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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 415 records · Page 23
All sourcesNASA NTRSDOE OSTI
DOE OSTI2020

Ring-opening and hydrodenitrogenation of indole under hydrothermal conditions over Ni, Pt, Ru, and Ni-Ru bimetallic catalysts

Here, the activity and selectivity of activated-carbon-supported Ni, Pt, Ru, and Ni-Ru bimetallic catalysts was examined for hydrothermal denitrogenation of indole. The molar yield of pyrrole ring-opening compounds, without an added hydrogen source, are in the order: Ni < Pt < Ni 90 Ru 10 < Ni 75 Ru 25 < Ni 50 Ru 50 ~ Ni 25 Ru 75 ~ Ru. Ru-containing catalysts facilitated production of hydrocarbons (hydrodenitrogenation (HDN) products) when used with added formic acid (hydrogen source). We elucidated catalytic hydrothermal HDN pathways for indole based on the product distributions and the variation of their yields with time. Hydrogenation of indole to indoline is the primary pathway and ring-opening of indoline to form alkyl anilines is faster than forming HDN products (alkyl benzenes). DFT calculations confirmed experimental activity trends, showing Ru is more active than Ni for indole ring opening and for o-toluidine deamination. If no hydrogen source is present, directly breaking the N-C bond in the pyrrole ring is more favorable than breaking the C-N bond with an aromatic carbon. If a H source is provided, the pyrrole ring hydrogenates first, forming indoline, followed by cleavage of the C-N bond.

42 ENGINEERING↗
DOE OSTI2025

CO Reduction to Ethylene and Cyclopropane via a Trappable Ruthenium Methylidene

Ruthenium complexes based on cis-[Ru(bpy) 2 (CO) 2 ] 2+ (bpy is 2,2′-bipyridine) can reduce CO 2 and CO to C 1 products including methanol, but access to products containing C–C bonds has been elusive. A reaction pathway to convert CO into multicarbon products ethylene and cyclopropane is presented here, along with mechanistic studies elucidating the key intermediates in C–C bond formation. The ruthenium hydroxymethyl complex [Ru(bpy′) 2 (CO)(CH 2 OH)] + (bpy′ = 5,5′-dimethyl-2,2′-bipyridine) undergoes protonolysis to generate the ethylene complex [Ru(bpy′) 2 (CO)(C 2 H 4 )] 2+ even at −80 °C, with free ethylene released upon warming to room temperature. Experimental evidence implicates a highly electrophilic methylidene complex [Ru(bpy′) 2 (CO)(CH 2 )] 2+ as the key intermediate. The methylidene was successfully trapped with nitriles and pyridine, forming adducts (ylide complexes) that each have a unique reactivity profile. With an appropriate nitrile, the adduct can be characterized at low temperature before warming generates ethylene. A more stable pyridine adduct [Ru(bpy′) 2 (CO)(CH 2 pyridine)] 2+ was crystallographically characterized. Even ethylene itself is sufficiently nucleophilic to react with the electrophilic methylidene, revealing a route from CO to the C 3 hydrocarbon cyclopropane. Furthermore, the methods for controlling the reactivity of hydroxymethyl and methylidene complexes toward C–C bond formation can inform the development of CO and CO 2 reduction catalysts.

Carbene Compounds↗
NASA NTRS2019

On Promoting the Use of Lidar Systems in Forest Ecosystem Research

Forest structure is an important driver of ecosystem dynamics, including the exchange of carbon, water and energy between canopies and the atmosphere. Structural descriptors are also used in numerous studies of eco-logical processes and ecosystem services. Over the last 20+ years, lidar technology has fundamentally changed the way we observe and describe forest structure, and it will continue to impact the ways in which we investigate and monitor the relations between forest structure and functions. Here we present the currently available lidar system types (ground, air, and space-based), we highlight opportunities and challenges associated with each system, as well as challenges associated with a wider use of lidar technology and wider availability of lidar derived products. We also suggest pathways for lidar to further contribute to addressing questions in forest ecosystem science and increase benefits to a wider community of researchers.

Lidar systems↗
DOE OSTI2026

Demonstration of GaAs-Based Photovoltaics on Acoustically Spalled Surfaces Grown by MOCVD

This study demonstrates the potential of sonic lift-off (SLO) technology for repeatable substrate reuse in gallium arsenide (GaAs)–based photovoltaics (PVs), offering a pathway toward cost effective production for terrestrial PV applications. Single-junction GaAs solar cells fabricated on acoustically spalled substrates achieved performance metrics comparable with those grown on standard commercial substrates, exhibiting short-circuit current densities (J sc ) of approximately 26.2–26.4 mA/cm 2 , open-circuit voltages (V oc ) around 1001 mV, fill factors (FFs) of 84%, and AM1.5G efficiencies near 22%. These results confirm that one reuse cycle can be realized without a performance penalty, offering a direct pathway to reducing substrate cost in fabrication. Furthermore, subsequent reuse of the substrates highlighted critical challenges, as devices fabricated from reused substrates showed significant degradation in performance (J sc of 15.5 mA/cm 2 , V oc of 748 mV, FF of 72%, and efficiency of 8%), primarily due to degradation of the epitaxial structure resulting from repeated spalling cycles.

Acoustic Spalling↗
DOE OSTI2025

Polyyne production is regulated by the transcriptional regulators PgnC and GacA in Pseudomonas protegens Pf-5

ABSTRACT Polyynes produced by bacteria have promising applications in agriculture and medicine due to their potent antimicrobial activities. Polyyne biosynthetic genes have been identified inPseudomonasandBurkholderia. However, the molecular mechanisms underlying the regulation of polyyne biosynthesis remain largely unknown. In this study, we used a soil bacteriumPseudomonas protegensPf-5, which was recently reported to produce polyyne called protegenin, as a model to investigate the regulation of bacterial polyyne production. Our results show that Pf-5 controls polyyne production at both the pathway-specific level and a higher global level. Mutation ofpgnC, a transcriptional regulatory gene located in the polyyne biosynthetic gene cluster, abolished polyyne production. Gene expression analysis revealed that PgnC directly activates the promoter of polyyne biosynthetic genes. The production of polyyne also requires a global regulator GacA. Mutation ofgacAdecreased the translation of PgnC, which is consistent with the result thatpgnCleader mRNA bound directly to RsmE, an RNA-binding protein negatively regulated by GacA. These results suggest that GacA induces the expression of the PgnC regulator, which in turn activates polyyne biosynthesis. Additionally, the polyyne-producing strain of Pf-5, but not the polyyne-nonproducing strain, could inhibit a broad spectrum of bacteria including both Gram-negative and Gram-positive bacteria. IMPORTANCE Antimicrobial metabolites produced by bacteria are widely used in agriculture and medicine to control plant, animal, and human pathogens. Although bacteria-derived polyynes have been identified as potent antimicrobials for decades, the molecular mechanisms by which bacteria regulate polyyne biosynthesis remain understudied. In this study, we found that polyyne biosynthesis is directly activated by a pathway-specific regulator PgnC, which is induced by a global regulator GacA through the RNA-binding protein RsmE inPseudomonas protegens. To our knowledge, this work is the first comprehensive study of the regulatory mechanisms of bacterial polyyne biosynthesis at both pathway-specific level and global level. The discovered molecular mechanisms can help us optimize polyyne production for agricultural or medical applications.

Biotechnology & Applied Microbiology↗
DOE OSTI2022

Exploration of the bio-analogous asymmetric C–C coupling mechanism in tandem CO 2 electroreduction

C–C coupling is a critical step of CO 2 fixation in constructing the carbon skeleton of value-added multicarbon products. The Wood–Ljungdahl pathway is an efficient natural process through which microbes transform CO 2 into methyl and carbonyl groups and subsequently couple them together. This asymmetric coupling mechanism remains largely unexplored in inorganic CO 2 electroreduction. Here we experimentally validate the asymmetric coupling pathway through isotope-labelled co-reduction experiments on a Cu surface where 13 CH 3 I and 12 CO are co-fed externally as the methyl and the carbonyl source, respectively. Isotope-labelled multicarbon oxygenates were detected, which confirms an electrocatalytic asymmetric coupling on the Cu surface. We further employed tandem Cu–Ag nanoparticle systems in which *CH x and *CO intermediates can be generated to achieve asymmetric C–C coupling for a practical CO 2 electroreduction. We found that the production of multicarbon oxygenates is correlated with the generation rate of two intermediate indicators, CH 4 and CO. By aligning their rates, the oxygenates generation rate can be maximized.

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

Proteome reallocation enables the selective de novo biosynthesis of non-linear, branched-chain acetate esters

The one-carbon recursive ketoacid elongation pathway is responsible for making various branched-chain amino acids, aldehydes, alcohols, and acetate esters in living cells. Controlling selective microbial biosynthesis of these target molecules at high efficiency is challenging due to enzyme promiscuity, regulation, and metabolic burden. In this study, we present a systematic modular design approach to control proteome reallocation for selective microbial biosynthesis of branched-chain acetate esters. Through pathway modularization, we partitioned the branched-chain ester pathways into four submodules including keto-isovalerate submodule for converting pyruvate to keto-isovalerate, ketoacid elongation submodule for producing longer carbon-chain keto-acids, ketoacid decarboxylase submodule for converting ketoacids to alcohols, and alcohol acyltransferase submodule for producing branched-chain acetate esters by condensing alcohols and acetyl-CoA. By systematic manipulation of pathway gene replication and transcription, enzyme specificity of the first committed steps of these submodules, and downstream competing pathways, we demonstrated selective microbial production of isoamyl acetate over isobutyl acetate. In this work, we found that the optimized isoamyl acetate pathway globally redistributed the amino acid fractions in the proteomes and required up to 23-31% proteome reallocation at the expense of other cellular resources, such as those required to generate precursor metabolites and energy for growth and amino acid biosynthesis. From glucose fed-batch fermentation, the engineered strains produced isoamyl acetate up to a titer of 8.8 g/L (> 0.25 g/L toxicity limit), a yield of 0.22 g/g (61% of maximal theoretical value), and 86% selectivity, achieving the highest titers, yields and selectivity of isoamyl acetate reported to date.

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

Precursor prioritization for p-cymene production through synergistic integration of biology and chemistry

Abstract The strategy of synergistic application of biological and chemical catalysis is an important approach for efficiently converting renewable biomass into chemicals and fuels. In particular, the method of determining the appropriate intermediate between the two catalytic methods is critical. In this work, we demonstrate p -cymene production through the integration of biosynthesis and heterogenous catalysis and show how a preferred biologically derived precursor could be determined. On the biological side, we performed the limonene and 1,8-cineole production through the mevalonate pathway. Titers of 0.605 g/L and a 1.052 g/L were achieved, respectively. This difference is in agreement with the toxicity of these compounds toward the producing microorganisms, which has implications for subsequent development of the microbial platform. On the heterogeneous catalysis side, we performed the reaction with both biological precursors to allow for direct comparison. Using hydrogenation/dehydrogenation metals on supports with acid sites, both limonene and 1,8-cineole were converted to p -cymene with similar yields under equivalent reaction conditions. Thus, we could determine that the most promising strategy would be to target 1,8-cineole, the higher titer and lower toxicity bio-derived precursor with subsequent catalytic conversion to p -cymene. We further optimized the biological production of 1,8-cineole via fed-batch fermentation and reached the titer of 4.37 g/L which is the highest known 1,8-cineole titer from microbial production. This work provides a valuable paradigm for early stage considerations to determine the best route for the high-efficiency production of a target biobased molecule using an integration of biology and chemistry.

09 BIOMASS FUELS↗
NASA NTRS2003

Dissociative Ionization and Product Distributions of Benzene and Pyridine by Electron Impact

We report a theoretical study of the dissociative ionization (DI) and product distributions of benzene (C6H6) and pyridine (C5H5N) from their low-lying ionization channels. Our approach makes use of the fact that electronic motion is much faster than nuclear motion allowing DI to be treated as a two-step process. The first step is the electron-impact ionization resulting in an ion with the same nuclear geometry as the neutral molecule. In the second step, the nuclei relax from the initial geometry and undergo unimolecular dissociation. For the ionization process we use the improved binary-encounter dipole (iBED) model [W.M. Huo, Phys. Rev. A64,042719-I (2001)]. For the unimolecular dissociation, we use multiconfigurational self-consistent field (MCSCF) methods to determine the steepest descent pathways to the possible product channels. More accurate methods are then used to obtain better energetics of the paths which are used to determine unimolecular dissociation probabilities and product distributions. Our analysis of the dissociation products and the thresholds of their productions for benzene are compared with the recent dissociative photoionization meausurements of benzene by Feng et al. [R. Feng, G. Cooper, C.E. Brion, J. Electron Spectrosc. Relat. Phenom. 123,211 (2002)] and the dissociative photoionization measurements of pyridine by Tixier et al. [S. Tixier, G. Cooper, R. Feng, C.E. Brion, J. Electron Spectrosc. Relat. Phenom. 123,185 (2002)] using dipole (e,e+ion) coincidence spectroscopy.

Dateo, Christopher E.↗
DOE OSTI2024

Technical and Economic Assessment and Gap Analysis of Advanced Nuclear Reactor Integration with a Reference Methanol Synthesis Plant

Efforts continue to identify the most-economic methods to decarbonize several sectors of the United States (U.S.) economy. Industrial processes such as synfuel synthesis and high value commodity chemicals rely heavily on energy-dense and easily stored and transported fossil fuels, which power and feed their operations. Steam methane reforming (SMR) is a widely used process for producing methanol. In this process, methane (CH 4 ) from natural gas (NG) reacts with steam (H 2 O) over a catalyst at high temperatures (700-1,000°C) to produce syngas, a mixture of hydrogen (H 2 ) and carbon monoxide (CO). The syngas is then converted into methanol (CH 3 OH) through a second catalytic reaction. This method is known for being an efficient and commonly employed pathway for industrial methanol production. The high-temperature heat needed for SMR, which is currently used in the natural-gas-to-methanol process, cannot be supplied by small modular nuclear reactor (SMNR) direct heating; the temperatures required for the SMR process exceed those of the main steam produced by near-market high-temperature gas reactors (HTGRs). For the conventional methanol process, this leaves possible nuclear-integration opportunities that include: (1) blending nuclear hydrogen into the SMR NG fuel, or (2) assessing alternative synthesis routes leveraging nuclear capabilities and steam electrolysis outputs. In the reference methanol plant, SMR provides the methanol-synthesis reactor with H 2 and co. In Case (2), the state-of-the-art reverse water gas shift (RWGS) pathway achieves the same, sourcing carbon from an industrial CO 2 source.

08 HYDROGEN↗
DOE OSTI2025

Technical and Economic Assessment and Gap Analysis of Advanced Nuclear Reactor Integration with a Reference Methanol Synthesis Plant

Efforts continue to identify the most-economic methods to decarbonize several sectors of the United States (U.S.) economy. Industrial processes such as synfuel synthesis and high value commodity chemicals rely heavily on energy-dense and easily stored and transported fossil fuels, which power and feed their operations. Steam methane reforming (SMR) is a widely used process for producing methanol. In this process, methane (CH 4 ) from natural gas (NG) reacts with steam (H 2 O) over a catalyst at high temperatures (700°1,000°C) to produce syngas, a mixture of hydrogen (H 2 ) and carbon monoxide (CO). The syngas is then converted into methanol (CH 3 OH) through a second catalytic reaction. This method is known for being an efficient and commonly employed pathway for industrial methanol production. The high-temperature heat needed for SMR, which is currently used in the natural-gas-to-methanol process, cannot be supplied by small modular nuclear reactor (SMNR) direct heating; the temperatures required for the SMR process exceed those of the main steam produced by near-market high-temperature gas reactors (HTGRs). For the conventional methanol process, this leaves possible nuclear-integration opportunities that include: (1) blending nuclear hydrogen into the SMR NG fuel, or (2) assessing alternative synthesis routes leveraging nuclear capabilities and steam electrolysis outputs. In the reference methanol plant, SMR provides the methanol-synthesis reactor with H 2 and co. In Case (2), the state-of-the-art reverse water gas shift (RWGS) pathway achieves the same, sourcing carbon from an industrial CO 2 source.

08 - HYDROGEN↗
DOE OSTI2024

Hydrogen-Steam Separation Using Mechanical Vapor Recompression Cycle

Solar thermochemical hydrogen and fuel production is a promising pathways for producing sustainable fuels and chemicals. One of the main challenges in the development of these systems is their low steam conversion extent, dictated by its restrictive thermodynamics requiring extremely high temperatures and low oxygen partial pressure to obtain conversions over 10 \%. While condensing the unreacted steam is technically simple, the latent heat is thus lost, which can be larger than the producd hydrogen higher heating value. We propose to use a modified mechanical vapor recompression cycle, allowing to recover the latent heat by compressing the steam-hydrogen mixture prior to the condensation process, thus creating a temperature different between the hot exhaust and cold inlet streams. We show that this method can recover over 90 % of the latent heat, thus increasing the viability of solar thermochemical hydrogen production cycles even under limited conversion conditions.

hydrogen technology↗
DOE OSTI2025

Climate-focused Life Cycle Assessments of Biochar Production by an ARTi Pyrolysis Reactor and an Air Burners CharBoss® Air Curtain Incinerator

This report presents a limited, dynamic, consequential life cycle assessment (LCA) to compare the climate impacts of two biochar production methods using wood as a feedstock. The two methods are a pyrolysis reactor supplied by ARTi (Des Moines, IA, https://www.arti.com/) and a T26 CharBoss® air curtain incinerator supplied by Air Burners, Inc. (Palm City, FL, https://airburners.com/). The underlying LCA methodology is described in a chapter by Singh et al. (2024) and implemented in the form of a workbook freely available as online Supplementary Material for the chapter. For the convenience of the reader, a pre-print version of the relevant portions of Singh et al. (2024) is attached as Appendix A to this report. Specific assumptions and calculations to obtain the input parameters used in this LCA for each production method are described in the Methodology section below. This implementation of the LCA considers emissions associated with biomass loading, comminution and conversion, biochar decay in soil, and the production and use of bioenergy generated during the conversion process. The LCA is “limited” in that upstream emissions associated with biomass production, harvest, transportation, and land-use change, as well as embodied emissions in equipment and facilities are not considered. Similarly, downstream emissions from biochar transport and incorporation into soil (i.e., tillage), and the impact of biochar soil amendments on soil greenhouse gas (GHG) emissions (other than CO 2 from biochar decay in soil), soil organic carbon stocks, crop response, and surface albedo are not considered. As the intent is to compare different biochar production methods in a simple unbiased manner, the primary alternative biomass pathway for the LCA is immaculate combustion, which is the hypothetical instantaneous and complete conversion of carbon in the biomass to CO 2 at time zero without generation of any other greenhouse gases or aerosols (GHGAs) or any useful bioenergy. Use of this pathway provides relative values for the production methods and, when the embodied emissions are similar and the same feedstock is used, these relative values are reasonable approximations for those attained with a full LCA.

09 BIOMASS FUELS↗
DOE OSTI2024

Expression of dehydroshikimate dehydratase in poplar induces transcriptional and metabolic changes in the phenylpropanoid pathway

Abstract Modification of lignin in feedstocks via genetic engineering aims to reduce biomass recalcitrance to facilitate efficient conversion processes. These improvements can be achieved by expressing exogenous enzymes that interfere with native biosynthetic pathways responsible for the production of the lignin precursors. In planta expression of a bacterial 3-dehydroshikimate dehydratase in poplar trees reduced lignin content and altered the monomer composition, which enabled higher yields of sugars after cell wall polysaccharide hydrolysis. Understanding how plants respond to such genetic modifications at the transcriptional and metabolic levels is needed to facilitate further improvement and field deployment. In this work, we acquired fundamental knowledge on lignin-modified poplar expressing 3-dehydroshikimate dehydratase using RNA-seq and metabolomics. The data clearly demonstrate that changes in gene expression and metabolite abundance can occur in a strict spatiotemporal fashion, revealing tissue-specific responses in the xylem, phloem, or periderm. In the poplar line that exhibited the strongest reduction in lignin, we found that 3% of the transcripts had altered expression levels and ~19% of the detected metabolites had differential abundance in the xylem from older stems. The changes affected predominantly the shikimate and phenylpropanoid pathways as well as secondary cell wall metabolism, and resulted in significant accumulation of hydroxybenzoates derived from protocatechuate and salicylate.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2026

Design and Optimization of Processing Pathways for Rare Earth Element Recovery from End-of-Life Products

In this presentation, we first discuss the benefits of targeting end-of-life (EOL) products as a feedstock. We then discuss the problem statement of designing a processing facility for recovering rare earth elements (REEs) from EOL HDDs and motors from EOL EVs/HEVs. We then discuss the most profitable pathway, and then discuss the environmental impacts associated with the processing pathway, comparing it to a processing facility in China, and find it to have lower impacts overall. We also investigate the trade-off solutions for other pathways that are not as profitable, but have lower impacts by generating Pareto Fronts. In the next portion of the presentation, Critical Materials Recycling, Inc. is discussed. We discuss how their process was found to be the optimal pathway after performing superstructure optimization, and how we are currently working with them to further optimize their process to increase profits. Finally, we wrap up with conclusions summarizing the main takeaways of the presentation.

36 MATERIALS SCIENCE↗
DOE OSTI2021

Partial inhibition of activin receptor-like kinase 4 alleviates bladder fibrosis caused by bladder outlet obstruction

The bladder undergoes profound structural alterations after bladder outlet obstruction (BOO), characterized by hypertrophy of the bladder wall and accumulation of extracellular matrix (ECM). Transforming growth factor-β (TGF-β) has been found to promote fibrosis of the bladder induced by partial bladder outlet obstruction (pBOO). Activin receptor-like kinase 4 (ALK4) is a downstream receptor of the TGF-β superfamily. However, the role of the ALK4–Smad2/3 pathway in the pathogenesis of bladder fibrosis caused by pBOO remains unknown. This study focused on learning the role of ALK4 in the process of bladder fibrosis caused by pBOO. The pBOO mice models showed that ALK4 expression was found to upregulate in the wild-type bladder 6 weeks after pBOO compared to control group. Then, mice with heterozygous knockout of the ALK4 gene (ALK4+/−) were generated. Histological analysis and Western blot (WB) results showed significant suppression of collagen expression in the bladders of ALK4+/− mice after pBOO compared with WT mice. WB also showed that ALK4+/− mice demonstrated significant suppression of phosphorylated Smad2/3 (p-Smad2/3) expression in the bladder 6 weeks after pBOO but not of phosphorylated extracellular signal-regulated kinase, c-Jun N-terminal kinase or protein 38 (p-ERK, p-JNK, p-P38) expression. This effect might have occurred through partial inactivation of the Smad2/3 signaling pathway. In vitro, ALK4 overexpression promoted collagen production in cultured BSMCs and activated the Smad2/3 signaling pathway. Taken together, our results demonstrated that ALK4 insufficiency alleviated bladder fibrosis in a mouse model of pBOO partly by suppressing Smad2/3 activity.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2025

Cell-Free-Based Thermophilic Biocatalyst for the Synthesis of Amino Acids from One-Carbon Feedstocks

Bioproduction from one-carbon compounds, such as formate, is an attractive prospect due to reduced energy requirements and the possibility for using CO 2 as a sustainable feedstock. Formate-fixing pathways engineered using Escherichia coli lysate-based cell-free expression (CFE) biocatalysts have the potential to route 100% of feedstock carbon toward chemical synthesis but are undermined by siphoning of in-pathway metabolites and cofactors by the CFE background metabolism. To address this limitation, we engineer a CFE-based thermophilic multienzyme biocatalyst for the synthesis of serine and glycine from formate, bicarbonate, and ammonia. After expression of the thermophilic formate-to-serine pathway in a one-pot reaction, the mesophilic E. coli CFE background machinery is removed by simple heat denaturation, eliminating the siphoning of cofactors, inpathway metabolites, and products. After bioprocess optimization, including pathway gene expression duration and chemical synthesis temperature, we achieve near stoichiometric conversion of formate and bicarbonate to serine and glycine, reaching 97% of stoichiometric yield. The use of a moderately thermophilic biocatalyst allowed chemical synthesis to take place at mesophilic temperatures, enabling the balance of optimal enzyme activity with minimal metabolite/cofactor thermal degradation. In a fed-batch experiment, the biocatalyst shows sustained chemical synthesis rates for 8 h, paving the way toward a continuous bioprocess. Finally, a sensitivity analysis of cofactor usage revealed that the most expensive cofactors, THF and NADPH, can be reduced by 5-fold without significantly lowering product yields. To the best of our knowledge, this is the first instance of expressing a thermophilic pathway in an E. coli lysate-based CFE system to generate a thermophilic biocatalyst for use at mesophilic temperatures. The CFEbased thermophilic formate-to-serine biocatalyst triples the combined serine and glycine yield previously obtained by a CFE-based mesophilic formate-to-serine biocatalyst (30%), and quadruple the yield obtained by a purified enzyme system (22%). Ultimately, this work opens the door to using E. coli lysate-based CFE for thermophilic biocatalyst generation to achieve high chemical synthesis yields.

bacteria↗
DOE OSTI2020

In vitro prototyping of limonene biosynthesis using cell-free protein synthesis

Metabolic engineering of microorganisms to produce sustainable chemicals has emerged as an important part of the global bioeconomy. Unfortunately, efforts to design and engineer microbial cell factories are challenging because design-build-test cycles, iterations of re-engineering organisms to test and optimize new sets of enzymes, are slow. To alleviate this challenge, we demonstrate a cell-free approach termed in vitro Prototyping and Rapid Optimization of Biosynthetic Enzymes (or iPROBE). In iPROBE, a large number of pathway combinations can be rapidly built and optimized. The key idea is to use cell-free protein synthesis (CFPS) to manufacture pathway enzymes in separate reactions that are then mixed to modularly assemble multiple, distinct biosynthetic pathways. As a model, we apply our approach to the 9-step heterologous enzyme pathway to limonene in extracts from Escherichia coli. In iterative cycles of design, we studied the impact of 54 enzyme homologs, multiple enzyme levels, and cofactor concentrations on pathway performance. In total, we screened over 150 unique sets of enzymes in 580 unique pathway conditions to increase limonene production in 24 h from 0.2 to 4.5 mM (23–610 mg/L). Finally, to demonstrate the modularity of this pathway, we also synthesized the biofuel precursors pinene and bisabolene. Finally, we anticipate that iPROBE will accelerate design-build-test cycles for metabolic engineering, enabling data-driven multiplexed cell-free methods for testing large combinations of biosynthetic enzymes to inform cellular design.

59 BASIC BIOLOGICAL SCIENCES↗