Engineering Papers⌕ Search

SEARCH · Engineering Papers

Results for “pathway engineering”

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.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 595 records · Page 33
All sourcesNASA NTRSDOE OSTI
NASA NTRS1995

Simulator Investigation of Pilot Aids for Helicopter Terminal Area Operations with One Engine Inoperative

Two recent piloted simulation experiments have investigated advanced display concepts applied to civil transport helicopter terminal area operations. Civil Category A helicopter operations apply to multi-engine helicopters wherein a safe recovery (land or fly out) is required in the event of a single engine failure. The investigation used the NASA Ames Research Center Vertical Motion Simulator, which has a full six degrees of freedom, to simulate the flight task as closely as possible. The goal of these experiments was to use advanced cockpit displays to improve flight safety and enhance the mission performance of Category A terminal area operations in confined areas. The first experiment investigated the use of military display formats to assist civil rotorcraft in performing a Category A takeoff in confined terminal areas. Specifically, it addressed how well a difficult hovering backup path could be followed using conventional instruments in comparison to panel mounted integrated displays. The hovering backup takeoff, which enables pilots to land back to the confined area pad in the event of an engine failure, was chosen since it is a difficult task to perform. Seven NASA and Army test pilots participated in the experiment. Evaluations, based on task performance and pilot workload, showed that an integrated display enabled the pilot to consistently achieve adequate or desired performance with reasonable pilot workload. Use of conventional instruments, however, frequently resulted in unacceptable performance (poor flight path tracking), higher pilot workload, and poor situational awareness. Although OEI landbacks were considered a visual task, the improved performance on the backup portion, in conjunction with increased situational awareness resulting from use of integrated displays, enabled the pilots to handle an engine failure and land back safely. In contrast, use of conventional instruments frequently led to excessive rates of sink at touchdown. A second simulation (in progress - July - August) is being conducted to investigate the use of advanced displays to perform vertical and short takeoffs and landings. One Engine Inoperative trajectories, which were optimized based on safety of flight restrictions, are utilized. Based on comments from the first experiment and further analytic development, appropriate fly out and approach guidance was added. Displays include conventional instruments with raw data, and the following integrated displays: multi-view and side-view hover displays based on the Apache Pilot Night Vision System, and variations of the pathway-in-the-sky displays with a flight-path-vector, a leader and flight director modifications. Panel mounted and head-up displays are being evaluated. Engine modifications have been incorporated to simulate 30 second and 2 minute contingency power ratings. Evaluations are based on task performance and pilot workload. NASA, Army, FAA, and industry test pilots participated. Details concerning the design, conduct, and the results of the experiment will be reported in the proposed paper.

Iseler, Laura↗
DOE OSTI2023

Improved Biofuel Production through Discovery and Engineering of Terpene Metabolism in Switchgrass

Project Objectives - Of the myriad specialized metabolites that plants deploy to adapt to environmental challenges, terpenes form the largest group. In many major crops, unique terpene blends serve as key stress defenses that directly impact plant fitness and yield. In addition, terpenes, such as bisabolene and pinene, are used for producing renewable biofuels. Essential to advancing a broader use of terpenes for biofuel feedstock engineering is a system-wide knowledge of the diverse biosynthetic machinery and defensive potential of often species-specific terpene blends. The proposed project would merge genome-wide enzyme discovery with comparative –omics, protein structural and plant microbiome studies to define the biosynthesis and stress-defensive functions of the switchgrass (Panicum virgatum) terpene network. These insights would be combined with developing and applying non-transgenic genome editing tools to design plants with desirable terpene blends for higher productivity and biofuel production on marginal lands. As a dedicated lignocellulosic feedstock for U.S. biofuel production with high net energy yield, stress tolerance, and available genome resources, switchgrass is well-suited for devising new avenues for biofuel production. Project Description – The diversity of plant terpene defenses is governed by species-specific families of terpene synthase (TPS) and cytochrome P450 monooxygenase (P450) enzymes. Mining of the switchgrass genome (genotype Alamo) identified ~100 TPS and P450 candidate genes, and combinatorial biochemical analysis of synthesized TPSs and P450s revealed more than a dozen enzymes with common and novel activities. In addition, several identified terpene metabolites and the corresponding transcripts were up-regulated in response to abiotic stressors. These findings demonstrate a unique switchgrass terpene network with probable importance to abiotic stress tolerance, thus providing a large chemical portfolio for optimizing crop resistance, yield, and biofuel composition. Leveraging these preliminary data, we propose to generate a genome-wide map of the switchgrass terpene metabolic network through multi-gene co-expression analyses that allow the efficient cross-validation of TPS and P450 functions. Key enzymes would further be applied to structure-function studies via X-ray protein crystallography, homology modeling and site-directed mutagenesis to gain mechanistic insight into the catalytic specificity of switchgrass terpene metabolism and provide gene and amino acid targets for genome editing. In tandem with terpene pathway discovery, system-wide metabolomics, transcriptomics and proteomics studies in switchgrass accessions of contrasting drought tolerance would define the role of switchgrass terpene metabolism in conferring abiotic stress resilience. Metabolic changes would be assessed in a combined approach of targeted (terpenes) and untargeted metabolite profiling using a high-resolution LC-MS/MS approach, differential gene expression analyses through multiplexed Illumina RNA sequencing, and quantitative analysis of high-priority pathway enzymes using multiple reaction monitoring (MRM). Drawing on these insights, knock-down/out mutants of stress-associated pathway nodes would be generated by optimizing transient virus-induced gene silencing (VIGS) and CRISPR/Cas9 systems under control of the Tobacco Rattle Virus (TRV). The resulting mutant lines would then be analyzed for stress susceptibility and the impact on the root microbiome to define gene functions in planta. Knowledge of terpene pathways, enzyme mechanisms and bioactivities would be applied to enhance switchgrass stress resilience and to tailor-make terpene blends for biofuel production. Here, TRV-enabled CRISPR/Cas9 genome editing, including allele-specific knock-out of redundant genes, engineering of enzyme specificity via structure-guided point mutations, and overexpression of terpene genes relevant to stress-protection or biofuel production, would be used to increase metabolic flux toward desired pathways. Broader Impacts - Integrating the system-wide discovery, mechanistic analysis and non-transgenic genome engineering of the switchgrass terpene network aligns the required steps to unlock the chemical potential of this important metabolite class to generate crops that are more resistant to stress and provide advanced biofuel production in light of rising climate pressures as foreseeable challenges for bioenergy crop cultivation. The proposed project would further offer interdisciplinary student training through active involvement in the project and integration of research concepts and outcomes into newly-developed graduate and undergraduate courses on Plant Biotechnology.

09 BIOMASS FUELS↗
DOE OSTI2020

Design and Implementation of a 1-3 MW th sCO 2 Support Loop for Gen3 CSP Primary Heat Exchangers

The generation 3 concentrating solar power, or Gen3CSP, campaign seeks to de-risk and deploy a CSP pilot plant through three parallel project tracks focused on solid, liquid, and gas-phase primary heat transfer fluids. Although the components between the sun and the primary heat exchanger from the thermal storage system differ with each track, the supercritical carbon dioxide (sCO 2 ) coolant system required to cool the primary heat exchanger in place of a complete power conversion system has very similar requirements regardless of the primary heat transfer fluid. In order to avoid duplicative efforts, this project will design, assemble, perform acceptance testing, and deploy a single sCO 2 coolant system design meeting the needs of any Gen3CSP topic 1 pathway pilot plant design.

14 SOLAR ENERGY↗
DOE OSTI2024

Catalyzing the future: recent advances in chemical synthesis using enzymes

Biocatalysis has the potential to address the need for more sustainable organic synthesis routes. Pro-tein engineering can tune enzymes to perform in cascade reactions and for efficient synthesis of en-antiomerically enriched compounds, using both natural and new-to-nature reaction pathways. This review highlights recent achievements in biocatal-ysis, especially the development of novel enzymatic syntheses to access versatile small molecule inter-mediates and complex biomolecules. Biocatalytic strategies for the degradation of persistent pollu-tants and approaches for biomass valorization are also discussed. Here, the transition of chemical synthesis to a greener future will be accelerated by imple-menting enzymes and engineering them for high performance and new activities.

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

Limits to the strain engineering of layered square-planar nickelate thin films

The layered square-planar nickelates, Nd n+1 Ni n O 2n+2 , are an appealing system to tune the electronic properties of square-planar nickelates via dimensionality; indeed, superconductivity was recently observed in Nd 6 Ni 5 O 12 thin films. Here, we investigate the role of epitaxial strain in the competing requirements for the synthesis of the n = 3 Ruddlesden-Popper compound, Nd 4 Ni 3 O 10 , and subsequent reduction to the square-planar phase, Nd 4 Ni 3 O 8 . We synthesize our highest quality Nd 4 Ni 3 O 10 films under compressive strain on LaAlO 3 (001), while Nd 4 Ni 3 O 10 on NdGaO 3 (110) exhibits tensile strain-induced rock salt faults but retains bulk-like transport properties. A high density of extended defects forms in Nd 4 Ni 3 O 10 on SrTiO 3 (001). Films reduced on LaAlO 3 become insulating and form compressive strain-induced c-axis canting defects, while Nd 4 Ni 3 O 8 films on NdGaO 3 are metallic. This work provides a pathway to the synthesis of Nd n+1 Ni n O 2n+2 thin films and sets limits on the ability to strain engineer these compounds via epitaxy.

75 CONDENSED MATTER PHYSICS, SUPERCONDUCTIVITY AND↗
DOE OSTI2021

L–malic acid production from xylose by engineered Saccharomyces cerevisiae

L-malic acid is widely used in the food, chemical, and pharmaceutical industries. Here, we report on production of malic acid from xylose, the second most abundant sugar in lignocellulosic hydrolysates, by engineered Saccharomyces cerevisiae. To enable malic acid production in a xylose-assimilating S. cerevisiae, we overexpressed PYC1 and PYC2, coding for pyruvate carboxylases, a truncated MDH3 coding for malate dehydrogenase, and SpMAE1, coding for a Schizosaccharomyces pombe malate transporter. Additionally, both the ethanol and glycerol-producing pathways were blocked to enhance malic acid production. The resulting strain produced malic acid from both glucose and xylose, but it produced much higher titers of malic acid from xylose than glucose. Interestingly, the engineered strain had higher malic acid yield from lower concentrations (10 g L –1 ) of xylose, with no ethanol production, than from higher xylose concentrations (20 and 40 g L –1 ). As such, a fed-batch culture maintaining xylose concentrations at low levels was conducted and 61.2 g L –1 of malic acid was produced, with a productivity of 0.32 g L –1 h. Furthermore, these results represent successful engineering of S. cerevisiae for the production of malic acid from xylose, confirming that that xylose offers the efficient production of various biofuels and chemicals by engineered S. cerevisiae.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

Data for L-Malic Acid Production from Xylose by Engineered Saccharomyces cerevisiae

L-malic acid is widely used in the food, chemical, and pharmaceutical industries. Here, we report on production of malic acid from xylose, the second most abundant sugar in lignocellulosic hydrolysates, by engineered Saccharomyces cerevisiae . To enable malic acid production in a xylose-assimilating S. cerevisiae , we overexpressed PYC1 and PYC2, coding for pyruvate carboxylases, a truncated MDH3 coding for malate dehydrogenase, and SpMAE1, coding for a Schizosaccharomyces pombe malate transporter. Additionally, both the ethanol- and glycerol-producing pathways were blocked to enhance malic acid production. The resulting strain produced malic acid from both glucose and xylose, but it produced much higher titers of malic acid from xylose than glucose. Interestingly, the engineered strain had higher malic acid yield from lower concentrations (10 g L‒1) of xylose, with no ethanol production, than from higher xylose concentrations (20 and 40 g L‒1). As such, a fed-batch culture maintaining xylose concentrations at low levels was conducted and 61.2 g L‒1 of malic acid was produced, with a productivity of 0.32 g L‒1 h. These results represent successful engineering of S. cerevisiae for the production of malic acid from xylose, confirming that that xylose offers the efficient production of various biofuels and chemicals by engineered S. cerevisiae .

Conversion↗
DOE OSTI2023

Threat Agnostic Virulence Assessment of Pathogens

Virulence assessment of new, emerging, and engineered pathogens is critical to mounting an appropriate response to a biothreat agent. The capacity of the pathogen to colonize human and harm tissues must be characterized to understand pathogenicity pathways and optimize diagnosis and treatment of resulting disease. Respiratory pathogens are of interest because they can have high transmissibility rates, as observed with the SARS-CoV-2 virus, the causative agent of Covid-19. Current technologies are insufficient to assess threats due to their reliance on systems with only one cell type and on sequencing the pathogen. However, it is known that sequence is not an accurate predictor of function, and sequencing can be unreliable for newly emerged or engineered pathogens. An ideal system would consist of relevant epithelial cell types and an assay sensitive enough to detect changes in host responses that do not rely on DNA sequencing. We chose a system consisting of host lung epithelial cells that can be used to assess the virulence of unknown respiratory pathogens. We interrogated pathogens using this model and assess features of pathogenicity. Our objective is to leverage PNNLs strengths in tissue engineering and proteomics capabilities to build a multiple reaction monitoring (MRM) or parallel reaction monitoring (PRM) liquid chromatography-tandem mass spectrometry assay for human host cell proteins whose abundance is influenced by infection. These responses can were then assessed for relative virulence using pathogen agnostic signatures. When confronted with a pathogen, cells activate dedicated signaling pathways, typically through phosphorylation of regulatory proteins and downstream activation of host cell networks.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2026

Microstructural Engineering of Cu-Rich Nanoprecipitate formation in NiCoFeCrCu0.12 High-Entropy Alloy via Severe Plastic Deformation for Enhanced Irradiation Tolerance

This study demonstrates a defect-engineering approach for controlling Cu-rich precipitates in FeNiCrCoCu0.2 high-entropy alloys (Cu-HEAs), delivering a novel pathway for next-generation nuclear reactor materials with superior irradiation resistance. This work establishes that severe plastic deformation (SPD) processing via Shear Assisted Processing and Extrusion (ShAPE) and Friction Stir Layer Deposition (FSLD) creates dense dislocation networks and subgrain boundaries that fundamentally alter precipitation behavior under identical thermal treatments. Atom probe tomography (APT) indicates that SPD produces a metastable, atomically homogeneous solid solution that, upon moderate heat treatment (500°C/10 hour), develops remarkedly stronger Cu clustering than the as-cast counterpart. High-temperature exposure (800°C/100 h) produces near-pure Cu precipitates (~90 at% Cu) with significantly enhanced defect-sink efficacy in SPD-processed alloys: precipitate sizes of 50-60 nm and number densities of 2.7-3.8 × 10¹7 m?³, compared to 89 nm and 0.44 × 10¹7 m?³ in as-cast materials. Collectively, the findings establish defect-mediated precipitation control as a scalable, high-impact route to tailor sink density and distribution in HEAs, enabling microstructures optimized for irradiation tolerance and mechanical robustness in nuclear reactor environments.

Meher, Subhashish↗
DOE OSTI2025

Strain-induced lead-free morphotropic phase boundary

Enhanced susceptibilities in ferroelectrics often arise near phase boundaries between competing ground states. While chemically-induced phase boundaries have enabled ultrahigh electrical and electromechanical responses in lead-based ferroelectrics, precise chemical tuning in lead-free alternatives, such as (K,Na)NbO 3 thin films, remains challenging due to the high volatility of alkali metals. Here, we demonstrate strain-induced morphotropic phase boundary-like polymorphic nanodomain structures in chemically simple, lead-free, epitaxial NaNbO 3 thin films. Combining ab initio simulations, thin-film epitaxy, scanning probe microscopy, synchrotron X-ray diffraction, and electron ptychography, we reveal a labyrinthine structure comprising coexisting monoclinic and bridging triclinic phases near a strain-induced phase boundary. The coexistence of energetically competing phases facilitates field-driven polarization rotation and phase transitions, giving rise to a multi-state polarization switching pathway and large enhancements in dielectric susceptibility and tunability across a broad frequency range. Our results open new possibilities for engineering lead-free thin films with enhanced functionalities for next-generation applications.

Electronic properties and materials↗
DOE OSTI2024

Isotopically nonstationary metabolic flux analysis of plants: recent progress and future opportunities

Metabolic flux analysis (MFA) is a valuable tool for quantifying cellular phenotypes and to guide plant metabolic engineering. By introducing stable isotopic tracers and employing mathematical models, MFA can quantify the rates of metabolic reactions through biochemical pathways. Recent applications of isotopically nonstationary MFA (INST–MFA) to plants have elucidated nonintuitive metabolism in leaves under optimal and stress conditions, described coupled fluxes for fast–growing algae, and produced a synergistic multi–organ flux map that is a first in MFA for any biological system. Finally, these insights could not be elucidated through other approaches and show the potential of INST–MFA to correct an oversimplified understanding of plant metabolism.

59 BASIC BIOLOGICAL SCIENCES↗
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

NNSA Minority Serving Institute Partnership Program (MSIPP)— Advanced Synergistic Program for Indigenous Research in Engineering (ASPIRE) (FY22 Q1 Progress Report)

In 2016, the National Nuclear Security Agency (NNSA) initiated the Minority Serving Institution Partnership Plan (MSIPP) targeting Tribal Colleges and Universities (TCUs) to offer programs that will prepare students for technical careers in NNSA’s laboratories and production plants. The MSIPP consortium’s approach is as follows: 1) align investments at the college and university level to develop a curriculum and workforce needed to support NNSA’s nuclear weapon enterprise mission, and 2) to enhance research and education at under-represented colleges and universities. The first TCU consortium that MSIPP launched was known as the Advanced Manufacturing Network Initiative (AMNI) whose purpose was to develop additive manufacturing (AM) learning opportunities. The AMNI consortium consisted of Bay Mills Community College, Cankdeska Cikana Community College, Navajo Tech University, Salish Kootenai Community College, Turtle Mountain Community College, and United Tribes Technical College. In 2016, the American Indian Higher Education Consortium (AIHEC), the AMNI consortium and the Southwestern Indian Polytechnic Institute (SIPI), in collaboration with Sandia National Labs, using a grant by NNSA hosted the first TCU Advanced Manufacturing Technology Summer Institute (TCU AMTSI). The AMNI consortium will officially end Sept. 2022. However, building on the successes of AMNI, in FY22 NNSA’s MSIPP launched three additional consortiums: (1) the Indigenous Mutual Partnership to Advanced Cybersecurity Technology (IMPACT), which focuses on STEM and cybersecurity, (2) the Advanced Synergistic Program for Indigenous Research in Engineering (ASPIRE), which focuses on STEM and the electrical and mechanical engineering skills set needed for renewable and distributed energy systems, and (3) the Partnership for Advanced Manufacturing Education and Research (PAMER), which focuses on developing and maintaining a sustainable pathway for a highly trained, next-generation additive manufacturing workforce and a corresponding community of subject matter experts for NNSA enterprises. The following report summarizes the status update during this quarter for the ASPIRE program.

42 ENGINEERING↗
DOE OSTI2025

Modulating Oxide‐Based Quantum Materials by Ion Implantation

Ion implantation has emerged as a powerful tool for manipulating and investigating oxide quantum materials, where strong electron correlations give rise to exotic phases and functionalities. This Review examines how controlled ion bombardment enables targeted modulation of structural, electronic, and magnetic properties in complex oxides. Recent advances in ion beam techniques is analyzed that provide unprecedented control over defect profiles, from point defects to extended damage cascades. In application, ion irradiation has demonstrated the ability to tune quantum phenomena including metal‐insulator transitions, magnetism, and superconductivity through selective doping, defect creation, and lattice strain. The ability to write quantum states with nanoscale precision using focused ion beams is opening new paths for quantum device fabrication. Challenges in understanding radiation damage mechanisms in strongly correlated systems is discussed, and identify opportunities for designing quantum functionality through defect‐engineering approaches. The technique's demonstrated capability to write quantum states with nanoscale precision, combined with its compatibility with existing semiconductor processing infrastructure, suggests a clear pathway for translating fundamental discoveries in quantum materials into practical technological applications.

defect engineering↗
DOE OSTI2023

Metabolic engineering to improve production of 3-hydroxypropionic acid from corn-stover hydrolysate in Aspergillus species

Fuels and chemicals derived from non-fossil sources are needed to lessen human impacts on the environment while providing a healthy and growing economy. 3-hydroxypropionic acid (3-HP) is an important chemical building block that can be used for many products. Biosynthesis of 3-HP is possible; however, low production is typically observed in those natural systems. Biosynthetic pathways have been designed to produce 3-HP from a variety of feedstocks in different microorganisms. In this study, the 3-HP β-alanine pathway consisting of aspartate decarboxylase, β-alanine-pyruvate aminotransferase, and 3-hydroxypropionate dehydrogenase from selected microorganisms were codon optimized for Aspergillus species and placed under the control of constitutive promoters. The pathway was introduced into Aspergillus pseudoterreus and subsequently into Aspergillus niger, and 3-HP production was assessed in both hosts. A. niger produced higher initial 3-HP yields and fewer co-product contaminants and was selected as a suitable host for further engineering. Proteomic and metabolomic analysis of both Aspergillus species during 3-HP production identified genetic targets for improvement of flux toward 3-HP including pyruvate carboxylase, aspartate aminotransferase, malonate semialdehyde dehydrogenase, succinate semialdehyde dehydrogenase, oxaloacetate hydrolase, and a 3-HP transporter. Overexpression of pyruvate carboxylase improved yield in shake-flasks from 0.09 to 0.12 C-mol 3-HP C-mol -1 glucose in the base strain expressing 12 copies of the β-alanine pathway. Deletion or overexpression of individual target genes in the pyruvate carboxylase overexpression strain improved yield to 0.22 C-mol 3-HP C-mol -1 glucose after deletion of the major malonate semialdehyde dehydrogenase. Further incorporation of additional β-alanine pathway genes and optimization of culture conditions (sugars, temperature, nitrogen, phosphate, trace elements) for 3-HP production from deacetylated and mechanically refined corn stover hydrolysate improved yield to 0.48 C-mol 3-HP C-mol -1 sugars and resulted in a final titer of 36.0 g/L 3-HP. The results of this study establish A. niger as a host for 3-HP production from a lignocellulosic feedstock in acidic conditions and demonstrates that 3-HP titer and yield can be improved by a broad metabolic engineering strategy involving identification and modification of genes participated in the synthesis of 3-HP and its precursors, degradation of intermediates, and transport of 3-HP across the plasma membrane.

09 BIOMASS FUELS↗
DOE OSTI2021

Biological upgrading of pyrolysis-derived wastewater: Engineering Pseudomonas putida for alkylphenol, furfural, and acetone catabolism and (methyl)muconic acid production

While biomass-derived carbohydrates have been predominant substrates for biological production of renewable fuels, chemicals, and materials, organic waste streams are growing in prominence as potential alternative feedstocks to improve the sustainability of manufacturing processes. Catalytic fast pyrolysis (CFP) is a promising approach to generate biofuels from lignocellulosic biomass, but it generates a complex, carbon-rich, and toxic wastewater stream that is challenging to process catalytically but could be biologically upgraded to valuable co-products. Here, we implemented modular, heterologous catabolic pathways in the Pseudomonas putida KT2440-derived EM42 strain along with the overexpression of native toxicity tolerance machinery to enable utilization of 89% (w/w) of carbon in CFP wastewater. The dmp monooxygenase and meta-cleavage pathway from Pseudomonas putida CF600 were constitutively expressed to enable utilization of phenol, cresols, 2- and 3-ethyl phenol, and methyl catechols, and the native chaperones clpB, groES, and groEL were overexpressed to improve toxicity tolerance to diverse aromatic substrates. Next, heterologous furfural and acetone utilization pathways were incorporated, and a native alcohol dehydrogenase was overexpressed to improve methanol utilization, generating reducing equivalents. All pathways (encoded by genes totaling ~30 kilobases of DNA) were combined into a single strain that can catabolize a mock CFP wastewater stream as a sole carbon source. Further engineering enabled conversion of all aromatic compounds in the mock wastewater stream to (methyl)muconates with a ~90% (mol/mol) yield. Biological upgrading of CFP wastewater as outlined in this work provides a roadmap for future applications in valorizing other heterogeneous waste streams.

(methyl)muconates↗
DOE OSTI2020

High–level β–carotene production from xylose by engineered Saccharomyces cerevisiae without overexpression of a truncated HMG1 (t HMG1 )

Beta-carotene is a natural pigment and health-promoting metabolite, and has been widely used in the nutraceutical, feed, and cosmetic industries. Here, we engineered a GRAS yeast Saccharomyces cerevisiae to produce beta-carotene from xylose, the sec- ond most abundant and inedible sugar component of lignocellulose biomass. Specifically, a beta-carotene biosynthetic pathway containing crtYB, crtI, and crtE from Xanthophyllomyces dendrorhous was introduced into a xylose-fermenting S. cerevisiae. Here, the resulting strain produced beta-carotene from xylose at a titer threefold higher than from glucose. Interestingly, overexpression of tHMG1, which has been reported as a critical genetic perturbation to enhance metabolic fluxes in the mevalonate pathway and beta-carotene production in yeast when glucose is used, did not further improve the production of beta-carotene from xylose. Through fermentation profiling, metabolites analysis, and transcriptional studies, we found the advantages of using xylose as a carbon source, instead of glucose, for beta-carotene production to be a more respiratory feature of xylose consumption, a larger cytosolic acetyl!CoA pool, and an upregulated expression level of rate-limiting genes in the beta-carotene!producing pathway, including ACS1 and HMG1. As a result, 772.8 mg/L of beta-carotene was ob- tained in a fed-batch bioreactor culture with xylose feeding. Considering the in- evitable large scale production of xylose when cellulosic biomass!based bioeconomy is implemented, our results suggest xylose utilization is a promising strategy for overproduction of carotenoids and other isoprenoids in engineered S. cerevisiae.

09 BIOMASS FUELS↗
DOE OSTI2022

A colorimetric method to measure in vitro nitrogenase functionality for engineering nitrogen fixation

Biological nitrogen fixation (BNF) is the reduction of N 2 into NH 3 in a group of prokaryotes by an extremely O 2 -sensitive protein complex called nitrogenase. Transfer of the BNF pathway directly into plants, rather than by association with microorganisms, could generate crops that are less dependent on synthetic nitrogen fertilizers and increase agricultural productivity and sustainability. In the laboratory, nitrogenase activity is commonly determined by measuring ethylene produced from the nitrogenase-dependent reduction of acetylene (ARA) using a gas chromatograph. The ARA is not well suited for analysis of large sample sets nor easily adapted to automated robotic determination of nitrogenase activities. Here, we show that a reduced sulfonated viologen derivative (S 2 V red ) assay can replace the ARA for simultaneous analysis of isolated nitrogenase proteins using a microplate reader. We used the S 2 V red to screen a library of NifH nitrogenase components targeted to mitochondria in yeast. Two NifH proteins presented properties of great interest for engineering of nitrogen fixation in plants, namely NifM independency, to reduce the number of genes to be transferred to the eukaryotic host; and O 2 resistance, to expand the half-life of NifH iron-sulfur cluster in a eukaryotic cell. This study established that NifH from Dehalococcoides ethenogenes did not require NifM for solubility, [Fe-S] cluster occupancy or functionality, and that NifH from Geobacter sulfurreducens was more resistant to O 2 exposure than the other NifH proteins tested. It demonstrates that nitrogenase components with specific biochemical properties such as a wider range of O 2 tolerance exist in Nature, and that their identification should be an area of focus for the engineering of nitrogen-fixing crops.

59 BASIC BIOLOGICAL SCIENCES↗