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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 433 records · Page 24
All sourcesNASA NTRSDOE OSTI
DOE OSTI2021

Engineering a Non‐Natural Photoenzyme for Improved Photon Efficiency**

Abstract Photoenzymes are biological catalysts that use light to convert starting materials into products. These catalysts require photon absorption for each turnover, making quantum efficiency an important optimization parameter. Flavin‐dependent “ene”‐reductases (EREDs) display latent photoenzymatic activity for synthetically valuable hydroalkylations; however, protein engineering has not been used to optimize this non‐natural function. We describe a protein engineering platform for the high throughput optimization of photoenzymes. A single round of engineering results in improved catalytic function toward the synthesis of γ, δ, ϵ‐lactams, and acyclic amides. Mechanistic studies show that key mutations can alter the enzyme's excited state dynamics, enhance its photon efficiency, and ultimately increase catalyst performance. Transient absorption spectroscopy reveals that engineered variants display dramatically decreased radical lifetimes, indicating an evolution toward a concerted mechanism.

Nicholls, Bryce T.↗
DOE OSTI2023

Mapping protein dynamics at high spatial resolution with temperature-jump X-ray crystallography

Understanding and controlling protein motion at atomic resolution is a hallmark challenge for structural biologists and protein engineers because conformational dynamics are essential for complex functions such as enzyme catalysis and allosteric regulation. Time-resolved crystallography offers a window into protein motions, yet without a universal perturbation to initiate conformational changes the method has been limited in scope. Here we couple a solvent-based temperature jump with time-resolved crystallography to visualize structural motions in lysozyme, a dynamic enzyme. We observed widespread atomic vibrations on the nanosecond timescale, which evolve on the submillisecond timescale into localized structural fluctuations that are coupled to the active site. An orthogonal perturbation to the enzyme, inhibitor binding, altered these dynamics by blocking key motions that allow energy to dissipate from vibrations into functional movements linked to the catalytic cycle. Because temperature jump is a universal method for perturbing molecular motion, the method demonstrated here is broadly applicable for studying protein dynamics.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2026

Nitrene generation and transfer for unnatural biosynthesis in living cells

Abstract Synthetic biology has enabled the production of natural and unnatural products from inexpensive sustainable feedstocks. Yet, the scope of available products has been limited largely to compounds accessible from nature’s chemical reactions. Evolved enzymes can catalyse reactions of unnatural substrates or reactions not found in nature but often require the addition of synthetic reagents to purified enzymes or to resting cells containing those enzymes. Here we show that chemical reactions of metabolic intermediates produced intracellularly in living cells can include intermolecular nitrene transfers. The biosynthesis of N -acetoxyanilines, in combination with the generation and transfer of N -aryl nitrene intermediates from them catalysed by a cytochrome P450, generates amino alcohols, diamines, diarylamines and aminoalkyl arenes from simple carbon feedstocks. These products—common substructures of pharmaceuticals and agrochemicals—are challenging to synthesize by standard organic chemistry and were produced from inexpensive, renewable feedstocks. Evolution of the enzymes in this pathway showed that titres can be increased by engineering and that the products can be synthesized with high enantioselectivity.

Donnell, Isaac↗
DOE OSTI2013

Production of extremophilic bacterial cellulase enzymes in aspergillus niger.

Enzymes can be used to catalyze a myriad of chemical reactions and are a cornerstone in the biotechnology industry. Enzymes have a wide range of uses, ranging from medicine with the production of pharmaceuticals to energy were they are applied to biofuel production. However, it is difficult to produce large quantities of enzymes, especially if they are non-native to the production host. Fortunately, filamentous fungi, such as Aspergillus niger, are broadly used in industry and show great potential for use a heterologous enzyme production hosts. Here, we present work outlining an effort to engineer A. niger to produce thermophilic bacterial cellulases relevant to lignocellulosic biofuel production.

Gladden, John Michael↗
DOE OSTI2018

Engineering cellulolytic bacterium Clostridium thermocellum to co-ferment cellulose- and hemicellulose-derived sugars simultaneously

Here, cellulose and hemicellulose are the most abundant components in plant biomass. A preferred Consolidated Bioprocessing (CBP) system is one which can directly convert both cellulose and hemicellulose into target products without adding the costly hydrolytic enzyme cocktail. In this work, the thermophilic, cellulolytic, and anaerobic bacterium, Clostridium thermocellum DSM 1313, was engineered to grow on xylose in addition to cellulose. Both xylA (encoding for xylose isomerase) and xylB (encoding for xylulokinase) genes from the thermophilic anaerobic bacterium Thermoanaerobacter ethanolicus were introduced to enable xylose utilization while still retaining its inherent ability to grow on 6-carbon substrates. Targeted integration of xylAB into C. thermocellum genome realized simultaneous fermentation of xylose with glucose, with cellobiose (glucose dimer), and with cellulose, respectively, without carbon catabolite repression. We also showed that the respective H 2 and ethanol production were twice as much when both xylose and cellulose were consumed simultaneously than when consuming cellulose alone. Moreover, the engineered xylose consumer can also utilize xylo-oligomers (with degree of polymerization of 2-7) in the presence of xylose. Isotopic tracer studies also revealed that the engineered xylose catabolism contributed to the production of ethanol from xylan which is a model hemicellulose in mixed sugar fermentation, demonstrating immense potential of this enhanced CBP strain in co-utilizing both cellulose and hemicellulose for the production of fuels and chemicals.

09 BIOMASS FUELS↗
NASA NTRS1986

ECUT (Energy Conversion and Utilization Technologies Program). Biocatalysis Project

Presented are the FY 1985 accomplishments, activities, and planned research efforts of the Biocatalysis Project of the U.S. Department of Energy, Energy Conversion and Utilization Technologies (ECUT) Program. The Project's technical activities were organized as follows: In the Molecular Modeling and Applied Genetics work element, research focused on (1) modeling and simulation studies to establish the physiological basis of high temperature tolerance in a selected enzyme and the catalytic mechanisms of three species of another enzyme, and (2) determining the degree of plasmid amplification and stability of several DNA bacterial strains. In the Bioprocess Engineering work element, research focused on (1) studies of plasmid propagation and the generation of models, (2) developing methods for preparing immobilized biocatalyst beads, and (3) developing an enzyme encapsulation method. In the Process Design and Analysis work element, research focused on (1) further refinement of a test case simulation of the economics and energy efficiency of alternative biocatalyzed production processes, (2) developing a candidate bioprocess to determine the potential for reduced energy consumption and facility/operating costs, and (3) a techno-economic assessment of potential advancements in microbial ammonia production.

Source record↗
DOE OSTI2020

Combining Random Mutagenesis and Metabolic Engineering for Enhanced Tryptophan Production in Synechocystis sp. Strain PCC 6803

Tryptophan (Trp) is an essential aromatic amino acid that has value as an animal feed supplement, as the amount found in plant-based sources is insufficient. An alternative to production by engineered microbial fermentation is to have tryptophan biosynthesized by a photosynthetic microorganism that could replace or supplement both the plant and industrially used microbes. We selected Synechocystis sp. strain PCC 6803, a model cyanobacterium, as the host and studied metabolic engineering and random mutagenesis approaches. Previous work on engineering heterotrophic microbes for improved Trp titers has targeted allosteric feedback regulation in enzymes 3-deoxy-d-arabinoheptulosonate 7-phosphate synthase (DAHPS) and anthranilate synthase (AS) as major bottlenecks in the shikimate pathway. In this work, the genes encoding feedback-resistant enzymes from Escherichia coli, aroG fbr and trpE fbr , were overexpressed in the host wild-type (WT) strain. Separately, the WT strain was subjected to random mutagenesis and selection using an amino acid analog to isolate tryptophan-overproducing strains. The randomly mutagenized strains were sequenced in order to identify the mutations that resulted in the desirable phenotypes. Interestingly, the tryptophan overproducers had mutations in the gene encoding chorismate mutase (CM), which catalyzes the conversion of chorismate to prephenate. The best tryptophan overproducer from random mutagenesis was selected as a host for metabolic engineering where aroG fbr and trpE fbr were overexpressed. The best strain developed produced 212 ± 23 mg/liter of tryptophan after 10 days of photoautotrophic growth under 3% (vol/vol) CO2. We demonstrated that a combination of random mutagenesis and metabolic engineering was superior to either individual approach.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2015

Development of a High Throughput Platform for Screening Glycoside Hydrolases Based on Oxime-NIMS

Cost-effective hydrolysis of biomass into sugars for biofuel production requires high-performance low-cost glycoside hydrolase (GH) cocktails that are active under demanding process conditions. Improving the performance of GH cocktails depends on knowledge of many critical parameters, including individual enzyme stabilities, optimal reaction conditions, kinetics, and specificity of reaction. With this information, rate- and/or yield-limiting reactions can be potentially improved through substitution, synergistic complementation, or protein engineering. Given the wide range of substrates and methods used for GH characterization, it is difficult to compare results across a myriad of approaches to identify high performance and synergistic combinations of enzymes. Here, we describe a platform for systematic screening of GH activities using automatic biomass handling, bioconjugate chemistry, robotic liquid handling, and nanostructure-initiator mass spectrometry (NIMS). Twelve well-characterized substrates spanning the types of glycosidic linkages found in plant cell walls are included in the experimental workflow. To test the application of this platform and substrate panel, we studied the reactivity of three engineered cellulases and their synergy of combination across a range of reaction conditions and enzyme concentrations. We anticipate that large-scale screening using the standardized platform and substrates will generate critical datasets to enable direct comparison of enzyme activities for cocktail design.

09 BIOMASS FUELS↗
DOE OSTI2013

Redirector: Designing Cell Factories by Reconstructing the Metabolic Objective

Advances in computational metabolic optimization are required to realize the full potential of new in vivo metabolic engineering technologies by bridging the gap between computational design and strain development. We present Redirector, a new Flux Balance Analysis-based framework for identifying engineering targets to optimize metabolite production in complex pathways. Previous optimization frameworks have modeled metabolic alterations as directly controlling fluxes by setting particular flux bounds. Redirector develops a more biologically relevant approach, modeling metabolic alterations as changes in the balance of metabolic objectives in the system. This framework iteratively selects enzyme targets, adds the associated reaction fluxes to the metabolic objective, thereby incentivizing flux towards the production of a metabolite of interest. These adjustments to the objective act in competition with cellular growth and represent up-regulation and down-regulation of enzyme mediated reactions. Using the iAF1260 E. coli metabolic network model for optimization of fatty acid production as a test case, Redirector generates designs with as many as 39 simultaneous and 111 unique engineering targets. These designs discover proven in vivo targets, novel supporting pathways and relevant interdependencies, many of which cannot be predicted by other methods. Redirector is available as open and free software, scalable to computational resources, and powerful enough to find all known enzyme targets for fatty acid production.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2019

Cell-free biosynthesis of limonene using enzyme-enriched Escherichia coli lysates

Abstract Isoprenoids are an attractive class of metabolites for enzymatic synthesis from renewable substrates. However, metabolic engineering of microorganisms for monoterpenoid production is limited by the need for time-consuming, and often non-intuitive, combinatorial tuning of biosynthetic pathway variations to meet design criteria. Towards alleviating this limitation, the goal of this work was to build a modular, cell-free platform for construction and testing of monoterpenoid pathways, using the fragrance and flavoring molecule limonene as a model. In this platform, multiple Escherichia coli lysates, each enriched with a single overexpressed pathway enzyme, are mixed to construct the full biosynthetic pathway. First, we show the ability to synthesize limonene from six enriched lysates with mevalonate substrate, an adenosine triphosphate (ATP) source, and cofactors. Next, we extend the pathway to use glucose as a substrate, which relies on native metabolism in the extract to convert glucose to acetyl-CoA along with three additional enzymes to convert acetyl-CoA to mevalonate. We find that the native E. coli farnesyl diphosphate synthase (IspA) is active in the lysate and diverts flux from the pathway intermediate geranyl pyrophospahte to farnesyl pyrophsophate and the byproduct farnesol. By adjusting the relative levels of cofactors NAD+, ATP and CoA, the system can synthesize 0.66 mM (90.2 mg l−1) limonene over 24 h, a productivity of 3.8 mg l−1 h−1. Our results highlight the flexibility of crude lysates to sustain complex metabolism and, by activating a glucose-to-limonene pathway with 9 heterologous enzymes encompassing 20 biosynthetic steps, expands an approach of using enzyme-enriched lysates for constructing, characterizing and prototyping enzymatic pathways.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

Current trends in waste valorization

This paper presents the scientific breakthroughs made in bioprocess engineering and microbial biotechnology for the conversion of wastes into products with added value and/or biofuels. The significant results obtained in the emerging fields of hybrid electrosynthesis, the role of enzymes in the degradation of plastics, polyhydroxyalkanoate and 5-aminolevulinic acid production, fermentation technology and the application of molecular engineering tools to bioprocess technology are highlighted.

09 BIOMASS FUELS↗
DOE OSTI2012

Characterization of xylan utilization and discovery of a new endoxylanase in Thermoanaerobacterium saccharolyticum through targeted gene deletions

The economical production of fuels and commodity chemicals from lignocellulose requires the utilization of both the cellulose and hemicellulose fractions. Xylanase enzymes allow greater utilization of hemicellulose while also increasing cellulose hydrolysis. Recent metabolic engineering efforts have resulted in a strain of Thermoanaerobacterium saccharolyticum that can convert C(5) and C(6) sugars, as well as insoluble xylan, into ethanol at high yield. To better understand the process of xylan solubilization in this organism, a series of targeted deletions were constructed in the homoethanologenic T. saccharolyticum strain M0355 to characterize xylan hydrolysis and xylose utilization in this organism. While the deletion of -xylosidase xylD slowed the growth of T. saccharolyticum on birchwood xylan and led to an accumulation of short-chain xylo-oligomers, no other single deletion, including the deletion of the previously characterized endoxylanase XynA, had a phenotype distinct from that of the wild type. This result indicates a multiplicity of xylanase enzymes which facilitate xylan degradation in T. saccharolyticum. Growth on xylan was prevented only when a previously uncharacterized endoxylanase encoded by xynC was also deleted in conjunction with xynA. Sequence analysis of xynC indicates that this enzyme, a low-molecular-weight endoxylanase with homology to glycoside hydrolase family 11 enzymes, is secreted yet untethered to the cell wall. Together, these observations expand our understanding of the enzymatic basis of xylan hydrolysis by T. saccharolyticum.

Podkaminer, Kara K↗
DOE OSTI2022

Divergent evolution of extreme production of variant plant monounsaturated fatty acids

Metabolic extremes provide opportunities to understand enzymatic and metabolic plasticity and biotechnological tools for novel biomaterial production. We discovered that seed oils of many Thunbergia species contain up to 92% of the unusual monounsaturated petroselinic acid (18:1Δ6), one of the highest reported levels for a single fatty acid in plants. Supporting the biosynthetic origin of petroselinic acid, we identified a Δ6-stearoyl-acyl carrier protein (18:0-ACP) desaturase from Thunbergia laurifolia , closely related to a previously identified Δ6-palmitoyl-ACP desaturase that produces sapienic acid (16:1Δ6)-rich oils in Thunbergia alata seeds. Guided by a T. laurifolia desaturase crystal structure obtained in this study, enzyme mutagenesis identified key amino acids for functional divergence of Δ6 desaturases from the archetypal Δ9-18:0-ACP desaturase and mutations that result in nonnative enzyme regiospecificity. Furthermore, we demonstrate the utility of the T. laurifolia desaturase for the production of unusual monounsaturated fatty acids in engineered plant and bacterial hosts. Through stepwise metabolic engineering, we provide evidence that divergent evolution of extreme petroselinic acid and sapienic acid production arises from biosynthetic and metabolic functional specialization and enhanced expression of specific enzymes to accommodate metabolism of atypical substrates.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2018

Cell-free biomanufacturing

Since its early development and use to decipher the genetic code, in vitro or ‘cell-free’ systems have been used as an important research tool to understand biochemical mechanisms and metabolic pathways. More recently, due to important engineering advances the technology is rapidly becoming a biomanufacturing platform for protein therapeutics, vaccines, enzyme biocatalysts, fuels, and commodity chemicals. Herein we report recent applications and advances in the cell-free biomanufacturing field and the potential of this emerging approach.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2015

Functional metabolomics: from biomarker discovery to metabolome reprogramming

Metabolomics is emerging as a powerful tool for studying metabolic processes, identifying crucial biomarkers responsible for metabolic characteristics and revealing metabolic mechanisms, which construct the content of discovery metabolomics. The crucial biomarkers can be used to reprogram a metabolome, leading to an aimed metabolic strategy to cope with alteration of internal and external environments, naming reprogramming metabolomics here. The striking feature on the similarity of the basic metabolic pathways and components among vastly different species makes the reprogramming metabolomics possible when the engineered metabolites play biological roles in cellular activity as a substrate of enzymes and a regulator to other molecules including proteins. The reprogramming metabolomics approach can be used to clarify metabolic mechanisms of responding to changed internal and external environmental factors and to establish a framework to develop targeted tools for dealing with the changes such as controlling and/or preventing infection with pathogens and enhancing host immunity against pathogens. This review introduces the current state and trends of discovery metabolomics and reprogramming metabolomics and highlights the importance of reprogramming metabolomics.

reprogramming metabolomics↗
DOE OSTI2010

Designing a highly active soluble PQQ-glucose dehydrogenase for efficient glucose biosensors and biofuel cells

Research highlights: {yields} A new mutant of PQQ-GDH designed for glucose biosensors application. {yields} First mutant of PQQ-GDH with higher activity for D-glucose than the Wild type. {yields} Position N428 is a key point to increase the enzyme activity. {yields} Molecular modeling shows that the N428 C mutant displays a better interaction for PQQ than the WT. -- Abstract: We report for the first time a soluble PQQ-glucose dehydrogenase that is twice more active than the wild type for glucose oxidation and was obtained by combining site directed mutagenesis, modelling and steady-state kinetics. The observed enhancement is attributed to a better interaction between the cofactor and the enzyme leading to a better electron transfer. Electrochemical experiments also demonstrate the superiority of the new mutant for glucose oxidation and make it a promising enzyme for the development of high-performance glucose biosensors and biofuel cells.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2025

Identification and characterization of substrate- and product-selective nylon hydrolases

Enzymes can rapidly and selectively hydrolyze diverse natural and anthropogenic polymers, but few have been shown to hydrolyze synthetic polyamides. Here, in this work, we synthesized and characterized a panel of 95 enzymes from the N-terminal nucleophile hydrolase superfamily with 30%–50% pairwise amino acid identity. We found that nearly 40% of the enzymes had substantial nylon hydrolase activity, but there was no relationship between phylogeny and activity, nor any evidence of prior evolutionary selection for nylon hydrolysis. Several newly identified hydrolases showed substrate selectivity, generating up to 20-fold higher product titers with nylon-6,6 versus nylon-6. However, the yield was still less than 1%, necessitating further optimization before potential applications. Finally, we determined the crystal structure and oligomerization state of a nylon-6,6-selective hydrolase to elucidate structural factors that could affect activity and selectivity. These new enzymes provide insights into nylon hydrolase evolution and opportunities for analysis and engineering of improved hydrolases.

nylon↗