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1.	Chen, Xin, 1980, et al. (författare) Dataset for suppressors of amyloid-beta toxicity and their functions in recombinant protein production in yeast 2022 Ingår i: Data in Brief. - : Elsevier BV. - 2352-3409. ; 42 Tidskriftsartikel (refereegranskat)abstract The production of recombinant proteins at high levels often induces stress-related phenotypes by protein misfolding or aggregation. These are similar to those of the yeast Alzheimer's disease (AD) model in which amyloid-beta peptides (A beta 42) were accumulated [1,2] . We have previously identified suppressors of A beta 42 cytotoxicity via the genome-wide synthetic genetic array (SGA) [3] and here we use them as metabolic engineering targets to evaluate their potentiality on recombinant protein production in yeast Saccharomyces cerevisiae. In order to investigate the mechanisms linking the genetic modifications to the improved recombinant protein production, we perform systems biology approaches (transcriptomics and proteomics) on the resulting strain and intermediate strains. The RNAseq data are preprocessed by the nf-core/RNAseq pipeline and analyzed using the Platform for Integrative Analysis of Omics (PIANO) package [4] . The quantitative proteome is analyzed on an Orbitrap Fusion Lumos mass spectrometer interfaced with an Easy-nLC1200 liquid chromatography (LC) system. LC-MS data files are processed by Proteome Discoverer version 2.4 with Mascot 2.5.1 as a database search engine. The original data presented in this work can be found in the research paper titled "Suppressors of Amyloid-beta Toxicity Improve Recombinant Protein Produc-tion in yeast by Reducing Oxidative Stress and Tuning Cellu-lar Metabolism", by Chen et al. [5] . (C) 2022 The Author(s). Published by Elsevier Inc.
2.	Chen, Xin, 1980, et al. (författare) Suppressors of amyloid-β toxicity improve recombinant protein production in yeast by reducing oxidative stress and tuning cellular metabolism 2022 Ingår i: Metabolic Engineering. - : Elsevier BV. - 1096-7176 .- 1096-7184. ; 72, s. 311-324 Tidskriftsartikel (refereegranskat)abstract High-level production of recombinant proteins in industrial microorganisms is often limited by the formation of misfolded proteins or protein aggregates, which consequently induce cellular stress responses. We hypothesized that in a yeast Alzheimer's disease (AD) model overexpression of amyloid-β peptides (Aβ42), one of the main peptides relevant for AD pathologies, induces similar phenotypes of cellular stress. Using this humanized AD model, we previously identified suppressors of Aβ42 cytotoxicity. Here we hypothesize that these suppressors could be used as metabolic engineering targets to alleviate cellular stress and improve recombinant protein production in the yeast Saccharomyces cerevisiae. Forty-six candidate genes were individually deleted and twenty were individually overexpressed. The positive targets that increased recombinant α-amylase production were further combined leading to an 18.7-fold increased recombinant protein production. These target genes are involved in multiple cellular networks including RNA processing, transcription, ER-mitochondrial complex, and protein unfolding. By using transcriptomics and proteomics analyses, combined with reverse metabolic engineering, we showed that reduced oxidative stress, increased membrane lipid biosynthesis and repressed arginine and sulfur amino acid biosynthesis are significant pathways for increased recombinant protein production. Our findings provide new insights towards developing synthetic yeast cell factories for biosynthesis of valuable proteins.
3.	Kreisel, Katrin, 1991, et al. (författare) DNA polymerase η contributes to genome-wide lagging strand synthesis. 2019 Ingår i: Nucleic acids research. - : Oxford University Press (OUP). - 1362-4962 .- 0305-1048. ; 47:5, s. 2425-2435 Tidskriftsartikel (refereegranskat)abstract DNA polymerase η (pol η) is best known for its ability to bypass UV-induced thymine-thymine (T-T) dimers and other bulky DNA lesions, but pol ηalso has other cellular roles. Here, we present evidence that pol η competes with DNA polymerases α and δfor the synthesis of the lagging strand genome-wide, where it also shows a preference for T-T in the DNA template. Moreover, we found that the C-terminus of pol η,which contains a PCNA-Interacting Protein motif is required for pol ηto function in lagging strand synthesis. Finally, we provide evidence that a pol η dependent signature is also found to be lagging strand specific in patients with skin cancer. Taken together, these findings provide insight into the physiological role of DNA synthesis by pol η and have implications for our understanding of how our genome is replicated to avoid mutagenesis, genome instability and cancer.
4.	Kroll, Alexander, et al. (författare) A general model to predict small molecule substrates of enzymes based on machine and deep learning 2023 Ingår i: Nature Communications. - 2041-1723 .- 2041-1723. ; 14:1 Tidskriftsartikel (refereegranskat)abstract For most proteins annotated as enzymes, it is unknown which primary and/or secondary reactions they catalyze. Experimental characterizations of potential substrates are time-consuming and costly. Machine learning predictions could provide an efficient alternative, but are hampered by a lack of information regarding enzyme non-substrates, as available training data comprises mainly positive examples. Here, we present ESP, a general machine-learning model for the prediction of enzyme-substrate pairs with an accuracy of over 91% on independent and diverse test data. ESP can be applied successfully across widely different enzymes and a broad range of metabolites included in the training data, outperforming models designed for individual, well-studied enzyme families. ESP represents enzymes through a modified transformer model, and is trained on data augmented with randomly sampled small molecules assigned as non-substrates. By facilitating easy in silico testing of potential substrates, the ESP web server may support both basic and applied science.
5.	Kroll, Alexander, et al. (författare) Deep learning allows genome-scale prediction of Michaelis constants from structural features 2021 Ingår i: PLoS Biology. - : Public Library of Science (PLoS). - 1544-9173 .- 1545-7885. ; 19:10 Tidskriftsartikel (refereegranskat)abstract AU The:Michaelis Pleaseconfirmthatallheadinglevelsarerepresentedcorrectly constant KM describes the affinity of an enzyme : for a specific substrate and is a central parameter in studies of enzyme kinetics and cellular physiology. As measurements of KM are often difficult and time-consuming, experimental estimates exist for only a minority of enzyme–substrate combinations even in model organisms. Here, we build and train an organism-independent model that successfully predicts KM values for natural enzyme–substrate combinations using machine and deep learning methods. Predictions are based on a task-specific molecular fingerprint of the substrate, generated using a graph neural network, and on a deep numerical representation of the enzyme’s amino acid sequence. We provide genome-scale KM predictions for 47 model organisms, which can be used to approximately relate metabolite concentrations to cellular physiology and to aid in the parameterization of kinetic models of cellular metabolism.
6.	Li, Gang, 1991, et al. (författare) Performance of Regression Models as a Function of Experiment Noise 2021 Ingår i: Bioinformatics and Biology Insights. - : SAGE Publications. - 1177-9322. ; 15 Tidskriftsartikel (refereegranskat)abstract Background: A challenge in developing machine learning regression models is that it is difficult to know whether maximal performance has been reached on the test dataset, or whether further model improvement is possible. In biology, this problem is particularly pronounced as sample labels (response variables) are typically obtained through experiments and therefore have experiment noise associated with them. Such label noise puts a fundamental limit to the metrics of performance attainable by regression models on the test dataset. Results: We address this challenge by deriving an expected upper bound for the coefficient of determination (R2) for regression models when tested on the holdout dataset. This upper bound depends only on the noise associated with the response variable in a dataset as well as its variance. The upper bound estimate was validated via Monte Carlo simulations and then used as a tool to bootstrap performance of regression models trained on biological datasets, including protein sequence data, transcriptomic data, and genomic data. Conclusions: The new method for estimating upper bounds for model performance on test data should aid researchers in developing ML regression models that reach their maximum potential. Although we study biological datasets in this work, the new upper bound estimates will hold true for regression models from any research field or application area where response variables have associated noise.
7.	Peng, Martin, et al. (författare) 3D-Printed Phenacrylate Decarboxylase Flow Reactors for the Chemoenzymatic Synthesis of 4-Hydroxystilbene 2019 Ingår i: Chemistry - A European Journal. - : Wiley. - 1521-3765 .- 0947-6539. ; 25:70, s. 15998-16001 Tidskriftsartikel (refereegranskat)abstract 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.
8.	Peng, Martin, et al. (författare) Modeling-Assisted Design of Thermostable Benzaldehyde Lyases from Rhodococcus erythropolis for Continuous Production of α-Hydroxy Ketones 2022 Ingår i: ChemBioChem. - : Wiley. - 1439-7633 .- 1439-4227. ; 23:7 Tidskriftsartikel (refereegranskat)abstract Enantiopure α-hydroxy ketones are important building blocks of active pharmaceutical ingredients (APIs), which can be produced by thiamine-diphosphate-dependent lyases, such as benzaldehyde lyase. Here we report the discovery of a novel thermostable benzaldehyde lyase from Rhodococcus erythropolis R138 (ReBAL). While the overall sequence identity to the only experimentally confirmed benzaldehyde lyase from Pseudomonas fluorescens Biovar I (PfBAL) was only 65 %, comparison of a structural model of ReBAL with the crystal structure of PfBAL revealed only four divergent amino acids in the substrate binding cavity. Based on rational design, we generated two ReBAL variants, which were characterized along with the wild-type enzyme in terms of their substrate spectrum, thermostability and biocatalytic performance in the presence of different co-solvents. We found that the new enzyme variants have a significantly higher thermostability (up to 22 °C increase in T50) and a different co-solvent-dependent activity. Using the most stable variant immobilized in packed-bed reactors via the SpyCatcher/SpyTag system, (R)-benzoin was synthesized from benzaldehyde over a period of seven days with a stable space-time-yield of 9.3 mmol ⋅ L-1 ⋅ d−1. Our work expands the important class of benzaldehyde lyases and therefore contributes to the development of continuous biocatalytic processes for the production of α-hydroxy ketones and APIs.
9.	Berglund, Anna-Karin, 1979, et al. (författare) Nucleotide pools dictate the identity and frequency of ribonucleotide incorporation in mitochondrial DNA. : Mapping ribonucleotides in mitochondrial DNA 2017 Ingår i: PLoS genetics. - : Public Library of Science (PLoS). - 1553-7404 .- 1553-7390. ; 13:2 Tidskriftsartikel (refereegranskat)abstract Previous work has demonstrated the presence of ribonucleotides in human mitochondrial DNA (mtDNA) and in the present study we use a genome-wide approach to precisely map the location of these. We find that ribonucleotides are distributed evenly between the heavy- and light-strand of mtDNA. The relative levels of incorporated ribonucleotides reflect that DNA polymerase γ discriminates the four ribonucleotides differentially during DNA synthesis. The observed pattern is also dependent on the mitochondrial deoxyribonucleotide (dNTP) pools and disease-causing mutations that change these pools alter both the absolute and relative levels of incorporated ribonucleotides. Our analyses strongly suggest that DNA polymerase γ-dependent incorporation is the main source of ribonucleotides in mtDNA and argues against the existence of a mitochondrial ribonucleotide excision repair pathway in human cells. Furthermore, we clearly demonstrate that when dNTP pools are limiting, ribonucleotides serve as a source of building blocks to maintain DNA replication. Increased levels of embedded ribonucleotides in patient cells with disturbed nucleotide pools may contribute to a pathogenic mechanism that affects mtDNA stability and impair new rounds of mtDNA replication.
10.	Börjesson, Johan, et al. (författare) Effect of poly(ethylene glycol) on enzymatic hydrolysis and adsorption of cellulase enzymes to pretreated lignocellulose 2007 Ingår i: Enzyme and Microbial Technology. - : Elsevier BV. - 0141-0229. ; 41:1-2, s. 186-195 Tidskriftsartikel (refereegranskat)abstract There is a need to develop the enzymatic hydrolysis of cellulose for production of ethanol from biomass. In recent years the inhibitory effects of lignin in lignocellulosic substrates has been the focus of several studies. This points to the importance of understanding the interactions between cellulose degrading enzymes and lignin. Surface active substances have been shown to adsorb to lignin surfaces resulting in reduction of unproductive enzyme binding. It is essential to understand the surface properties of both enzymes and lignin to develop pretreatment methods, surface active additives and engineering of cellulose degrading enzyme systems. This study investigates the PEG-lignin interaction as well as interactions between lignin and the enzyme modules of the Hypocrea jecorina (Trichoderma reesei) enzymes Cel7A and Cel7B. Interactions were monitored with C-14 labelled PEG 4000 and by measuring the enzymatic activity in solution. It was found that the dominating driving force of PEG adsorption on lignin is hydrophobic interaction. The effect of PEG addition on enzyme conversion of lignocellulose increased with higher temperature due to increased adsorption of PEG on lignin, thus resulting in a higher surface density of PEG on the surface. The hydrophobic adsorption of enzymes to lignin induces denaturation of enzymes on lignin surfaces. The addition of PEG to the enzyme hydrolysis at a temperature of 50 degrees C is suggested to hinder deactivation of enzymes by exclusion of enzymes from lignin surfaces. The adsorption of full-length Cel7B to lignin was stronger than for Cel7A. A more hydrophobic surface on the flat face of the cellulose binding module (CBM) together with an additional exposed aromatic residue on the rough face of Cel7B CBM compared to Cel7A CBM gives a higher affinity to lignin for the Cel7B enzyme. (c) 2007 Elsevier Inc. All rights reserved.
11.	Casey, John R., et al. (författare) Basin-scale biogeography of marine phytoplankton reflects cellular-scale optimization of metabolism and physiology 2022 Ingår i: Science advances. - : American Association for the Advancement of Science (AAAS). - 2375-2548. ; 8:3 Tidskriftsartikel (refereegranskat)abstract Extensive microdiversity within Prochlorococcus, the most abundant marine cyanobacterium, occurs at scales from a single droplet of seawater to ocean basins. To interpret the structuring role of variations in genetic potential, as well as metabolic and physiological acclimation, we developed a mechanistic constraint-based modeling framework that incorporates the full suite of genes, proteins, metabolic reactions, pigments, and biochemical compositions of 69 sequenced isolates spanning the Prochlorococcus pangenome. Optimizing each strain to the local, observed physical and chemical environment along an Atlantic Ocean transect, we predicted variations in strain-specific patterns of growth rate, metabolic configuration, and physiological state, defining subtle niche subspaces directly attributable to differences in their encoded metabolic potential. Predicted growth rates covaried with observed ecotype abundances, affirming their significance as a measure of fitness and inferring a nonlinear density dependence of mortality. Our study demonstrates the potential to interpret global-scale ecosystem organization in terms of cellular-scale processes.
12.	Engqvist, Martin, 1983, et al. (författare) ANT: Software for Generating and Evaluating Degenerate Codons for Natural and Expanded Genetic Codes 2015 Ingår i: ACS Synthetic Biology. - : American Chemical Society (ACS). - 2161-5063. ; 4:8, s. 935-938 Tidskriftsartikel (refereegranskat)abstract The Ambiguous Nucleotide Tool (ANT) is a desktop application that generates and evaluates degenerate codons. Degenerate codons are used to represent DNA positions that have multiple possible nucleotide alternatives. This is useful for protein engineering and directed evolution, where primers specified with degenerate codons are used as a basis for generating libraries of protein sequences. ANT is intuitive and can be used in a graphical user interface or by interacting with the code through a defined application programming interface. ANT comes with full support for nonstandard, user-defined, or expanded genetic codes (translation tables), which is important because synthetic biology is being applied to an ever widening range of natural and engineered organisms. The Python source code for ANT is freely distributed so that it may be used without restriction, modified, and incorporated in other software or custom data pipelines.
13.	Engqvist, Martin, 1983, et al. (författare) Applications of protein engineering and directed evolution in plant research 2019 Ingår i: Plant Physiology. - : Oxford University Press (OUP). - 1532-2548 .- 0032-0889. ; 179:3, s. 907-917 Tidskriftsartikel (refereegranskat)abstract Protein engineering and directed evolution are powerful technologies for probing protein sequencefunction relationships. Thesemethods have been used to engineer both plant-derived proteins and exogenous proteins heterologously expressed in plants. In this review, we aim to further increase the interdisciplinary crossover between the disciplines of protein engineering and plant biology by first introducing protein engineering in some detail. This introduction is key to understanding current limitations to protein engineering when applied to plants. Subsequently, we provide an overview of the recent methodological progress in, and novel applications of, protein engineering and directed evolution in plant research.
14.	Engqvist, Martin, 1983 (författare) Correlating enzyme annotations with a large set of microbial growth temperatures reveals metabolic adaptations to growth at diverse temperatures 2018 Ingår i: BMC Microbiology. - : Springer Science and Business Media LLC. - 1471-2180. ; 18:1 Tidskriftsartikel (refereegranskat)abstract Background: The ambient temperature of all habitats is a key physical property that shapes the biology of microbes inhabiting them. The optimal growth temperature (OGT) of a microbe, is therefore a key piece of data needed to understand evolutionary adaptations manifested in their genome sequence. Unfortunately there is no growth temperature database or easily downloadable dataset encompassing the majority of cultured microorganisms. We are thus limited in interpreting genomic data to identify temperature adaptations in microbes. Results: In this work I significantly contribute to closing this gap by mining data from major culture collection centres to obtain growth temperature data for a nonredundant set of 21,498 microbes. The dataset (https://doi.org/10.5281/zenodo.1175608) contains mainly bacteria and archaea and spans psychrophiles, mesophiles, thermophiles and hyperthermophiles. Using this data a full 43% of all protein entries in the UniProt database can be annotated with the growth temperature of the species from which they originate. I validate the dataset by showing a Pearson correlation of up to 0.89 between growth temperature and mean enzyme optima, a physiological property directly influenced by the growth temperature. Using the temperature dataset I correlate the genomic occurance of enzyme functional annotations with growth temperature. I identify 319 enzyme functions that either increase or decrease in occurrence with temperature. Eight metabolic pathways were statistically enriched for these enzyme functions. Furthermore, I establish a correlation between 33 domains of unknown function (DUFs) with growth temperature in microbes, four of which (DUF438, DUF1524, DUF1957 and DUF3458-C) were significant in both archaea and bacteria. Conclusions: The growth temperature dataset enables large-scale correlation analysis with enzyme function- and domain-level annotations. Growth-temperature dependent changes in their occurrence highlight potential evolutionary adaptations. A few of the identified changes are previously known, such as the preference for menaquinone biosynthesis through the futalosine pathway in bacteria growing at high temperatures. Others represent important starting points for future studies, such as DUFs where their occurrence change with temperature. The growth temperature dataset should become a valuable community resource and will find additional, important, uses in correlating genomic, transcriptomic, proteomic, metabolomic, phenotypic or taxonomic properties with temperature in future studies.
15.	Engqvist, Martin, 1983, et al. (författare) Directed evolution of gloeobacter violaceus rhodopsin spectral properties 2015 Ingår i: Journal of Molecular Biology. - : Elsevier BV. - 0022-2836 .- 1089-8638. ; 427:1, s. 205-220 Tidskriftsartikel (refereegranskat)abstract 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 violaceus 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 incurred 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 eight 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 WT (wild-type) protein.
16.	Engqvist, Martin, 1983, et al. (författare) GLYCOLATE OXIDASE3, a Glycolate Oxidase Homolog of Yeast l-Lactate Cytochrome c Oxidoreductase, Supports l-Lactate Oxidation in Roots of Arabidopsis 2015 Ingår i: Plant Physiology. - : Oxford University Press (OUP). - 1532-2548 .- 0032-0889. ; 169:2, s. 1042-1061 Tidskriftsartikel (refereegranskat)abstract In roots of Arabidopsis (Arabidopsis thaliana), L-lactate is generated by the reduction of pyruvate via L-lactate dehydrogenase, but this enzyme does not efficiently catalyze the reverse reaction. Here, we identify the Arabidopsis glycolate oxidase (GOX) paralogs GOX1, GOX2, and GOX3 as putative L-lactate-metabolizing enzymes based on their homology to CYB2, the L-lactate cytochrome c oxidoreductase from the yeast Saccharomyces cerevisiae. We found that GOX3 uses L-lactate with a similar efficiency to glycolate; in contrast, the photorespiratory isoforms GOX1 and GOX2, which share similar enzymatic properties, use glycolate with much higher efficiencies than L-lactate. The key factor making GOX3 more efficient with L-lactate than GOX1 and GOX2 is a 5- to 10-fold lower Km for the substrate. Consequently, only GOX3 can efficiently metabolize L-lactate at low intracellular concentrations. Isotope tracer experiments as well as substrate toxicity tests using GOX3 loss-offunction and overexpressor plants indicate that L-lactate is metabolized in vivo by GOX3. Moreover, GOX3 rescues the lethal growth phenotype of a yeast strain lacking CYB2, which cannot grow on L-lactate as a sole carbon source. GOX3 is predominantly present in roots and mature to aging leaves but is largely absent from young photosynthetic leaves, indicating that it plays a role predominantly in heterotrophic rather than autotrophic tissues, at least under standard growth conditions. In roots of plants grown under normoxic conditions, loss of function of GOX3 induces metabolic rearrangements that mirror wild-type responses under hypoxia. Thus, we identified GOX3 as the enzyme that metabolizes L-lactate to pyruvate in vivo and hypothesize that it may ensure the sustainment of low levels of L-lactate after its formation under normoxia.
17.	Engqvist, Martin, 1983 (författare) Highlighting the need for systems-level experimental characterization of plant metabolic enzymes 2016 Ingår i: Frontiers in Plant Science. - : Frontiers Media SA. - 1664-462X. ; 7:JULY2016 Tidskriftsartikel (refereegranskat)abstract 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.
18.	Engqvist, Martin, 1983, et al. (författare) Metabolic engineering of photorespiration 2017 Ingår i: Methods in Molecular Biology. - New York, NY : Springer New York. - 1940-6029 .- 1064-3745. ; 1653, s. 137-155 Bokkapitel (övrigt vetenskapligt/konstnärligt)abstract The introduction of two alternative glycolate catabolic pathways in the chloroplasts of Arabidopsis thaliana rendered plants with increased biomass. To introduce these synthetic pathways, the selected genes were stepwise integrated in the nuclear genome of wild-type plants. These plants were transformed by Agrobacterium tumefaciens carrying the binary vectors using the floral dip method. Selection of transformants was conducted using different selection agents and the expression of the transgenes was confirmed by PCR and enzyme activity measurements.
19.	Engqvist, Martin, 1983, et al. (författare) Mitochondrial 2-hydroxyglutarate metabolism 2014 Ingår i: Mitochondrion. - : Elsevier BV. - 1567-7249. ; 19:Part B, s. 275-281 Tidskriftsartikel (refereegranskat)abstract 2-Hydroxyglutarate (2-HG) is a five-carbon dicarboxylic acid with a hydroxyl group at the alpha position, which forms a stereocenter in this molecule. Although the existence of mitochondrial D- and L-2HG metabolisms has long been known in different eukaryotes, the biosynthetic pathways, especially in plants, have not been completely elucidated. While D-2HG is involved in intermediary metabolism, L-2HG may not have a cellular function but it needs to be recycled through a metabolic repair reaction. Independent of their metabolic origin, D- and L-2HG are oxidized in plant mitochondria to 2-ketoglutarate through the action of two stereospecific enzymes, d- and l-2-hydroxyacid dehydrogenases. While plants are to a large extent unaffected by high cellular concentrations of D-2HG, deficiencies in the metabolism of D- and L-2HG result in fatal disorders in humans. We present current data gathered on plant D- and L-2HG metabolisms and relate it to existing knowledge on 2HG metabolism in other organisms. We focus on the metabolic origin of these compounds, the mitochondrial catabolic steps catalyzed by the stereospecific dehydrogenases, and phylogenetic relationships between different studied 2-hydroxyacid dehydrogenases.
20.	Engqvist, Martin, 1983, et al. (författare) Plant D-2-hydroxyglutarate dehydrogenase participates in the catabolism of lysine especially during senescence 2011 Ingår i: Journal of Biological Chemistry. - 0021-9258 .- 1083-351X. ; 286:13, s. 11382-11390 Tidskriftsartikel (refereegranskat)abstract D-2-Hydroxyglutarate dehydrogenase (D-2HGDH) catalyzes the specific and efficient oxidation of D-2-hydroxyglutarate (D-2HG) to 2-oxoglutarate using FAD as a cofactor. In this work, we demonstrate that D-2HGDH localizes to plant mitochondria and that its expression increases gradually during developmental and dark-induced senescence in Arabidopsis thaliana, indicating an enhanced demand of respiration of alternative substrates through this enzymatic system under these conditions. Using loss-of-function mutants in D-2HGDH(d2hgdh1) and stable isotope dilution LC-MS/MS, we found that the D-isomer of 2HG accumulated in leaves of d2hgdh1 during both forms of carbon starvation. In addition to this, d2hgdh1 presented enhanced levels of most TCA cycle intermediates and free amino acids. In contrast to the deleterious effects caused by a deficiency in D-2HGDH in humans, d2hgdh1 and overexpressing lines of D-2HGDH showed normal developmental and senescence phenotypes, indicating a mild role of D-2HGDH in the tested conditions. Moreover, metabolic fingerprinting of leaves of plants grown in media supplemented with putative precursors indicated that D-2HG most probably originates during the catabolism of lysine. Finally, the L-isomer of 2HG was also detected in leaf extracts, indicating that both chiral forms of 2HG participate in plant metabolism.
21.	Engqvist, Martin, 1983, et al. (författare) Two D-2-hydroxy-acid dehydrogenases in arabidopsis thaliana with catalytic capacities to participate in the last reactions of the methylglyoxal and β-oxidation pathways 2009 Ingår i: Journal of Biological Chemistry. - 0021-9258 .- 1083-351X. ; 284:37, s. 25026-25037 Tidskriftsartikel (refereegranskat)abstract The Arabidopsis thaliana locus At5g06580 encodes an ortholog to Saccharomyces cerevisiae D-lactate dehydrogenase (AtD-LDH). The recombinant protein is a homodimer of 59-kDa subunits with one FAD per monomer. A substrate screen indicated that AtD-LDH catalyzes the oxidation of D- and L-lactate, D-2-hydroxybutyrate, glycerate, and glycolate using cytochrome c as an electron acceptor. AtD-LDH shows a clear preference for D-lactate, with a catalytic efficiency 200- and 2000-fold higher than that for L-lactate and glycolate, respectively, and a Km value for D-lactate of ∼160 μM. Knock-out mutants showed impaired growth in the presence of D-lactate or methylglyoxal. Collectively, the data indicated that the protein is a D-LDH that participates in planta in the methylglyoxal pathway. Web-based bioinformatic tools revealed the existence of a paralogous protein encoded by locus At4g36400. The recombinant protein is a homodimer of 61-kDa subunits with one FAD per monomer. A substrate screening revealed highly specific D-2-hydroxyglutarate (D-2HG) conversion in the presence of an organic cofactor with a Km value of ∼580 μM. Thus, the enzyme was characterized as a D-2HG dehydrogenase (AtD-2HGDH). Analysis of knock-out mutants demonstrated that AtD-2HGDH is responsible for the total D-2HGDH activity present in A. thaliana. Gene coexpression analysis indicated that AtD-2HGDH is in the same network as several genes involved in β-oxidation and degradation of branched-chain amino acids and chlorophyll. It is proposed that AtD-2HGDH participates in the catabolism of D-2HG most probably during the mobilization of alternative substrates from proteolysis and/or lipid degradation.
22.	Flytzanis, N.C., et al. (författare) Archaerhodopsin variants with enhanced voltage-sensitive fluorescence in mammalian and Caenorhabditis elegans neurons 2014 Ingår i: Nature Communications. - : Springer Science and Business Media LLC. - 2041-1723 .- 2041-1723. ; 5 Tidskriftsartikel (refereegranskat)abstract 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. Archer1s characteristics contribute to the goal of all-optical detection and modulation of activity in neuronal networks in vivo.
23.	Gajdoš, Matúš, et al. (författare) Chiral Alcohols from Alkenes and Water: Directed Evolution of a Styrene Hydratase 2023 Ingår i: Angewandte Chemie - International Edition. - : Wiley. - 1433-7851 .- 1521-3773. ; 62:7 Tidskriftsartikel (refereegranskat)abstract 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. Herein we report the identification and directed evolution of a fatty acid hydratase from Marinitoga hydrogenitolerans for the highly enantioselective hydration of styrenes to yield 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 provided access to chiral alcohols from simple alkenes and water with high enantioselectivity (>99 : 1 e.r.) and could be applied on a preparative scale.
24.	Gast, Veronica, 1992, et al. (författare) Engineering Saccharomyces cerevisiae for the production and secretion of Affibody molecules 2022 Ingår i: Microbial Cell Factories. - : Springer Science and Business Media LLC. - 1475-2859. ; 21:1 Tidskriftsartikel (refereegranskat)abstract BACKGROUND: Affibody molecules are synthetic peptides with a variety of therapeutic and diagnostic applications. To date, Affibody molecules have mainly been produced by the bacterial production host Escherichia coli. There is an interest in exploring alternative production hosts to identify potential improvements in terms of yield, ease of production and purification advantages. In this study, we evaluated the feasibility of Saccharomyces cerevisiae as a production chassis for this group of proteins. RESULTS: We examined the production of three different Affibody molecules in S. cerevisiae and found that these Affibody molecules were partially degraded. An albumin-binding domain, which may be attached to the Affibody molecules to increase their half-life, was identified to be a substrate for several S. cerevisiae proteases. We tested the removal of three vacuolar proteases, proteinase A, proteinase B and carboxypeptidase Y. Removal of one of these, proteinase A, resulted in intact secretion of one of the targeted Affibody molecules. Removal of either or both of the two additional proteases, carboxypeptidase Y and proteinase B, resulted in intact secretion of the two remaining Affibody molecules. The produced Affibody molecules were verified to bind their target, human HER3, as potently as the corresponding molecules produced in E. coli in an in vitro surface-plasmon resonance binding assay. Finally, we performed a fed-batch fermentation with one of the engineered protease-deficient S. cerevisiae strains and achieved a protein titer of 530 mg Affibody molecule/L. CONCLUSION: This study shows that engineered S. cerevisiae has a great potential as a production host for recombinant Affibody molecules, reaching a high titer, and for proteins where endotoxin removal could be challenging, the use of S. cerevisiae obviates the need for endotoxin removal from protein produced in E. coli.
25.	Gast, Veronica, 1992, et al. (författare) The Yeast eIF2 Kinase Gcn2 Facilitates H 2 O 2 -Mediated Feedback Inhibition of Both Protein Synthesis and Endoplasmic Reticulum Oxidative Folding during Recombinant Protein Production 2021 Ingår i: Applied and Environmental Microbiology. - 1098-5336 .- 0099-2240. ; 87:15, s. e0030121-16 Tidskriftsartikel (refereegranskat)abstract Recombinant protein production is a known source of oxidative stress. However, knowledge of which reactive oxygen species are involved or the specific growth phase in which stress occurs remains lacking. Using modern, hypersensitive genetic H2O2-specific probes, microcultivation, and continuous measurements in batch culture, we observed H2O2 accumulation during and following the diauxic shift in engineered Saccharomyces cerevisiae, correlating with peak α-amylase production. In agreement with previous studies supporting a role of the translation initiation factor kinase Gcn2 in the response to H2O2, we find that Gcn2-dependent phosphorylation of eIF2α increases alongside translational attenuation in strains engineered to produce large amounts of α-amylase. Gcn2 removal significantly improved α-amylase production in two previously optimized high-producing strains but not in the wild type. Gcn2 deficiency furthermore reduced intracellular H2O2 levels and the Hac1 splicing ratio, while expression of antioxidants and the endoplasmic reticulum (ER) disulfide isomerase PDI1 increased. These results suggest protein synthesis and ER oxidative folding are coupled and subject to feedback inhibition by H2O2. IMPORTANCE Recombinant protein production is a multibillion dollar industry. Optimizing the productivity of host cells is, therefore, of great interest. In several hosts, oxidants are produced as an unwanted side product of recombinant protein production. The buildup of oxidants can result in intracellular stress responses that could compromise the productivity of the host cell. Here, we document a novel protein synthesis inhibitory mechanism that is activated by the buildup of a specific oxidant (H2O2) in the cytosol of yeast cells upon the production of recombinant proteins. At the center of this inhibitory mechanism lies the protein kinase Gcn2. By removing Gcn2, we observed a doubling of recombinant protein productivity in addition to reduced H2O2 levels in the cytosol. In this study, we want to raise awareness of this inhibitory mechanism in eukaryotic cells to further improve protein production and contribute to the development of novel protein-based therapeutic strategies.

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