Highlighting the Need for Systems-Level Experimental Characterization of Plant Metabolic Enzymes

The biology of living organisms is determined by the action and interaction of a large number of individual gene products, each with specific functions. Discovering and annotating the function of gene products is key to our understanding of these organisms. Controlled experiments and bioinformatic predictions both contribute to functional gene annotation. For most species it is difficult to gain an overview of what portion of gene annotations are based on experiments and what portion represent predictions. Here, I survey the current state of experimental knowledge of enzymes and metabolism in Arabidopsis thaliana as well as eleven economically important crops and forestry trees – with a particular focus on reactions involving organic acids in central metabolism. I illustrate the limited availability of experimental data for functional annotation of enzymes in most of these species. Many enzymes involved in metabolism of citrate, malate, fumarate, lactate, and glycolate in crops and forestry trees have not been characterized. Furthermore, enzymes involved in key biosynthetic pathways which shape important traits in crops and forestry trees have not been characterized. I argue for the development of novel high-throughput platforms with which limited functional characterization of gene products can be performed quickly and relatively cheaply. I refer to this approach as systems-level experimental characterization. The data collected from such platforms would form a layer intermediate between bioinformatic gene function predictions and in-depth experimental studies of these functions. Such a data layer would greatly aid in the pursuit of understanding a multiplicity of biological processes in living organisms

3D‐Printed Phenacrylate Decarboxylase Flow Reactors for the Chemoenzymatic Synthesis of 4‐Hydroxystilbene

Continuous flow systems for chemical synthesis are becoming a major focus in organic chemistry and there is a growing interest in the integration of biocatalysts due to their high regio‐ and stereoselectivity. Methods established for 3D bioprinting enable the fast and simple production of agarose‐based modules for biocatalytic reactors if thermally stable enzymes are available. We report here on the characterization of four different cofactor‐free phenacrylate decarboxylase enzymes suitable for the production of 4‐vinylphenol and test their applicability for the encapsulation and direct 3D printing of disk‐shaped agarose‐based modules that can be used for compartmentalized flow microreactors. Using the most active and stable phenacrylate decarboxylase from Enterobacter spec. in a setup with four parallel reactors and a subsequent palladium(II) acetate‐catalysed Heck reaction, 4‐hydroxystilbene was synthesized from p‐coumaric acid with a total yield of 14.7 % on a milligram scale. We believe that, due to the convenient direct immobilization of any thermostable enzyme and straightforward tuning of the reaction sequence by stacking of modules with different catalytic activities, this simple process will facilitate the establishment and use of cascade reactions and will therefore be of great advantage for many research approaches

Directed Evolution of Gloeobacter violaceus Rhodopsin Spectral Properties

Proton-pumping rhodopsins (PPRs) are photoactive retinal-binding proteins that transport ions across biological membranes in response to light. These proteins are interesting for light-harvesting applications in bioenergy production, in optogenetics applications in neuroscience, and as fluorescent sensors of membrane potential. Little is known, however, about how the protein sequence determines the considerable variation in spectral properties of PPRs from different biological niches or how to engineer these properties in a given PPR. Here we report a comprehensive study of amino acid substitutions in the retinal binding pocket of Gloeobacter violacaeus rhodopsin (GR) that tune its spectral properties. Directed evolution generated 70 GR variants with absorption maxima shifted by up to +/- 80 nm, extending the protein’s light absorption significantly beyond the range of known natural PPRs. While proton pumping activity was disrupted in many of the spectrally shifted variants, we identified single tuning mutations that incurrred blue and red shifts of 42 nm and 22 nm, respectively, that did not disrupt proton pumping. Blue-shifting mutations were distributed evenly along the retinal molecule while red-shifting mutations were clustered near the residue K257, which forms a covalent bond with retinal through a Schiff base linkage. Thirty-four of the identified tuning mutations are not found in known microbial rhodopsins. We discovered a subset of red-shifted GRs that exhibit high levels of fluorescence relative to the wild-type protein

Transgenic Introduction of a Glycolate Oxidative Cycle into A. thaliana Chloroplasts Leads to Growth Improvement

The photorespiratory pathway helps illuminated C3-plants under conditions of limited CO2 availability by effectively exporting reducing equivalents in form of glycolate out of the chloroplast and regenerating glycerate-3-P as substrate for RubisCO. On the other hand, this pathway is considered as probably futile because previously assimilated CO2 is released in mitochondria. Consequently, a lot of effort has been made to reduce this CO2 loss either by reducing fluxes via engineering RubisCO or circumventing mitochondrial CO2 release by the introduction of new enzyme activities. Here we present an approach following the latter route, introducing a complete glycolate catabolic cycle in chloroplasts of Arabidopsis thaliana comprising glycolate oxidase (GO), malate synthase (MS), and catalase (CAT). Results from plants bearing both GO and MS activities have already been reported (Fahnenstich et al., 2008). This previous work showed that the H2O2 produced by GO had strongly negative effects. These effects can be prevented by introducing a plastidial catalase activity, as reported here. Transgenic lines bearing all three transgenic enzyme activities were identified and some with higher CAT activity showed higher dry weight, higher photosynthetic rates, and changes in glycine/serine ratio compared to the wild type. This indicates that the fine-tuning of transgenic enzyme activities in the chloroplasts seems crucial and strongly suggests that the approach is valid and that it is possible to improve the growth of A. thaliana by introducing a synthetic glycolate oxidative cycle into chloroplasts

Archaerhodopsin variants with enhanced voltage-sensitive fluorescence in mammalian and Caenorhabditis elegans neurons

Probing the neural circuit dynamics underlying behaviour would benefit greatly from improved genetically encoded voltage indicators. The proton pump ​Archaerhodopsin-3 (​Arch), an optogenetic tool commonly used for neuronal inhibition, has been shown to emit voltage-sensitive fluorescence. Here we report two ​Arch variants with enhanced radiance (Archers) that in response to 655 nm light have 3–5 times increased fluorescence and 55–99 times reduced photocurrents compared with ​Arch WT. The most fluorescent variant, Archer1, has 25–40% fluorescence change in response to action potentials while using 9 times lower light intensity compared with other ​Arch-based voltage sensors. Archer1 is capable of wavelength-specific functionality as a voltage sensor under red light and as an inhibitory actuator under green light. As a proof-of-concept for the application of ​Arch-based sensors in vivo, we show fluorescence voltage sensing in behaving Caenorhabditis elegans. Archer1’s characteristics contribute to the goal of all-optical detection and modulation of activity in neuronal networks in vivo

Directed evolution of a far-red fluorescent rhodopsin

Microbial rhodopsins are a diverse group of photoactive transmembrane proteins found in all three domains of life. A member of this protein family, Archaerhodopsin-3 (Arch) of halobacterium Halorubrum sodomense, was recently shown to function as a fluorescent indicator of membrane potential when expressed in mammalian neurons. Arch fluorescence, however, is very dim and is not optimal for applications in live-cell imaging. We used directed evolution to identify mutations that dramatically improve the absolute brightness of Arch, as confirmed biochemically and with live-cell imaging (in Escherichia coli and human embryonic kidney 293 cells). In some fluorescent Arch variants, the pK_a of the protonated Schiff-base linkage to retinal is near neutral pH, a useful feature for voltage-sensing applications. These bright Arch variants enable labeling of biological membranes in the far-red/infrared and exhibit the furthest red-shifted fluorescence emission thus far reported for a fluorescent protein (maximal excitation/emission at ∼620 nm/730 nm)

Surface softening in metal-ceramic sliding contacts: An experimental and numerical investigation

This study investigates the tribolayer properties at the interface of ceramic/metal (i.e., WC/W) sliding contacts using various experimental approaches and classical atomistic simulations. Experimentally, nanoindentation and micropillar compression tests, as well as adhesion mapping by means of atomic force microscopy, are used to evaluate the strength of tungsten?carbon tribolayers. To capture the influence of environmental conditions, a detailed chemical and structural analysis is performed on the worn surfaces by means of XPS mapping and depth profiling along with transmission electron microscopy of the debris particles. Experimentally, the results indicate a decrease in hardness and modulus of the worn surface compared to the unworn one. Atomistic simulations of nanoindentation on deformed and undeformed specimens are used to probe the strength of the WC tribolayer and despite the fact that the simulations do not include oxygen, the simulations correlate well with the experiments on deformed and undeformed surfaces, where the difference in behavior is attributed to the bonding and structural differences of amorphous and crystalline W-C. Adhesion mapping indicates a decrease in surface adhesion, which based on chemical analysis is attributed to surface passivation

Cancro e actividade profissional: contributo para o estudo nos cancros de cabeça e pescoço e pulmão, brônquios e traqueia na região Centro, de 2002 a 2006

Dissertação de mestrado em Saúde Ocupacional, apresentada à Fac. de Medicina da Universidade de CoimbraO cancro tem um impacto cada vez mais emergente nas sociedades em desenvolvimento e no mundo em geral, surgindo muitas vezes associado aos hábitos de vida das populações, inclusive no que se refere à sua ocupação. Centrados nos indivíduos residentes na região centro do país com diagnóstico de Cancro de Cabeça e Pescoço e Cancro do Pulmão, Brônquios e Traqueia diagnosticado entre 2002 e 2006, definimos como objectivos centrais para o presente estudo analisar possíveis relações entre o tipo de tumor e sua incidência e algumas variáveis sócio-demográficas e a profissão/ocupação. No estudo foram utilizados os dados de 1011 doentes, predominando o género masculino (83,3%). À data do diagnóstico do tumor, as idades situavam-se entre os 18.90 anos e 93.60 anos, sendo a média de 63.47. No género masculino os tumores foram diagnosticados com idades inferiores aos do género feminino, sendo esta diferença estatisticamente significativa (p < 0.001), o que sugere que os doentes de género masculino tendem a desenvolver mais cedo o tipo de tumores em estudo. Relativamente à localização topográfica do tumor, verificamos que predominam os brônquios e pulmões (29.8%), a laringe (16.3%) e partes da língua não especificadas (10.5%). Verificaram-se também diferenças estatisticamente significativas (p < 0.001) no que se refere à incidência de tumores em função da sua localização anatómica e a profissão/ocupação dos doentes, o que parece sugerir que os tumores de cabeça e pescoço têm maior incidência nas profissões que exigem maior esforço físico, enquanto que os tumores do Pulmão, Brônquios e Traqueia apresentam maior incidência nas profissões fisicamente menos exigentes. Com base nestes resultados são apresentadas sugestões de trabalho e de investigação, certos de que conhecer melhor este processo, contribuirá para uma maior sensibilização dos Serviços Ocupacionais no sentido de que, os factores de risco profissionais sejam equacionados numa perspectiva global e integradora que caracteriza as acções de promoção da saúde.Cancer has an ever emerging impact on developing societies and on the world. It is often associated to a population’s lifestyle, in particular to their occupation/profession. Based on a population resident in the central zone of the country, with the diagnosis of an Head and Neck, Lung, Bronchus and/or Tracheal cancer, diagnosed between 2002 and 2006, we defined as the main aim of this study, analyze the possible relationship between the type and incidence of the various tumors and their social-demographic and occupational variables. One thousand and eleven (1011) patients were studied, the majority were male (83,3%), with an average age of 63,47 years, varying between 18,90 to 93,60 years . There was a statistical difference in the age incidence between the male and female groups (p < 0.001). We found that male individuals were significantly younger than their female counterparts. This suggests that males tend to develop these kinds of tumors earlier. Regarding their anatomical distribution, we found a predominance of lung and bronqueal tumors (29.8%), larynx (16.3%) and tongue (10.5%). We verified significant statistical differences (p < 0.001) between the anatomical distribution and the occupation of the patients. It seems that Head and neck tumors tend to appear mostly in individuals whose occupation involves greater physical stress, in contrast to Lung, Bronchus and Traqueal tumors. Based on these findings and results, we present suggestions of studies and investigations that can be conducted in this area. We are sure that a greater understanding of this entity, will contribute to a greater awareness by the Occupational Services of the professional risk factors and their integration in health promotional and preventive actions

Chiral Alcohols from Alkenes and Water: Directed Evolution of a Styrene Hydratase

Gajdos M, Wagner J, Ospina Sánchez F, Köhler A, Engqvist MKM, Hammer S. Chiral Alcohols from Alkenes and Water: Directed Evolution of a Styrene Hydratase. Angewandte Chemie International Edition. 2022.Enantioselective synthesis of chiral alcohols through asymmetric addition of water across an unactivated alkene is a highly sought-after transformation and a big challenge in catalysis. Here we report the identification and directed evolution of a fatty acid hydratase fromMarinitoga hydrogenitoleransfor highly enantioselective hydration of styrenes yielding chiral 1-arylethanols. While directed evolution for styrene hydration was performed in the presence of heptanoic acid to mimic fatty acid binding, the engineered enzyme displayed remarkable asymmetric styrene hydration activity in the absence of the small molecule activator. The evolved styrene hydratase provides access to chiral alcohols with high enantioselectivity (>99:1 e.r.), yield (up to 98%) and on preparative scale using simple alkenes and water as starting material. © 2022 Wiley-VCH GmbH