Functional characterisation of in vitro synthesised G-protein coupled receptors in polymersomes

Membrane proteins play an indispensable role in physiological processes. It is, therefore, not surprising that many diseases are based on the malfunction of membrane proteins. Hence membrane proteins and especially G-protein coupled receptors(GPCRs)- the largest subfamily- have become an important drug target. Due to their high selectivity and sensitivity membrane proteins are also feasible for the detection of small quantities of substances with biosensors. Despite this widespread interest in GPCRs due to their importance as drug targets and biosensors there is still a lack of knowledge of structure, function and endogenous ligands for quiet a few of the previously identified receptors.rnBottlenecks in over-expression, purification, reconstitution and handling of membrane proteins arise due to their hydrophobic nature. Therefore the production of reasonable amounts of functional membrane proteins for structural and functional studies is still challenging. Also the limited stability of lipid based membrane systems hampers their application as platforms forrnscreening applications and biosensors.rnIn recent years the in vitro protein synthesis became a promising alternative to gain better yields for expression of membrane proteins in bio-mimetic membrane systems. These expression systems are based on cell extracts. Therefore cellular effects on protein expression are reduced. The open nature of the cell-free expression systems easily allows for the adjustment of reactionrnconditions for the protein of interest. The cell-free expression in the presence of bio-mimetic membrane systems allows the direct incorporation of the membrane proteins and therefore skips the time-consuming purification and reconstitution processes. Amphiphilic block-copolymers emerged as promising alternative for the less stable lipid-based membrane systems. They, likernlipids, form membraneous structures in aqueous solutions but exhibit increased mechanical and chemical stability.rnThe aim of this work was the generation of a GPCR-functionalised membrane system by combining both promising alternatives: in vitro synthesis and polymeric membrane systems. This novel platform should be feasible for the characterisation of the incorporated GPCR. Immunodetection of Dopamine receptor 1 and 2 expressed in diblock- and triblock-polymersomes demonstrated the successful in vitro expression of GPCRs in polymeric membranes. Antibodyrnbinding studies suggested a favoured orientation of dopamine receptors in triblockpolymersomes.rnA dopamine-replacement assay on DRD2-functionalised immobilised triblockpolymersomes confirmed functionality of the receptor in the polymersomes. The altered binding curve suggests an effect of the altered hydrophobic environment presented by the polymer membrane on protein activity.

Mikrobielle Synthese von Biopolymeren aus der nachwachsenden Rohstoffquelle Weizenstroh

Weizenstroh als erneuerbare Ressource zur Produktion von Biopolymeren und wichtigen Grundchemikalien stellt eine ökologisch sinnvolle Alternative dar. Durch die vom PFI durchgeführte Thermodruckhydrolyse konnte das Weizenstroh und die darin enthaltenen Zucker fast vollständig mobilisiert werden. Ein umfangreiches Screening nach Organismen, welche die Zucker des Weizenstrohs verwerten konnten, ergab, dass einige wenige Stämme zur PHB-Bildung aus Xylose befähigt waren (10 %). Zur PHB-Synthese aus Glucose waren indes ca. doppelt so viele Organismen in der Lage (20 %). Zwei der insgesamt 118 untersuchten Organismen zeigten besonders gute PHB-Bildung sowohl mit Xylose als auch mit Glucose als Substrat. Dabei handelte es sich um die hauseigenen Stämme Bacillus licheniformis KHC 3 und Bacillus megaterium KNaC 2. Nach Enttoxifizierung der hemicellulosischen Fraktion konnte diese als C-Quelle im Mineral Medium eingesetzt werden. Burkholderia sacchari DSM 17165 und Hydrogenophaga pseudoflava DSM 1034, sowie die hauseigenen Isolate Bacillus licheniformis KHC 3 und Bacillus megaterium KNaC 2 wurden für die Synthese von PHB aus der hemicellulosischen Fraktion verwendet. Die Zucker der hemicellulosischen Fraktion (Xylose, Glucose, Arabinose) konnten durch diese Organismen zur PHB-Synthese genutzt werden. Hierbei stellte sich heraus, dass die beiden Bacillus-Stämme besser zur Produktion von PHB aus dem hemicellulosischen Hydrolysat geeignet waren als die Stämme der DSMZ. Die alternative Umsetzung der im hemicellulosischem Hydrolysat enthaltenen Zucker (Xylose, Glucose und Arabinose) in die wichtigen Grundchemikalien Lactat und Acetat konnte durch die Verwendung von heterofermentativen Milchsäurebakterien verwirklicht werden. Die Bildung dieser wichtigen Grundchemikalien stellt eine interessante Alternative zur PHB-Synthese dar. Die Menge an teuren Zusätzen wie Tomatensaft, welcher für das Wachstum der MSB essentiell war, konnte reduziert werden. Die Glucose der zweiten Fraktion des Weizenstrohs, der cellulosischen Fraktion, konnte ebenfalls durch den Einsatz von Mikroorganismen in PHB umgewandelt werden. Kommerzielle Cellulasen der Firma Novozymes konnten große Mengen an Glucose (≥10 g/l) aus der cellulosischen Fraktion freisetzen. Diese freie Glucose wurde mit Hilfe von Cupriavidus necator DSM 545, Cupriavidus necator NCIMB 11599, Bacillus licheniformis KHC 3 und Bacillus megaterium KNaC 2 zu PHB fermentiert. Wie auch beim hemicellulosischen Hydrolysat konnten hier die beiden Bacillus-Stämme die besten Ergebnisse erzielen. Bei ihnen machte die PHB mehr als die Hälfte der Trockenmasse aus. Die Abtrennung des Zielprodukts ohne die Verwendung von umweltschädlichen Lösungsmitteln wurde durch die Lyse der Zielzellen durch eigens isolierte Enzyme aus Streptomyceten verwirklicht. Die Zelllyse durch die Enzyme aus Streptomyces globisporus subsp. caucasius DSM 40814 und Streptomyces albidoflavus DSM 40233 war erfolgreich und zeigte vor allem bei den Bacillen hohe Wirkung (83 % und 99 % Zelllyse). Bei dem Gram-negativen Organismus Cupriavidus necator DSM 428 konnte die anfangs niedrige Zelllyse von 38 % durch Ultraschallbehandlung auf ca. 75 % erhöht werden.

Engineering the synthesis of lantibiotics in e. Coli by combining the cynnamicin and nisin modification systems.

I lantibiotici sono molecole peptidiche prodotte da un gran numero di batteri Gram-positivi, posseggono attività antibatterica contro un ampio spettro di germi, e rappresentano una potenziale soluzione alla crescente problematica dei patogeni multi-resistenti. La loro attività consiste nel legame alla membrana del bersaglio, che viene quindi destabilizzata mediante l’induzione di pori che determinano la morte del patogeno. Tipicamente i lantibiotici sono formati da un “leader-peptide” e da un “core-peptide”. Il primo è necessario per il riconoscimento della molecola da parte di enzimi che effettuano modifiche post-traduzionali del secondo - che sarà la regione con attività battericida una volta scissa dal “leader-peptide”. Le modifiche post-traduzionali anticipate determinano il contenuto di amminoacidi lantionina (Lan) e metil-lantionina (MeLan), caratterizzati dalla presenza di ponti-tioetere che conferiscono maggior resistenza contro le proteasi, e permettono di aggirare la principale limitazione all’uso dei peptidi in ambito terapeutico. La nisina è il lantibiotico più studiato e caratterizzato, prodotto dal batterio L. lactis che è stato utilizzato per oltre venti anni nell’industria alimentare. La nisina è un peptide lungo 34 amminoacidi, che contiene anelli di lantionina e metil-lantionina, introdotti dall’azione degli enzimi nisB e nisC, mentre il taglio del “leader-peptide” è svolto dall’enzima nisP. Questo elaborato affronta l’ingegnerizzazione della sintesi e della modifica di lantibiotici nel batterio E.coli. In particolare si affronta l’implementazione dell’espressione eterologa in E.coli del lantibiotico cinnamicina, prodotto in natura dal batterio Streptomyces cinnamoneus. Questo particolare lantibiotico, lungo diciannove amminoacidi dopo il taglio del leader, subisce modifiche da parte dell’enzima CinM, responsabile dell’introduzione degli aminoacidi Lan e MeLan, dell’enzima CinX responsabile dell’idrossilazione dell’acido aspartico (Asp), e infine dell’enzima cinorf7 deputato all’introduzione del ponte di lisinoalanina (Lal). Una volta confermata l’attività della cinnamicina e di conseguenza quella dell’enzima CinM, si è deciso di tentare la modifica della nisina da parte di CinM. A tal proposito è stato necessario progettare un gene sintetico che codifica nisina con un leader chimerico, formato cioè dalla fusione del leader della cinnamicina e del leader della nisina. Il prodotto finale, dopo il taglio del leader da parte di nisP, è una nisina completamente modificata. Questo risultato ne permette però la modifica utilizzando un solo enzima invece di due, riducendo il carico metabolico sul batterio che la produce, e inoltre apre la strada all’utilizzo di CinM per la modifica di altri lantibiotici seguendo lo stesso approccio, nonché all’introduzione del ponte di lisinoalanina, in quanto l’enzima cinorf7 necessita della presenza di CinM per svolgere la sua funzione.

Structure-Function Relations in the PI-PLC/GDPD Enzyme Superfamily

Phosphatidylinositol-specific phospholipases C (PI-PLC) are known to participate in many eukaryotic signal transduction pathways and act as virulence factors in lower organisms. Glycerophosphoryl diester phosphodiesterase (GDPD) enzymes are involved in phosphate homeostasis and phospholipid catabolism for energy production. Streptomyces antibioticus phosphatidylinositol-specific phospholipase C (SaPLC1) is a 38 kDa enzyme that displays characteristics of both enzyme superfamilies, representing an evolutionary link between these divergent enzyme classes. SaPLC1 also boasts a unique catalytic mechanism that involves a trans 1,6-cyclic inositol phosphate intermediate instead of the typical cis 1,2-cyclic inositol phosphate. The mechanism by which this occurs is still unclear. To attack this problem, we established a wide mutagenesis scan of the active site and measured activities of alanine mutants. A chemical rescue assay was developed to verify that the activity loss was due to the removal of the functional role of the mutated residue. 31P-NMR was employed in characterizing and quantifying intermediates in mutants that slowed the reaction sufficiently. We found that the H37A and H76A mutations support the hypothesis that these structurally conserved residues are also conserved in terms of their catalytic roles. H37 was found to be the general base (GB), while H76 plays the role of general acid (GA). K131 was identified as a semi-conserved key positive charge donor found at the entrance of the active site. By elucidating the SaPLC1 mechanism in relation to its active site architecture, we have increased our understanding of the structure-function relations that support catalysis in the PI-PLC/GDPD superfamily. These findings provide groundwork for in vivo studies of SaPLC1 function and its possible role in novel signaling or metabolism in Streptomyces.

THE POTENTIAL OF INDUSTRIAL WASTE AND AGRICULTURAL FEEDSTOCK TOWARDS SUSTAINABLE BIOFUELS PRODUCTION: TECHNO-ECONOMIC AND ENVIRONMENTAL IMPACT PERSPECTIVES

This Ph.D. research is comprised of three major components; (i) Characterization study to analyze the composition of defatted corn syrup (DCS) from a dry corn mill facility (ii) Hydrolysis experiments to optimize the production of fermentable sugars and amino acid platform using DCS and (iii) Sustainability analyses. Analyses of DCS included total solids, ash content, total protein, amino acids, inorganic elements, starch, total carbohydrates, lignin, organic acids, glycerol, and presence of functional groups. Total solids content was 37.4% (± 0.4%) by weight, and the mass balance closure was 101%. Total carbohydrates [27% (± 5%) wt.] comprised of starch (5.6%), soluble monomer carbohydrates (12%) and non-starch carbohydrates (10%). Hemicellulose components (structural and non-structural) were; xylan (6%), xylose (1%), mannan (1%), mannose (0.4%), arabinan (1%), arabinose (0.4%), galatactan (3%) and galactose (0.4%). Based on the measured physical and chemical components, bio-chemical conversion route and subsequent fermentation to value added products was identified as promising. DCS has potential to serve as an important fermentation feedstock for bio-based chemicals production. In the sugar hydrolysis experiments, reaction parameters such as acid concentration and retention time were analyzed to determine the optimal conditions to maximize monomer sugar yields while keeping the inhibitors at minimum. Total fermentable sugars produced can reach approximately 86% of theoretical yield when subjected to dilute acid pretreatment (DAP). DAP followed by subsequent enzymatic hydrolysis was most effective for 0 wt% acid hydrolysate samples and least efficient towards 1 and 2 wt% acid hydrolysate samples. The best hydrolysis scheme DCS from an industry's point of view is standalone 60 minutes dilute acid hydrolysis at 2 wt% acid concentration. The combined effect of hydrolysis reaction time, temperature and ratio of enzyme to substrate ratio to develop hydrolysis process that optimizes the production of amino acids in DCS were studied. Four key hydrolysis pathways were investigated for the production of amino acids using DCS. The first hydrolysis pathway is the amino acid analysis using DAP. The second pathway is DAP of DCS followed by protein hydrolysis using proteases [Trypsin, Pronase E (Streptomyces griseus) and Protex 6L]. The third hydrolysis pathway investigated a standalone experiment using proteases (Trypsin, Pronase E, Protex 6L, and Alcalase) on the DCS without any pretreatment. The final pathway investigated the use of Accellerase 1500® and Protex 6L to simultaneously produce fermentable sugars and amino acids over a 24 hour hydrolysis reaction time. The 3 key objectives of the techno-economic analysis component of this PhD research included; (i) Development of a process design for the production of both the sugar and amino acid platforms with DAP using DCS (ii) A preliminary cost analysis to estimate the initial capital cost and operating cost of this facility (iii) A greenhouse gas analysis to understand the environmental impact of this facility. Using Aspen Plus®, a conceptual process design has been constructed. Finally, both Aspen Plus Economic Analyzer® and Simapro® sofware were employed to conduct the cost analysis as well as the carbon footprint emissions of this process facility respectively. Another section of my PhD research work focused on the life cycle assessment (LCA) of commonly used dairy feeds in the U.S. Greenhouse gas (GHG) emissions analysis was conducted for cultivation, harvesting, and production of common dairy feeds used for the production of dairy milk in the U.S. The goal was to determine the carbon footprint [grams CO2 equivalents (gCO2e)/kg of dry feed] in the U.S. on a regional basis, identify key inputs, and make recommendations for emissions reduction. The final section of my Ph.D. research work was an LCA of a single dairy feed mill located in Michigan, USA. The primary goal was to conduct a preliminary assessment of dairy feed mill operations and ultimately determine the GHG emissions for 1 kilogram of milled dairy feed.

DivIVA is required for polar growth in the MreB-lacking rod-shaped actinomycete Corynebacterium glutamicum.

The actinomycete Corynebacterium glutamicum grows as rod-shaped cells by zonal peptidoglycan synthesis at the cell poles. In this bacterium, experimental depletion of the polar DivIVA protein (DivIVA(Cg)) resulted in the inhibition of polar growth; consequently, these cells exhibited a coccoid morphology. This result demonstrated that DivIVA is required for cell elongation and the acquisition of a rod shape. DivIVA from Streptomyces or Mycobacterium localized to the cell poles of DivIVA(Cg)-depleted C. glutamicum and restored polar peptidoglycan synthesis, in contrast to DivIVA proteins from Bacillus subtilis or Streptococcus pneumoniae, which localized at the septum of C. glutamicum. This confirmed that DivIVAs from actinomycetes are involved in polarized cell growth. DivIVA(Cg) localized at the septum after cell wall synthesis had started and the nucleoids had already segregated, suggesting that in C. glutamicum DivIVA is not involved in cell division or chromosome segregation.

Covalent immobilisation of VEGF on plasma-coated electrospun scaffolds for tissue engineering applications.

Recent findings in the field of biomaterials and tissue engineering provide evidence that surface immobilised growth factors display enhanced stability and induce prolonged function. Cell response can be regulated by material properties and at the site of interest. To this end, we developed scaffolds with covalently bound vascular endothelial growth factor (VEGF) and evaluated their mitogenic effect on endothelial cells in vitro. Nano- (254±133 nm) or micro-fibrous (4.0±0.4 μm) poly(ɛ-caprolactone) (PCL) non-wovens were produced by electrospinning and coated in a radio frequency (RF) plasma process to induce an oxygen functional hydrocarbon layer. Implemented carboxylic acid groups were converted into amine-reactive esters and covalently coupled to VEGF by forming stable amide bonds (standard EDC/NHS chemistry). Substrates were analysed by X-ray photoelectron spectroscopy (XPS), enzyme-linked immuno-assays (ELISA) and immunohistochemistry (anti-VEGF antibody and VEGF-R2 binding). Depending on the reaction conditions, immobilised VEGF was present at 127±47 ng to 941±199 ng per substrate (6mm diameter; concentrations of 4.5 ng mm(-2) or 33.3 ng mm(-2), respectively). Immunohistochemistry provided evidence for biological integrity of immobilised VEGF. Endothelial cell number of primary endothelial cells or immortalised endothelial cells were significantly enhanced on VEGF-functionalised scaffolds compared to native PCL scaffolds. This indicates a sustained activity of immobilised VEGF over a culture period of nine days. We present a versatile method for the fabrication of growth factor-loaded scaffolds at specific concentrations.

Immobilisation data of juvenile and adult male and female Weddell seals at Drescher Inlet from expeditions DRE1990 and DRE1992

Fourten Weddell seals (Leptonychotes weddellii) and two crabeater seals (Lobodon carcinophaga) were immobilised at Drescher Inlet (Riiser Larsen Ice Shelf), eastern Weddell Sea coast, between January and February 1990 using a combination of ketamine, xylazine, and diazepam. Eleven Weddell seals were drugged once, and two and one were drugged two and three times each, coming to a total of 18 immobilisation procedures. Another 16 seals were immobilised between January and February 1992. Ten seals were drugged once, and three and two were drugged two and three times each, coming to a total of 25 immobilisation procedures. Narcoses were terminated with yohimbine. Data as given by doi:10.1594/PANGAEA.438920 were selected for publication. Data sets doi:10.1594/PANGAEA.438921 and doi:10.1594/PANGAEA.438926 followed the same methods and dose regimes.

Diversity and identification of heterotrophic bacteria from Antarctic Rocks of the McMurdo Dry Valleys (Ross Desert)

Diversity of endolithic Dry Valley rock microorganisms was studied by evaluating the presence of morphotypes in enrichments. Storage of rock samples for 16 h over dry ice affected the diversity of endolithic organisms, especially that of algae and fungi. Diversity in various samples depended on rock location and exposure, on the rock type, and to some extent on the pH of the pulverized rock samples. In most cases sandstone contained more morphotypes than dolerite or granite. Presence of many different phototrophs resulted in greater diversity of the heterotrophs in the enrichments. Samples from Linnaeus Terrace and Battleship Promontory had higher morphotype (MT) numbers than those from more exposed sites such as New Mountain, University Valley, Dais, or Mt. Fleming. Beacon sandstone (13 samples) from Linnaeus Terrace varied greatly with respect to MT numbers, although the pH values ranged only from 4.2-5.3. The highest MT number of 24 per sample was obtained from the upper surface of a flat boulder tilted to the North. Only two MT's were found in a hard sandstone sample from the wind-exposed and more shaded east side of the Terrace. 15 sandstone samples from Battleship Promontory contained more diverse populations: there occurred a total of 131 different MT's in these samples as compared to only 68 in Linnaeus Terrace samples. Cysts of colorless flagellates were found in some Battleship Promontory samples; rnost samples were populated with a wealth of different cyanobacteria. Studies on the distribution of actinomycete morphotypes in Linnaeus Terrace sandstone revealed great differences between individual boulders. Identification tests and lipid analyses made with representative strains of the isolated 1500 pure cultures led to genus names such as Caulobacter, Blastobacter, Hyphomicrobium, Micrococcus, Arthrobacter, Brevibacterium, Corynebacterium, Bifidobacterium, Mycobacterium, Nocardia (Amycolata), Micromonospora, Streptomyces, Blastococcus, and Deinococcus. Our data demonstrate the great diversity of Antarctic endolithic microbial populations.

Dive track profile, intermandibular angel sensor profile and immobilisation data of adult female Weddell seals at Drescher Inlet from expedition DRE2003

Determination of when and where animals feed and how much they consume is fundamental to understand their ecology and role in ecosystems. However, the lack of reliable data on feeding habits of wild animals, and particularly in marine endotherms, attests to the difficulty in doing this. A promising recent development proposes using a Hall sensor-magnet System - the inter-mandibular angle sensor (IMASEN) attached to animals' jaws to elucidate feeding events. We conducted trials on captive pinnipeds by feeding IMASEN-equipped animals with prey to examine the utility of this system. Most feeding events were clearly distinguishable from other jaw movements; only small prey items might not be resolved adequately. Based on the results of this study we examined feeding events from free-ranging Weddell seals fitted with IMASENs and dead-reckoners during December 2003 at Drescher Inlet (Riiser Larsen Ice Shelf, eastern Weddell Sea coast), and present data on prey capture and ingestion in relation to the three-dimensionalmovement patterns of the seals. A total of 19 Weddell seals were immobilised by using a combination of ketamine, xylazine, and diazepam. Eight seals were drugged once, six two times, and two and three were drugged three and four times each, coming to a total of 38 immobilisation procedures. Narcoses were terminated with yohimbine (doi:10.1594/PANGAEA.438931).

Dive depth profile and immobilisation data of adult male and female Weddell seals at Drescher Inlet from expedition DRE1998

Adult male and female Weddell seals (Leptonychotes weddellii) were fitted with Time-depth recorders (TDR) at Drescher Inlet (Riiser Larsen Ice Shelf), eastern Weddell Sea coast, in February 1998. Eight of 15 data sets were selected for analyses to investigate the seals' foraging behaviour (doi:10.1594/PANGAEA.511465, doi:10.1594/PANGAEA.511454, doi:10.1594/PANGAEA.511456, doi:10.1594/PANGAEA.511457, doi:10.1594/PANGAEA.511459, doi:10.1594/PANGAEA.511462, doi:10.1594/PANGAEA.511466, doi:10.1594/PANGAEA.511467). These data sets provided simultaneous dive records of eight seals over eight days. The seals primarily foraged within two depth layers, these being from the sea surface to 160 m where temperature and salinity varied considerably, and from 340 to 450 m near the bottom where temperature was lowest and salinity highest, with little variation. While pelagic and benthic diving occurred during daylight, the seals foraged almost exclusively in the upper water column at night. Trawling during daytime confirmed that Pleuragramma antarcticum were by far the most abundant fish both in the pelagial and close to the bottom. Pelagic night-hauls at 110-170 m depth showed highly variable biomass of P. antarcticum with a peak at around midnight. The temporal changes in the local abundance of P. antarcticum, particularly in the pelagial, may explain the trends in the seals' pelagic and benthic foraging activities. This is the first study which describes the jaw movements of a hunting seal which are presumably indicative of feeding events. Trophic links from the Weddell seal to fish, zooplankton and krill, Euphausia superba, are discussed. Another seven data sets did not overlap substantially with the selected time frame (doi:10.1594/PANGAEA.511458, doi:10.1594/PANGAEA.511460, doi:10.1594/PANGAEA.511464, doi:10.1594/PANGAEA.511468, doi:10.1594/PANGAEA.511469, doi:10.1594/PANGAEA.511453, doi:10.1594/PANGAEA.511463). A total of 25 Weddell seals were immobilised during the study period using a combination of ketamine, xylazine, and diazepam. Seven seals were drugged once, 15 seals two times, and three were drugged three times, coming to a total of 46 immobilisation procedures. Narcoses were terminated with yohimbine (doi:10.1594/PANGAEA.438933).

Individual codes, GenBank accession numbers and GenBank accession links for Mediterranean sponges

Metabolomic analysis has shown the chemical richness of the sponge-associated actinomycetes Streptomyces sp. SBT349, Nonomureae sp. SBT364, and Nocardiopsis sp. SBT366. The genomes of these actinomycetes were sequenced and the genomic potential for secondary metabolism was evaluated. Their draft genomes have sizes of 8.0, 10, and 5.8Mb having 687, 367, and 179 contigs with a GC content of 71.6, 70.7, and 72.7%, respectively. Moreover, antiSMASH 3.0 predicted 108, 149, and 75 secondary metabolite gene clusters, respectively which highlight the metabolic capacity of the three actinomycete species to produce diverse classes of natural products.

Geochemical fingerprints and controls in the sediments of an urban river: River Manzanares, Madrid (Spain)

The geochemical fingerprint of sediment retrieved from the banks of the River Manzanares as it passes through the City of Madrid is presented here. The river collects the effluent water from several Waste Water Treatment (WWT) plants in and around the city, such that, at low flows, up to 60% of the flow has been treated. A total of 18 bank-sediment cores were collected along the course of the river, down to its confluence with the Jarama river, to the south–east of Madrid. Trace and major elements in each sample were extracted following a double protocol: (a) “Total” digestion with HNO3, HClO4 and HF; (b) “Weak” digestion with sodium acetate buffered to pH=5 with acetic acid, under constant stirring. The digests thus obtained were subsequently analysed by ICP-AES, except for Hg which was extracted with aqua regia and sodium chloride-hydroxylamine sulfate, and analysed by Cold Vapour-AAS. X-ray diffraction was additionally employed to determine the mineralogical composition of the samples. Uni- and multivariate analyses of the chemical data reveal the influence of Madrid on the geochemistry of Manzanares' sediments, clearly manifested by a marked increase in the concentration of typically “urban” elements Ag, Cr, Cu, Pb and Zn, downstream of the intersection of the river with the city's perimeter. The highest concentrations of these elements appear to be associated with illegal or accidental dumping of waste materials, and with the uncontrolled incorporation of untreated urban runoff to the river. The natural matrix of the sediment is characterised by fairly constant concentrations of Ce, La and Y, whereas changes in the lithology intersected by the river cause corresponding variations in Ca–Mg and Al–Na contents. In the final stretch of the river, the presence of carbonate materials seems to exert a strong geochemical control on the amount of Zn and, to a lesser extent, Cu immobilised in the sediments. This fact suggests that a variable but significant proportion of both elements may be susceptible to reincorporation in the aqueous phase under realistic environmental conditions.

Ectopic expression of the minimal whiE polyketide synthase generates a library of aromatic polyketides of diverse sizes and shapes

The single recombinant expressing the Streptomyces coelicolor minimal whiE (spore pigment) polyketide synthase (PKS) is uniquely capable of generating a large array of well more than 30 polyketides, many of which, so far, are novel to this recombinant. The characterized polyketides represent a diverse set of molecules that differ in size (chain length) and shape (cyclization pattern). This combinatorial biosynthetic library is, by far, the largest and most complex of its kind described to date and indicates that the minimal whiE PKS does not independently control polyketide chain length nor dictate the first cyclization event. Rather, the minimal PKS enzyme complex must rely on the stabilizing effects of additional subunits (i.e., the cyclase whiE-ORFVI) to ensure that the chain reaches the full 24 carbons and cyclizes correctly. This dramatic loss of control implies that the growing polyketide chain does not remain enzyme bound, resulting in the spontaneous cyclization of the methyl terminus. Among the six characterized dodecaketides, four different first-ring cyclization regiochemistries are represented, including C7/C12, C8/C13, C10/C15, and C13/C15. The dodecaketide TW93h possesses a unique 2,4-dioxaadamantane ring system and represents a new structural class of polyketides with no related structures isolated from natural or engineered organisms, thus supporting the claim that engineered biosynthesis is capable of producing novel chemotypes.