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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 343 records · Page 19
All sourcesNASA NTRSDOE OSTI
DOE OSTI2020

Prospects for Engineering Biophysical CO 2 Concentrating Mechanisms into Land Plants to Enhance Yields

Although cyanobacteria and algae represent a small fraction of the biomass of all primary producers, their photosynthetic activity accounts for roughly half of the daily CO 2 fixation that occurs on Earth. These microorganisms are able to accomplish this feat by enhancing the activity of the CO 2 -fixing enzyme Rubisco using biophysical CO 2 concentrating mechanisms (CCMs). Biophysical CCMs operate by concentrating bicarbonate and converting it into CO 2 in a compartment that houses Rubisco (in contrast with other CCMs that concentrate CO 2 via an organic intermediate, such as malate in the case of C 4 CCMs). This activity provides Rubisco with a high concentration of its substrate, thereby increasing its reaction rate. The genetic engineering of a biophysical CCM into land plants is being pursued as a strategy to increase crop yields. This review focuses on the progress toward understanding the molecular components of cyanobacterial and algal CCMs, as well as recent advances toward engineering these components into land plants.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2020

Fatty Acid and Alcohol Metabolism in Pseudomonas putida: Functional Analysis Using Random Barcode Transposon Sequencing.

With its ability to catabolize a wide variety of carbon sources and a growing engineering toolkit, Pseudomonas putida KT2440 is emerging as an important chassis organism for metabolic engineering. Despite advances in our understanding of the organism, many gaps remain in our knowledge of the genetic basis of its metabolic capabilities. The gaps are particularly noticeable in our understanding of both fatty acid and alcohol catabolism, where many paralogs putatively coding for similar enzymes coexist, making biochemical assignment via sequence homology difficult. To rapidly assign function to the enzymes responsible for these metabolisms, we leveraged random barcode transposon sequencing (RB-Tn-Seq). Global fitness analyses of transposon libraries grown on 13 fatty acids and 10 alcohols produced strong phenotypes for hundreds of genes. Fitness data from mutant pools grown on fatty acids of varying chain lengths indicated specific enzyme substrate preferences and enabled us to hypothesize that DUF1302/DUF1329 family proteins potentially function as esterases. From the data, we also postulate catabolic routes for the two biogasoline molecules isoprenol and isopentanol, which are catabolized via leucine metabolism after initial oxidation and activation with coenzyme A (CoA). Because fatty acids and alcohols may serve as both feedstocks and final products of metabolic-engineering efforts, the fitness data presented here will help guide future genomic modifications toward higher titers, rates, and yields. IMPORTANCE To engineer novel metabolic pathways into P. putida , a comprehensive understanding of the genetic basis of its versatile metabolism is essential. Here, we provide functional evidence for the putative roles of hundreds of genes involved in the fatty acid and alcohol metabolism of the bacterium. These data provide a framework facilitating precise genetic changes to prevent product degradation and to channel the flux of specific pathway intermediates as desired.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

Lung and liver editing by lipid nanoparticle delivery of a stable CRISPR–Cas9 ribonucleoprotein

Lipid nanoparticle (LNP) delivery of clustered regularly interspaced short palindromic repeat (CRISPR) ribonucleoproteins (RNPs) could enable high-efficiency, low-toxicity and scalable in vivo genome editing if efficacious RNP–LNP complexes can be reliably produced. Here we engineer a thermostable Cas9 from Geobacillus stearothermophilus (GeoCas9) to generate iGeoCas9 variants capable of >100× more genome editing of cells and organs compared with the native GeoCas9 enzyme. Furthermore, iGeoCas9 RNP–LNP complexes edit a variety of cell types and induce homology-directed repair in cells receiving codelivered single-stranded DNA templates. Using tissue-selective LNP formulations, we observe genome-editing levels of 16-37% in the liver and lungs of reporter mice that receive single intravenous injections of iGeoCas9 RNP–LNPs. In addition, iGeoCas9 RNPs complexed to biodegradable LNPs edit the disease-causing SFTPC gene in lung tissue with 19% average efficiency, representing a major improvement over genome-editing levels observed previously using viral or nonviral delivery strategies. These results show that thermostable Cas9 RNP–LNP complexes can expand the therapeutic potential of genome editing.

59 BASIC BIOLOGICAL SCIENCES↗
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↗
DOE OSTI2023

Engineering Nannochloropsis oceanica for the production of diterpenoid compounds

Abstract Photosynthetic microalgae like Nannochloropsis hold enormous potential as sustainable, light‐driven biofactories for the production of high‐value natural products such as terpenoids. Nannochloropsis oceanica is distinguished as a particularly robust host with extensive genomic and transgenic resources available. Its capacity to grow in wastewater, brackish, and sea waters, coupled with advances in microalgal metabolic engineering, genome editing, and synthetic biology, provides an excellent opportunity. In the present work, we demonstrate how N. oceanica can be engineered to produce the diterpene casbene—an important intermediate in the biosynthesis of pharmacologically relevant macrocyclic diterpenoids. Casbene accumulated after stably expressing and targeting the casbene synthase from Daphne genkwa (DgTPS1) to the algal chloroplast. The engineered strains yielded production titers of up to 0.12 mg g −1 total dry cell weight (DCW) casbene. Heterologous overexpression and chloroplast targeting of two upstream rate‐limiting enzymes in the 2‐C‐methyl‐ d ‐erythritol 4‐phosphate pathway, Coleus forskohlii 1‐deoxy‐ d ‐xylulose‐5‐phosphate synthase and geranylgeranyl diphosphate synthase genes, further enhanced the yield of casbene to a titer up to 1.80 mg g −1 DCW. The results presented here form a basis for further development and production of complex plant diterpenoids in microalgae.

Du, Zhi‐Yan↗
DOE OSTI2022

Transgenic manipulation of triacylglycerol biosynthetic enzymes in B. napus alters lipid-associated gene expression and lipid metabolism

Abstract Oilseed rape ( Brassica napus ) is an important crop that is cultivated for the oil (mainly triacylglycerol; TAG) it produces in its seeds. TAG synthesis is controlled mainly by key enzymes in the Kennedy pathway, such as glycerol 3-phosphate acyltransferase (GPAT), lysophosphatidate acyltransferase (LPAT) and diacylglycerol acyltransferase (DGAT) but can also be produced from phosphoglycerides such as phosphatidylcholine (PC) by the activity of the enzyme phospholipid: diacylglycerol acyltransferase (PDAT). To evaluate the potential for these enzymes to alter oil yields or composition, we analysed transgenic B. napus lines which overexpressed GPAT, LPAT or PDAT using heterologous transgenes from Arabidopsis and Nasturtium and examined lipid profiles and changes in gene expression in these lines compared to WT. Distinct changes in PC and TAG abundance and spatial distribution in embryonic tissues were observed in some of the transgenic lines, together with altered expression of genes involved generally in acyl-lipid metabolism. Overall our results show that up-regulation of these key enzymes differentially affects lipid composition and distribution as well as lipid-associated gene expression, providing important information which could be used to improve crop properties by metabolic engineering.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

Single-molecule tracking reveals dual front door/back door inhibition of Cel7A cellulase by its product cellobiose

Degrading cellulose is a key step in the processing of lignocellulosic biomass into bioethanol. Cellobiose, the disaccharide product of cellulose degradation, has been shown to inhibit cellulase activity, but the mechanisms underlying product inhibition are not clear. We combined single-molecule imaging and biochemical investigations with the goal of revealing the mechanism by which cellobiose inhibits the activity of Trichoderma reesei Cel7A, a well-characterized exo-cellulase. We find that cellobiose slows the processive velocity of Cel7A and shortens the distance moved per encounter; effects that can be explained by cellobiose binding to the product release site of the enzyme. Cellobiose also strongly inhibits the binding of Cel7A to immobilized cellulose, with a K i of 2.1 mM. The isolated catalytic domain (CD) of Cel7A was also inhibited to a similar degree by cellobiose, and binding of an isolated carbohydrate-binding module to cellulose was not inhibited by cellobiose, suggesting that cellobiose acts on the CD alone. Finally, cellopentaose inhibited Cel7A binding at micromolar concentrations without affecting the enzyme’s velocity of movement along cellulose. Together, these results suggest that cellobiose inhibits Cel7A activity both by binding to the “back door” product release site to slow activity and to the “front door” substrate-binding tunnel to inhibit interaction with cellulose. These findings point to strategies for engineering cellulases to reduce product inhibition and enhance cellulose degradation, supporting the growth of a sustainable bioeconomy.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2026

Advancing Protein Display on Bacterial Spores through an Extensive Survey of Coat Components

The profound stability of bacterial spores makes them a promising platform for biotechnological applications like biocatalysis, bioremediation, drug delivery, etc. However, though the Bacillus subtilis spore is composed of >40 types of proteins, only ∼12 have been explored as fusion carriers for protein display. Here, we assessed the suitability of 33 spore proteins (SPs) as enzyme display carriers by direct allele tagging at native genomic loci. Of the 33 SPs investigated, 26 formed functional fusions with β-glucuronidase (GUS)─a ∼272 kDa homotetramer. This almost triples the number of SPs assessed for enzyme display and doubles the number of functional fusions documented in the literature. We quantitatively assessed 1) SP promoter activation dynamics, 2) GUS activity on spores, 3) surface availability, and 4) protection from thermal and proteolytic degradation. Multicopy expression and pairwise coexpression of the most promising SP-GUS fusions highlighted the complexity of spore structure/assembly and the difficulty in predicting compatibility between different SP fusions. We also assessed the suitability of engineered spores to degrade PET (polyethylene terephthalate) films and found that surface-exposed SPs were most effective. Beyond the broad survey, a key outcome of our work was the identification of SscA (small spore coat assembly protein A) as an effective spore display carrier. SscA supported enzyme activity at least 4-fold higher than any other SP, including the well-established anchor, CotY. We attribute this to its promoter, which demonstrated early and sustained activation relative to other SPs and its small size (∼3 kDa), which likely minimally interferes with enzyme folding, oligomerization, and activity. Labeling and genetic studies, its hydrophobic nature, and low surface availability suggest that SscA assembles within the inner spore coat, which makes it stabilizing and suitable for many biocatalytic applications. Overall, this work serves as a knowledge base to advance the biotechnological utility of B. subtilis spores.

Bacillus subtilis↗
DOE OSTI2020

Catalytic bias in oxidation–reduction catalysis

Cataytic bias refers to the propensity of a reaction catalyst to effect a different rate acceleration in one direction versus the other in a chemical reaction under non-equilibrium conditions. In biocatalysis, the inherent bias of an enzyme is often advantagous to augment the innate thermodynamics of a reaction to promote efficiency and fidelity in the coordination of catabolic and anabolic pathways. In industrial chemical catalysis a directional cataltyic bias is a sought after property in facilitating the engineering of systems that couple catalysis with harvest and storage of for example fine chemicals or energy compounds. Interestingly, there is little information about catalytic bias in biocatalysis likely in large part due to difficulties in developing tractible assays sensitive enough to study detailed kinetics. For oxidation–reduction reactions, colorimetric redox indicators exist in a range of reduction potentials to provide a mechanism to study both directions of reactions in a fairly facile manner. The current short review attempts to define catalytic bias conceptually and to develop model systems for defining the parameters that control catalytic bias in enzyme catalyzed oxidation–reduction catalysis.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2023

Microorganism with knock-in at acetolactate decarboxylase gene locus

Provided herein is a genetically engineered microorganism comprising knock-in of DNA at an acetolactate decarboxylase gene locus. Replacement of the acetolactate decarboxylase gene with DNA encoding one or more native or nonnative enzymes confers certain advantages, including fermentation stability and increased production of native and nonnative products from gaseous substrates.

Leang, Ching↗
DOE OSTI2025

Microorganism with knock-in at acetolactate decarboxylase gene locus

Provided herein is a genetically engineered microorganism comprising knock-in of DNA at an acetolactate decarboxylase gene locus. Replacement of the acetolactate decarboxylase gene with DNA encoding one or more native or nonnative enzymes confers certain advantages, including fermentation stability and increased production of native and nonnative products from gaseous substrates.

Leang, Ching↗
DOE OSTI2022

Metabolic engineering strategies to produce medium-chain oleochemicals via acyl-ACP:CoA transacylase activity

Abstract Microbial lipid metabolism is an attractive route for producing oleochemicals. The predominant strategy centers on heterologous thioesterases to synthesize desired chain-length fatty acids. To convert acids to oleochemicals (e.g., fatty alcohols, ketones), the narrowed fatty acid pool needs to be reactivated as coenzyme A thioesters at cost of one ATP per reactivation - an expense that could be saved if the acyl-chain was directly transferred from ACP- to CoA-thioester. Here, we demonstrate such an alternative acyl-transferase strategy by heterologous expression of PhaG, an enzyme first identified in Pseudomonads , that transfers 3-hydroxy acyl-chains between acyl-carrier protein and coenzyme A thioester forms for creating polyhydroxyalkanoate monomers. We use it to create a pool of acyl-CoA’s that can be redirected to oleochemical products. Through bioprospecting, mutagenesis, and metabolic engineering, we develop three strains of Escherichia coli capable of producing over 1 g/L of medium-chain free fatty acids, fatty alcohols, and methyl ketones.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2023

Breast tumor-targeted drug delivery via polymer nanocarriers: Endogenous and exogenous strategies

Breast cancer, one of the most common types of cancer worldwide, has a high degree of malignancy while lacking efficient treatments. The off-target events frequently occurring in drug administration have been a major obstacle, which causes severe side effects to patients and reduces the therapeutic efficacy. An ideal medication should precisely target the specific site with precise control over concentration and duration. In recent years, there has been immense progress in polymer nanocarriers that demonstrate great potential in the targeted delivery. These nanocarriers are constructed from materials sensitive to a wide range of endogenous (e.g., pH, enzyme, redox) and exogenous stimuli (e.g., light, ultrasound, magnetic waves), driving cargo release to the targeted tissue or cells. Here, we elucidate the mechanism of current targeting strategies and the engineering methods to construct these targeting systems or to improve their targeting efficiency. Here we capture the recent progress in the targeted nanocarriers derived from polymers which span hydrogels and micelles, demonstrating their design and applications in chemo, hyperthermia, immuno, and gene therapies.

36 MATERIALS SCIENCE↗
DOE OSTI2024

Complete biosynthesis of QS-21 in engineered yeast

QS-21 is a potent vaccine adjuvant and remains the only saponin-based adjuvant that has been clinically approved for use in humans. However, owing to the complex structure of QS-21, its availability is limited. Today, the supply depends on laborious extraction from the Chilean soapbark tree or on low-yielding total chemical synthesis. Here we demonstrate the complete biosynthesis of QS-21 and its precursors, as well as structural derivatives, in engineered yeast strains. The successful biosynthesis in yeast requires fine-tuning of the host’s native pathway fluxes, as well as the functional and balanced expression of 38 heterologous enzymes. The required biosynthetic pathway spans seven enzyme families—a terpene synthase, P450s, nucleotide sugar synthases, glycosyltransferases, a coenzyme A ligase, acyl transferases and polyketide synthases—from six organisms, and mimics in yeast the subcellular compartmentalization of plants from the endoplasmic reticulum membrane to the cytosol. Finally, by taking advantage of the promiscuity of certain pathway enzymes, we produced structural analogues of QS-21 using this biosynthetic platform. This microbial production scheme will allow for the future establishment of a structure–activity relationship, and will thus enable the rational design of potent vaccine adjuvants.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2026

Transgenic Mixed‐Linkage‐Glucan Enhancement Affects Root Characteristics and Decomposition in Soils of Contrasting Vegetation History

ABSTRACT Development of transgenic bioenergy sorghum [ Sorghum bicolor (L.) Moench] with increased contents of mixed‐linkage (1,3;1,4)‐β‐glucan (MLG) is an important step towards enhancing quality of bioenergy feedstocks. Since MLG‐enhancement leads to greater biomass digestibility, our overarching hypothesis is that root residues of MLG‐enhanced plants may be more readily decomposed in the soil, potentially creating new opportunities for optimizing soil carbon (C) sequestration, nutrient cycling, and overall agricultural sustainability. The study examined morphological, chemical, and enzymatic characteristics of fine and coarse roots of four bioenergy sorghum genotypes. Then, we incubated the roots within soils with contrasting vegetation histories while measuring C mineralization, microbial biomass C (MBC), and activity of hydrolytic enzymes and calculating vector length and vector angle enzymatic stoichiometry parameters. The results indicated that MLG‐enhancing transformations increased root total nitrogen (N) contents, decreased C/N ratios, and were associated with higher MLG concentrations in fine than in coarse roots. Incubations with transgenic roots led to 16%–38% higher MBC and 19%–41% lower microbial metabolic quotient (qCO 2 ). While enzyme activity differed markedly among the studied genotypes, it did not directly respond to MLG levels in root tissues. The increase in MBC without concurrent increases in C mineralization or hydrolytic enzyme activities in transgenic genotypes suggests that MLG enhancement promoted microbial anabolic retention of root‐derived C rather than stimulating catabolic decomposition. Enzymatic vector results indicated that these parameters reflect a variety of drivers behind microbial enzyme production, including availability of specific substrates, such as MLG here, and/or deficiency in specific nutrients, such as phosphorus (P). The study confirms the positive impacts from the roots of engineered MLG‐enhanced bioenergy plants on soil microbial activity and highlights the interactive influences on the MLG‐enhancement effects from root size and inherent soil properties.

Mahmoodabadi, Majid [Department of Plant, Soil, an↗
DOE OSTI2024

Towards using bacterial microcompartments as a platform for spatial metabolic engineering in the industrially important and metabolically versatile Zymomonas mobilis

Advances in synthetic biology have enabled the incorporation of novel biochemical pathways for the production of high-value products into industrially important bacterial hosts. However, attempts to redirect metabolic fluxes towards desired products often lead to the buildup of toxic or undesirable intermediates or, more generally, unwanted metabolic cross-talk. The use of shells derived from self-assembling protein-based prokaryotic organelles, referred to as bacterial microcompartments (BMCs), as a scaffold for metabolic enzymes represents a sophisticated approach that can both insulate and integrate the incorporation of challenging metabolic pathways into industrially important bacterial hosts. Here we took a synthetic biology approach and introduced the model shell system derived from the myxobacterium Haliangium ochraceum (HO shell) into the industrially relevant organism Zymomonas mobilis with the aim of constructing a BMC-based spatial scaffolding platform. SDS-PAGE, transmission electron microscopy, and dynamic light scattering analyses collectively demonstrated the ability to express and purify empty capped and uncapped HO shells from Z. mobilis . As a proof of concept to internally load or externally decorate the shell surface with enzyme cargo, we have successfully targeted fluorophores to the surfaces of the BMC shells. Overall, our results provide the foundation for incorporating enzymes and constructing BMCs with synthetic biochemical pathways for the future production of high-value products in Z. mobilis .

59 BASIC BIOLOGICAL SCIENCES↗