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PBAT (poly butylene adipate-co-terephthalate) is a widely used biodegradable plastic, but the knowledge about its metabolization in anaerobic environments is very limited. In this study, the anaerobic digester sludge from a municipal wastewater treatment plant was used as inoculum to investigate the biodegradability of PBAT monomers in thermophilic conditions. The research employs a combination of 13C-labelled monomers and proteogenomics to track the labelled carbon and identify the microorganisms involved. A total of 122 labelled peptides of interest were identified for adipic acid (AA) and 1,4-butanedio (BD). Through the time-dependent isotopic enrichment and isotopic profile distributions, Bacteroides, Ichthyobacterium, and Methanosarcina were proven to be directly involved in the metabolization of at least one monomer. This study provides a first insight into the identity and genomic potential of microorganisms responsible for biodegradability of PBAT monomers during anaerobic digestion under thermophilic conditions.

Methicillin-resistant Staphylococcus aureus (MRSA) is a major human pathogen that causes a wide range of infections. Its resistance to β-lactam antibiotics complicates treatment due to the limited number of antibiotics with activity against MRSA. To investigate development of alternative therapeutics, the mechanisms that mediate antibiotic resistance in MRSA need to be fully understood. In the present study, MRSA cells were subjected to antibiotic stress from methicillin in combination with three cannabinoid compounds and analyzed using proteomics to assess the changes in physiology. Subjecting MRSA to nonlethal levels of methicillin resulted in an increased production of penicillin-binding protein 2 (PBP2). Exposure to cannabinoids showed antibiotic activity against MRSA, and differential proteomics revealed reduced levels of proteins involved in the energy production as well as PBP2 when used in combination with methicillin.

Peptide emulsifiers derived from plant proteins are gathering growing interest as green and sustainable replacements for chemical additives in food. Potato (Solanum tuberosum) is one of the most important crops for both human consumption and industrial processing. Globally, the annual production of potato starch exceeds 3,000,000 MT with more than 200,000 MT of potato protein isolated as a side-stream, providing an enormous source of raw protein. The direct isolation of food-grade protein is in many cases regarded as cost-ineffective. Nevertheless, potato proteins may be a valuable source of functional peptides.Previously, we demonstrated amphiphilicity-based bioinformatic prediction of peptide emulsifiers embedded in potato proteins1,2. Amongst the predicted peptides, especially one (γ1), derived from the storage protein patatin, showed exceptional emulsifying activity in vitro. Although patatin is the most abundant protein in potatoes, it is not a single protein, but a family of highly homologous isoforms. Using bottom-up proteomics in combination with multiple sequence alignment and in silico digestion, we identified several γ1 variants. The variants consist of both full-length isoforms with single amino acid substitutions and tryptic variants/truncations from different patatin isoforms. The emulsifying activity of the γ1 variants, physical stability of the emulsions during storage, and interfacial properties were investigated3. Furthermore, the interfacial conformation of the peptides was investigated by SRCD and supplemented by NMR and benchtop CD for selected peptides in micellar model systems. Based on these results, we are able to i) evaluate the full potential of using γ1 variants as peptide emulsifiers in food; ii) provide novel insight on the structure/function relationship of amphipathic, α-helical peptide emulsifiers; iii) combine in vitro functional validation with in silico proteolysis to design a scalable and targeted enzymatic hydrolysis, resulting in a hydrolysate with improved emulsifying properties; and iv) apply the hydrolysates as stabilizers for encapsulation of fish oil for foods.

Additional file 1: Table S1. This table contains the primer sequences of both the primers used for gene-amplification and the primer used for initial sanger-sequencing in fragments of around 700 bp, containing at least 50 bp overlap between each fragment. Table S2. This table contains the different plasmids utilized in the project, both the native plasmids used as expression vectors, but also plasmids purchased containing the synthetically derived codon optimized genes. Figure S1. Phylogenetic tree of the PPTases used in the present study (bold) together with 22 additional published PPTases. Bootstrap values (> 70%) from 1000 replications are indicated at the respective nodes. Figure S2. Predicted structure of sfp/ACP interaction with the CoA and Mg2+ ion highlighted by arrows. Figure S3. Production levels of bikaverin and bostrycoidin in the individual strains (relative to OD at 48 h) in the supernatant and pellets. The mean of the supernatant from BY4743::FvPPT1 was set to 100 for both compounds.

Natural products display a large structural variation and different uses within a broad spectrum of industries. In this study, we investigate the influence of carbohydrates and nitrogen sources on the production and selectivity of production of four different polyketides produced by Fusarium solani, fusarubin, javanicin, bostrycoidin and anhydrofusarubin. We introduce four different carbohydrates and two types of nitrogen sources. Hereafter, a full factorial design was applied using combinations of three levels of sucrose and three levels of the two types of nitrogen. Each combination displayed different selectivity and production yields for all the compounds of interest. Response surface design was utilized to investigate possible maximum yields for the surrounding combinations of media. It was also shown that the maximum yields were not always the ones illustrating high selectivity, which is an important factor for making purification steps easier. We visualized the production over time for one of the media types, illustrating high yields and selectivity.

Lytic polysaccharide monooxygenases (LPMOs) have a unique ability to activate molecular oxygen for subsequent oxidative cleavage of glycosidic bonds. To provide insight into the mode of action of these industrially important enzymes, we have performed an integrated NMR/electron paramagnetic resonance (EPR) study into the detailed aspects of an AA10 LPMO–substrate interaction. Using NMR spectroscopy, we have elucidated the solution-phase structure of apo-BlLPMO10A from Bacillus licheniformis, along with solution-phase structural characterization of the Cu(I)-LPMO, showing that the presence of the metal has minimal effects on the overall protein structure. We have, moreover, used paramagnetic relaxation enhancement (PRE) to characterize Cu(II)-LPMO by NMR spectroscopy. In addition, a multifrequency continuous-wave (CW)-EPR and 15N-HYSCORE spectroscopy study on the uniformly isotope-labeled 63Cu(II)-bound 15N-BlLPMO10A along with its natural abundance isotopologue determined copper spin-Hamiltonian parameters for LPMOs to markedly improved accuracy. The data demonstrate that large changes in the Cu(II) spin-Hamiltonian parameters are induced upon binding of the substrate. These changes arise from a rearrangement of the copper coordination sphere from a five-coordinate distorted square pyramid to one which is four-coordinate near-square planar. There is also a small reduction in metal–ligand covalency and an attendant increase in the d(x2−y2) character/energy of the singly occupied molecular orbital (SOMO), which we propose from density functional theory (DFT) calculations predisposes the copper active site for the formation of a stable Cu–O2 intermediate. This switch in orbital character upon addition of chitin provides a basis for understanding the coupling of substrate binding with O2 activation in chitin-active AA10 LPMOs.

Fusarium pigmentation is dictated by a set of two polyketide synthase (PKS) gene clusters where one is expressed during mycelial growth and the other during perithecial development. In the vast majority of Fusarium species, perithecial pigmentation relies on the PKS3 gene cluster responsible for biosynthesis of fusarubins and bostrycoidins. In these species, mycelial pigmentation is mediated by bikaverin or aurofusarin. However, the situation is different for F. solani where mycelial pigmentation is controlled by the PKS3 gene cluster, while the less common PKS35 is responsible for the perithecial pigmentation, although no actual compound(s) has ever been associated with the latter. We set out to characterize the polyketide pigments of F. solani. The PKS3 gene cluster of F. solani shares seven genes with the previously characterized clusters in F. graminearum and F. fujikuroi. However, it differs from the previously described clusters by containing additional genes, some with predicted enzymatic function related to secondary metabolism. When we overexpressed the cluster specific transcription factor in F. solani, we observed a massive increase in production of javanicin, bostrycoidin, fusarubin and dihydrofusarubin. In order to investigate the perithecial pigment chemistry we performed heterologous expression of the PKS35 in Saccharomyces cerevisiae, yielding prephenalenone and dehydroxyprephenalenone as the initial polyketide intermediates. Secondly, we overexpressed the local transcription factor gene in F. solani, resulting in a dark green and orange/red phenotype. We identified three new candidate compounds by mass spectrometry. To our surprise, the mutant also produced elevated levels of javanicin and bostrycoidin obscuring the observation and isolation of PKS35-related compounds. Interestingly, this co-expression indicates the regulation of both PKS3 and PKS35 gene clusters is somehow connected. Fusarium pigmentation is dictated by a set of two polyketide synthase (PKS) gene clusters where one is expressed during mycelial growth and the other during perithecial development. In the vast majority of Fusarium species, perithecial pigmentation relies on the PKS3 gene cluster responsible for biosynthesis of fusarubins and bostrycoidins. In these species, mycelial pigmentation is mediated by bikaverin or aurofusarin. However, the situation is different for F. solani where mycelial pigmentation is controlled by the PKS3 gene cluster, while the less common PKS35 is responsible for the perithecial pigmentation, although no actual compound(s) has ever been associated with the latter. We set out to characterize the polyketide pigments of F. solani. The PKS3 gene cluster of F. solani shares seven genes with the previously characterized clusters in F. graminearum and F. fujikuroi. However, it differs from the previously described clusters by containing additional genes, some with predicted enzymatic function related to secondary metabolism. When we overexpressed the cluster specific transcription factor in F. solani, we observed a massive increase in production of javanicin, bostrycoidin, fusarubin and dihydrofusarubin. In order to investigate the perithecial pigment chemistry we performed heterologous expression of the PKS35 in Saccharomyces cerevisiae, yielding prephenalenone and dehydroxyprephenalenone as the initial polyketide intermediates. Secondly, we overexpressed the local transcription factor gene in F. solani, resulting in a dark green and orange/red phenotype. We identified three new candidate compounds by mass spectrometry. To our surprise, the mutant also produced elevated levels of javanicin and bostrycoidin obscuring the observation and isolation of PKS35-related compounds. Interestingly, this co-expression indicates the regulation of both PKS3 and PKS35 gene clusters is somehow connected.

Dissertation, Rheinisch-Westfälische Technische Hochschule Aachen, 2020; Aachen 1 Online-Ressource (167 Seiten) : Illustrationen, Diagramme (2020). = Dissertation, Rheinisch-Westfälische Technische Hochschule Aachen, 2020, In this thesis, BIM is examined as part of the integral planning methodology. Specifically, the BIM goal in this thesis is to develop the thermal-energy simulation of HVAC systems using the harmonized Information Delivery Manual (IDM) and Model View Definition (MVD) method, using various application examples. Based thereon, the requirements for a given software architecture are derived and several data models are interconnected. The connection of the different data models is accomplished via specially defined transformation logics. The approaches based on the object and data level allow the definition of the associated Level of Development (LOD)s. Through these extensive elaborations future tools and methods can be established and tailored according to practical requirements. With the development of an IDM, the goal-oriented modeling is described component by component. In the first step, the user level and the technical level are initially considered separately, but in thesecond step they are considered holistically . At the user level, a process diagram is defined from the perspective of management, coordinators, and modelers. While creating the process diagram, the individual tasks, the sequence of execution, and their interfaces are identified. Within the interfaces, the data and information needed for the exchange is specified. This preliminary work allows the definition of a data exchange requirement model at the technical level. Such a model records not only the method of transmission, but also the type of data and information subject to exchange in the respective interfaces.After this formalization, a MVD will be developed. The purpose of the MVD is to tailor the individual interfaces for future applications. The IDM, in particular the process diagram and the data exchange requirements model, form the basis for the definition of the LODs. The LODs define the coordination of the model versions to be delivered during the course of the collaboration on the building project. The specification of the period-related degrees of each model’s maturity allows the coordination of the project. A loss-free transfer in the different interfaces requires a further specification of the transformation of the data and information in the course of the generated output. For this purpose, the relationship structure of the individual models is examined and a method for the transformationof the different data is presented. The methods and results presented in this thesis were elaborated and examined during the course of the EnEff: BIM research project. This research project, funded by the Federal Ministry for Economic Affairs and Energy, was carried out from the end of 2013 to November 2016 and bears the title:ËnEff: BIM planning and operational optimization of energy-efficient buildings through building information models", with the funding code: 03ET1177A-F. To channel further international activities, this project was bundled and edited together with an international research consortium in the IEA EBC Annex 60: "New generation of computational tools for building and community energy systems based on the Modelica and Functional Mockup Interface standards"coordinated by the Energy and Buildings Communities Program., Published by Aachen

Additional file 5. pSHUT4-fsr6 plasmid validation.

Additional file 7. NMR table of javanicin isolated from Fs OE::fsr6.

Additional file 3. pSHUT4-eYFP plasmid validation.

Additional file 1. List of primers.

Additional file 4. F. solani OE::eYFP mutant colony PCR validation.

Additional file 6. F. solani OE::fsr6 mutant validation by PCR and TubeSeq sequencing.

Additional file 8. NMR table of bostrycoidin isolated from Fs OE::fsr6.

Additional file 2. fsr6 nucleotide sequence.

OpenBIM, by definition, uses open data models to allow collaborative work practices. Industry Foundation Classes (IFC) is an open source data model for sharing planning, design, construction, and facility management data across various applications used in the AECOO industry. IFC is therefore suitable for storing significant volumes of data required for advanced Building Energy Performance Simulation (BEPS). This comprehensive nature of the data model makes IFC the key technology for storage and distribution of data but requires extensive infrastructure for data management. The use of Model View Definition (MVD) methodology refines data management by enabling lean data management through process specific subsets of the IFC data model. This paper describes the development of an MVD for advanced BEPS. European Commission - Seventh Framework Programme (FP7) International Energy Agency BMWi (German Federal Ministry of Economic Affairs and Energy) Conselho Nacional de Desenvolvimento Científico and Tecnológico (CNPq)

NMR spectroscopy is a well-established technique to determine the structure of peptides and small proteins in solution, also when bound to detergent micelles or phospholipid bicelles. The structure of the peptide alone is, however, not conveying the full picture, if the peptide is bound to a micelle, since it does not tell anything about the orientation of the peptide in the micelle. This article describes how to obtain that information together with information on peptide structure.

The activated sludge in wastewater treatment plants (WWTP) designed for enhanced biological phosphorus removal (EBPR) experiences periodically changing nutrient and oxygen availability. Tetrasphaera is the most abundant genus in Danish WWTP and represents up to 20&ndash, 30% of the activated sludge community based on 16S rRNA amplicon sequencing and quantitative fluorescence in situ hybridization analyses, although the genus is in low abundance in the influent wastewater. Here we investigated how Tetrasphaera can successfully out-compete most other microorganisms in such highly dynamic ecosystems. To achieve this, we analyzed the physiological adaptations of the WWTP isolate T. elongata str. LP2 during an aerobic to anoxic shift by label-free quantitative proteomics and NMR-metabolomics. Escherichia coli was used as reference organism as it shares several metabolic capabilities and is regularly introduced to wastewater treatment plants without succeeding there. When compared to E. coli, only minor changes in the proteome of T. elongata were observed after the switch to anoxic conditions. This indicates that metabolic pathways for anaerobic energy harvest were already expressed during the aerobic growth. This allows continuous growth of Tetrasphaera immediately after the switch to anoxic conditions. Metabolomics furthermore revealed that the substrates provided were exploited far more efficiently by Tetrasphaera than by E. coli. These results suggest that T. elongata prospers in the dynamic WWTP environment due to adaptation to the changing environmental conditions.

The extrinsic proteins of photosystem II of higher plants and green algae PsbO, PsbP, PsbQ, and PsbR are essential for stable oxygen production in the oxygen evolving center. In the available X-ray crystallographic structure of higher plant PsbQ residues S14-Y33 are missing. Building on the backbone NMR assignment of PsbQ, which includes this "missing link", we report the extended resonance assignment including side chain atoms. Based on nuclear Overhauser effect spectra a high resolution solution structure of PsbQ with a backbone RMSD of 0.81 Å was obtained from torsion angle dynamics. Within the N-terminal residues 1-45 the solution structure deviates significantly from the X-ray crystallographic one, while the four-helix bundle core found previously is confirmed. A short α-helix is observed in the solution structure at the location where a β-strand had been proposed in the earlier crystallographic study. NMR relaxation data and unrestrained molecular dynamics simulations corroborate that the N-terminal region behaves as a flexible tail with a persistent short local helical secondary structure, while no indications of forming a β-strand are found. Proteins 2015; 83:1677-1686. © 2015 The Authors. Proteins: Structure, Function, and Bioinformatics Published by Wiley Periodicals, Inc.

The interaction of lipolytic enzymes with anionic surfactants is of great interest with respect to industrially produced detergents. Here, we report the interaction of cutinase from the thermophilic fungus Humicola insolens with the anionic surfactant SDS, and show the enzyme specifically binds a single SDS molecule under nondenaturing concentrations. Protein interaction with SDS was investigated by NMR, ITC and molecular dynamics simulations. The NMR resonances of the protein were assigned, with large stretches of the protein molecule not showing any detectable resonances. SDS is shown to specifically interact with the loops surrounding the catalytic triad with medium affinity (Ka ≈ 105 M−1). The mode of binding is closely similar to that seen previously for binding of amphiphilic molecules and substrate analogues to cutinases, and hence SDS acts as a substrate mimic. In addition, the structure of the enzyme has been solved by X-ray crystallography in its apo form and after cocrystallization with diethyl p-nitrophenyl phosphate (DNPP) leading to a complex with monoethylphosphate (MEP) esterified to the catalytically active serine. The enzyme has the same fold as reported for other cutinases but, unexpectedly, esterification of the active site serine is accompanied by the ethylation of the active site histidine which flips out from its usual position in the triad.

The interaction of lipolytic enzymes with anionic surfactants is of great interest with respect to industrially produced detergents. Here, we report the interaction of cutinase from the thermophilic fungus Humicola insolens with the anionic surfactant SDS, and show the enzyme specifically binds a single SDS molecule under nondenaturing concentrations. Protein interaction with SDS was investigated by NMR, ITC and molecular dynamics simulations. The NMR resonances of the protein were assigned, with large stretches of the protein molecule not showing any detectable resonances. SDS is shown to specifically interact with the loops surrounding the catalytic triad with medium affinity (Ka ≈ 10(5) M(-1) ). The mode of binding is closely similar to that seen previously for binding of amphiphilic molecules and substrate analogues to cutinases, and hence SDS acts as a substrate mimic. In addition, the structure of the enzyme has been solved by X-ray crystallography in its apo form and after cocrystallization with diethyl p-nitrophenyl phosphate (DNPP) leading to a complex with monoethylphosphate (MEP) esterified to the catalytically active serine. The enzyme has the same fold as reported for other cutinases but, unexpectedly, esterification of the active site serine is accompanied by the ethylation of the active site histidine which flips out from its usual position in the triad.

Fungi produce a wide array of secondary metabolites, with interesting bioactivities by help of a number of enzyme complexes. Polyketide synthases (PKS) area class of multidomain enzymes, producing a class of secondary metabolites called polyketides1,2. Only few structures of PKS’s have been described, evenfewer from fungi and none from Fusarium species. Multidomain proteins can be quite challenging to work with, which is why the project intends to solve the3D-structures of single domains of PKS’s. In this project, the plan is to clone, express and purify the Acyl-carrier protein (ACP) domain from PKS6 in Fusariumgraminearum for structural analysis.

Antimicrobial peptides are a new class of antibiotics that are promising for pharmaceutical applications because they have retained efficacy throughout evolution. One class of antimicrobial peptides are the defensins, which have been found in different species. Here we describe a new fungal defensin, eurocin. Eurocin acts against a range of Gram-positive human pathogens but not against Gram-negative bacteria. Eurocin consists of 42 amino acids, forming a cysteine-stabilized α/β-fold. The thermal denaturation data point shows the disulfide bridges being responsible for the stability of the fold. Eurocin does not form pores in cell membranes at physiologically relevant concentrations; it does, however, lead to limited leakage of a fluorophore from small unilamellar vesicles. Eurocin interacts with detergent micelles, and it inhibits the synthesis of cell walls by binding equimolarly to the cell wall precursor lipid II.

Like many other filamentous fungi, Fusarium graminearum has the genetic potential to produce a vast array of unknown secondary metabolites. A promising approach to determine the nature of these is to activate silent secondary metabolite gene clusters through constitutive expression of cluster specific transcription factors. We have developed a system in which an expression cassette containing the transcription factor from the targeted PKS cluster disrupts the production of the red mycelium pigment aurofusarin. This aids with identification of mutants as they appear as white colonies and metabolite analyses where aurofusarin and its intermediates are normally among the most abundant compounds. The system was used for constitutive expression of the local transcription factor from the PKS9 cluster (renamed FSL) leading to production of three novel fusarielins, F, G and H. This group of compounds has not previously been reported from F. graminearum or linked to a biosynthetic gene in any fungal species. The toxicity of the three novel fusarielins was examined against colorectal cancer cell lines where fusarielin H was more potent than fusarielin F and G.

In this paper we describe a novel method for quantification of amino acids. First, α-hydroxy acid derivatives of amino acids were formed after reaction with dinitrogen trioxide by the van Slyke reaction. Second, the α-hydroxy acid derivatives were separated on an Aminex HPX-87H column (Bio-Rad) eluted isocratically with 5 mM H2SO4 and quantified by refractive index detection. We were able to measure the reaction products of 13 of the 20 classical amino acids: glycine, l-alanine, l-valine, l-leucine, l-isoleucine, l-methionine, l-serine, l-threonine, l-asparagine, l-glutamine, l-aspartic acid, l-glutamic acid, and l-proline. We obtained linear relationships between the product peak areas and initial amino acid concentration, whereby the concentrations of these amino acids could be quantified on the basis of the quantification of their products. The method can be used to analyze amino acids in parallel with other small molecules, such as sugars or short chain fatty acids, and was used for parallel quantification of glycine, l-alanine, or l-glutamic acid, and glucose uptake in cultures of the heterotrophic dinoflagellate Crypthecodinium cohnii. The method can also be used to quantify other amines, as demonstrated by detection of Tris (2-amino-2-(hydroxymethyl)propane-1,3-diol).

Udgivelsesdato: MAR 17