulating existing biochemical pathways or introducing new functions. Genetic malleability, and regulatory approval for human consumption, coupled with robust fermentation performance at industrial scale, establishes Auxenochlorella and Prototheca as prime candidates for algal production of biochemicals and biomaterials. The examples presented here highlight strain improvement and engineering for synthesis of hydroxylated fatty acids for biomaterials, structured triglycerides resembling human milk fat for infant nutrition, very-long-chain mono- and polyunsaturated fatty acids with nutraceutical or therapeutic potential, and cannabinoids for pharmacological applications.

Moseley, Jeffrey L. [University of California, Ber↗