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At least 55 records · Page 3
All sourcesNASA NTRSDOE OSTI
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 OSTI2020

In situ investigation of the deformation behaviors of Fe 20 Co 30 Cr 25 Ni 25 and Fe 20 Co 30 Cr 30 Ni 20 high entropy alloys by high-energy X-ray diffraction

In situ synchrotron-based high-energy X-ray diffraction (HE-XRD) technique was employed to investigate the mechanical behaviors and microstructural evolution of face-centered cubic (FCC) Fe 20 Co 30 Cr 25 Ni 25 and Fe 20 Co 30 Cr 30 Ni 20 high entropy alloys (HEAs) during tensile deformation. Fe 20 Co 30 Cr 30 Ni 20 HEA has a good combination of strength (ultimate tensile strength of 864 ± 35 MPa) and ductility (elongation of 0.627 ± 0.021). The HE-XRD investigation reveals that Fe 20 Co 30 Cr 30 Ni 20 HEA has the transformation-induced plasticity (TRIP) effect, which starts at a critical stress of ~555 MPa. Furthermore, transmission electron microscopy confirmed this deformation-induced new phase is hexagonal-close-packed structured ε-martensite, which follows an orientation relationship of {111} γ //(0001) ε , and (110) γ //[11 2 ¯ 0] ε with the FCC γ matrix. The observation of deformation twins in the deformed samples of the two studied HEAs proves that twinning-induced plasticity (TWIP) effect occurs in both HEAs. The combination of TRIP and TWIP effects lead to the high strength, large ductility and improved strain hardening behavior of Fe 20 Co 30 Cr 30 Ni 20 HEA.

36 MATERIALS SCIENCE↗
DOE OSTI2023

Construction of a Compact Array of Microplasma Jet Devices and Its Application for Random Mutagenesis of Rhodosporidium toruloides

A small and efficient DNA mutation-inducing machine was constructed with an array of microplasma jet devices (7 × 1) that can be operated at atmospheric pressure for microbial mutagenesis. Using this machine, we report disruption of a plasmid DNA and generation of mutants of an oleaginous yeast Rhodosporidium toruloides. Specifically, a compact-sized microplasma channel (25 × 20 × 2 mm 3 ) capable of generating an electron density of greater than 10 13 cm –3 was constructed to produce reactive species (N 2 *, N 2 +, O, OH, and H α ) under helium atmospheric conditions to induce DNA mutagenesis. The length of microplasma channels in the device played a critical role in augmenting both the volume of plasma and the concentration of reactive species. First, we confirmed that microplasma treatment can linearize a plasmid by creating nicks in vitro. Second, we treated R. toruloides cells with a jet device containing 7 microchannels for 5 min; 94.8% of the treated cells were killed, and 0.44% of surviving cells showed different colony colors as compared to their parental colony. Microplasma-based DNA mutation is energy-efficient and can be a safe alternative for inducing mutations compared to conventional methods using toxic mutagens. In conclusion, this compact and scalable device is amenable for industrial strain improvement involving large-scale mutagenesis.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2025

Addressing genome scale design tradeoffs in Pseudomonas putida for bioconversion of an aromatic carbon source

Genome-scale metabolic models (GSMM) are commonly used to identify gene deletion sets that result in growth coupling and pairing product formation with substrate utilization and can improve strain performance beyond levels typically accessible using traditional strain engineering approaches. However, sustainable feedstocks pose a challenge due to incomplete high-resolution metabolic data for non-canonical carbon sources required to curate GSMM and identify implementable designs. Here we address a four-gene deletion design in the Pseudomonas putida KT2440 strain for the lignin-derived non-sugar carbon source, p-coumarate (p-CA), that proved challenging to implement. We examine the performance of the fully implemented design for p-coumarate to glutamine, a useful biomanufacturing intermediate. In this study glutamine is then converted to indigoidine, an alternative sustainable pigment and a model heterologous product that is commonly used to colorimetrically quantify glutamine concentration. Through proteomics, promoter-variation, and growth characterization of a fully implemented gene deletion design, we provide evidence that aromatic catabolism in the completed design is rate-limited by fumarase hydratase (FUM) enzyme activity in the citrate cycle and requires careful optimization of another fumarate hydratase protein (PP_0897) expression to achieve growth and production. A double sensitivity analysis also confirmed a strict requirement for fumarate hydratase activity in the strain where all genes in the growth coupling design have been implemented. Metabolic cross-feeding experiments were used to examine the impact of complete removal of the fumarase hydratase reaction and revealed an unanticipated nutrient requirement, suggesting additional functions for this enzyme. While a complete implementation of the design was achieved, this study highlights the challenge of completely inactivating metabolic reactions encoded by under-characterized proteins, especially in the context of multi-gene edits.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

Picochlorum celeri as a model system for robust outdoor algal growth in seawater

Abstract With fast growth rates, broad halotolerance and the ability to thrive at high temperatures, algae in the genus Picochlorum are emerging as promising biomass producers. Recently, we isolated a remarkably productive strain, Picochlorum celeri , that attains > 40 g m −2 day −1 productivities using simulated outdoor light. To test outdoor productivities, Picochlorum celeri was cultivated in 820 L raceway ponds at the Arizona Center for Algae Technology and Innovation. Picochlorum celeri demonstrated the highest outdoor biomass productivities reported to date at this testbed averaging ~ 31 g m −2 day −1 over four months with a monthly (August) high of ~ 36 g m −2 day −1 . Several single day productivities were > 40 g m −2 day −1 . Importantly for sustainability, Picochlorum celeri achieved these productivities in saline water ranging from seawater to 50 parts per thousand sea salts, without any biocides or pond crashes, for over 143 days. Lastly, we report robust genetic engineering tools for future strain improvements.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

Development of dual‐inducible duet‐expression vectors for tunable gene expression control and CRISPR interference‐based gene repression in Pseudomonas putida KT2440

Summary The development of P. putida as an industrial host requires a sophisticated molecular toolbox for strain improvement, including vectors for gene expression and repression. To augment existing expression plasmids for metabolic engineering, we developed a series of dual‐inducible duet‐expression vectors for P. putida KT2440. A number of inducible promoters (P lac , P tac , P tetR/tetA and P bad ) were used in different combinations to differentially regulate the expression of individual genes. Protein expression was evaluated by measuring the fluorescence of reporter proteins (GFP and RFP). Our experiments demonstrated the use of compatible plasmids, a useful approach to coexpress multiple genes in P. putida KT2440. These duet vectors were modified to generate a fully inducible CRISPR interference system using two catalytically inactive Cas9 variants from S. pasteurianus (dCas9) and S. pyogenes (spdCas9). The utility of developed CRISPRi system(s) was demonstrated by repressing the expression of nine conditionally essential genes, resulting in growth impairment and prolonged lag phase for P. putida KT2440 growth on glucose. Furthermore, the system was shown to be tightly regulated, tunable and to provide a simple way to identify essential genes with an observable phenotype.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2020

Quantitative Proteome Profiling Reveals Cellobiose-Dependent Protein Processing and Export Pathways for the Lignocellulolytic Response in Neurospora crassa

Filamentous fungi are intensively used for producing industrial enzymes, including lignocellulases. Employing insoluble cellulose to induce the production of lignocellulases causes some drawbacks, e.g., a complex fermentation operation, which can be overcome by using soluble inducers such as cellobiose. Here, a triple β-glucosidase mutant of Neurospora crassa, which prevents rapid turnover of cellobiose and thus allows the disaccharide to induce lignocellulases, was applied to profile the proteome responses to cellobiose and cellulose (Avicel). Our results revealed a shared proteomic response to cellobiose and Avicel, whose elements included lignocellulases and cellulolytic product transporters. While the cellulolytic proteins showed a correlated increase in protein and mRNA levels, only a moderate correlation was observed on a proteomic scale between protein and mRNA levels (R 2 = 0.31). Ribosome biogenesis and rRNA processing were significantly overrepresented in the protein set with increased protein but unchanged mRNA abundances in response to Avicel. Ribosome biogenesis, as well as protein processing and protein export, was also enriched in the protein set that showed increased abundance in response to cellobiose. NCU05895, a homolog of yeast CWH43, is potentially involved in transferring a glycosylphosphatidylinositol (GPI) anchor to nascent proteins. This protein showed increased abundance but no significant change in mRNA levels. Disruption of CWH43 resulted in a significant decrease in cellulase activities and secreted protein levels in cultures grown on Avicel, suggesting a positive regulatory role for CWH43 in cellulase production. The findings should have an impact on a systems engineering approach for strain improvement for the production of lignocellulases.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

Data for Construction of a Compact Array of Microplasma Jet Devices and Its Application for Random Mutagenesis of Rhodosporidium toruloides

A small and efficient DNA mutation-inducing machine was constructed with an array of microplasma jet devices (7 × 1) that can be operated at atmospheric pressure for microbial mutagenesis. Using this machine, we report disruption of a plasmid DNA and generation of mutants of an oleaginous yeast Rhodosporidium toruloides . Specifically, a compact-sized microplasma channel (25 × 20 × 2 mm3) capable of generating an electron density of greater than 1013 cm–3 was constructed to produce reactive species (N2*, N2+, O, OH, and Hα) under helium atmospheric conditions to induce DNA mutagenesis. The length of microplasma channels in the device played a critical role in augmenting both the volume of plasma and the concentration of reactive species. First, we confirmed that microplasma treatment can linearize a plasmid by creating nicks in vitro. Second, we treated R. toruloides cells with a jet device containing 7 microchannels for 5 min; 94.8% of the treated cells were killed, and 0.44% of surviving cells showed different colony colors as compared to their parental colony. Microplasma-based DNA mutation is energy-efficient and can be a safe alternative for inducing mutations compared to conventional methods using toxic mutagens. This compact and scalable device is amenable for industrial strain improvement involving large-scale mutagenesis.

Conversion↗
DOE OSTI2023

Multi-parameter distributed fiber optic sensing using double-Brillouin peak fiber in Brillouin optical time domain analysis

In this paper, we demonstrate a multi-parameter fiber sensing system based on stimulated Brillouin scattering in a double-Brillouin peak specialty fiber with enhanced Brillouin gain response. The amplitude level of the second Brillouin gain peak, which originated from the higher-order acoustic modes, has been improved with an approximately similar amplitude level to the first Brillouin gain peak from the fundamental acoustic mode. Compared to other multi-Brillouin peak fibers presented in the literature, the proposed fiber significantly reduces the measured Brillouin frequency shift error, thus improving strain and temperature accuracies. By utilizing the sensitivity values of the strain and temperature associated with each Brillouin gain spectrum (BGS) peak, a successful discriminative measurement of strain and temperature is performed with an accuracy of ±13 μɛ, and ±0.5 °C, respectively. The proposed double-Brillouin peak fiber appears to be a possible alternative to other multi-BGS peak fibers, for instance, large effective area fiber and dispersion compensating fibers, which are inherently accompanied by large measurement errors due to the weak Brillouin gain values originating from the higher-order acoustic modes. The demonstrated results show different strain and temperature coefficients of 47 kHz/µɛ, 1.15 MHz/°C for peak 1 and 51 kHz/µɛ, 1.37 MHz/°C for peak 2. Moreover, the enhanced BGS peak gains having nearly the same amplitude levels enable the discriminative measurement of strain and temperature. Such fibers in Brillouin interrogation eliminate the need for complex monitoring setups and reduce measurement errors. We recommend that for long-distance natural gas pipeline monitoring, where discriminative strain and temperature measurement is crucial, the proposed double-Brillouin peak fiber can be highly beneficial.

47 OTHER INSTRUMENTATION↗
NASA NTRS2014

Application of Synthetic Biology to Bioregenerative Life Support for Human Spaceflight

The conversion of carbon dioxide into higher value products is a key challenge for the development of closed-loop life support systems for human space flight. Much of the past research on bioregenerative life support systems has focused on plant growth chambers as a solution for CO2 removal and O2 generation, but photosynthetic microorganisms may also have a role to play in these functions. Cyanobacteria have the advantages of relatively high CO2 fixation rates and fairly well-developed molecular biology tools, allowing for genetic engineering approaches to strain improvement. Manned missions to Mars or other targets beyond low Earth orbit will require advances in the nutritional systems for life support on these longer duration missions. A key challenge will likely be supplementing pre-packaged meals with specific nutrients that will be deficient due to problems in long-term storage or low abundance. Vitamin K is one such nutrient that may be important as a supplement. Production of vitamin K for nutrient supplementation during spaceflight will likely require genetic engineering of microorganisms to increase vitamin titers. A microbial bioreactor system that could efficiently convert CO2 to nutritional supplements would be a valuable component for a future advanced life support system. We are exploring biological systems to determine the feasibility of using bioreactors to convert CO2 to higher-value products. We are examining the performance of photosynthetic bacteria engineered to produce sugars, determining rates of production and reliability. We are also engineering microbes to produce higher titers of vitamin K and other potentially important nutrients. The results of this research will offer demonstrations of potential technologies that could be developed further in the future. This work will also provide valuable information for understanding basic science questions about the use of genetically engineered microbes in the microgravity environment.

Bioregenerative life support↗
DOE OSTI2022

Additive manufacturing of ferritic/martensitic steel with improved high temperature strength

Techniques for using additive manufacturing (AM) to fabricate creep resistant ferritic/martensitic steel with improved high temperature strength are described. AM processing may be performed on Grade 91 steel powder. Beam powers from about 221 W to about 270 W may be used. Traverse rates from about 675 mm/s to about 825 mm/s may be used. Heat inputs ranging from about 55.7 J/mm3 to about 83.2 J/mm 3 may be produced. Creep resistant ferritic/martensitic steel, produced according to the present disclosure, has improved strain yield strength and ductility as compared to wrought steel.

Lienert, Thomas J.↗
DOE OSTI2021

A new procedure for implementing the modified inherent strain method with improved accuracy in predicting both residual stress and deformation for laser powder bed fusion

As a metal additive manufacturing (AM) process, laser powder bed fusion (L-PBF) has been widely used to produce parts with complex geometries. The large thermal gradient caused by the fast, intense, and repeated laser scanning induces significant residual deformation and stress to the as-built parts, which increase manufacturing difficulty and geometrical inaccuracy as a result. The modified inherent strain (MIS) method exploiting multiscale process simulations was developed to simulate residual deformation accurately and efficiently. However, the existing procedure of implementing the MIS method is found to give inaccurate residual stress prediction. Here in this work, a new implementation procedure for the MIS method is proposed to improve the simulation accuracy of residual stress without degrading the residual deformation prediction. The new procedure concerns the application of inherent strains to the part-scale layer-by-layer finite element model to obtain residual stress and deformation field. While the existing implementation of the part-scale MIS model involves only mechanical properties at ambient temperature, the new procedure adds one more solution step employing mechanical properties at an elevated temperature determined from the inherent strain extraction step. Both numerical and experimental studies are conducted to validate the proposed new implementation procedure. It shows that by using the new procedure, the MIS-based simulation can predict both residual stress and deformation of as-built L-PBF metal parts with good accuracy.

36 MATERIALS SCIENCE↗
NASA NTRS1993

Investigation of high-speed free shear flows using improved pressure-strain correlated Reynolds stress turbulence model

A high-speed shear layer is studied using compressibility corrected Reynolds stress turbulence model which employs newly developed model for pressure-strain correlation. MacCormack explicit prediction-corrector method is used for solving the governing equations and the turbulence transport equations. The stiffness arising due to source terms in the turbulence equations is handled by a semi-implicit numerical technique. Results obtained using the new model show a sharper reduction in growth rate with increasing convective Mach number. Some improvements were also noted in the prediction of the normalized streamwise stress and Reynolds shear stress. The computed results are in good agreement with the experimental data.

Tiwari, S. N.↗
NASA NTRS2003

Flame Spray Strain Gages with Improved Durability and Lifetimes

The focus of this APP research program was to improve the bond coats used in the fabrication of flame sprayed instrumentation. Typically. a bond coat is applied to a superalloy surface prior to the application of a thin dielectric coating onto which instrumentation is placed. After affixing the instrumentation, a much thicker ceramic topcoat is typically applied to protect the instrumentation from harsh environments. The fatigue life of NiCoCrAlY coated superalloys was extended beyond current state-of-the-art by relatively simple and cost effective means. Heat treatment in reduced oxygen partial pressures at 1750 to 1800 F effectively doubled the fatigue life of NiCoCrAlY coated substrates relative to as-sprayed substrates and when used in conjunction with platinum diffusion barriers yielded a four fold increase in the fatigue life of NiCoCrAlY coated substrates. Further improvements in the fatigue life of thermally sprayed coatings were made by employing intermediate coatings, which minimized thermal expansion differences between the bond coat and top coat. Combinatorial chemistry experiments yielded an optimum composition for an intermediate TCE matching coating that showed considerable promise in extending the fatigue life of thermal spray instrumentation. The intermediate coating had two functions: to reduce the surface roughness of the peaks and valleys associated with the as-sprayed NiCoCrAlY bond coat, and to produce a thin layer of a mixture of Al2O3 and NiCoCrAlY that exhibited an intermediate TCE. The optimal composition of the intermediate coating consisted of 60 wt% Al2O3 and 40 wt% NiCoCrAlY, as determined by energy dispersive analysis of x-rays (EDS). Intermediate coatings having this composition were prepared by physical vapor deposition and the resulting coating systems are being evaluated in our test facility.

Fralick, Gustave↗
DOE OSTI2021

Redundancy in Aromatic O -Demethylation and Ring-Opening Reactions in Novosphingobium aromaticivorans and Their Impact in the Metabolism of Plant-Derived Phenolics

ABSTRACT Lignin is a plant heteropolymer composed of phenolic subunits. Because of its heterogeneity and recalcitrance, the development of efficient methods for its valorization still remains an open challenge. One approach to utilize lignin is its chemical deconstruction into mixtures of monomeric phenolic compounds, followed by biological funneling into a single product. Novosphingobium aromaticivorans DSM 12444 has been previously engineered to produce 2-pyrone-4,6-dicarboxylic acid (PDC) from depolymerized lignin by simultaneously metabolizing multiple aromatics through convergent routes involving the intermediates 3-methoxygallic acid (3-MGA) and protocatechuic acid (PCA). We investigated enzymes predicted to be responsible for O -demethylation and oxidative aromatic ring opening, two critical reactions involved in the metabolism of phenolic compounds by N. aromaticivorans . The results showed the involvement of DesA in O -demethylation of syringic and vanillic acids, LigM in O- demethylation of vanillic acid and 3-MGA, and a new O- demethylase, DmtS, in the conversion of 3-MGA into gallic acid (GA). In addition, we found that LigAB was the main aromatic ring-opening dioxygenase involved in 3-MGA, PCA, and GA metabolism and that a previously uncharacterized dioxygenase, LigAB2, had high activity with GA. Our results indicate a metabolic route not previously identified in N. aromaticivorans that involves O -demethylation of 3-MGA to GA. We predict that this pathway channels ∼15% of the carbon flow from syringic acid, with the rest following ring opening of 3-MGA. The new knowledge obtained in this study allowed for the creation of an improved engineered strain for the funneling of aromatic compounds that exhibits stoichiometric conversion of syringic acid into PDC. IMPORTANCE For lignocellulosic biorefineries to effectively contribute to reduction of fossil fuel use, they need to become efficient at producing chemicals from all major components of plant biomass. Making products from lignin will require engineering microorganisms to funnel multiple phenolic compounds to the chemicals of interest, and N. aromaticivorans is a promising chassis for this technology. The ability of N. aromaticivorans to efficiently and simultaneously degrade many phenolic compounds may be linked to having functionally redundant aromatic degradation pathways and enzymes with broad substrate specificity. A detailed knowledge of aromatic degradation pathways is thus essential to identify genetic engineering targets to maximize product yields. Furthermore, knowledge of enzyme substrate specificity is critical to redirect flow of carbon to desired pathways. This study described an uncharacterized pathway in N. aromaticivorans and the enzymes that participate in this pathway, allowing the engineering of an improved strain for production of PDC from lignin.

Biotechnology & Applied Microbiology↗