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Results for “transaminases”

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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13 records
All sourcesNASA NTRSDOE OSTI
DOE OSTI2021

Integration of proteomics and metabolomics into the Design, Build, Test, Learn cycle to improve 3-hydroxypropionic acid production in Aspergillus pseudoterreus

Biological engineering of microorganisms to produce value-added chemicals is a promising route to sustainable manufacturing. However, overproduction of metabolic intermediates at high titer, rate, and yield from inexpensive substrates is challenging in non-model systems where limited information is available regarding metabolic flux and its control in production conditions. Integrated multi-omic analyses of engineered strains offers an in-depth look at metabolites and proteins directly involved in growth and production of target and non-target bioproducts. Here we applied multi-omic analyses to overproduction of the polymer precursor 3-hydroxypropionic acid (3HP) in the filamentous fungus Aspergillus pseudoterreus. A synthetic pathway consisting of aspartate decarboxylase, beta-alanine pyruvate transaminase, and 3HP dehydrogenase was designed and built for A. pseudoterreus. Strains with single- and multi-copy integration events were isolated and multi-omics analysis consisting of intracellular and extracellular metabolomics and targeted and global proteomics was used to interrogate the strains in shake-flask and bioreactor conditions. Production of a variety of co-products (organic acids and glycerol) and oxidative degradation of 3HP were identified as metabolic pathways competing with 3HP production. Intracellular accumulation of nitrogen as 2,4-diaminobutanoate was identified as an off-target nitrogen sink that may also limit flux through the engineered 3HP pathway. Elimination of the high-expression oxidative 3HP degradation pathway by deletion of a putative malonate semialdehyde dehydrogenase improved the yield of 3HP by 3.4× after 10 days in shake-flask culture. This is the first report of 3HP production in a filamentous fungus amenable to industrial scale biomanufacturing of organic acids at high titer and low pH.

Pomraning, Kyle R.↗
DOE OSTI2024

Enzymatic Routes to Designer Hemicelluloses for Use in Biobased Materials

Various enzymes can be used to modify the structure of hemicelluloses directly in vivo or following extraction from biomass sources, such as wood and agricultural residues. Generally, these enzymes can contribute to designer hemicelluloses through four main strategies: (1) enzymatic hydrolysis such as selective removal of side groups by glycoside hydrolases (GH) and carbohydrate esterases (CE), (2) enzymatic cross-linking, for instance, the selective addition of side groups by glycosyltransferases (GT) with activated sugars, (3) enzymatic polymerization by glycosynthases (GS) with activated glycosyl donors or transglycosylation, and (4) enzymatic functionalization, particularly via oxidation by carbohydrate oxidoreductases and via amination by amine transaminases. Thus, this Perspective will first highlight enzymes that play a role in regulating the degree of polymerization and side group composition of hemicelluloses, and subsequently, it will explore enzymes that enhance cross-linking capabilities and incorporate novel chemical functionalities into saccharide structures. These enzymatic routes offer a precise way to tailor the properties of hemicelluloses for specific applications in biobased materials, contributing to the development of renewable alternatives to conventional materials derived from fossil fuels.

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

Reductive amination cascades in cell‐free and resting whole cell formats for valorization of lignin deconstruction products

Abstract The selective introduction of amine groups within deconstruction products of lignin could provide an avenue for valorizing waste biomass while achieving a green synthesis of industrially relevant building blocks from sustainable sources. Here, we built and characterized enzyme cascades that create aldehydes and subsequently primary amines from diverse lignin‐derived carboxylic acids using a carboxylic acid reductase (CAR) and an ω‐transaminase (TA). Unlike previous studies that have paired CAR and TA enzymes, here we examine multiple homologs of each of these enzymes and a broader set of candidate substrates. In addition, we compare the performance of these systems in cell‐free and resting whole‐cell biocatalysis formats using the conversion of vanillate to vanillyl amine as model chemistry. We also demonstrate that resting whole cells can be recycled for multiple batch reactions. We used the knowledge gained from this study to produce several amines from carboxylic acid precursors using one‐pot biocatalytic reactions, several of which we report for the first time. These results expand our knowledge of these industrially relevant enzyme families to new substrates and contexts for environmentally friendly and potentially low‐cost synthesis of diverse aryl aldehydes and amines.

Biotechnology & Applied Microbiology↗
DOE OSTI2026

Biocatalytic Carboxylic Acid Reduction and Transamination in Cell‐Free Lysates at High Substrate Loading

Chemoselective reduction of stable carboxylic acids to reactive aldehydes is of interest across many industries. While carboxylic acid reductases (CARs) are promising biocatalysts for this chemistry, poor chemoselectivity and low yield are commonly obtained when using less expensive crude lysate preparations and prerequisite ATP and NADPH regeneration systems. Here, in this work, we developed a highly chemoselective multienzyme cascade featuring a CAR and an ω-transaminase (TA) in crude lysate format, with conversion of the dicarboxylic acid terephthalic acid (TPA) into the diamine para -xylylenediamine (pXDA) as the model chemistry. We improved chemoselectivity for pXDA using engineered aldehyde-stabilizing Escherichia coli strains, though desired product yields remained modest. We next found that CAR activity was limited at high substrate loadings and overcame this bottleneck by modulating the ratio of polyphosphate (polyP 6 ) to Mg 2+ , enabling volumetric scaling and increased substrate loading up to 50 mM TPA. We then showcased the portability of this platform across substrates, resulting in the synthesis of four other high-value amines from carboxylate precursors. The combination of high carboxyl group turnover, up to 93.5 mM under the tested conditions, and the simplicity of crude enzyme preparation is a promising platform for sustainable functional group interconversion.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2020

Pyridoxal 5'-phosphate dependent reactions: Analyzing the mechanism of aspartate aminotransferase

Enzyme catalysis is the primary activity in energy and information metabolism and enzyme cofactors are key to the catalytic ability of most enzymes. Pyridoxal 5'-phosphate (PLP) cofactor, derived from Vitamin B6, is widely distributed in nature and has significant latitude in catalytic diversity. X-ray crystallography has revealed the structures of diverse PLP dependent enzymes from multiple families. But these structures are incomplete, lacking the positions of protons essential for understanding enzymatic mechanisms. Here, we review the diversity of PLP and discuss the use of neutron crystallography and joint X-ray/neutron refinement of Fold Type I aspartate aminotransferase to visualize the positions of protons in both the internal and external aldimine forms. Strategies used to prepare extremely large crystals required for neutron diffraction and the approach to data refinement including the PLP cofactor are discussed. The observed positions of protons, including one located in a previously unknown low-barrier hydrogen bond, have been used to create more accurate models for computational analysis. The results revealed a new mechanism for the transaminase reaction where hyperconjugation is key to reducing the energy barrier which finally provides a clear explanation of the Dunathan alignment.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2021

Tetramethylpyrazine inhibits neutrophil extracellular traps formation and alleviates hepatic ischemia/reperfusion injury in rat liver transplantation

Hepatic ischemia/reperfusion injury (IRI) is an adverse effect for liver transplantation which is characterized by immune response mediated inflammation. Recent studies report that neutrophil extracellular traps (NETs) are implicated in hepatic IRI. The aim of this study was to explore the mechanism of action of tetramethylpyrazine (TMP), the main chemical composition of Ligusticum chuanxiong in treatment of ischemic related diseases. Data showed that hepatic IRI increases the leak of alanine aminotransferase (ALT) and aspartate transaminase (AST), and stimulates formation of NETs. Extracellular DNA/NETs assay, hematoxylin-eosin (HE) staining, immunofluorescence assay, terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) and Western blot assay, showed that TMP significantly reduces formation of NETs and alleviates hepatic IRI. Moreover, TMP and Diphenyleneiodonium (DPI) suppressed ROS production in neutrophils. In addition, analysis showed that activation of NADPH oxidase plays a role in formation of NETs triggered by hepatic IRI. Notably, TMP inhibited formation of NETs though inhibition of NADPH oxidase. Additionally, Combination treatment using TMP and DPI was more effective compared with monotherapy of either of the two drugs. These findings show that combination therapy using TMP and DPI is a promising method for treatment hepatic IRI.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2020

Reaction Energetics and 13 C Fractionation of Alanine Transamination in the Aqueous and Gas Phases

The alanine transaminase enzyme catalyzes the transfer of an amino group from alanine to α-ketoglutarate to produce pyruvate and glutamate. Isotope fractionation factors (IFFs) for the reaction +H 3 NCH(CH 3 )COO – + – OOCCH 2 CH 2 C(O)COO – ↔ CH 3 C(O)COO – + + H 3 NCH(CH 2 CH 2 COO – )COO – (zwitterionic neutral alanine + doubly deprotonated α-ketoglutarate ↔ pyruvate + zwitterionic glutamate anion) were calculated from the partition functions of explicitly and implicitly solvated molecules at 298 K. Calculations were done for alanine (noncharge separated, zwitterion, deprotonated), pyruvic acid (neutral, deprotonated), glutamic acid (noncharge separated, zwitterion, deprotonated, doubly deprotonated), and α-ketoglutaric acid (neutral, deprotonated, doubly deprotonated). The computational results, calculated from gas phase- and aqueous-optimized clusters with explicit H 2 O molecules at the MP2/aug-cc-pVDZ and MP2/aug-cc-pVDZ/COSMO levels, respectively, predict that substitution of 13 C at the C2 position of alanine and pyruvic acid and their various forms leads to the C2 position of pyruvic acid/pyruvate being enriched in 13 C/ 12 C ratio by 9‰. Simpler approaches that estimate the IFFs based solely on changes in the zero-point energies (ZPEs) are consistent with the higher-level model. ZPE-based IFFs calculated for simple analogues formaldehyde and methylamine (analogous to the C2 positions of pyruvate and alanine, respectively) predict a 13 C enrichment in formaldehyde of 7–8‰ at the MP2/aug-cc-pVDZ and aug-cc-pVTZ levels. A simple predictive model using canonical functional group frequencies and reduced masses for 13 C exchange between R 2 C=O and R 2 CH–NH 2 predicted enrichment in R 2 C=O that is too large by a factor of two but is qualitatively accurate compared with the more sophisticated models. Our models are all in agreement with the expectation that pyruvate and formaldehyde will be preferentially enriched in 13 C because of the strength of their >C=O bond relative to that of ≡C–NH 2 in alanine and methylamine. 13 C/ 12 C substitution is also modeled at the methyl and carboxylic acid sites of alanine and pyruvic acid, respectively.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2025

A polyketide-based biosynthetic platform for diols, amino alcohols and hydroxy acids

Medium- and branched-chain diols and amino alcohols are important industrial solvents, polymer building blocks, cosmetics and pharmaceutical ingredients, yet biosynthetically challenging to produce. Here we present an approach that uses a modular polyketide synthase (PKS) platform for the efficient production of these compounds. This platform takes advantage of a versatile loading module from the rimocidin PKS and nicotinamide adenine dinucleotide phosphate-dependent terminal thioreductases. Reduction of the terminal aldehyde with alcohol dehydrogenases enables the production of diols, oxidation enables the production of hydroxy acids and specific transaminases allow the production of various amino alcohols. Furthermore, replacement of the malonyl-coenzyme A-specific acyltransferase in the extension module with methyl- or ethylmalonyl-coenzyme A-specific acyltransferase enables the production of branched-chain diols, amino alcohols and carboxylic acids in high titres. Use of our PKS platform in Streptomyces albus demonstrated the high tunability and efficiency of the platform.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2025

Pathogenesis of Chapare Virus in Cynomolgus Macaques

Chapare virus (CHAPV) is an emerging New World arenavirus that is the causative agent of Chapare hemorrhagic fever (CHHF) responsible for recent outbreaks with alarmingly high case fatality rates in Bolivia near the Brazilian border. Here, we describe a nonhuman primate (NHP) model of CHHF infection which represents an essential tool to understand this emerging biological threat agent. Cynomolgus macaques challenged intravenously with CHAPV develop clinical disease, which recapitulates several key features of human CHHF. All subjects lost weight and had clinical scores following CHAPV challenge. Notably, one of four NHPs developed lethal disease with viral hepatitis and hemorrhagic features. Clinical chemistry and hematology revealed leukopenia, anemia, thrombocytopenia, and increased transaminase levels. In all four subjects, viremia was detectable for the first week following challenge and viral RNA was detectable in serum and many tissues persisting 35 days-post challenge. Several medical countermeasures (MCM) have efficacy against CHAPV infection in vitro, but the current model for MCM testing and approval of new drugs is reliant on the availability of animal models. This work lays the foundation for future CHHF MCM development.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2020

Co-catabolism of arginine and succinate drives symbiotic nitrogen fixation

Biological nitrogen fixation emerging from the symbiosis between bacteria and crop plants holds promise to increase the sustainability of agriculture. One of the biggest hurdles for the engineering of nitrogen-fixing organisms is an incomplete knowledge of metabolic interactions between microbe and plant. In contrast to the previously assumed supply of only succinate, we describe here the CATCH-N cycle as a novel metabolic pathway that co-catabolizes plant provided arginine and succinate to drive the energy-demanding process of symbiotic nitrogen fixation in endosymbiotic rhizobia. Using systems biology, isotope labeling studies and transposon sequencing in conjunction with biochemical characterization, we uncovered highly redundant network components of the CATCH-N cycle including transaminases that interlink the co-catabolism of arginine and succinate. The CATCH-N cycle uses N2 as an additional sink for reductant and therefore delivers up to 25% higher yields of nitrogen than classical arginine catabolism—two alanines and three ammonium ions are secreted for each input of arginine and succinate. We argue that the CATCH-N cycle has evolved as part of a synergistic interaction to sustain bacterial metabolism in the microoxic and highly acid environment of symbiosomes. Thus, the CATCHN cycle entangles the metabolism of both partners to promote symbiosis. Our results provide a theoretical framework and metabolic blueprint for the rational design of plants and plant-associated organisms with new properties to improve nitrogen fixation.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

Postbiotic Gamma-Aminobutyric Acid and Camel Milk Intervention as Innovative Trends Against Hyperglycemia and Hyperlipidemia in Streptozotocin-Induced C57BL/6J Diabetic Mice

Diabetes is a serious disease that threatens human health worldwide. The study hypothesis is to investigate the novel trends that may aid in the prevention of diabetic complications. Camel milk was presented as traditional functional food, and Lactobacillus brevis KLDS 1.0727 and KLDS 1.0373 strains were shown to synthesize postbiotic Gamma-aminobutyric acid as a potential food additive, which can therapeutically intervene against hyperglycemia and hyperlipidemia in streptozotocin-induced C 57 BL/6J mice. During a four-week timeframe, body weight and postprandial blood glucose levels were monitored. Post-euthanasia, blood plasma was obtained to investigate hyperlipidemia, insulin concentrations, liver, and renal functions. The liver, pancreas, kidney, and spleen underwent histopathological examinations. The results demonstrated that KLDS 1.0727 and KLDS 1.0373 ( LAC S1 , LAC S2 ) and camel milk treatments all had a significant influence on hypoglycemic activity, as evidenced by reduced postprandial blood glucose levels. LAC S1 , LAC S2 , and camel milk therapy significantly reduced blood hypolipidemic, and some liver enzymes such as (alanine aminotransferase and aspartate transaminase) levels. Therefore, we recommend consuming camel milk regularly and expanding its use with fermented foods containing L. brevis , one of the probiotics capable of producing gamma-aminobutyric acid (GABA) as future food additives that can improve human health and reduce the prevalence of several diseases disorders.

Abdelazez, Amro↗
DOE OSTI2024

Constructing the Nitrogen Flux Maps (NFMs) of Plants

The main objectives of this project are to construct plant N flux maps (NFMs) from plant genomes and to determine functionality of AT enzymes and plant N metabolic network. To address this grand challenge, this project made use of rapidly growing numbers of plant genomes, high-throughput functional characterization platforms, and computational modeling to deduce both biochemical and systems level functionality of ATs and NFMs. The obtained NFMs will provide a novel framework to advance basic understanding of plant N metabolism and facilitate rational engineering of plants with high productivity even under limited N input.

59 BASIC BIOLOGICAL SCIENCES↗