Biotransformation Of l-Tryptophan To Produce Arcyriaflavin A With Pseudomonas putida KT2440.

Natural products such as indolocarbazoles are a valuable source of highly bioactive compounds with numerous potential applications in the pharmaceutical industry. Arcyriaflavin A, isolated from marine invertebrates and slime molds, is one representative of this group and acts as a cyclin D1-cyclin-dependent kinase 4 inhibitor. To date, access to this compound has mostly relied on multi-step total synthesis. In this study, biosynthetic access to arcyriaflavin A was explored using recombinant Pseudomonas putida KT2440 based on a previously generated producer strain. We used a Design of Experiment approach to analyze four key parameters, which led to the optimization of the bioprocess. By engineering the formation of outer membrane vesicles and using an adsorbent in the culture broth, we succeeded to increase the yield of arcyriaflavin A in the cell-free supernatant, resulting in a nearly eight-fold increase in the overall production titers. Finally, we managed to scale up the bioprocess leading to a final yield of 4.7 mg arcyriaflavin A product isolated from 1 L of bacterial culture. Thus, this study showcases an integrative approach to improve biotransformation and moreover also provides starting points for further optimization of indolocarbazole production in P. putida.

Simple Enzyme Immobilization for Flow Chemistry? An Assessment of Available Strategies for an Acetaldehyde-Dependent Aldolase.

Enzyme immobilization is a technology that enables (bio-)catalysts to be applied in continuous-flow systems. However, there is a plethora of immobilization methods available with individual advantages and disadvantages. Here, we assessed the influence of simple and readily available methods with respect to the performance of 2-deoxy-d-ribose-5-phosphate aldolase (DERA) in continuous-flow conditions. The investigated immobilization strategies cover the unspecific attachment to carriers via epoxides, affinity-based attachment via metal ion affinity, StrepTag™-StrepTactin™ interaction as well as the covalent affinity attachment of an enzyme to a matrix tethered by the HaloTag®. The metal-ion-affinity-based approach outperformed the other methods in terms of immobilized activity and stability under applied conditions. As most enzymes examined today already have a HisTag for purification purposes, effective immobilization may be applied, as simple as a standard purification, if needed.

Biochemical and Initial Structural Characterization of the Monocot Chimeric Jacalin OsJAC1.

The monocot chimeric jacalin OsJAC1 from Oryza sativa consists of a dirigent and a jacalin-related lectin domain. The corresponding gene is expressed in response to different abiotic and biotic stimuli. However, there is a lack of knowledge about the basic function of the individual domains and their contribution to the physiological role of the entire protein. In this study, we have established a heterologous expression in Escherichia coli with high yields for the full-length protein OsJAC1 as well as its individual domains. Our findings showed that the secondary structure of both domains is dominated by ß-strand elements. Under reducing conditions, the native protein displayed clearly visible transition points of thermal unfolding at 59 and 85 °C, which could be attributed to the lectin and the dirigent domain, respectively. Our study identified a single carbohydrate-binding site for each domain with different specificities towards mannose and glucose (jacalin domain), and galactose moieties (dirigent domain), respectively. The recognition of different carbohydrates might explain the ability of OsJAC1 to respond to different abiotic and biotic factors. This is the first report of specific carbohydrate-binding activity of a DIR domain, shedding new light on its function in the context of this monocot chimeric jacalin.

Biocatalytic C3-Indole Methylation-A Useful Tool for the Natural-Product-Inspired Stereoselective Synthesis of Pyrroloindoles.

Enantioselective synthesis of bioactive compounds bearing a pyrroloindole framework is often laborious. In contrast, there are several S-adenosyl methionine (SAM)-dependent methyl transferases known for stereo- and regioselective methylation at the C3 position of various indoles, directly leading to the formation of the desired pyrroloindole moiety. Herein, the SAM-dependent methyl transferase PsmD from Streptomyces griseofuscus, a key enzyme in the biosynthesis of physostigmine, is characterized in detail. The biochemical properties of PsmD and its substrate scope were demonstrated. Preparative scale enzymatic methylation including SAM regeneration was achieved for three selected substrates after a design-of-experiment optimization.

Urban road traffic noise and noise annoyance-a study on perceived noise control and its value among the elderly.

Noise annoyance may reflect a pro-participatory attitude towards public information and consultation according to the European Environmental Noise Directive. However, noise annoyance is also indicative of a stress response to perceived uncontrollable noise exposure. Using cross-sectional data on a sample of elderly citizens (n = 1772), we investigated whether the value residents ascribed to being able to control noise exposure at home moderated the potential indirect effect of road traffic noise on annoyance through perceived noise control. Our results confirmed the presence of such a moderated mediation, which may justify studying the impact of residents' valuing perceived noise control on participation readiness.

Halogenating Enzymes for Active Agent Synthesis: First Steps Are Done and Many Have to Follow.

Halogens can be very important for active agents as vital parts of their binding mode, on the one hand, but are on the other hand instrumental in the synthesis of most active agents. However, the primary halogenating compound is molecular chlorine which has two major drawbacks, high energy consumption and hazardous handling. Nature bypassed molecular halogens and evolved at least six halogenating enzymes: Three kind of haloperoxidases, flavin-dependent halogenases as well as α-ketoglutarate and S-adenosylmethionine (SAM)-dependent halogenases. This review shows what is known today on these enzymes in terms of biocatalytic usage. The reader may understand this review as a plea for the usage of halogenating enzymes for fine chemical syntheses, but there are many steps to take until halogenating enzymes are reliable, flexible, and sustainable catalysts for halogenation.

Preparation of Cyclic Prodiginines by Mutasynthesis in Pseudomonas putida KT2440.

Prodiginines are a group of naturally occurring pyrrole alkaloids produced by various microorganisms and known for their broad biological activities. The production of nature-inspired cyclic prodiginines was enabled by combining organic synthesis with a mutasynthesis approach based on the GRAS (generally recognized as safe) certified host strain Pseudomonas putida KT2440. The newly prepared prodiginines exerted antimicrobial effects against relevant alternative biotechnological microbial hosts whereas P. putida itself exhibited remarkable tolerance against all tested prodiginines, thus corroborating the bacterium's exceptional suitability as a mutasynthesis host for the production of these cytotoxic secondary metabolites. Moreover, the produced cyclic prodiginines proved to be autophagy modulators in human breast cancer cells. One promising cyclic prodiginine derivative stood out, being twice as potent as prodigiosin, the most prominent member of the prodiginine family, and its synthetic derivative obatoclax mesylate.

Complex molecules, clever solutions - Enzymatic approaches towards natural product and active agent syntheses.

Natural compounds are often structurally complex and their synthesis is still highly challenging. The review intends to give an overview on developments in biotechnology and their role for the production of natural products and active agents. In vitro and in vivo methods are presented side by side beginning with rather simple but smart single step conversions, followed by cascade reactions, and finishing with complex bio-, semi- and mutasynthesis endeavours. All the enzymatic approaches do obviously complement traditional synthetic methods; with their particular strengths, the combined repertoire will lead to an increased efficiency in natural product synthesis as well as in providing analogues.

[Contribution of natural spaces to human health and wellbeing]. / Einfluss von Naturräumen auf die Gesundheit ­ Evidenzlage und Konsequenzen für Wissenschaft und Praxis.

Natural spaces and especially urban green and blue spaces have been recognised for a long time as spaces with great potential for protecting and promoting human health and well-being. They may affect human physical, mental and social health and well-being in various ways. On one hand, this comes to pass through reduction and moderation of potential environmental health risks (e. g. noise, particulate matter, heat), psycho-physiological effects of nature experience, as well as physical effects of exposure to natural compounds and elements. On the other hand, natural spaces can affect health and well-being indirectly e. g. through motivation of health promoting behaviour (e. g. more physical activity) and through use as outdoor meeting spaces, by decreasing aggression, and through the resulting positive effects on social well-being. Yet, some potential adverse health effects of nature and landscapes have been reported, too (e. g. insecurity or fear in confusing or unmaintained natural spaces, potential rivalry in usage, allergies or skin irritations due to natural elements, risk of communicable diseases from vectors). Against the background of positive effects of natural spaces, creating, restoring and enhancing urban green and blue spaces are often claimed in terms of sustainable and integrated urban development. But which associations and impacts exist between natural spaces and health? What are the resulting demands when integrating natural spaces for a health-promoting implementation practice? This overview article provides some answers to these questions.

Alternative hosts for functional (meta)genome analysis.

Microorganisms are ubiquitous on earth, often forming complex microbial communities in numerous different habitats. Most of these organisms cannot be readily cultivated in the laboratory using standard media and growth conditions. However, it is possible to gain access to the vast genetic, enzymatic, and metabolic diversity present in these microbial communities using cultivation-independent approaches such as sequence- or function-based metagenomics. Function-based analysis is dependent on heterologous expression of metagenomic libraries in a genetically amenable cloning and expression host. To date, Escherichia coli is used in most cases; however, this has the drawback that many genes from heterologous genomes and complex metagenomes are expressed in E. coli either at very low levels or not at all. This review emphasizes the importance of establishing alternative microbial expression systems consisting of different genera and species as well as customized strains and vectors optimized for heterologous expression of membrane proteins, multigene clusters encoding protein complexes or entire metabolic pathways. The use of alternative host-vector systems will complement current metagenomic screening efforts and expand the yield of novel biocatalysts, metabolic pathways, and useful metabolites to be identified from environmental samples.

Application of the C3-Methyltransferase StspM1 for the Synthesis of the Natural Pyrroloindole Motif.

Even though pyrroloindoles are widely present in natural products with different kinds of biological activities, their selective synthesis remains challenging with existing tools in organic chemistry, and there is furthermore a demand for stereoselective and mild methods to access this structural motif. Nature uses C3-methyltransferases to form the pyrroloindole framework, starting from the amino acid tryptophan. In the present study, the SAM-dependent methyltransferase StspM1 from Streptomyces sp. HPH0547 is used to build the pyrroloindole structural motif in tryptophan-based diketopiperazines (DKP). The substrate scope of the enzyme regarding different Trp-Trp-DKP isomers was investigated on an experimental and computational level. After further characterization and optimization of the methylation reaction with a design of experiment approach, a preparative scale reaction with the immobilized enzyme including a SAM regeneration system was performed to show the synthetic use of this biocatalytic tool to access the pyrroloindole structural motif.

Room Temperature Lattice Thermal Conductivity of GeSn Alloys.

CMOS-compatible materials for efficient energy harvesters at temperatures characteristic for on-chip operation and body temperature are the key ingredients for sustainable green computing and ultralow power Internet of Things applications. In this context, the lattice thermal conductivity (κ) of new group IV semiconductors, namely Ge1-xSnx alloys, are investigated. Layers featuring Sn contents up to 14 at.% are epitaxially grown by state-of-the-art chemical-vapor deposition on Ge buffered Si wafers. An abrupt decrease of the lattice thermal conductivity (κ) from 55 W/(m·K) for Ge to 4 W/(m·K) for Ge0.88Sn0.12 alloys is measured electrically by the differential 3ω-method. The thermal conductivity was verified to be independent of the layer thickness for strained relaxed alloys and confirms the Sn dependence observed by optical methods previously. The experimental κ values in conjunction with numerical estimations of the charge transport properties, able to capture the complex physics of this quasi-direct bandgap material system, are used to evaluate the thermoelectric figure of merit ZT for n- and p-type GeSn epitaxial layers. The results highlight the high potential of single-crystal GeSn alloys to achieve similar energy harvest capability as already present in SiGe alloys but in the 20 °C-100 °C temperature range where Si-compatible semiconductors are not available. This opens the possibility of monolithically integrated thermoelectric on the CMOS platform.

A new multicopper oxidase from Gram-positive bacterium Rhodococcus erythropolis with activity modulating methionine rich tail.

Multicopper oxidases are involved in a wide variety of physiological tasks in nature. They are part of the lignin formation/decomposition system in plants and fungi. In bacteria they are part of developmental processes and the heavy metal resistance apparatus. A well characterised example is the copper tolerance protein CueO of Escherichia coli (CueO(EC)). Here, we report the heterologous expression of the apo- and holo-form of CueO(RE), a homologue to CueO(EC) from Rhodococcus erythropolis. Upon incubation with copper(II) ions, low active apo-CueO(RE) was converted into the active holo-CueO(RE) in vivo. The holo-form was physico-chemically characterised using a copper(I) BCA complex and the model substrate 2,6-dimethoxyphenol. The spectroscopic and catalytic properties are different from CueO(EC), revealing a high catalytic efficiency (k(cat)/K(m)) of 115 min(-1)mM(-1) with physiological K(m) of 80 µM for the cuprous oxidase activity. At the C-terminus of CueO(RE) a methionine rich tail region was identified which can be found in a variety of actinobacteria. Chimeras of the E. coli and R. erythropolis enzymes were constructed to investigate the influence of this tail regarding kinetic parameters. It was shown that the tail did not have the same function as the corresponding methionine rich loop in CueO(EC). However, it modulated the kinetic properties of the enzyme.

Specific health status has an impact on the willingness to use telemonitoring: data from a 2009 health survey in north rhine-westphalia, Germany.

OBJECTIVE: Demographic changes in Germany are expected to cause a rising need for medical care and therapy while capacities are declining. Telemedicine offers the option of minimizing some aspects of these problems, but so far, many telemedicine projects in Germany (including telemonitoring projects) are not applied to the clinical routine. This study was done to assess the influence of health factors on potential willingness to use telemonitoring devices at home. MATERIALS AND METHODS: Health status and other health-relevant factors were determined using individual and medical factors (e.g., reported diseases). Principal-component analysis was used to identify groups with a specific response behavior. This study was based on a representative telephone survey conducted in the German state of North Rhine-Westphalia in 2009. RESULTS: Willingness to use telemonitoring was high in North Rhine-Westphalia but decreased with age. Men showed a significantly greater willingness to use telemonitoring than did women. Also, there was an effect associated with the subjects' health status (e.g., cardiovascular diseases caused a decrease of 9.7% in the level of acceptance, whereas musculoskeletal disorders caused a decrease of 5.1%). CONCLUSIONS: The target groups for telemonitoring consisted mainly of elderly persons and those with certain diseases. This study showed that being diseased lowered the willingness to use telemonitoring. People need to understand better how telemonitoring can help to improve controlling their health status and coping with the disease. It is necessary to reflect on these specific needs if telemonitoring is to become routine in the German healthcare system.

The Crystal Structure of the Defense Conferring Rice Protein OsJAC1 Reveals a Carbohydrate Binding Site on the Dirigent-like Domain.

Pesticides are routinely used to prevent severe losses in agriculture. This practice is under debate because of its potential negative environmental impact and selection of resistances in pathogens. Therefore, the development of disease resistant plants is mandatory. It was shown that the rice (Oryza sativa) protein OsJAC1 enhances resistance against different bacterial and fungal plant pathogens in rice, barley, and wheat. Recently we reported possible carbohydrate interaction partners for both domains of OsJAC1 (a jacalin-related lectin (JRL) and a dirigent (DIR) domain), however, a mechanistic understanding of its function is still lacking. Here, we report crystal structures for both individual domains and the complex of galactobiose with the DIR domain, which revealed a new carbohydrate binding motif for DIR proteins. Docking studies of the two domains led to a model of the full-length protein. Our findings offer insights into structure and binding properties of OsJAC1 and its possible function in pathogen resistance.

Pathogen Resistance Depending on Jacalin-Dirigent Chimeric Proteins Is Common among Poaceae but Absent in the Dicot Arabidopsis as Evidenced by Analysis of Homologous Single-Domain Proteins.

MonocotJRLs are Poaceae-specific two-domain proteins that consist of a jacalin-related lectin (JRL) and a dirigent (DIR) domain which participate in multiple developmental processes, including disease resistance. For OsJAC1, a monocotJRL from rice, it has been confirmed that constitutive expression in transgenic rice or barley plants facilitates broad-spectrum disease resistance. In this process, both domains of OsJAC1 act cooperatively, as evidenced from experiments with artificially separated JRL- or DIR-domain-containing proteins. Interestingly, these chimeric proteins did not evolve in dicotyledonous plants. Instead, proteins with a single JRL domain, multiple JRL domains or JRL domains fused to domains other than DIR domains are present. In this study, we wanted to test if the cooperative function of JRL and DIR proteins leading to pathogen resistance was conserved in the dicotyledonous plant Arabidopsis thaliana. In Arabidopsis, we identified 50 JRL and 24 DIR proteins, respectively, from which seven single-domain JRL and two single-domain DIR candidates were selected. A single-cell transient gene expression assay in barley revealed that specific combinations of the Arabidopsis JRL and DIR candidates reduced the penetration success of barley powdery mildew. Strikingly, one of these pairs, AtJAX1 and AtDIR19, is encoded by genes located next to each other on chromosome one. However, when using natural variation and analyzing Arabidopsis ecotypes that express full-length or truncated versions of AtJAX1, the presence/absence of the full-length AtJAX1 protein could not be correlated with resistance to the powdery mildew fungus Golovinomyces orontii. Furthermore, an analysis of the additional JRL and DIR candidates in a bi-fluorescence complementation assay in Nicotiana benthamiana revealed no direct interaction of these JRL/DIR pairs. Since transgenic Arabidopsis plants expressing OsJAC1-GFP also did not show increased resistance to G. orontii, it was concluded that the resistance mediated by the synergistic activities of DIR and JRL proteins is specific for members of the Poaceae, at least regarding the resistance against powdery mildew. Arabidopsis lacks the essential components of the DIR-JRL-dependent resistance pathway.

Recent Advances in Biocatalysis for Drug Synthesis.

Biocatalysis is constantly providing novel options for the synthesis of active pharmaceutical ingredients (APIs). In addition to drug development and manufacturing, biocatalysis also plays a role in drug discovery and can support many active ingredient syntheses at an early stage to build up entire scaffolds in a targeted and preparative manner. Recent progress in recruiting new enzymes by genome mining and screening or adapting their substrate, as well as product scope, by protein engineering has made biocatalysts a competitive tool applied in academic and industrial spheres. This is especially true for the advances in the field of nonribosomal peptide synthesis and enzyme cascades that are expanding the capabilities for the discovery and synthesis of new bioactive compounds via biotransformation. Here we highlight some of the most recent developments to add to the portfolio of biocatalysis with special relevance for the synthesis and late-stage functionalization of APIs, in order to bypass pure chemical processes.

Optimized prodigiosin production with Pseudomonas putida KT2440 using parallelized noninvasive online monitoring.

The red pigment prodigiosin is of high pharmaceutical interest, due to its potential applications as an antitumor drug and antibiotic agent. As previously demonstrated, Pseudomonas putida KT2440 is a suitable host for prodigiosin production, as it exhibits high tolerance toward the antimicrobial properties of prodigiosin. So far, prodigiosin concentrations of up to 94 mg/L have been achieved in shake flask cultivations. For the characterization and optimization of the prodigiosin production process, the scattered light of P. putida and fluorescence of prodigiosin was measured. The excitation and emission wavelengths for prodigiosin measurement were analyzed by recording 2D fluorescence spectra. The strongest prodigiosin fluorescence was obtained at a wavelength combination of 535/560 nm. By reducing the temperature to 18 °C and using 16 g/L glucose, the prodigiosin concentration was more than doubled compared with the initial cultivation conditions. The obtained results demonstrate the capabilities of parallelized microscale cultivations combined with noninvasive online monitoring of fluorescence for rapid bioprocess development, using prodigiosin as a molecule of current biotechnological interest.

A Fluorescence-Based Assay System for the Determination of Haloperoxidase-Activity Using a Two-Dimensional Calibration Ap-proach.

Screening for an interesting biocatalyst and its subsequent kinetic characterization depends on a reliable activity assay. In this work, a fluorometric assay based on the halogenation of 4-methyl-7-diethylamino-coumarin was established to monitor haloperoxidase-activity. Since haloperoxidases utilize hydrogen peroxide and halide ions to halogenate a broad range of substrates by releasing hypohalous acids, a direct quantification of haloperoxidase-activity remains difficult. With the system presented here, 3-bromo-4-methyl-7-diethylaminocoumarin is preferentially formed and monitored by fluorescence measurements. As starting material and product share similar spectroscopical properties, a two-dimensional calibration ap-proach was utilized to allow for quantification of each compound within a single measurement. To validate the system, the two-dimensional Michaelis-Menten kinetics of a vanadium-dependent chloroperoxidase from Curvularia inaequalis were recorded, yielding the first overall kinetic parameters for this enzyme. With limits of detection and quantification in the low µm range, this assay may provide a reliable alternative system for the quantification of haloperoxidase-activity.

Novel Prodiginine Derivatives Demonstrate Bioactivities on Plants, Nematodes, and Fungi.

Bacterial metabolites represent an invaluable source of bioactive molecules which can be used as such or serve as chemical frameworks for developing new antimicrobial compounds for various applications including crop protection against pathogens. Prodiginines are tripyrrolic, red-colored compounds produced by many bacterial species. Recently, due to the use of chemical-, bio-, or mutasynthesis, a novel group of prodiginines was generated. In our study, we perform different assays to evaluate the effects of prodigiosin and five derivatives on nematodes and plant pathogenic fungi as well as on plant development. Our results showed that prodigiosin and the derivatives were active against the bacterial feeding nematode Caenorhabditis elegans in a concentration- and derivative-dependent manner while a direct effect on infective juveniles of the plant parasitic nematode Heterodera schachtii was observed for prodigiosin only. All compounds were found to be active against the plant pathogenic fungi Phoma lingam and Sclerotinia sclerotiorum. Efficacy varied depending on compound concentration and chemical structure. We observed that prodigiosin (1), the 12 ring- 9, and hexenol 10 derivatives are neutral or even positive for growth of Arabidopsis thaliana depending on the applied compound concentration, whereas other derivatives appear to be suppressive. Our infection assays revealed that the total number of developed H. schachtii individuals on A. thaliana was decreased to 50% in the presence of compounds 1 or 9. Furthermore, female nematodes and their associated syncytia were smaller in size. Prodiginines seem to indirectly inhibit H. schachtii parasitism of the plant. Further research is needed to elucidate their mode of action. Our results indicate that prodiginines are promising metabolites that have the potential to be developed into novel antinematodal and antifungal agents.