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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 145 records · Page 8
All sourcesNASA NTRSDOE OSTI
DOE OSTI2023

A randomized multiplex CRISPRi-Seq approach for the identification of critical combinations of genes

Identifying virulence-critical genes from pathogens is often limited by functional redundancy. To rapidly interrogate the contributions of combinations of genes to a biological outcome, we have developed a mu ltiplex, r andomized C RISPR i nterference s equencing (MuRCiS) approach. At its center is a new method for the randomized self-assembly of CRISPR arrays from synthetic oligonucleotide pairs. When paired with PacBio long-read sequencing, MuRCiS allowed for near-comprehensive interrogation of all pairwise combinations of a group of 44 Legionella pneumophila virulence genes encoding highly conserved transmembrane proteins for their role in pathogenesis. Both amoeba and human macrophages were challenged with L. pneumophila bearing the pooled CRISPR array libraries, leading to the identification of several new virulence-critical combinations of genes. lpg2888 and lpg3000 were particularly fascinating for their apparent redundant functions during L. pneumophila human macrophage infection, while lpg3000 alone was essential for L. pneumophila virulence in the amoeban host Acanthamoeba castellanii . Thus, MuRCiS provides a method for rapid genetic examination of even large groups of redundant genes, setting the stage for application of this technology to a variety of biological contexts and organisms.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

A randomized multiplex CRISPRi-Seq approach for the identification of critical combinations of genes

Identifying virulence-critical genes from pathogens is often limited by functional redundancy. To rapidly interrogate the contributions of combinations of genes to a biological outcome, we have developed a multiplex, randomized CRISPR interference sequencing (MuRCiS) approach. At its center is a new method for the randomized self-assembly of CRISPR arrays from synthetic oligonucleotide pairs. When paired with PacBio long-read sequencing, MuRCiS allowed for near-comprehensive interrogation of all pairwise combinations of a group of 44 Legionella pneumophila virulence genes encoding highly conserved transmembrane proteins for their role in pathogenesis. Both amoeba and human macrophages were challenged with L. pneumophila bearing the pooled CRISPR array libraries, leading to the identification of several new virulence-critical combinations of genes. lpg2888 and lpg3000 were particularly fascinating for their apparent redundant functions during L. pneumophila human macrophage infection, while lpg3000 alone was essential for L. pneumophila virulence in the amoeban host Acanthamoeba castellanii. Thus, MuRCiS provides a method for rapid genetic examination of even large groups of redundant genes, setting the stage for application of this technology to a variety of biological contexts and organisms.

79 ASTRONOMY AND ASTROPHYSICS↗
DOE OSTI2023

A randomized multiplex CRISPRi-Seq approach for the identification of critical combinations of genes

Identifying virulence-critical genes from pathogens is often limited by functional redundancy. To rapidly interrogate the contributions of combinations of genes to a biological outcome, we have developed a mu ltiplex, r andomized C RISPR i nterference s equencing (MuRCiS) approach. At its center is a new method for the randomized self-assembly of CRISPR arrays from synthetic oligonucleotide pairs. When paired with PacBio long-read sequencing, MuRCiS allowed for near-comprehensive interrogation of all pairwise combinations of a group of 44 Legionella pneumophila virulence genes encoding highly conserved transmembrane proteins for their role in pathogenesis. Both amoeba and human macrophages were challenged with L. pneumophila bearing the pooled CRISPR array libraries, leading to the identification of several new virulence-critical combinations of genes. lpg2888 and lpg3000 were particularly fascinating for their apparent redundant functions during L. pneumophila human macrophage infection, while lpg3000 alone was essential for L. pneumophila virulence in the amoeban host Acanthamoeba castellanii . Thus, MuRCiS provides a method for rapid genetic examination of even large groups of redundant genes, setting the stage for application of this technology to a variety of biological contexts and organisms.

Ellis, Nicole A. (ORCID:0000000195928415)↗
NASA NTRS1994

Planar rotational magnetic micromotors with integrated shaft encoder and magnetic rotor levitation

Deep x-ray lithography and electroplating may be combined to form a fabrication tool for micromechanical devices with large structural heights, to 500 micron, and extreme edge acuities, less than 0.1 micron-run-out per 100 micron of height. This process concept which originated in Germany as LIGA may be further extended by adding surface micromachining. This extension permits the fabrication of precision metal and plastic parts which may be assembled into three-dimensional micromechanical components and systems. The processing tool may be used to fabricate devices from ferromagnetic material such as nickel and nickel-iron alloys. These materials when properly heat treated exhibit acceptable magnetic behavior for current to flux conversion and marginal behavior for permanent magnet applications. The tool and materials have been tested via planar, magnetic, rotational micromotor fabrication. Three phase reluctance machines of the 6:4 configuration with 280 micron diameter rotors have been tested and analyzed. Stable rotational speeds to 34,000 rpm with output torques above 10 x 10(exp -9) N-m have been obtained. The behavior is monitored with integrated shaft encoders which are photodiodes which measure the rotor response. Magnetic levitation of the rotor via reluctance forces has been achieved and has reduced frictional torque losses to less than 1 percent of the available torque. The results indicate that high speed limits of these actuators are related to torque ripple. Hysteresis motors with magnetic bearings are under consideration and will produce high speed rotational machines with excellent sensor application potential.

Guckel, Henry↗
DOE OSTI2020

Enhancing nuclear data validation analysis by using machine learning

In this work, we demonstrate how machine learning (ML) techniques can be used as an auxiliary tool for nuclear data validation analysis. The output of the ML analysis can inform evaluators and validators on the quality, or lack thereof, of specific nuclear data and benchmark experiments with respect to simulating these experimental benchmark values. To this end, measured and simulated effective neutron multiplication factors, k eff , of 875 selected ICSBEP critical assemblies and the latter's sensitivities with respect to nuclear data as well as benchmarks' features (e.g., material nuclide, core geometry) are used as input for a random forest (RF) regression model. The RF is used to encode the complex inter-dependencies between thousands of nuclear data features (i.e., sensitivity profiles and aspects describing the measurements) and 875 simulated and experimental k eff values in order to understand what nuclear data features are most informative for predicting bias. The complexity of relationships and high-dimensional space is difficult-to-impossible to search fully using simply expert judgment. As a first proof-of-concept—step, it is demonstrated that this technique is able to correctly trace large discrepancies between simulated and experimental k eff back to fabricated shortcomings in nuclear data that were perturbed to simulated k eff values. In a second, real-case scenario, step, the RF algorithm is used to validate the ENDF/B-VIII.0 library in comparison to ENDF/B-VII.1 nuclear data. One case is showcased where the chosen ML algorithms highlighted nuclear data (the 19 F(n,inl) cross section from 0.4–0.9 MeV) that are shown to be problematic by comparing them to associated differential experimental data and nuclear data from other libraries. In addition to that, it is shown that the RF results point towards poor benchmark experiments and associated underestimated uncertainties (e.g., the PU-SOL-THERM-028 series). However, using the RF algorithm for validating nuclear data with respect to k eff is currently limited to pinpointing groups of questionable nuclear data due to the inherent correlations between features introduced by the nuclear data themselves and how k eff is simulated. Due to this, we recommend that the ML methods presented be used to augment—rather than replace—the expert knowledge of evaluators and validators.

96 KNOWLEDGE MANAGEMENT AND PRESERVATION↗
DOE OSTI2021

Activity-based, genome-resolved metagenomics uncovers key populations and pathways involved in subsurface conversions of coal to methane

Microbial metabolisms and interactions that facilitate subsurface conversions of recalcitrant carbon to methane are poorly understood. We deployed an in situ enrichment device in a subsurface coal seam in the Powder River Basin (PRB), USA, and used BONCAT-FACS-Metagenomics to identify translationally active populations involved in methane generation from a variety of coal-derived aromatic hydrocarbons. From the active fraction, high-quality metagenome-assembled genomes (MAGs) were recovered for the acetoclastic methanogen, Methanothrix paradoxum, and a novel member of the Chlorobi with the potential to generate acetate via the Pta-Ack pathway. Members of the Bacteroides and Geobacter also encoded Pta-Ack and together, all four populations had the putative ability to degrade ethylbenzene, phenylphosphate, phenylethanol, toluene, xylene, and phenol. Metabolic reconstructions, gene analyses, and environmental parameters also indicated that redox fluctuations likely promote facultative energy metabolisms in the coal seam. The active "Chlorobi PRB" MAG encoded enzymes for fermentation, nitrate reduction, and multiple oxygenases with varying binding affinities for oxygen. "M. paradoxum PRB" encoded an extradiol dioxygenase for aerobic phenylacetate degradation, which was also present in previously published Methanothrix genomes. Finaly, these observations outline underlying processes for bio-methane from subbituminous coal by translationally active populations and demonstrate activity-based metagenomics as a powerful strategy in next generation physiology to understand ecologically relevant microbial populations.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

Integrating genomics and metabolomics for scalable non-ribosomal peptide discovery

Non-Ribosomal Peptides (NRPs) represent a biomedically important class of natural products that include a multitude of antibiotics and other clinically used drugs. NRPs are not directly encoded in the genome but are instead produced by metabolic pathways encoded by biosynthetic gene clusters (BGCs). Since the existing genome mining tools predict many putative NRPs synthesized by a given BGC, it remains unclear which of these putative NRPs are correct and how to identify post-assembly modifications of amino acids in these NRPs in a blind mode, without knowing which modifications exist in the sample. To address this challenge, here we report NRPminer, a modification-tolerant tool for NRP discovery from large (meta)genomic and mass spectrometry datasets. We show that NRPminer is able to identify many NRPs from different environments, including four previously unreported NRP families from soil-associated microbes and NRPs from human microbiota. Furthermore, in this work we demonstrate the anti-parasitic activities and the structure of two of these NRP families using direct bioactivity screening and nuclear magnetic resonance spectrometry, illustrating the power of NRPminer for discovering bioactive NRPs.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

Large-scale prediction of outer-membrane multiheme cytochromes uncovers hidden diversity of electroactive bacteria and underlying pathways

Multi-heme cytochromes (MHCs), together with accessory proteins like porins and periplasmic cytochromes, enable microbes to transport electrons between the cytoplasmic membrane and extracellular substrates (e.g., minerals, electrodes, other cells). Extracellular electron transfer (EET) has been described in multiple systems; yet, the broad phylogenetic and mechanistic diversity of these pathways is less clear. One commonality in EET-capable systems is the involvement of MHCs, in the form of porin-cytochrome complexes, pili-like cytochrome polymers, and lipid-anchored extracellular cytochromes. Here, we put forth MHCscan—a software tool for identifying MHCs and identifying potential EET capability. Using MHCscan, we scanned ~60,000 bacterial and 2,000 archaeal assemblies, and identify a diversity of MHCs, many of which represent enzymes with no known function, and many found within organisms not previously known to be electroactive. In total, our scan identified ~1,400 unique enzymes, each encoding more than 10 heme-binding motifs. In our analysis, we also find evidence for modularity and flexibility in MHC-dependent EET pathways, and suggest that MHCs may be far more common than previously recognized, with many facets yet to be discovered. We present MHCscan as a lightweight and user-friendly software tool that is freely available: https://github.com/Arkadiy-Garber/MHCscan.

59 BASIC BIOLOGICAL SCIENCES↗
NASA NTRS1989

Imaging characteristics of the Extreme Ultraviolet Explorer microchannel plate detectors

The Extreme Ultraviolet Explorer (EUVE) satellite will conduct an all-sky survey over the wavelength range from 70 A to 760 A using four grazing-incidence telescopes and seven microchannel-plate (MCP) detectors. The imaging photon-counting MCP detectors have active areas of 19.6 cm2. Photon arrival position is determined using a wedge-and-strip anode and associated pulse-encoding electronics. The imaging characteristics of the EUVE flight detectors are presented including image distortion, flat-field response, and spatial differential nonlinearity. Also included is a detailed discussion of image distortions due to the detector mechanical assembly, the wedge-and-strip anode, and the electronics. Model predictions of these distortions are compared to preflight calibration images which show distortions less than 1.3 percent rms of the detector diameter of 50 mm before correction. The plans for correcting these residual detector image distortions to less than 0.1 percent rms are also presented.

Vallerga, J. V.↗
DOE OSTI2026

The relationship between gene traits and transcription in soil microbial communities varies by environmental stimulus

Codon and nucleotide frequencies are known to relate to the rate of gene transcription, yet how these traits shape transcriptional profiles of soil microbial communities remains unclear. Here we test the prediction that functional genes with high codon optimization and energetically lower cost nucleotides (i.e., nucleotides requiring less adenosine triphosphate (ATP) for synthesis) have higher transcriptional expression in a soil microbial community. In laboratory incubations, we subjected an agricultural soil to two separate short-term environmental changes: labile carbon (glucose) addition or a sudden 30-min increase in temperature from 20 °C to 60 °C. Using the total genomic codon frequencies to predict preferred codon usage for each taxon, we then estimated codon optimization for each transcript. On the community level, we found a higher average level of codon optimization after the addition of glucose. Synonymous nucleotide composition in the transcript pool also shifted towards energetically cheaper nucleotides, favoring uracil (U) over adenine (A) and cytosine (C) over guanine (G). Similarly, we found that encoded amino acid usage shifted towards energetically cheaper amino acids in response to labile carbon. In contrast, in communities responding to heat shock, there were no significant differences in the averaged gene traits of expressed transcripts. We used metagenome-assembled-genomes to further examine the ability of gene traits to predict transcriptional responses within and between taxa. We found that traits of individual genes could not reliably predict the level of transcription of a gene within or between taxa—highlighting the limits of this approach. However, we did find that when traits were averaged across several related genes, codon optimization was able to predict levels of transcription in metabolic pathways associated with growth and nutrient uptake in response to glucose. Similar relationships were not observed in response to heat, or for functions associated with stress—such as genes associated with sporulation or heat shock. These results demonstrate that gene traits, such as codon usage, nucleotide selection, and amino acid selection, relate to the transcriptional expression of genes in soil microbial communities and suggests that these relationships may be dependent on both gene function and the specific type of environmental stimuli.

Biological and medical sciences↗
DOE OSTI2024

Novel, active, and uncultured hydrocarbon-degrading microbes in the ocean

ABSTRACT Given the vast quantity of oil and gas input to the marine environment annually, hydrocarbon degradation by marine microorganisms is an essential ecosystem service. Linkages between taxonomy and hydrocarbon degradation capabilities are largely based on cultivation studies, leaving a knowledge gap regarding the intrinsic ability of uncultured marine microbes to degrade hydrocarbons. To address this knowledge gap, metagenomic sequence data from the Deepwater Horizon (DWH) oil spill deep-sea plume was assembled to which metagenomic and metatranscriptomic reads were mapped. Assembly and binning produced new DWH metagenome-assembled genomes that were evaluated along with their close relatives, all of which are from the marine environment (38 total). These analyses revealed globally distributed hydrocarbon-degrading microbes with clade-specific substrate degradation potentials that have not been reported previously. For example, methane oxidation capabilities were identified in all Cycloclasticus . Furthermore, all Bermanella encoded and expressed genes for non-gaseous n -alkane degradation; however, DWH Bermanella encoded alkane hydroxylase, not alkane 1-monooxygenase. All but one previously unrecognized DWH plume member in the SAR324 and UBA11654 have the capacity for aromatic hydrocarbon degradation. In contrast, Colwellia were diverse in the hydrocarbon substrates they could degrade. All clades encoded nutrient acquisition strategies and response to cold temperatures, while sensory and acquisition capabilities were clade specific. These novel insights regarding hydrocarbon degradation by uncultured planktonic microbes provides missing data, allowing for better prediction of the fate of oil and gas when hydrocarbons are input to the ocean, leading to a greater understanding of the ecological consequences to the marine environment. IMPORTANCE Microbial degradation of hydrocarbons is a critically important process promoting ecosystem health, yet much of what is known about this process is based on physiological experiments with a few hydrocarbon substrates and cultured microbes. Thus, the ability to degrade the diversity of hydrocarbons that comprise oil and gas by microbes in the environment, particularly in the ocean, is not well characterized. Therefore, this study aimed to utilize non-cultivation-based ‘omics data to explore novel genomes of uncultured marine microbes involved in degradation of oil and gas. Analyses of newly assembled metagenomic data and previously existing genomes from other marine data sets, with metagenomic and metatranscriptomic read recruitment, revealed globally distributed hydrocarbon-degrading marine microbes with clade-specific substrate degradation potentials that have not been previously reported. This new understanding of oil and gas degradation by uncultured marine microbes suggested that the global ocean harbors a diversity of hydrocarbon-degrading bacteria, which can act as primary agents regulating ecosystem health.

Howe, Kathryn L.↗
NASA NTRS1987

Flexible structure control laboratory development and technology demonstration

An experimental structure is described which was constructed to demonstrate and validate recent emerging technologies in the active control and identification of large flexible space structures. The configuration consists of a large, 20 foot diameter antenna-like flexible structure in the horizontal plane with a gimballed central hub, a flexible feed-boom assembly hanging from the hub, and 12 flexible ribs radiating outward. Fourteen electrodynamic force actuators mounted to the hub and to the individual ribs provide the means to excite the structure and exert control forces. Thirty permanently mounted sensors, including optical encoders and analog induction devices provide measurements of structural response at widely distributed points. An experimental remote optical sensor provides sixteen additional sensing channels. A computer samples the sensors, computes the control updates and sends commands to the actuators in real time, while simultaneously displaying selected outputs on a graphics terminal and saving them in memory. Several control experiments were conducted thus far and are documented. These include implementation of distributed parameter system control, model reference adaptive control, and static shape control. These experiments have demonstrated the successful implementation of state-of-the-art control approaches using actual hardware.

Vivian, H. C.↗
DOE OSTI2023

Identification and Characterization of ten Escherichia coli Strains Encoding Novel Shiga Toxin 2 Subtypes, Stx2n as Well as Stx2j, Stx2m, and Stx2o, in the United States

The sharing of genome sequences in online data repositories allows for large scale analyses of specific genes or gene families. This can result in the detection of novel gene subtypes as well as the development of improved detection methods. Here, we used publicly available WGS data to detect a novel Stx subtype, Stx2n in two clinical E. coli strains isolated in the USA. During this process, additional Stx2 subtypes were detected; six Stx2j, one Stx2m strain, and one Stx2o, were all analyzed for variability from the originally described subtypes. Complete genome sequences were assembled from short- or long-read sequencing and analyzed for serotype, and ST types. The WGS data from Stx2n- and Stx2o-producing STEC strains were further analyzed for virulence genes pro-phage analysis and phage insertion sites. Nucleotide and amino acid maximum parsimony trees showed expected clustering of the previously described subtypes and a clear separation of the novel Stx2n subtype. WGS data were used to design OMNI PCR primers for the detection of all known stx1 (283 bp amplicon), stx2 (400 bp amplicon), intimin encoded by eae (221 bp amplicon), and stx2f (438 bp amplicon) subtypes. These primers were tested in three different laboratories, using standard reference strains. An analysis of the complete genome sequence showed variability in serogroup, virulence genes, and ST type, and Stx2 pro-phages showed variability in size, gene composition, and phage insertion sites. The strains with Stx2j, Stx2m, Stx2n, and Stx2o showed toxicity to Vero cells. Stx2j carrying strain, 2012C-4221, was induced when grown with sub-inhibitory concentrations of ciprofloxacin, and toxicity was detected. Taken together, these data highlight the need to reinforce genomic surveillance to identify the emergence of potential new Stx2 or Stx1 variants. The importance of this surveillance has a paramount impact on public health. Per our description in this study, we suggest that 2017C-4317 be designated as the Stx2n type-strain.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

A bistable and reconfigurable molecular system with encodable bonds

Molecular systems with ability to controllably transform between different conformations play pivotal roles in regulating biochemical functions. Here, we report the design of a bistable DNA origami four-way junction (DOJ) molecular system that adopts two distinct stable conformations with controllable reconfigurability by using conformation-controlled base stacking. Exquisite control over DOJ’s conformation and transformation is realized by programming the stacking bonds (quasi–blunt-ends) within the junction to induce prescribed coaxial stacking of neighboring junction arms. A specific DOJ conformation may be achieved by encoding the stacking bonds with binary stacking sequences based on thermodynamic calculations. Dynamic transformations of DOJ between various conformations are achieved by using specific environmental and molecular stimulations to reprogram the stacking codes. This work provides a useful platform for constructing self-assembled DNA nanostructures and nanomachines and insights for future design of artificial molecular systems with increasing complexity and reconfigurability.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2021

Disentangling Rotational Dynamics and Ordering Transitions in a System of Self-Organizing Protein Nanorods via Rotationally Invariant Latent Representations

The dynamics of complex ordering systems with active rotational degrees of freedom exemplified by protein self-assembly is explored using a machine learning workflow that combines deep learning-based semantic segmentation and rotationally invariant variational autoencoder-based analysis of orientation and shape evolution. The latter allows for disentanglement of the particle orientation from other degrees of freedom and compensates for lateral shifts. The disentangled representations in the latent space encode the rich spectrum of local transitions that can now be visualized and explored via continuous variables. The time dependence of ensemble averages allows insight into the time dynamics of the system and, in particular, illustrates the presence of the potential ordering transition. Finally, analysis of the latent variables along the single-particle trajectory allows tracing these parameters on a single-particle level. The proposed approach is expected to be universally applicable for the description of the imaging data in optical, scanning probe, and electron microscopy seeking to understand the dynamics of complex systems where rotations are a significant part of the process.

representation learning↗
DOE OSTI2023

Heterologous expression of a fully active Azotobacter vinelandii nitrogenase Fe protein in Escherichia coli

ABSTRACT The functional versatility of the Fe protein, the reductase component of nitrogenase, makes it an appealing target for heterologous expression, which could facilitate future biotechnological adaptations of nitrogenase-based production of valuable chemical commodities. Yet, the heterologous synthesis of a fully active Fe protein of Azotobacter vinelandii ( Av NifH) in Escherichia coli has proven to be a challenging task. Here, we report the successful synthesis of a fully active Av NifH protein upon co-expression of this protein with Av IscS/U and Av NifM in E. coli . Our metal, activity, electron paramagnetic resonance, and X-ray absorption spectroscopy/extended X-ray absorption fine structure (EXAFS) data demonstrate that the heterologously expressed Av NifH protein has a high [Fe 4 S 4 ] cluster content and is fully functional in nitrogenase catalysis and assembly. Moreover, our phylogenetic analyses and structural predictions suggest that Av NifM could serve as a chaperone and assist the maturation of a cluster-replete Av NifH protein. Given the crucial importance of the Fe protein for the functionality of nitrogenase, this work establishes an effective framework for developing a heterologous expression system of the complete, two-component nitrogenase system; additionally, it provides a useful tool for further exploring the intricate biosynthetic mechanism of this structurally unique and functionally important metalloenzyme. IMPORTANCE The heterologous expression of a fully active Azotobacter vinelandii Fe protein (AvNifH) has never been accomplished. Given the functional importance of this protein in nitrogenase catalysis and assembly, the successful expression of AvNifH in Escherichia coli as reported herein supplies a key element for the further development of heterologous expression systems that explore the catalytic versatility of the Fe protein, either on its own or as a key component of nitrogenase, for nitrogenase-based biotechnological applications in the future. Moreover, the “clean” genetic background of the heterologous expression host allows for an unambiguous assessment of the effect of certain nif-encoded protein factors, such as AvNifM described in this work, in the maturation of AvNifH, highlighting the utility of this heterologous expression system in further advancing our understanding of the complex biosynthetic mechanism of nitrogenase.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

Promising approaches for the assembly of the catalytically active, recombinant Desulfomicrobium baculatum hydrogenase with substitutions at the active site

Background: Hydrogenases (H2ases) are metalloenzymes capable of the reversible conversion of protons and electrons to molecular hydrogen. Exploiting the unique enzymatic activity of H2ases can lead to advancements in the process of biohydrogen evolution and green energy production. Results: Here we created of a functional, optimized operon for rapid and robust production of recombinant [NiFe] Desulfomicrobium baculatum hydrogenase (Dmb H2ase). The conversion of the [NiFeSe] Dmb H2ase to [NiFe] type was performed on genetic level by site-directed mutagenesis. The native dmb operon includes two structural H2ase genes, coding for large and small subunits, and an additional gene, encoding a specific maturase (protease) that is essential for the proper maturation of the enzyme. Dmb, like all H2ases, needs intricate bio-production machinery to incorporate its crucial inorganic ligands and cofactors. Strictly anaerobic, sulfate reducer D. baculatum bacteria are distinct, in terms of their biology, from E. coli. Thus, we introduced a series of alterations within the native dmb genes. As a result, more than 100 elements, further compiled into 32 operon variants, were constructed. The initial requirement for a specific maturase was omitted by the artificial truncation of the large Dmb subunit. The assembly of the produced H2ase subunit variants was investigated both, in vitro and in vivo. This approach resulted in 4 recombinant [NiFe] Dmb enzyme variants, capable of H 2 evolution. The aim of this study was to overcome the gene expression, protein biosynthesis, maturation and ligand loading bottlenecks for the easy, fast, and cost-effective delivery of recombinant [NiFe] H2ase, using a commonly available E. coli strains. Conclusion: The optimized genetic constructs together with the developed growth and purification procedures appear to be a promising platform for further studies toward fully-active and O 2 tolerant, recombinant [NiFeSe] Dmb H2ase, resembling the native Dmb enzyme. It could likely be achieved by selective cysteine to selenocysteine substitution within the active site of the [NiFe] Dmb variant.

59 BASIC BIOLOGICAL SCIENCES↗