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At least 1,225 records · Page 68

Discovery of cyanophycin dipeptide hydrolase enzymes suggests widespread utility of the natural biopolymer cyanophycin

Cyanophycin is a bacterial polymer mainly used for nitrogen storage. It is composed of a peptide backbone of L-aspartate residues with L-arginines attached to their side chains through isopeptide bonds. Cyanophycin is degraded in two steps: Cyanophycinase cleaves the polymer into β-Asp-Arg dipeptides, which are hydrolyzed into free Asp and Arg by enzymes possessing isoaspartyl dipeptide hydrolase activity. Two unrelated enzymes with this activity, isoaspartyl dipeptidase (IadA) and isoaspartyl aminopeptidase (IaaA) have been shown to degrade β-Asp-Arg dipeptides, but bacteria which encode cyanophycin-metabolizing genes can lack iaaA and iadA genes. In this study, we investigate a previously uncharacterized enzyme whose gene can cluster with cyanophycin-metabolizing genes. This enzyme, which we name cyanophycin dipeptide hydrolase (CphZ), is specific for dipeptides derived from cyanophycin degradation. Accordingly, a co-complex structure of CphZ and β-Asp-Arg shows that CphZ, unlike IadA or IaaA, recognizes all portions of its β-Asp-Arg substrate. Bioinformatic analyses showed that CphZ is found in very many proteobacteria and is homologous to an uncharacterized protein encoded in the “arginine/ornithine transport” (aot) operon of many pseudomonas species, including Pseudomonas aeruginosa . In vitro assays show that AotO is indeed a CphZ, and in cellulo growth experiments show that this enzyme and the aot operon allow P. aeruginosa to take up and use β-Asp-Arg as a sole carbon and nitrogen source. Together the results establish the novel, highly specific enzyme subfamily of CphZs, suggesting that cyanophycin is potentially used by a much wider range of bacteria than previously appreciated.

Science & Technology - Other Topics↗

High-throughput enzyme screening platform for the IPP-bypass mevalonate pathway for isopentenol production

Isopentenol (or isoprenol, 3-methyl-3-buten-1-ol) is a drop-in biofuel and a precursor for commodity chemicals such as isoprene. Biological production of isopentenol via the mevalonate pathway has been optimized extensively in Escherichia coli , yielding 70% of its theoretical maximum. However, high ATP requirements and isopentenyl diphosphate (IPP) toxicity pose immediate challenges for engineering bacterial strains to overproduce commodities utilizing IPP as an intermediate. To overcome these limitations, we developed an “IPP-bypass” isopentenol pathway using the promiscuous activity of a mevalonate diphosphate decarboxylase (PMD) and demonstrated improved performance under aeration-limited conditions. However, relatively low activity of PMD toward the non-native substrate (mevalonate monophosphate, MVAP) was shown to limit flux through this new pathway. By inhibiting all IPP production from the endogenous non-mevalonate pathway, we developed a high-throughput screening platform that correlated promiscuous PMD activity toward MVAP with cellular growth. Successful identification of mutants that altered PMD activity demonstrated the sensitivity and specificity of the screening platform. Strains with evolved PMD mutants and the novel IPP-bypass pathway increased titers up to 2.4-fold. Further enzymatic characterization of the evolved PMD variants suggested that higher isopentenol titers could be achieved either by altering residues directly interacting with substrate and cofactor or by altering residues on nearby α-helices. These altered residues could facilitate the production of isopentenol by tuning either k cat or K i of PMD for the non-native substrate. The synergistic modification made on PMD for the IPP-bypass mevalonate pathway is expected to significantly facilitate the industrial scale production of isopentenol.

09 BIOMASS FUELS↗

Structure-function analysis of microRNA 3'-end trimming by Nibbler

Nibbler (Nbr) is a 3'-to-5' exoribonuclease whose catalytic 3'-end trimming activity impacts microRNA (miRNA) and PIWI-interacting RNA (piRNA) biogenesis. Here, we report on structural and functional studies to decipher the contributions of Nbr’s N-terminal domain (NTD) and exonucleolytic domain (EXO) in miRNA 3'-end trimming. We have solved the crystal structures of the NTD core and EXO domains of Nbr, both in the apo-state. The NTD-core domain of Aedes aegypti Nbr adopts a HEAT-like repeat scaffold with basic patches constituting an RNA-binding surface exhibiting a preference for binding double-strand RNA (dsRNA) over single-strand RNA (ssRNA). Structure-guided functional assays in Drosophila S2 cells confirmed a principal role of the NTD in exonucleolytic miRNA trimming, which depends on basic surface patches. Gain-of-function experiments revealed a potential role of the NTD in recruiting Nbr to Argonaute-bound small RNA substrates. The EXO domain of A. aegypti and Drosophila melanogaster Nbr adopt a mixed α/β-scaffold with a deep pocket lined by a DEDDy catalytic cleavage motif. We demonstrate that Nbr’s EXO domain exhibits Mn2+-dependent ssRNA-specific 3'-to-5' exoribonuclease activity. Modeling of a 3' terminal Uridine into the catalytic pocket of Nbr EXO indicates that 2'-O-methylation of the 3'-U would result in a steric clash with a tryptophan side chain, suggesting that 2'-O-methylation protects small RNAs from Nbr-mediated trimming. Overall, our data establish that Nbr requires its NTD as a substrate recruitment platform to execute exonucleolytic miRNA maturation, catalyzed by the ribonuclease EXO domain.

59 BASIC BIOLOGICAL SCIENCES↗

Mechanistic modeling study on process optimization and precursor utilization with atmospheric spatial atomic layer deposition

Spatial atomic layer deposition (SALD) is a promising technology with the aim of combining the advantages of excellent uniformity and conformity of temporal atomic layer deposition (ALD), and an industrial scalable and continuous process. In this manuscript, an experimental and numerical combined model of atmospheric SALD system is presented. To establish the connection between the process parameters and the growth efficiency, a quantitative model on reactant isolation, throughput, and precursor utilization is performed based on the separation gas flow rate, carrier gas flow rate, and precursor mass fraction. The simulation results based on this model show an inverse relation between the precursor usage and the carrier gas flow rate. With the constant carrier gas flow, the relationship of precursor usage and precursor mass fraction follows monotonic function. The precursor concentration, regardless of gas velocity, is the determinant factor of the minimal residual time. The narrow gap between precursor injecting heads and the substrate surface in general SALD system leads to a low Péclet number. In this situation, the gas diffusion act as a leading role in the precursor transport in the small gap rather than the convection. Fluid kinetics from the numerical model is independent of the specific structure, which is instructive for the SALD geometry design as well as its process optimization.

42 ENGINEERING↗

Revisiting alkaline aerobic lignin oxidation

Lignin conversion to renewable chemicals is a promising means to improve the economic viability of lignocellulosic biorefineries. Alkaline aerobic oxidation of lignin has long been employed for production of aromatic compounds such as vanillin and syringaldehyde, but this approach primarily focuses on condensed substrates such as Kraft lignin and lignosulfonates. Conversely, emerging lignocellulosic biorefinery schemes enable the production of more native-like, reactive lignin. Here, we revisit alkaline aerobic oxidation of highly reactive lignin substrates to understand the impact of reaction conditions and catalyst choice on product yield and distribution. The oxidation of native poplar lignin was studied as a function of temperature, NaOH loading, reaction time, and oxygen partial pressure. Besides vanillin and syringaldehyde, other oxidation products include acetosyringone and vanillic, syringic, and p-hydroxybenzoic acids. Reactions with vanillin and syringaldehyde indicated that these compounds are further oxidized to non-aromatic carboxylic acids during alkaline aerobic oxidation, with syringaldehyde being substantially more reactive than vanillin. The production of phenolic compounds from lignin is favored by high NaOH loadings and temperatures, but short reaction times, as the products degrade rapidly, which is further exacerbated by the presence of oxygen. Under optimal conditions, a phenolic monomer yield of 30 wt% was obtained from poplar lignin. Testing a range of catalysts showed that Cu-containing catalysts, such as CuSO 4 and LaMn 0.8 Cu 0.2 O 3 , accelerate product formation; specifically, the catalyst does not increase the maximum yield, but expands the operating window in which high product yields are obtainable. We also demonstrate that other native and isolated lignin substrates that are significantly chemically modified are effectively converted to phenolic compounds. Finally, alkaline aerobic oxidation of native lignins was compared to nitrobenzene oxidation and reductive catalytic fractionation, as these methods constitute suitable benchmarks for lignin depolymerization. While nitrobenzene oxidation achieved a somewhat higher yield, similar monomer yields were obtained through RCF and alkaline aerobic oxidation, especially for lignins with a high guaiacyl- and/or p-hydroxyphenyl-content, as syringyl units are more unstable during oxidation. Overall, this study highlights the potential for aerobic lignin oxidation revisited on native-like lignin substrates.

09 BIOMASS FUELS↗

Near band edge emission characteristics of sputtered nano-crystalline ZnO films

Sputtered zinc oxide (ZnO) thin films deposited on unheated glass substrate under different sputtering gas mixtures (Ar+O{sub 2}) have been investigated using X-ray diffraction and photo luminescence spectroscopy. Earlier reported studies on ZnO films prepared by different techniques exhibit either a sharp/broad near band edge (NBE) emission peak depending on the crystalline quality of the film. In the present study zinc oxide films, grown on unheated substrates, are seen to possess a preferred (002) orientation with a microstructure consisting of clustered nano-sized crystallites. The splitting in the near band edge emission (NBE) into three characteristic peaks is attributed to quantum confinement effect, and is observed specifically under an excitation of 270 nm. Deep level emission (DLE) in the range 400 to 700 nm is not observed indicating absence of deep level radiative defects.

75 CONDENSED MATTER PHYSICS, SUPERCONDUCTIVITY AND↗

Gold Catalyzed Polymerization Reactions of Unsaturated Substrates: Towards New Functional, Recyclable, and Upcycled Aromatic Polymers (Final Report)

The overarching goal of this project lied in the translation of gold (Au) catalyzed small molecule synthetic transformations to the field of polymer science, the generation of new macromolecular structures, and the application of the materials and methodologies produced toward sustainable polymer development and chemical upcycling. Specifically, this project aimed to polymerize monomers based on unsaturated precursors and access novel polymeric materials through the extension of Au catalyzed intermolecular processes. The specific objectives of this project comprised: (1) the extension of Au-catalyzed intermolecular reactions to bifunctional monomers and the polycondensation of diverse nucleophiles; (2) to gain fundamental mechanistic insight into Au-catalyzed polymerizations; (3) the application of these reactions to the synthesis of new classes of aromatic polymers that can be readily and/or selectively deconstructed; and (4) to apply these chemistries to post-polymerization modification (PPM) of commercially relevant polyaromatic substrates, including post-industrial plastics and post-consumer waste.

36 MATERIALS SCIENCE↗

Does plasma jet sintering follow an Arrhenius-type expression?

Atmospheric pressure, ambient temperature plasma jets have become a promising candidate for material processing in parallel with developments in additive manufacturing. Recent work has shown that plasma jets can be used to sinter printed nanoparticles at temperatures much lower than typically required for conventional thermal sintering. In this report we conduct a mechanistic study on plasma jet sintering that correlates specific energy input with the electrical conductivity of printed silver films after sintering. Increasing the specific energy input accelerated the sintering process following an Arrhenius-like exponential trend across a large range of conditions, including both helium and argon plasma jets. Although an exponential relationship is also found with the plasma heated substrate temperature, independent studies indicate that heating is not the primary mechanism. These results suggest there is a general behavior that couples the plasma jet with the surface.

70 PLASMA PHYSICS AND FUSION TECHNOLOGY↗

Enhancement of the magnetic interfacial exchange energy at a specific interface in NiFe/CoO/Co trilayer thin films via ion-beam modification

A series of ferromagnetic Ni{sub 80}Fe{sub 20}(55 nm)/antiferromagnetic CoO (25 to 200 nm)/ferromagnetic Co (55 nm)/SiO{sub 2}(substrate) trilayer thin films were fabricated by ion-beam assisted deposition in order to understand the role of ion beam modification on the interfacial and interlayer coupling. The microstructural study using transmission electron microscopy, X-ray reflectometry, and polarised neutron reflectometry showed that ion-beam modification during the deposition process led to an oxygen-rich Co/CoO nanocomposite interface region at the bottom layer. This interface caused a high exchange bias field for the ferromagnetic cobalt. However, the exchange bias for top permalloy ferromagnet remained low, in line with expectations from the literature for the typical interfacial energy. This suggest that the ion-beam enhancement of the magnetic exchange bias is localized to the Co/CoO interface where local microstructural effects provide the dominant mechanism.

75 CONDENSED MATTER PHYSICS, SUPERCONDUCTIVITY AND↗

mRNA bound to the 30S subunit is a HigB toxin substrate

Activation of bacterial toxins during stress results in cleavage of mRNAs in the context of the ribosome. These toxins are thought to function as global translational inhibitors yet recent studies suggest each may have distinct mRNA specificities that result in selective translation for bacterial survival. Here we demonstrate that mRNA in the context of a bacterial 30S subunit is sufficient for ribosome-dependent toxin HigB endonucleolytic activity, suggesting that HigB interferes with the initiation step of translation. We determined the X-ray crystal structure of HigB bound to the 30S, revealing that two solvent-exposed clusters of HigB basic residues directly interact with 30S 16S rRNA helices 18, 30, and 31. We further show that these HigB residues are essential for ribosome recognition and function. Comparison with other ribosome-dependent toxins RelE and YoeB reveals that each interacts with similar features of the 30S aminoacyl (A) site yet does so through presentation of diverse structural motifs.

59 BASIC BIOLOGICAL SCIENCES↗

Metagenomics Reveals Diet-Specific Specialization of Bacterial Communities in Fungus Gardens of Grass- and Dicot-Cutter Ants

Leaf-cutter ants in the genus Atta are dominant herbivores in the Neotropics. While most species of Atta cut dicots to incorporate into their fungus gardens, some species specialize on grasses. Here we examine the bacterial community associated with the fungus gardens of grass- and dicot-cutter ants to examine how changes in substrate input affect the bacterial community. We sequenced the metagenomes of 12 Atta fungus gardens, across four species of ants, with a total of 5.316 Gbp of sequence data. We show significant differences in the fungus garden bacterial community composition between dicot- and grass-cutter ants, with grass-cutter ants having lower diversity. Reflecting this difference in community composition, the bacterial functional profiles between the fungus gardens are significantly different. Specifically, grass-cutter ant fungus garden metagenomes are particularly enriched for genes responsible for amino acid, siderophore, and terpenoid biosynthesis while dicot-cutter ant fungus gardens metagenomes are enriched in genes involved in membrane transport. Differences between community composition and functional capacity of the bacteria in the two types of fungus gardens reflect differences in the substrates that the ants incorporated. These results show that different substrate inputs matter for fungus garden bacteria and shed light on the potential role of bacteria in mediating the ants’ transition to the use of a novel substrate.

59 BASIC BIOLOGICAL SCIENCES↗

Instrument for evaluating the electrical resistance and wavelength-resolved transparency of stretchable electronics during strain

A complete analysis of strain tolerance in a stretchable transparent conductor (TC) should include tracking of both electrical conductivity and transparency during strain; however, transparency is generally neglected in contemporary analyses. In this paper, we describe an apparatus that tracks both parameters while TCs of arbitrary composition are deformed under stretching-mode strain. We demonstrate the tool by recording the electrical resistance and light transmission spectra for indium tin oxide-coated plastic substrates under both linearly increasing strain and complex cyclic strain processes. The optics are sensitive across the visible spectrum and into the near-infrared region (∼400-900 nm), and without specifically optimizing for sampling speed, we achieve a time resolution of ∼200 ms. In our automated analysis routine, we include a calculation of a common TC figure of merit (FOM), and because solar cell electrodes represent a key TC application, we also weigh both our transparency and FOM results against the solar power spectrum to determine “solar transparency” and “solar FOM.” Finally, we demonstrate how the apparatus may be adapted to measure the basic performance metrics for complete solar cells under uniaxial strain.

46 INSTRUMENTATION RELATED TO NUCLEAR SCIENCE AND ↗

Rapid Tooling for Functional Prototype of Metal Mold Processes Final Report CRADA No. TC-1032-98

Production inserts for die-casting were generally fabricated from materials with sufficient strength and· good wear properties at casting temperatures for long life. Frequently tool steels were used and machining was done with a combination of. conventional and Electric Discharge Machining (EDM) with some handwork, an expensive and time consuming process, partilly for prototype work. We proposed electron beam physical vapor deposition (EBPVD) as a process for rapid fabrication of dies. Metals, ranging from low melting point to refractory metals (Ta, Mo, etc.), would be evaporated and deposited at high rates (-2mm/hr.). Alloys could be easily evaporated and deposited if their constituent vapor pressures were similar and with more difficulty if they were not. Of course, layering of different materials was possible if required for a specific application. For example, a hard surface layer followed by a tough steel and backed by a high thermal conductivity (possibly cooled) copper layer could be fabricated. Electron-beam deposits exhibited 100% density and lull strength when deposited at a substrate (mandrel) temperature that was a substantial fraction of the deposited material's melting point. There were several materials that could have the required high temperature properties and ease of fabrication required for such a mandrel. We had successfully used graphite, machined from free formed objects with a replicator, to produce aluminum-bronze test molds. There were several parting layer materials of interest, but the ideal material depended upon the specific application.

36 MATERIALS SCIENCE↗

Which way does the dendrite grow? Competition among epitaxy, preferred growth direction, and thermal gradients in powder bed fusion additive manufacturing

The as-processed microstructure of metal alloy parts manufactured through laser powder bed fusion (LPBF) is heavily derived from the cellular dendritic solidification. The growth direction of dendrites within the melt pool is determined through competition among epitaxial growth, preferred growth directions, and maximum thermal gradients. However, the dominant factor and the specific role of each in developing melt pool microstructures remain unknown. Here, in this study, we performed single laser track scans on an SS316L single crystal substrate and combined experimental characterization of microstructure and crystal orientations with Computational Fluid Dynamics simulations of thermal gradients to evaluate the role of each factor in determining dendritic growth direction and evolution. Our results reveal that epitaxial growth dominates microstructure development by preferentially growing along a single 〈100〉 variant of the single crystal substrate adjacent to the melt pool boundary. Under LPBF’s highly curved and rapidly evolving thermal field, this preferential dendrite variant selection and its continued growth from the melt pool boundary to the centerline are governed by the local temperature gradient magnitude at the solid-liquid interface, rather than by the instantaneous maximum temperature gradient direction alone. Using these findings, we successfully predict changes in the dendrite growth direction with changing laser scan direction on a single crystal substrate, and show that the geometric melt pool centerline can deviate from the microstructural centerline because asymmetric local temperature gradient magnitudes transiently limit growth, resulting in different dendrite travel distances on each side of the melt pool.

36 MATERIALS SCIENCE↗

The unique architecture of umbrella toxins permits a two-tiered molecular bet-hedging strategy for interbacterial antagonism

Bacteria exist in competitive and rapidly changing environments in which the nature of future threats cannot be easily predicted. Streptomyces coelicolor produces three antibacterial umbrella particles that harbor distinct polymorphic toxin domains and an overlapping set of six diversified lectins. Here, we show that the exquisite specificity of umbrella particles derives from lectin-mediated species-specific binding to previously undescribed hypervariable surface glycoconjugates. A cryo-electron microscopy (cryo-EM) structure of one such lectin in complex with its oligosaccharide substrate defines the molecular basis for targeting through the coordinated recognition of multiple glycan features. Biochemical and genetic studies of several target species, in conjunction with lectin-swapping experiments, support a model whereby S. coelicolor umbrella toxin diversification at the levels of lectin composition and toxin polymorphism represents a unique, two-tiered bet-hedging strategy. Bioinformatic analyses support this as a means by which the unusual architecture of umbrella toxins offers Streptomyces a generalizable strategy to antagonize an unpredictable array of competitors.

59 BASIC BIOLOGICAL SCIENCES↗

Growth-coupled bioconversion of levulinic acid to butanone

Common strategies for conversion of lignocellulosic biomass to chemical products center on deconstructing biomass polymers into fermentable sugars. Here, we demonstrate an alternative strategy, a growth-coupled, high-yield bioconversion, by feeding cells a non-sugar substrate, by-passing central metabolism, and linking a key metabolic step to generation of acetyl-CoA that is required for biomass and energy generation. Specifically, we converted levulinic acid (LA), an established degradation product of lignocellulosic biomass, to butanone (a.k.a. methyl-ethyl ketone - MEK), a widely used industrial solvent. Our strategy combines a catabolic pathway from Pseudomonas putida that enables conversion of LA to 3-ketovaleryl-CoA, a CoA transferase that generates 3-ketovalerate and acetyl-CoA, and a decarboxylase that generates 2-butanone. By removing the ability of E. coli to consume LA and supplying excess acetate as a carbon source, we built a strain of E. coli that could convert LA to butanone at high yields, but at the cost of significant acetate consumption. Using flux balance analysis as a guide, we built a strain of E. coli that linked acetate assimilation to production of butanone. This strain was capable of complete bioconversion of LA to butanone with a reduced acetate requirement and increased specific productivity. To demonstrate the bioconversion on real world feedstocks, we produced LA from furfuryl alcohol, a compound readily obtained from biomass. These LA feedstocks were found to contain inhibitors that prevented cell growth and bioconversion of LA to butanone. We used a combination of column chromatography and activated carbon to remove the toxic compounds from the feedstock, resulting in LA that could be completely converted to butanone. In conclusion, this work motivates continued collaboration between chemical and biological catalysis researchers to explore alternative conversion pathways and the technical hurdles that prevent their rapid deployment.

59 BASIC BIOLOGICAL SCIENCES↗

Biochemical and Structural Characterization of Selective Allosteric Inhibitors of the Plasmodium falciparum Drug Target, Prolyl-tRNA-synthetase

Plasmodium falciparum (Pf) prolyl-tRNA synthetase (ProRS) is one of the few chemical-genetically validated drug targets for malaria, yet highly selective inhibitors have not been described. In this paper, approximately 40,000 compounds were screened to identify compounds that selectively inhibit PfProRS enzyme activity vs. H. sapiens (Hs) ProRS. X-ray crystallography structures were solved for apo, as well as substrate, and inhibitor bound forms of PfProRS. We identified two new inhibitors of PfProRS that bind outside the active site. These two allosteric inhibitors showed >100X specificity for PfProRS compared to HsProRS, demonstrating this class of compounds could overcome the toxicity related to HsProRS inhibition by halofuginone and its analogs. Initial medicinal chemistry was performed on one of the two compounds, guided by the co-crystallography of the compound with PfProRS, and the results can instruct future medicinal chemistry work to optimize these promising new leads for drug development against malaria.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗

Dissecting the cytochrome c2–reaction centre interaction in bacterial photosynthesis using single molecule force spectroscopy

The reversible docking of small, diffusible redox proteins onto a membrane protein complex is a common feature of bacterial, mitochondrial and photosynthetic electron transfer (ET) chains. Spectroscopic studies of ensembles of such redox partners have been used to determine ET rates and dissociation constants. Here, we report a single-molecule analysis of the forces that stabilise transient ET complexes. We examined the interaction of two components of bacterial photosynthesis, cytochrome c 2 and the reaction centre (RC) complex, using dynamic force spectroscopy and PeakForce quantitative nanomechanical imaging. RC–LH1–PufX complexes, attached to silicon nitride AFM probes and maintained in a photo-oxidised state, were lowered onto a silicon oxide substrate bearing dispersed, immobilised and reduced cytochrome c 2 molecules. Microscale patterns of cytochrome c 2 and the cyan fluorescent protein were used to validate the specificity of recognition between tip-attached RCs and surface-tethered cytochrome c 2 . Following the transient association of photo-oxidised RC and reduced cytochrome c 2 molecules, retraction of the RC-functionalised probe met with resistance, and forces between 112 and 887 pN were required to disrupt the post-ET RC– c 2 complex, depending on the retraction velocities used. If tip-attached RCs were reduced instead, the probability of interaction with reduced cytochrome c 2 molecules decreased 5-fold. Thus, the redox states of the cytochrome c 2 haem cofactor and RC ‘special pair’ bacteriochlorophyll dimer are important for establishing a productive ET complex. The millisecond persistence of the post-ET cytochrome c 2 [oxidised]–RC[reduced] ‘product’ state is compatible with rates of cyclic photosynthetic ET, at physiologically relevant light intensities.

Biochemistry & Molecular Biology↗