Search NASA⌕ Search

SEARCH · Search NASA

Results for “biocatalysis”

Search indexed NASA NTRS and DOE OSTI research on propulsion, heat transfer, battery materials and energy systems. Follow report and document links to the original sources.

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

At least 91 records · Page 5
All sourcesNASA NTRSDOE OSTI
DOE OSTI2024

teemi: An open-source literate programming approach for iterative design-build-test-learn cycles in bioengineering

Synthetic biology dictates the data-driven engineering of biocatalysis, cellular functions, and organism behavior. Integral to synthetic biology is the aspiration to efficiently find, access, interoperate, and reuse high-quality data on genotype-phenotype relationships of native and engineered biosystems under FAIR principles, and from this facilitate forward-engineering strategies. However, biology is complex at the regulatory level, and noisy at the operational level, thus necessitating systematic and diligent data handling at all levels of the design, build, and test phases in order to maximize learning in the iterative design-build-test-learn engineering cycle. To enable user-friendly simulation, organization, and guidance for the engineering of biosystems, we have developed an open-source python-based computer-aided design and analysis platform operating under a literate programming user-interface hosted on Github. The platform is called teemi and is fully compliant with FAIR principles. In this study we apply teemi for i) designing and simulating bioengineering, ii) integrating and analyzing multivariate datasets, and iii) machine-learning for predictive engineering of metabolic pathway designs for production of a key precursor to medicinal alkaloids in yeast. The teemi platform is publicly available at PyPi and GitHub.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

Absolute Reactive Oxygen Species Densities in the Effluent of the COST Reference Source and Plasma-generated Atomic Oxygen Density Measurements in Liquid using TALIF

Reactive oxygen species are one of the most important species produced in a plasma. In particular the role of atomic oxygen and the hydroxyl radical OH has attracted many studies ranging from plasma medicine to plasma-driven biocatalysis and water treatment. Atomic oxygen and the hydroxyl radical OH are very reactive and react unselectively with many different substrates. The quantification of atomic oxygen and OH is challenging because of their reactivity and unknown reactions and rate constants.

70 PLASMA PHYSICS AND FUSION TECHNOLOGY↗
DOE OSTI2021

Anaerobic Fungal Mevalonate Pathway Genomic Biases Lead to Heterologous Toxicity Underpredicted by Codon Adaptation Indices

Anaerobic fungi are emerging biotechnology platforms with genomes rich in biosynthetic potential. Yet, the heterologous expression of their biosynthetic pathways has had limited success in model hosts like E. coli. We find one reason for this is that the genome composition of anaerobic fungi like P. indianae are extremely AT-biased with a particular preference for rare and semi-rare AT-rich tRNAs in E coli, which are not explicitly predicted by standard codon adaptation indices (CAI). Native P. indianae genes with these extreme biases create drastic growth defects in E. coli (up to 69% reduction in growth), which is not seen in genes from other organisms with similar CAIs. However, codon optimization rescues growth, allowing for gene evaluation. In this manner, we demonstrate that anaerobic fungal homologs such as PI.atoB are more active than S. cerevisiae homologs in a hybrid pathway, increasing the production of mevalonate up to 2.5 g/L (more than two-fold) and reducing waste carbon to acetate by ~90% under the conditions tested. This work demonstrates the bioproduction potential of anaerobic fungal enzyme homologs and how the analysis of codon utilization enables the study of otherwise difficult to express genes that have applications in biocatalysis and natural product discovery.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2025

Machine Learning-Guided Identification of PET Hydrolases from Natural Diversity

The enzymatic depolymerization of poly(ethylene terephthalate) (PET) is emerging as a leading chemical recycling technology for waste polyester. As part of this endeavor, new candidate enzymes identified from natural diversity can serve as useful starting points for enzyme evolution and engineering. In this study, we improved upon HMM searches by applying an iterative machine learning strategy to identify 400 putative PET-degrading enzymes (PET hydrolases) from naturally occurring homologs. Using high-throughput (HTP) experimental techniques, we successfully expressed and purified >200 enzyme candidates and assayed them for PET hydrolysis activity as a function of pH, temperature, and substrate crystallinity. From this library, we discovered 91 previously unknown PET hydrolases, 35 of which retain activity at pH 4.5 on crystalline material, which are conditions relevant to developing more efficient commercial processes. Notably, four enzymes showed equal to or higher activity than LCC-ICCG, a benchmark PET hydrolase, at this challenging condition in our screening assay, and 11 of which have pH optima <7. Using these data, we identified regions of PETases statistically correlated to activity at lower pH. We additionally investigated the effect of condition-specific activity data on trained machine learning predictors and found a precision (putative hit rate) improvement of up to 30% compared to a Hidden Markov Model alone. Our findings show that by pointing enzyme discovery toward conditions of interest with multiple rounds of experimental and machine learning, we can discover large sets of active enzymes and explore factors associated with activity at those conditions.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2026

Engineering and Application of a Thermostable MHETase for PET Depolymerization

Enzymatic hydrolysis of poly(ethylene terephthalate) (PET) releases mono(2-hydroxyethyl) terephthalate (MHET) as a major product, the accumulation of which can prolong reactor residence times and complicate downstream monomer separations. The use of a MHETase enzyme can enable MHET hydrolysis to the monomers, terephthalic acid and ethylene glycol, but industrial PETases typically operate at thermophilic temperatures and the well-known MHETase from Ideonella sakaiensis is a mesophilic enzyme, thus warranting the development of thermophilic MHETases. Here, we characterize thermostable MHET-active enzymes from a natural diversity screen by applying a hidden Markov model based on the previously reported, archaeal ferulic acid esterase, PET46. We identified enzymes with higher thermostability than PET46 and quantified their MHETase activity in reactions at 70 °C. The crystal structure of MHT077, the homologue with the highest MHETase activity and an apparent melting temperature (T m,app ) of 94.6 °C, informed site saturation mutagenesis in the active site and lid-domain interface. MHT077 exhibited a ∼100-fold slower unfolding rate at 65 °C than PET46, indicating substantially greater kinetic stability. In parallel, we applied evolution-informed design, a probabilistic model that leverages coevolutionary patterns in large multiple sequence alignments, to improve the activity and thermostability of five ferulic acid esterases. One design, EV-MHT043–5 was identified with a comparable thermostability (T m,app = 96.1 °C) and a 3-fold improvement in its MHETase activity relative to the wildtype enzyme, MHT043. Combination variants of beneficial mutations were screened and afforded a variant, MHT077 LFK , which reduced MHET accumulation in bioreactor experiments with postconsumer PET waste. Overall, this study expands the known MHET-hydrolyzing protein scaffolds available for enzymatic PET recycling.

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

Protein–Protein Complex Stability Controls Substrate Scope in a β‐Ketoacyl‐ACP Reductase Specific for Medium Chains

Assembly-line enzymes carry out multistep synthesis of important metabolites by using acyl carrier proteins (ACPs) to shuttle intermediates along defined sequences of active sites. Despite longstanding interest in reprogramming these systems for metabolic engineering and biosynthetic chemistry, the mechanisms underlying their reaction order remain poorly understood and difficult to control. In this work, we describe a β-ketoacyl-ACP reductase from Pseudomonas putida (PpFabG4) with an unusual selectivity for medium chains and use it to explore the molecular basis of substrate specificity in enzymes that pull intermediates from fatty acid synthesis, a common route to specialized products. X-ray crystallography shows no obvious barriers to short-chain binding. Molecular simulations and supporting mutational analyses indicate that substrate preference arises instead from a weak enzyme–ACP interaction that is stabilized by medium acyl chains but not by short chains. Indeed, mutations that strengthen this interaction for PpFabG4 or weaken it for EcFabG, an Escherichia coli β-ketoacyl-ACP reductase with a broad substrate specificity, can enhance or reduce activity on short-chain substrates by over 100-fold. Our findings show how the stability of enzyme-ACP interactions can control substrate scope in promiscuous enzymes and guide the exchange of intermediates between (and within) assembly-line systems.

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

Engineering a Non–Natural Photoenzyme for Improved Photon Efficiency

We developed a novel HTS engineering platform to optimize photoenzymatic activity. The improvements in variants were correlated to an increase in enzymatic photon efficiency. Here, transient absorption spectroscopy revealed a shift from a stepwise to a concerted mechanism. The platform was expanded to improve the synthesis of γ, δ, ϵ-lactams, and acyclic amides.

32 ENERGY CONSERVATION, CONSUMPTION, AND UTILIZATI↗
DOE OSTI2026

A Chemoselective and Stereodivergent Platform of Heme‐Nitrene Transferases to Access Chiral Aryl‐β‐Amino Esters and An Investigation of the Sequence‐Activity Landscape

Engineered biocatalysts can utilize nitrene precursors to access enantioenriched amination products, yet they have not been applied to produce valuable, enantiomerically enriched noncanonical β-amino esters. Current approaches to synthesizing β-amino acids rely on pre-oxidized precursors and multistep synthetic approaches involving various protecting groups. We engineered a platform of heme enzymes for stereoselective C–H bond amination of readily available carboxylic ester derivatives to install primary amines. A directed evolution campaign coupled with sequencing of over 1000 variants enabled us to develop engineered variants that use either O-pivaloylhydroxylamine triflic acid (PONT) or hydroxylamine hydrochloride (H 2 NOH∙HCl) as aminating reagents. An analysis of the resulting sequence–activity dataset revealed additional improvements that could be made to the final variant, highlighting the utility of sequencing data to guide future steps in directed evolution campaigns. Furthermore, the evolved nitrene transferases expand the scope of accessible chiral β-amino acid building blocks for peptidomimetic applications and provide new starting points for the design and synthesis of enantioenriched β-amino acid motifs.

amino ester building blocks↗
DOE OSTI2022

M379A Mutant Tyrosine Phenol–lyase from Citrobacter freundii Has Altered Conformational Dynamics

The M379A mutant of Citrobacter freundii tyrosine phenol-lyase (TPL) has been prepared. M379A TPL is a robust catalyst to prepare a number of tyrosines substituted at the 3-position with bulky groups that cannot be made with wild type TPL. The three dimensional structures of M379A TPL complexed with L-methionine and 3-bromo-DL-phenylalanine have been determined by X-ray crystallography. Methionine is bound as a quinonoid complex in a closed active site in 3 of 4 chains of homotetrameric M379A TPL. M379A TPL reacts with L-methionine about 8-fold slower than wild type TPL. The temperature dependence shows that the slower reaction is due to less positive activation entropy. The structure of the M379A TPL complex of 3-bromo-DL-phenylalanine has a quinonoid complex in two subunits, with an open active site conformation. The effects of the M379A mutation on TPL suggest that the mutant enzyme has altered the conformational dynamics of the active site.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2020

Emerging Strategies for Modifying Lignin Chemistry to Enhance Biological Lignin Valorization

Biological lignin valorization represents a promising approach contributing to sustainable and economic biorefineries. Here, the low level of valuable lignin–derived products remains a major challenge hindering the implementation of microbial lignin conversion. Lignin's properties play a significant role in determining the efficiency of lignin bioconversion. To date, despite significant progress in the development of biomass pretreatment, lignin fractionation, and fermentation over the last few decades, little efforts have gone into identifying the ideal lignin substrates for an efficient microbial metabolism. In this Minireview, emerging and state–of–the–art strategies for biomass pretreatment and lignin fractionation are summarized to elaborate their roles in modifying lignin structure for bioconversion. Fermentation strategies aimed at enhancing lignin depolymerization for microbial utilization are systematically reviewed as well. With an improved understanding of the ideal lignin structure elucidated by comprehensive metabolic pathways and/or big data analysis, modifying lignin chemistry could be more directional and effective. Ultimately, together with the progress of fermentation process optimization, biological lignin valorization will become more competitive in biorefineries.

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

Comparative performance of PETase as a function of reaction conditions, substrate properties, and product accumulation

There is keen interest to develop new technologies to recycle the plastic poly(ethylene terephthalate) (PET). To this end, the use of PET-hydrolyzing enzymes has shown promise for PET deconstruction to its monomers, terephthalate (TPA) and ethylene glycol (EG). Here, we compare the Ideonella sakaiensis PETase wild-type enzyme to a previously reported improved variant (W159H/S238F). We compare the thermostability of each enzyme and describe a 1.45 Å resolution structure of the mutant, highlighting changes in the substrate binding cleft compared to the wild-type enzyme. Subsequently, the performance of the wild-type and variant enzyme was compared as a function of temperature, substrate morphology, and reaction mixture composition. These studies show that reaction temperature has the strongest influence on performance between the two enzymes. We also show that both enzymes achieve higher levels of PET conversion for substrates with moderate crystallinity relative to amorphous substrates. Finally, we assess the impact of product accumulation on reaction progress for the hydrolysis of both PET and bis(2-hydroxyethyl) terephthalate (BHET). Each enzyme displays different inhibition profiles to mono(2-hydroxyethyl) terephthalate (MHET) and TPA, while both are sensitive to inhibition by EG. Overall, this work highlights the importance of reaction conditions, substrate selection, and product accumulation for catalytic performance of PET-hydrolyzing enzymes, which have implications for enzyme screening in the development of enzyme-based polyester recycling.

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

Concentration‐Dependent Inhibition of Mesophilic PETases on Poly(ethylene terephthalate) Can Be Eliminated by Enzyme Engineering

Abstract Enzyme‐based depolymerization is a viable approach for recycling of poly(ethylene terephthalate) (PET). PETase from Ideonella sakaiensis ( Is PETase) is capable of PET hydrolysis under mild conditions but suffers from concentration‐dependent inhibition. In this study, this inhibition is found to be dependent on incubation time, the solution conditions, and PET surface area. Furthermore, this inhibition is evident in other mesophilic PET‐degrading enzymes to varying degrees, independent of the level of PET depolymerization activity. The inhibition has no clear structural basis, but moderately thermostable Is PETase variants exhibit reduced inhibition, and the property is completely absent in the highly thermostable HotPETase, previously engineered by directed evolution, which simulations suggest results from reduced flexibility around the active site. This work highlights a limitation in applying natural mesophilic hydrolases for PET hydrolysis and reveals an unexpected positive outcome of engineering these enzymes for enhanced thermostability.

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

A high throughput assay to detect enzymatic polyethylene oxidation

Biological plastics deconstruction and upcycling have emerged as sustainable alternatives to traditional recycling technologies for plastics waste. The discovery and engineering of efficient thermostable poly(ethylene terephthalate) (PET) hydrolases have made biological PET recycling possible at scale; however, enzymes for non-PET plastics, which account for approximately 70% of all plastics produced, remain largely undiscovered. To accelerate the discovery of such enzymes, we develop a high-throughput screen to detect initial polymer oxidation, specifically that of the C-H bond to an aldehyde. We test 4-hydrazino-7-nitro-2,1,3-benxoxadiozole hydrazine (NBD-H), which reacts with generated aldehydes to form a fluorescent hydrazone on plasma oxidized low-density polyethylene (LDPE) films. Hydrazone generation correlated well with the area of aldehyde peaks as measured by Fourier Transform Infrared Spectroscopy (FTIR) (R2 = 0.92). Moreover, we demonstrate that the probe reliably identifies LDPE-active dye decolorizing peroxidases (DyPs) that generate aldehydes on LDPE films (1.7 – 3.0 fold change relative to background), serving as an effective screen as demonstrated by receiver operating characteristic area under the curve of 0.95. Furthermore, this assay offers an LDPE oxidation screening platform that can be readily parallelized and automated for accelerated discovery of enzymes involved in polyolefin deconstruction.

biocatalysis↗
DOE OSTI2022

Biocatalytic gateway to convert glycerol into 3-hydroxypropionic acid in waste-based biorefineries: Fundamentals, limitations, and potential research strategies

Microbial conversion of bioenergy-derived waste glycerol into value-added chemicals has emerged as an important bioprocessing technology due to its eco-friendliness, feasible technoeconomics, and potential to provide sustainability in biodiesel and bioethanol production. Glycerol is an abundant liquid waste from bioenergy plants with a projected volume of 6 million tons by 2025, accounting for about 10% of biodiesel and 2.5% of bioethanol yields. 3-Hydroxypropionic acid (3-HP) is a major product of glycerol bioconversion, which is the third largest biobased platform compound with expected market size and value of 3.6 million tons/year and USD 10 billion/year, respectively. Despite these biorefinery values, 3-HP biosynthesis from glycerol is still at an immature stage of commercial exploitation. The main challenges behind this immaturity are the toxic effects of 3-HPA on cells, the distribution of carbon flux to undesirable pathways, low tolerance of cells to glycerol and 3-HP, co-factor dependence of enzymes, low enzyme activity and stability, and the problems of substrate inhibition and specificity of enzymes. To address these challenges, it is necessary to understand the fundamentals of glycerol bioconversion and 3-HP production in terms of metabolic pathways, related enzymes, cell factories, midstream process configurations, and downstream 3-HP recovery, as discussed in this review critically and comprehensively. It is equally important to know the current challenges and limitations in 3-HP production, which are discussed in detail along with recent research efforts and remaining gaps. Finally, possible research strategies are outlined considering the recent technological advances in microbial biosynthesis, aiming to attract further research efforts to achieve a sustainable and industrially exploitable 3-HP production technology. Here, by discussing the use of advanced tools and strategies to overcome the existing challenges in 3-HP biosynthesis, this review will attract researchers from many other similar biosynthesis technologies and provide a common gateway for their further development.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

Discovery, characterization, and metabolic engineering of Rieske non-heme iron monooxygenases for guaiacol O-demethylation

Aryl-O-demethylation is a common rate-limiting step in the catabolism of lignin-related compounds, including guaiacol. In this work, we used randomly barcoded transposon insertion sequencing (RB-TnSeq) in the bacterium Novosphingobium aromaticivorans to identify a Rieske-type guaiacol O-demethylase, GdmA. Similarity searches identified GdmA homologs in other bacteria, along with candidate reductase partners, denoted GdmB. GdmAB combinations were biochemically characterized for activity with several lignin-related substrates. Structural and sequence comparisons of vanillate- and guaiacol-specific O-demethylase active sites revealed conserved hallmarks of substrate specificity. GdmAB combinations were also evaluated in Pseudomonas putida KT2440, which does not natively utilize guaiacol. GdmAB from Cupriavidus necator N-1 demonstrated the highest rate of guaiacol turnover in vitro and in engineered P. putida strains and notably higher catalytic efficiency than a cytochrome P450 system (GcoAB) and the vanillate Rieske-type O-demethylase from P. putida (VanAB). The GdmAB O-demethylases described here expand the suite of options for microbial conversion of a model lignin-derived substrate.

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

The role of binding modules in enzymatic poly(ethylene terephthalate) hydrolysis at high-solids loadings

In nature, enzymes that deconstruct biological polymers, such as cellulose and chitin, often exhibit multi-domain architectures, comprising a catalytic domain and a non-catalytic binding module; the latter serves to increase the enzyme concentration at the substrate surface. This multi-domain architecture has been shown to improve the hydrolysis of poly(ethylene terephthalate) (PET) using engineered cutinase enzymes. Here, we examine the role of accessory binding modules at industrially relevant PET solids loadings necessary for cost-effective enzymatic recycling. Using a thermostable variant of leaf compost cutinase (LCC), we produced synthetic fusion constructs of LCC with five type A carbohydrate-binding modules (CBMs). At solids loadings below 10 wt%, the CBMs improve aromatic monomer yield from PET, but above this threshold, conversion extents up to 97% are reached with no added benefits from the presence of CBM fusions. This suggests that fusion constructs with the herein studied CBMs are not necessary for industrial enzymatic PET recycling.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2024

Engineering a photoenzyme to use red light

Photoenzymatic reactions involving flavin-dependent “ene”-reductases (EREDs) rely on protein-templated charge transfer (CT) complexes between the cofactor and substrate for radical initiation. These complexes typically absorb in the blue region of the electromagnetic spectrum. Here, we engineered an ERED to form CT complexes that absorb red light. Mechanistic studies indicate that red-light activity is due to the growth of a red-absorbing shoulder off the previously identified cyan absorption feature. Molecular dynamics simulations, docking, and excited-state calculations suggest that the cyan feature involves a π→π* transition on flavin, whereas the red-light absorption is a π→π* transition between flavin and the substrate. Furthermore, differences in the electronic transition are due to changes in the substrate-binding conformation and allosteric tuning of the electronic structure of the cofactor-substrate complex. Microenvironment tuning of the CT complex for red-light activity is observed with other engineered photoenzymatic reactions, highlighting this effect’s generality.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗