Enzyme cascades for in vitro and in vivo FMN prenylation and UbiD (de)carboxylase activation under aerobic conditions.

Microbial carboxylases and decarboxylases play important roles in the global carbon cycle and have many potential applications in biocatalysis and synthetic biology. The widespread family of reversible UbiD-like (de)carboxylases are of particular interest because these enzymes are active against a diverse range of substrates. Several characterized UbiD enzymes have been shown to catalyze reversible (de)carboxylation of aromatic and aliphatic substrates using the recently discovered prenylated FMN (prFMN) cofactor, which is produced by the associated family of UbiX FMN prenyltransferases.

Polyesterase activity is widespread in the family IV carboxylesterases from bacteria.

Enzyme-based depolymerization of plastics, including polyesters, has emerged as a promising approach for plastic waste recycling and reducing environmental plastic pollution. Currently, most of the known polyester-degrading enzymes are represented by a few natural and engineered PETases from the carboxylesterase family V. To identify novel groups of polyesterases, we selected 25 proteins from the carboxylesterase family IV, which share 22 % to 80 % sequence identity to the metagenomic thermophilic polyesterase IS12.

Biochemical plasticity of the Escherichia coli CRISPR Cascade revealed by in vitro reconstitution of Cascade activities from purified Cas proteins.

The most abundant clustered regularly interspaced short palindromic repeats (CRISPR) type I systems employ a multisubunit RNA-protein effector complex (Cascade), with varying protein composition and activity. The Escherichia coli Cascade complex consists of 11 protein subunits and functions as an effector through CRISPR RNA (crRNA) binding, protospacer adjacent motif (PAM)-specific double-stranded DNA targeting, R-loop formation, and Cas3 helicase-nuclease recruitment for target DNA cleavage. Here, we present a biochemical reconstruction of the E.

Charging and Mobilization of Dust Particles on a Surface in Plasma.

We present first experimental results showing that single dust particles on a dielectric surface are mobilized and lofted due to exposure to an electron beam or ultraviolet radiation. It is shown that secondary electrons and/or photoelectrons emitted from a substrate surface are recollected on the surfaces within microcavities between a dust particle and the substrate surface, resulting in large negative charges and subsequently causing mobilization of the dust particle due to Coulomb repulsion. Dust mobility tested against the electron beam energy is shown to follow the secondary electron yield curve of the substrate surface in both the experimental and modeling results.

Moderately thermostable GH1 β-glucosidases from hyperacidophilic archaeon Cuniculiplasma divulgatum S5.

Family GH1 glycosyl hydrolases are ubiquitous in prokaryotes and eukaryotes and are utilized in numerous industrial applications, including bioconversion of lignocelluloses. In this study, hyperacidophilic archaeon Cuniculiplasma divulgatum (S5T=JCM 30642T) was explored as a source of novel carbohydrate-active enzymes. The genome of C.

A random mutagenesis screen enriched for missense mutations in bacterial effector proteins.

To remodel their hosts and escape immune defenses, many pathogens rely on large arsenals of proteins (effectors) that are delivered to the host cell using dedicated translocation machinery. Effectors hold significant insight into the biology of both the pathogens that encode them and the host pathways that they manipulate. One of the most powerful systems biology tools for studying effectors is the model organism, Saccharomyces cerevisiae.

Gold/cobalt ferrite nanocomposite as a potential agent for photothermal therapy.

The study encompasses an investigation of optical, photothermal and biocompatibility properties of a composite consisting of golden cores surrounded by superparamagnetic CoFeO nanoparticles. Accompanied with the experiment, the computational modeling reveals that each adjusted magnetic nanoparticle redshifts the plasmon resonance frequency in gold and nonlinearly increases the extinction cross-section at ~800 nm. The concentration dependent photothermal study demonstrates a temperature increase of 8.

Ni(II)-binding affinity of CcNikZ-II and its homologs: the role of the HH-prong and variable loop revealed by structural and mutational studies.

Extracytoplasmic Ni(II)-binding proteins (NiBPs) are molecular shuttles involved in cellular nickel uptake. Here, we determined the crystal structure of apo CcNikZ-II at 2.38 Å, which revealed a Ni(II)-binding site comprised of the double His (HH-)prong (His511, His512) and a short variable (v-)loop nearby (Thr59-Thr64, TEDKYT).

Structural insights into hydrolytic defluorination of difluoroacetate by microbial fluoroacetate dehalogenases.

Fluorine forms the strongest single bond to carbon with the highest bond dissociation energy among natural products. However, fluoroacetate dehalogenases (FADs) have been shown to hydrolyze this bond in fluoroacetate under mild reaction conditions. Furthermore, two recent studies demonstrated that the FAD RPA1163 from Rhodopseudomonas palustris can also accept bulkier substrates.

Engineered nickel bioaccumulation in by NikABCDE transporter and metallothionein overexpression.

Mine wastewater often contains dissolved metals at concentrations too low to be economically extracted by existing technologies, yet too high for environmental discharge. The most common treatment is chemical precipitation of the dissolved metals using limestone and subsequent disposal of the sludge in tailing impoundments. While it is a cost-effective solution to meet regulatory standards, it represents a lost opportunity.

Quantifying metal-binding specificity of CcNikZ-II from Clostridium carboxidivorans in the presence of competing metal ions.

Many proteins bind transition metal ions as cofactors to carry out their biological functions. Despite binding affinities for divalent transition metal ions being predominantly dictated by the Irving-Williams series for wild-type proteins, in vivo metal ion binding specificity is ensured by intracellular mechanisms that regulate free metal ion concentrations. However, a growing area of biotechnology research considers the use of metal-binding proteins in vitro to purify specific metal ions from wastewater, where specificity is dictated by the protein's metal binding affinities.

Harnessing extremophilic carboxylesterases for applications in polyester depolymerisation and plastic waste recycling.

The steady growth in industrial production of synthetic plastics and their limited recycling have resulted in severe environmental pollution and contribute to global warming and oil depletion. Currently, there is an urgent need to develop efficient plastic recycling technologies to prevent further environmental pollution and recover chemical feedstocks for polymer re-synthesis and upcycling in a circular economy. Enzymatic depolymerization of synthetic polyesters by microbial carboxylesterases provides an attractive addition to existing mechanical and chemical recycling technologies due to enzyme specificity, low energy consumption, and mild reaction conditions.

CRISPR-induced DNA reorganization for multiplexed nucleic acid detection.

Nucleic acid sensing powered by the sequence recognition of CRIPSR technologies has enabled major advancement toward rapid, accurate and deployable diagnostics. While exciting, there are still many challenges facing their practical implementation, such as the widespread need for a PAM sequence in the targeted nucleic acid, labile RNA inputs, and limited multiplexing. Here we report FACT (Functionalized Amplification CRISPR Tracing), a CRISPR-based nucleic acid barcoding technology compatible with Cas12a and Cas13a, enabling diagnostic outputs based on cis- and trans-cleavage from any sequence.

Thermophilic Carboxylesterases from Hydrothermal Vents of the Volcanic Island of Ischia Active on Synthetic and Biobased Polymers and Mycotoxins.

Hydrothermal vents are geographically widespread and host microorganisms with robust enzymes useful in various industrial applications. We examined microbial communities and carboxylesterases of two terrestrial hydrothermal vents of the volcanic island of Ischia (Italy) predominantly composed of , , and . High-temperature enrichment cultures with the polyester plastics polyhydroxybutyrate and polylactic acid (PLA) resulted in an increase of and species and to some extent and species.

Systems engineering of Escherichia coli for n-butane production.

Rising concerns about climate change and sustainable energy have attracted efforts towards developing environmentally friendly alternatives to fossil fuels. Biosynthesis of n-butane, a highly desirable petro-chemical, fuel additive and diluent in the oil industry, remains a challenge. In this work, we first engineered enzymes Tes, Car and AD in the termination module to improve the selectivity of n-butane biosynthesis, and ancestral reconstruction and a synthetic RBS significantly improved the AD abundance.

Metabolite Damage and Damage Control in a Minimal Genome.

Analysis of the genes retained in the minimized JCVI-Syn3A genome established that systems that repair or preempt metabolite damage are essential to life. Several genes known to have such functions were identified and experimentally validated, including 5-formyltetrahydrofolate cycloligase, coenzyme A (CoA) disulfide reductase, and certain hydrolases. Furthermore, we discovered that an enigmatic YqeK hydrolase domain fused to NadD has a novel proofreading function in NAD synthesis and could double as a MutT-like sanitizing enzyme for the nucleotide pool.

Photoemission of Plasmonic Gold Nanostars in Laser-Controlled Electron Current Devices for Technical and Biomedical Applications.

The main goal of this work was to modify the previously developed blade-type planar structure using plasmonic gold nanostars in order to stimulate photofield emission and provide efficient laser control of the electron current. Localization and enhancement of the field at the tips of gold nanostars provided a significant increase in the tunneling electron current in the experimental sample (both electrical field and photofield emission). Irradiation at a wavelength in the vicinity of the plasmon resonance (red laser) provided a gain in the photoresponse value of up to 5 times compared to irradiation far from the resonance (green laser).

REVOLVER: A low-cost automated protein purifier based on parallel preparative gravity column workflows.

Protein purification is a ubiquitous procedure in biochemistry and the life sciences, and represents a key step in the protein production pipeline. The need for scalable and parallel protein purification systems is driven by the demands for increasing the throughput of recombinant protein characterization. Therefore, automating the process to simultaneously handle multiple samples with minimal human intervention is highly desirable, yet there are only a handful of such systems that have been developed, all of which are closed source and expensive.

Defluorination Capability of l-2-Haloacid Dehalogenases in the HAD-Like Hydrolase Superfamily Correlates with Active Site Compactness.

l-2-Haloacid dehalogenases, industrially and environmentally important enzymes that catalyse cleavage of the carbon-halogen bond in S-2-halocarboxylic acids, were known to hydrolyse chlorinated, brominated and iodinated substrates but no activity towards fluorinated compounds had been reported. A screen for novel dehalogenase activities revealed four l-2-haloacid dehalogenases capable of defluorination. We now report crystal structures for two of these enzymes, Bpro0530 and Rha0230, as well as for the related proteins PA0810 and RSc1362, which hydrolyse chloroacetate but not fluoroacetate, all at ∼2.

Structural and biochemical insights into CRISPR RNA processing by the Cas5c ribonuclease SMU1763 from Streptococcus mutans.

The cariogenic pathogen Streptococcus mutans contains two CRISPR systems (type I-C and type II-A) with the Cas5c protein (SmuCas5c) involved in processing of long CRISPR RNA transcripts (pre-crRNA) containing repeats and spacers to mature crRNA guides. In this study, we determined the crystal structure of SmuCas5c at a resolution of 1.72 Å, which revealed the presence of an N-terminal modified RNA recognition motif and a C-terminal twisted β-sheet domain with four bound sulphate molecules.

Automation assisted anaerobic phenotyping for metabolic engineering.

Background: Microorganisms can be metabolically engineered to produce a wide range of commercially important chemicals. Advancements in computational strategies for strain design and synthetic biological techniques to construct the designed strains have facilitated the generation of large libraries of potential candidates for chemical production. Consequently, there is a need for high-throughput laboratory scale techniques to characterize and screen these candidates to select strains for further investigation in large scale fermentation processes.

The HydS C-terminal domain of the Thiocapsa bogorovii HydSL hydrogenase is involved in membrane anchoring and electron transfer.

Thiocapsa bogorovii BBS (former name Thiocapsa roseopersicina) contains HydSL hydrogenase belonging to 1e subgroup of NiFe hydrogenases (isp-type). The operon of these hydrogenases contains gene for small subunit (hydS), gene for large subunit (hupL), and genes isp1 and isp2 between them. It is predicted that last two genes code electron transport careers for electron transfer from/to HydSL hydrogenase.

Modeling of Laser-Induced Plasmon Effects in GNS-DLC-Based Material for Application in X-ray Source Array Sensors.

An important direction in the development of X-ray computed tomography sensors in systems with increased scanning speed and spatial resolution is the creation of an array of miniature current sources. In this paper, we describe a new material based on gold nanostars (GNS) embedded in nanoscale diamond-like carbon (DLC) films (thickness of 20 nm) for constructing a pixel current source with photoinduced electron emission. The effect of localized surface plasmon resonance in GNS on optical properties in the wavelength range from UV to near IR, peculiarities of localization of field and thermal sources, generation of high-energy hot electrons, and mechanisms of their transportation in vacuum are investigated.

Lignin-oxidizing activity of bacterial laccases characterized using soluble substrates and polymeric lignin.

Efficient biotransformation of lignin requires the activity of different oxidative enzymes. In this work, 19 bacterial multi-copper oxidases were screened for oxidase activity against 19 soluble substrates and revealed the highest activity in the laccase CotA (BSU0630) from Bacillus subtilis. Structure-based site-directed mutagenesis of CotA identified four conserved residues (His419, Cys492, His497, and Met502) as critical for activity against 2,2'-azinobis(3-ethylbenzthiazoline-6-sulfonate) (ABTS).