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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 1,657 records · Page 92
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DOE OSTI2022

Grape ASR Regulates Glucose Transport, Metabolism and Signaling

To decipher the mediator role of the grape Abscisic acid, Stress, Ripening (ASR) protein, VvMSA, in the pathways of glucose signaling through the regulation of its target, the promoter of hexose transporter VvHT1, we overexpressed and repressed VvMSA in embryogenic and non-embryogenic grapevine cells. The embryogenic cells with organized cell proliferation were chosen as an appropriate model for high sensitivity to the glucose signal, due to their very low intracellular glucose content and low glycolysis flux. In contrast, the non-embryogenic cells displaying anarchic cell proliferation, supported by high glycolysis flux and a partial switch to fermentation, appeared particularly sensitive to inhibitors of glucose metabolism. By using different glucose analogs to discriminate between distinct pathways of glucose signal transduction, we revealed VvMSA positioning as a transcriptional regulator of the glucose transporter gene VvHT1 in glycolysis-dependent glucose signaling. The effects of both the overexpression and repression of VvMSA on glucose transport and metabolism via glycolysis were analyzed, and the results demonstrated its role as a mediator in the interplay of glucose metabolism, transport and signaling. The overexpression of VvMSA in the Arabidopsis mutant abi8 provided evidence for its partial functional complementation by improving glucose absorption activity.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

Examination of Genetic Control Elements in the Phototrophic Firmicute Heliomicrobium modesticaldum

Heliomicrobium modesticaldum has been used as a model organism for the Heliobacteria, the only phototrophic family in the Firmicutes. It is a moderately thermophilic anoxygenic phototrophic bacterium that is capable of fermentative growth in the dark. The genetic manipulation of H. modesticaldum is still in its infancy. Methods to introduce genes through the use of exogenous plasmids and to delete genes from the chromosome through the use of the native CRISPR/Cas system have been developed in the last several years. To expand our genetic toolkit, it was necessary to control gene expression. In this study, we analyzed constitutive and inducible promoters developed for clostridia for their use in H. modesticaldum and further tested two reporters, adhB and lacZ, as indicators of promoter strength. Alcohol dehydrogenase (AdhB) was unsuitable as a reporter in this species due to high endogenous activity and/or low activity of the reporter, but a thermostable LacZ worked well as a reporter. A set of constitutive promoters previously reported to work in Clostridium thermocellum was found to be reliable for controlling the expression of the lacZ reporter gene in H. modesticaldum at a range of activities spanning an order of magnitude. An anhydrotetracycline-inducible promoter was created by inserting tetO operators into a strong constitutive promoter, but it was not fully repressible. The implementation of a xylose-inducible promoter resulted in complete repression of β-gal in the absence of xylose, and reliable expression tunable through the concentration of xylose added to the culture.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2020

EMS-Induced Mutagenesis of Clostridium carboxidivorans for Increased Atmospheric CO 2 Reduction Efficiency and Solvent Production

Clostridium carboxidivorans (P7) is one of the most important solvent-producing bacteria capable of fermenting syngas (CO, CO 2 , and H 2 ) to produce chemical commodities when grown as an autotroph. This study aimed to develop ethyl methanesulfonate (EMS)-induced P7 mutants that were capable of growing in the presence of CO 2 as a unique source of carbon with increased solvent formation and atmospheric CO 2 reduction to limit global warming. Phenotypic analysis including growth and end product characterization of the P7 wild type (WT) demonstrated that this strain grew better at 25 °C than 37 °C when CO 2 served as the only source of carbon. In the current study, 55 mutagenized P7-EMS mutants were developed by using 100 mM and 120 mM EMS. Interestingly, using a forward genetic approach, three out of the 55 P7-EMS mutants showed a significant increase in ethanol, butyrate, and butanol production. The three P7-EMS mutants presented on average a 4.68-fold increase in concentrations of ethanol when compared to the P7-WT. Butyric acid production from 3 P7-EMS mutants contained an average of a 3.85 fold increase over the levels observed in the P7-WT cultures under the same conditions (CO 2 only). In addition, one P7-EMS mutant presented butanol production (0.23 ± 0.02 g/L), which was absent from the P7-WT under CO 2 conditions. Most of the P7-EMS mutants showed stability of the obtained end product traits after three transfers. Most importantly, the amount of reduced atmospheric CO 2 increased up to 8.72 times (0.21 g/Abs) for ethanol production and up to 8.73 times higher (0.16 g/Abs) for butyrate than the levels contained in the P7-WT. Additionally, to produce butanol, the P7-EMSIII-J mutant presented 0.082 g/Abs of CO 2 reduction. This study demonstrated the feasibility and effectiveness of employing EMS mutagenesis in generating solvent-producing anaerobic bacteria mutants with improved and novel product formation and increased atmospheric CO 2 reduction efficiency.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2025

Effect of CO 2 Concentration on the Microbial Activity of Orenia metallireducens (Strain Z6) in Surface Inert Materials

Carbon dioxide (CO 2 ) sequestration has garnered widespread attention as a key strategy for mitigating CO 2 emissions and combating the greenhouse effect. However, the mechanisms underlying the interactions between CO 2 , widespread siliceous minerals and biological processes remain unclear. The present study explored the potential impacts of different CO 2 concentrations on microbial activity, environmental conditions and their feedback on the fate of CO 2 . A total of 20 experimental conditions was created, with the variables including different natural and synthetic siliceous minerals (e.g., quartz sand and a type of commercial glass beads), the presence or absence of the iron-reducing microorganism Orenia metallireducens (strain Z6) and varying CO 2 concentrations (0%, 20%, 50%, 100%) in the presence of ferrihydrite and pyruvate. Geochemical, microbial and mineralogical analyses revealed that elevated CO 2 concentrations significantly inhibited microbial Fe(III) reduction and pyruvate metabolism. Interestingly, compared to cultures without mineral amendments or those with glass beads alone, the addition of quartz sand enabled strain Z6 to better withstand the environmental stress caused by elevated CO 2 , promoting pyruvate fermentation and iron reduction. In addition to an increased pH, the formation of siderite, hematite and vivianite was also observed in the bioactive systems. Although both glass beads and quartz sand were primarily composed of silica, differences in the mineral structure, elemental composition and acid neutralization capacity rendered quartz sand more chemically active and unexpectedly led to greater CO 2 sequestration.

CO2 stress↗
DOE OSTI2024

Photosynthetic Electron Flows and Networks of Metabolite Trafficking to Sustain Metabolism in Photosynthetic Systems

Photosynthetic eukaryotes have metabolic pathways that occur in distinct subcellular compartments. However, because metabolites synthesized in one compartment, including fixed carbon compounds and reductant generated by photosynthetic electron flows, may be integral to processes in other compartments, the cells must efficiently move metabolites among the different compartments. This review examines the various photosynthetic electron flows used to generate ATP and fixed carbon and the trafficking of metabolites in the green alga Chlamydomomas reinhardtii; information on other algae and plants is provided to add depth and nuance to the discussion. We emphasized the trafficking of metabolites across the envelope membranes of the two energy powerhouse organelles of the cell, the chloroplast and mitochondrion, the nature and roles of the major mobile metabolites that move among these compartments, and the specific or presumed transporters involved in that trafficking. These transporters include sugar-phosphate (sugar-P)/inorganic phosphate (Pi) transporters and dicarboxylate transporters, although, in many cases, we know little about the substrate specificities of these transporters, how their activities are regulated/coordinated, compensatory responses among transporters when specific transporters are compromised, associations between transporters and other cellular proteins, and the possibilities for forming specific ‘megacomplexes’ involving interactions between enzymes of central metabolism with specific transport proteins. Finally, we discuss metabolite trafficking associated with specific biological processes that occur under various environmental conditions to help to maintain the cell’s fitness. These processes include C4 metabolism in plants and the carbon concentrating mechanism, photorespiration, and fermentation metabolism in algae.

Plant Sciences↗
DOE OSTI2024

A Multi-Streamline Approach for Upcycling PET into a Biodiesel and Asphalt Modifier

The non-degradable nature of petroleum-based plastics and the dependence on petroleum-based products in daily life and production are dilemmas of human development today. We hereby developed a plastic waste upcycling process to address these challenges. A multi-stream fraction strategy was developed to process poly (ethylene terephthalate) (PET) plastics into soluble and insoluble fractions. The soluble fraction was used as a sole carbon source for microbial fermentation to produce biodiesel precursor lipids with an appreciable bioconversion yield. The insoluble fraction containing fractionated polymers was used as the asphalt binder modifiers. The downsized PET additive improved the high-temperature performance of the asphalt binder by 1 performance grade (PG) without decreasing the low-temperature PG. Subsequent SEM imaging unveiled alterations in the micromorphology induced by PET incorporation. Further FTIR and 1 H NMR analysis highlighted the aromatic groups of PET polymers as a crucial factor influencing performance enhancement. The results demonstrated the multi-stream fraction as a promising approach for repurposing plastic waste to produce biodiesel and modify asphalt. Finally, this approach holds the potential to tackle challenges in fuel supply and enhance infrastructure resilience to global warming.

09 BIOMASS FUELS↗
DOE OSTI2025

Circular Perspective for Utilization of Industrial Wastewaters via Phytoremediation

Wastewater generated in municipal rendering facilities requires multi-step treatment, but it may also serve as a source of nutrients and water and thus may be valorized before or instead of the necessary wastewater treatment operations. In this work, wastewaters from a composting plant were utilized to support the growth of Miscanthus x giganteus, known as both a remediation plant and an energy biomass source. A pot experiment was established to compare the effects of different wastewater doses (0, 50, 100, and 200 mL per pot per week) on the miscanthus biomass yield, phytoextraction of heavy metals, biomass heat of combustion, and plant condition. The increase in the wastewater dose resulted in increases in both biomass yield (from about 44 to 139%) and biomass heat of combustion (from 7 to 17%) when compared to the control sample, with no adverse effects on plant physiological parameters. The highest concentrations of metals were found in miscanthus grown with the highest dose of wastewaters. It was found that higher wastewater dose correlates to both higher phytoextraction and phytorecovery of metals from plant substrate and wastewaters. The highest metal uptake was identified for Fe (431 mg·pot −1 ), followed by Al, Zn, Mn, Cu, Ni, Cr. The lowest metal uptake was noted for Pb, Co and Cd (0.88, 0.11, and 0.95 mg·pot −1 , respectively). The results indicate that miscanthus can be recommended for industrial wastewater treatment. In addition, due to high absorption efficiency of the substrate components, miscanthus can be used as a remediation tool, e.g., for the ecological stabilization of remediation of metal-polluted soils, especially in municipal facilities like rendering plants. This presents a circular perspective for the valorization of post-fermentation wastewaters with subsequent growth of energy crops, with other potential benefits for the environment, such as soil treatment, absorption of CO 2 , and air purification.

Miscanthus x giganteus↗
DOE OSTI2012

Complete genome sequence of Syntrophobacter fumaroxidans strain (MPOBT)

Syntrophobacter fumaroxidans strain MPOBT is the best-studied species of the genus Syntrophobacter. The species is of interest because of its anaerobic syntrophic lifestyle, its involvement in the conversion of propionate to acetate, H2 and CO2 during the overall degradation of organic matter, and its release of products that serve as substrates for other microorganisms. The strain is able to ferment fumarate in pure culture to CO2 and succinate, and is also able to grow as a sulfate reducer with propionate as an electron donor. This is the first complete genome sequence of a member of the genus Syntrophobacter and a member genus in the family Syntrophobacteraceae. Here we describe the features of this organism, together with the complete genome sequence and annotation. The 4,990,251 bp long genome with its 4,098 protein-coding and 81 RNA genes is a part of the Microbial Genome Program (MGP) and the Genomes to Life (GTL) Program project.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2013

Metagenomes of tropical soil-derived anaerobic switchgrass-adapted consortia with and without iron

Tropical forest soils decompose litter rapidly with frequent episodes of anoxia, making it likely that bacteria using alternate terminal electron acceptors (TEAs) such as iron play a large role in supporting decomposition under these conditions. The prevalence of many types of metabolism in litter deconstruction makes these soils useful templates for improving biofuel production. To investigate how iron availability affects decomposition, we cultivated feedstock-adapted consortia (FACs) derived from iron-rich tropical forest soils accustomed to experiencing frequent episodes of anaerobic conditions and frequently fluctuating redox. One consortium was propagated under fermenting conditions, with switchgrass as the sole carbon source in minimal media (SG only FACs), and the other consortium was treated the same way but received poorly crystalline iron as an additional terminal electron acceptor (SG + Fe FACs). We sequenced the metagenomes of both consortia to a depth of about 150 Mb each, resulting in a coverage of 26× for the more diverse SG + Fe FACs, and 81× for the relatively less diverse SG only FACs. Both consortia were able to quickly grow on switchgrass, and the iron-amended consortium exhibited significantly higher microbial diversity than the unamended consortium. We found evidence of higher stress in the unamended FACs and increased sugar transport and utilization in the ironamended FACs. This work provides metagenomic evidence that supplementation of alternative TEAs may improve feedstock deconstruction in biofuel production.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2013

Complete genome sequence of Enterobacter sp. IIT-BT 08: A potential microbial strain for high rate hydrogen production

Enterobacter sp. IIT-BT 08 belongs to Phylum: Proteobacteria, Class: Gammaproteobacteria, Order: Enterobacteriales, Family: Enterobacteriaceae. The organism was isolated from the leaves of a local plant near the Kharagpur railway station, Kharagpur, West Bengal, India. It has been extensively studied for fermentative hydrogen production because of its high hydrogen yield. For further enhancement of hydrogen production by strain development, complete genome sequence analysis was carried out. Sequence analysis revealed that the genome was linear, 4.67 Mbp long and had a GC content of 56.01%. The genome properties encode 4,393 protein-coding and 179 RNA genes. Additionally, a putative pathway of hydrogen production was suggested based on the presence of formate hydrogen lyase complex and other related genes identified in the genome. Thus, in the present study we describe the specific properties of the organism and the generation, annotation and analysis of its genome sequence as well as discuss the putative pathway of hydrogen production by this organism.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2015

Genetic engineering and improvement of a Zymomonas mobilis for arabinose utilization and its performance on pretreated corn stover hydrolyzate

In this paper, a glucose, xylose and arabinose utilizing Zymomonas mobilis strain was constructed by incorporating arabinose catabolic pathway genes, araBAD encoding L-ribulokinase, L-arabinose isomerase and L-ribulose-5-phosphate- 4-epimerase in a glucose, xylose co-fermenting host, 8b, using a transposition integration approach. Further improvement on this arabinose-capable integrant, 33C was achieved by applying a second transposition to create a genomic knockout (KO) mutant library. Using arabinose as a sole carbon source and a selection pressure, the KO library was subjected to a growth-enrichment process involving continuous sub-culturing for over 120 generations. Strain 13-1-17, isolated from such process demonstrated significant improvement in metabolizing arabinose in a dilute acid pretreated, saccharified corn stover slurry. Through Next Generation Sequencing (NGS) analysis, integration sites of the transposons were identified. Furthermore, multiple additional point mutations (SNPs: Single Nucleotide Polymorphisms) were discovered in 13-1-17, affecting genes araB and RpiB in the genome. Finally, we speculate that these mutations may have impacted the expression of the enzymes coded by these genes, ribulokinase and Ribose 5-P-isomerase, thus attributing to the improvement of the arabinose utilization.

09 BIOMASS FUELS↗
DOE OSTI2016

Biogeochemical modeling of CO 2 and CH 4 production in anoxic Arctic soil microcosms

Abstract. Soil organic carbon turnover to CO2 and CH4 is sensitive to soil redox potential and pH conditions. However, land surface models do not consider redox and pH in the aqueous phase explicitly, thereby limiting their use for making predictions in anoxic environments. Using recent data from incubations of Arctic soils, we extend the Community Land Model with coupled carbon and nitrogen (CLM-CN) decomposition cascade to include simple organic substrate turnover, fermentation, Fe(III) reduction, and methanogenesis reactions, and assess the efficacy of various temperature and pH response functions. Incorporating the Windermere Humic Aqueous Model (WHAM) enables us to approximately describe the observed pH evolution without additional parameterization. Although Fe(III) reduction is normally assumed to compete with methanogenesis, the model predicts that Fe(III) reduction raises the pH from acidic to neutral, thereby reducing environmental stress to methanogens and accelerating methane production when substrates are not limiting. The equilibrium speciation predicts a substantial increase in CO2 solubility as pH increases, and taking into account CO2 adsorption to surface sites of metal oxides further decreases the predicted headspace gas-phase fraction at low pH. Without adequate representation of these speciation reactions, as well as the impacts of pH, temperature, and pressure, the CO2 production from closed microcosms can be substantially underestimated based on headspace CO2 measurements only. Our results demonstrate the efficacy of geochemical models for simulating soil biogeochemistry and provide predictive understanding and mechanistic representations that can be incorporated into land surface models to improve climate predictions.

54 ENVIRONMENTAL SCIENCES↗
DOE OSTI2019

Modeling anaerobic soil organic carbon decomposition in Arctic polygon tundra: insights into soil geochemical influences on carbon mineralization

Abstract. Rapid warming of Arctic ecosystems exposes soil organic matter (SOM) to accelerated microbial decomposition, potentially leading to increased emissions of carbon dioxide ( CO 2 ) and methane ( CH 4 ) that have a positive feedback on global warming. Current estimates of the magnitude and form of carbon emissions from Earth system models include significant uncertainties, partially due to the oversimplified representation of geochemical constraints on microbial decomposition. Here, we coupled modeling principles developed in different disciplines, including a thermodynamically based microbial growth model for methanogenesis and iron reduction, a pool-based model to represent upstream carbon transformations, and a humic ion-binding model for dynamic pH simulation to build a more versatile carbon decomposition model framework that can be applied to soils under varying redox conditions. This new model framework was parameterized and validated using synthesized anaerobic incubation data from permafrost-affected soils along a gradient of fine-scale thermal and hydrological variabilities across Arctic polygonal tundra. The model accurately simulated anaerobic CO 2 production and its temperature sensitivity using data on labile carbon pools and fermentation rates as model constraints. CH 4 production is strongly influenced by water content, pH, methanogen biomass, and presence of competing electron acceptors, resulting in high variability in its temperature sensitivity. This work provides new insights into the interactions of SOM pools, temperature increase, soil geochemical feedbacks, and resulting CO 2 and CH 4 production. The proposed anaerobic carbon decomposition framework presented here builds a mechanistic link between soil geochemistry and carbon mineralization, making it applicable over a wide range of soils under different environmental settings.

54 ENVIRONMENTAL SCIENCES↗
DOE OSTI2018

Modeling Anaerobic Soil Organic Carbon Decomposition in Arctic Polygon Tundra: Insights into Soil Geochemical Influences on Carbon Mineralization: Modeling Archive

This anaerobic carbon decomposition model framework was developed with explicit representation of fermentation, methanogenesis and iron reduction by combining three well-known modeling approaches developed in different disciplines. A pool-based model to represent upstream carbon transformations and replenishment of dissolved organic carbon (DOC) pool, a thermodynamically-based model to calculate rate kinetics and biomass growth for methanogenesis and Fe(III) reduction, and a humic ion-binding model for aqueous phase speciation and pH calculations. The combined models are implemented in the open source geochemical model PHREEQC (V3.0). Installation of PHREEQC is required to run this model. This dataset contains one zipped file and one pdf.The Next-Generation Ecosystem Experiments: Arctic (NGEE Arctic), was a research effort to reduce uncertainty in Earth System Models by developing a predictive understanding of carbon-rich Arctic ecosystems and feedbacks to climate. NGEE Arctic was supported by the Department of Energy's Office of Biological and Environmental Research.The NGEE Arctic project had two field research sites: 1) located within the Arctic polygonal tundra coastal region on the Barrow Environmental Observatory (BEO) and the North Slope near Utqiagvik (Barrow), Alaska and 2) multiple areas on the discontinuous permafrost region of the Seward Peninsula north of Nome, Alaska.Through observations, experiments, and synthesis with existing datasets, NGEE Arctic provided an enhanced knowledge base for multi-scale modeling and contributed to improved process representation at global pan-Arctic scales within the Department of Energy's Earth system Model (the Energy Exascale Earth System Model, or E3SM), and specifically within the E3SM Land Model component (ELM).

54 ENVIRONMENTAL SCIENCES↗
DOE OSTI2017

Chapter 11 - Biomass Compositional Analysis for Conversion to Renewable Fuels and Chemicals. In the book Biomass Volume Estimation and Valorization for Energy.

As the world continues to deplete its non-renewable resources there has begun a shift towards using renewable materials for the production of fuels and chemicals. Terrestrial biomass, as well as municipal solid wastes, provide renewable feedstocks for fuel and chemical production. However, one of the major challenges to using biomass as a feedstock for fuels and chemicals production is the great amount of innate variability between different biomass types and within individual biomass species. This inconsistency arises from varied growth and harvesting conditions and presents challenges for conversion processes, which frequently require physically and chemically uniform materials. This chapter will examine intrinsic biomass compositional characteristics including: cellulose, hemicellulose, lignin, extractives/volatiles, and ash for a wide array of biomass types. Additionally, extrinsic properties, like moisture content and particle grind size, will be examined for their effect on biomass conversion to fuels using four major conversion processes: direct combustion, pyrolysis, hydrothermal liquefaction, and fermentation. A brief discussion on recent research for the production of building block chemicals from biomass will also be presented.

09 BIOMASS FUELS↗
DOE OSTI2026

The anaerobic fungus Caecomyces churrovis produces H 2 via a non-bifurcating NADH-dependent enzyme complex

ABSTRACT Hydrogenosomes are mitochondria-derived organelles that produce ATP and H 2 to support energy metabolism in anaerobic eukaryotes. H 2 production allows reoxidation of reduced cofactors generated during fermentative metabolism; however, the metabolic mechanisms for H 2 production in anaerobic eukaryotes remains incompletely understood. In particular, it remains unclear whether anaerobic fungi (AF) hydrogenosomes use a ferredoxin-dependent pathway or a distinct mechanism to regenerate NAD(P) + and link electron transfer to H 2 formation. Here, by combining genomic search, proteomic analysis, and enzymology, we reveal the molecular mechanism for H 2 production in the AF strain Caecomyces churrovis . Our enzyme assays on the organelle fraction of C. churrovis revealed the activity of H 2 :NAD + oxidoreductase but not pyruvate:ferredoxin oxidoreductase, which is usually linked to H 2 formation. We identified genes encoding [FeFe] hydrogenase (Hyd) and NADH dehydrogenase subunits E and F (NuoE, NuoF) in C. churrovis , and confirmed their expression in the isolated hydrogenosomal fractions by proteomic analysis. Combining the individually purified enzymes, we found Hyd and NuoEF proteins formed H 2 directly from NADH independently of ferredoxin, functioning as a non-bifurcating NADH-dependent enzyme rather than an electron-bifurcating enzyme. We identified homologs of hydrogenosomal NuoE, NuoF, and Hyd in many other AF, indicating this pathway is commonly shared among the AF. This work demonstrates the existence of a non-bifurcating NADH-dependent enzyme complex in eukaryotes. Moreover, this complex could potentially be exploited as a target for controlling AF H 2 production and altering fungal metabolism. IMPORTANCE H 2 production is a prominent feature of anaerobic energy metabolism, yet our understanding of eukaryotic mechanisms remains limited. Anaerobic fungi (AF) are key decomposers of lignocellulose and contribute to hydrogen flux in anaerobic environments. Although it has been more than 40 years since the H 2 production from AF was first reported, the molecular mechanism for hydrogenosomal H 2 production and redox balance remains unclear. We demonstrate that AF produce H 2 from NADH utilizing a non-bifurcating NADH-dependent enzyme complex rather than an electron-bifurcating, ferredoxin-dependent variant. We show that this enzyme complex is conserved across multiple AF lineages and thus demonstrate the occurrence of a non-bifurcating NADH-dependent enzyme in eukaryotes. This discovery expands our understanding of eukaryotic hydrogenosomal metabolism, reveals a previously unknown strategy for redox balancing, and highlights potential targets for manipulating H 2 production. These insights have broad implications for microbial energy metabolism, anaerobic ecosystems, and bioengineering of H 2 -producing systems.

Zhang, Bo [Department of Chemical Engineering, Uni↗
DOE OSTI2026

Catalytic Bias of NADH-Dependent Reduced Ferredoxin: NADP+ Oxidoreductase (Nfn) and its Relevance to Ethanol Production in Thermoanaerobacterium Saccharolyticum

NADH-dependent reduced ferredoxin: NADP+ oxidoreductase (Nfn) enzyme catalyzes an energy-conserving flavin-based electron bifurcation (FBEB) reaction. In microbial metabolism, Nfn links redox pools of three electron carriers - ferredoxin (Fd), NAD(H), and NADP(H) - through the following FBEB reaction: 2 NADPH + NAD+ + 2 Fdox 2 NADP+ + NADH + 2 Fdred + H+ The forward reaction is termed electron bifurcation, and the reverse reaction is electron confurcation. Catalytic bias describes an enzyme's tendency to favor one direction of a reversible reaction over the other and is expressed as the ratio of activities in the two directions. Thermoanaerobacterium saccharolyticum (Tsac) is a thermophilic, ethanologenic bacterium that ferments hemicellulose to ethanol at yields above 90%. Its Nfn enzyme is known to support ethanol production, presumably by operating in the confurcating direction to balance cellular cofactors, but this has not previously been demonstrated. To investigate the Tsac Nfn further, we heterologously expressed, purified, and reconstituted the proteins NfnS (NfnA), NfnL (NfnB), and the putative partner Fd with iron-sulfur cluster and/or FAD cofactors. Activity assays monitoring the oxidation or reduction of Fd showed that, across pH 5-10, Tsac Nfn is catalytically biased towards the confurcating direction, favoring NADPH generation over NADPH oxidation by at least fivefold. Using protein electrochemistry, we also determined the reduction potentials of the cofactors in NfnL and Fd. These results indicate that the energetic landscape of FBEB in Tsac Nfn is similar to that in an ortholog. However, Tsac Fd has redox properties distinct from previously assayed Fds, suggesting that the identity and redox properties of Fd may help determine the catalytic bias of Nfn. Additionally, we confirmed the standalone ferredoxin: NADP+ oxidoreductase (FNOR) activity of NfnL but found it to be low and likely insignificant for in vivo redox conversion. We also show how the catalytic bias of Nfn integrates with the hydrogen cycling mechanism proposed in Tsac to better explain cofactor balancing for ethanol production. Our work advances the understanding of electron transfer processes within metabolic networks for the generation of valuable bioproducts.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2014

Selection of chromosomal DNA libraries using a multiplex CRISPR system

The directed evolution of biomolecules to improve or change their activity is central to many engineering and synthetic biology efforts. However, selecting improved variants from gene libraries in living cells requires plasmid expression systems that suffer from variable copy number effects, or the use of complex marker-dependent chromosomal integration strategies. We developed quantitative gene assembly and DNA library insertion into the Saccharomyces cerevisiae genome by optimizing an efficient single-step and marker-free genome editing system using CRISPR-Cas9. With this Multiplex CRISPR (CRISPRm) system, we selected an improved cellobiose utilization pathway in diploid yeast in a single round of mutagenesis and selection, which increased cellobiose fermentation rates by over 10-fold. Mutations recovered in the best cellodextrin transporters reveal synergy between substrate binding and transporter dynamics, and demonstrate the power of CRISPRm to accelerate selection experiments and discoveries of the molecular determinants that enhance biomolecule function.

59 BASIC BIOLOGICAL SCIENCES↗