Gamle, Denmark
Gamle, Denmark

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Petersen I.L.,Copenhagen University | Petersen I.L.,Carlsberg Research Center | Tomasi G.,Copenhagen University | Sorensen H.,Copenhagen University | And 3 more authors.
Environmental Pollution | Year: 2011

Metabolic profiling in plants can be used to differentiate between treatments and to search for biomarkers for exposure. A methodology for processing Ultra-High-Performance Liquid Chromatography-Diode-Array-Detection data is devised. This methodology includes a scheme for selecting informative wavelengths, baseline removal, retention time alignment, selection of relevant retention times, and principal component analysis (PCA). Plant crude extracts from rapeseed seedling exposed to sublethal concentrations of glyphosate are used as a study case. Through this approach, plants exposed to concentrations down to 5 μM could be distinguished from the controls. The compounds responsible for this differentiation were partially identified and were different from those specific for high exposure samples, which suggests that two different responses to glyphosate are elicited in rapeseed depending on the level of exposure. The PCA loadings indicate that a combination of other metabolites could be more sensitive than the response of shikimate to detect glyphosate exposure. © 2010 Published by Elsevier Ltd.


Sanchez R.G.,Carlsberg Laboratory | Sanchez R.G.,Carlsberg Research Center | Solodovnikova N.,Carlsberg Laboratory | Wendland J.,Carlsberg Laboratory
Yeast | Year: 2012

Lager beer brewing relies on strains collectively known as Saccharomyces carlsbergensis, which are hybrids between S. cerevisiae and S. eubayanus-like strains. Lager yeasts are particularly adapted to low-temperature fermentations. Selection of new yeast strains for improved traits or fermentation performance is laborious, due to the allotetraploid nature of lager yeasts. Initially, we have generated new F1 hybrids by classical genetics, using spore clones of lager yeast and S. cerevisiae and complementation of auxotrophies of the single strains upon mating. These hybrids were improved on several parameters, including growth at elevated temperature and resistance against high osmolarity or high ethanol concentrations. Due to the uncertainty of chromosomal make-up of lager yeast spore clones, we introduced molecular markers to analyse mating-type composition by PCR. Based on these results, new hybrids between a lager and an ale yeast strain were isolated by micromanipulation. These hybrids were not subject to genetic modification. We generated and verified 13 hybrid strains. All of these hybrid strains showed improved stress resistance as seen in the ale parent, including improved survival at the end of fermentation. Importantly, some of the strains showed improved fermentation rates using 18°Plato at 18-25°C. Uniparental mitochondrial DNA inheritance was observed mostly from the S. cerevisiae parent. © 2012 John Wiley & Sons, Ltd.


Pesnot T.,University of East Anglia | Palcic M.M.,Carlsberg Research Center | Wagner G.K.,University of East Anglia | Wagner G.K.,King's College London
ChemBioChem | Year: 2010

Glycosyltransferases (GTs) are a large class of carbohydrate-active enzymes that are involved, in both pro- and eukaryotic organisms, in numerous important biological processes, from cellular adhesion to carcinogenesis. GTs have enormous potential as molecular targets for chemical biology and drug discovery. For the full realisation of this potential, operationally simple and generally applicable GT bioassays, especially for inhibitor screening, are indispensable tools. In order to facilitate the development of GT high-throughput screening assays for the identification of GT inhibitors, we have developed novel, fluorescent derivatives of UDP-galactose (UDP-Gal) that are recognised as donor analogues by several different retaining galactosyltransferases (GalTs). We demonstrate for one of these derivatives that fluorescence emission is quenched upon specific binding to individual GalTs, and that this effect can be used as the read-out in ligand-displacement experiments. The novel fluorophore acts as an excellent sensor for several different enzymes and is suitable for the development of a new type of GalT bioassay, whose modular nature and operational simplicity will significantly facilitate inhibitor screening. Importantly, the structural differences between the natural donor UDP-Gal and the new fluorescent derivatives are minimal, and the general assay principle described herein may therefore also be applicable to other GalTs and/or proteins that use nucleotides or nucleotide conjugates as their cofactor. © 2010 Wiley-VCH Verlag GmbH & Co. KGaA.


Sim L.,Protein Chemistry Group | Groes M.,Carlsberg Research Center | Olesen K.,Carlsberg Research Center | Olesen K.,Novo Nordisk AS | And 2 more authors.
FEBS Journal | Year: 2013

The mechanism of yeast flocculation is generally considered to be mediated through the interaction of cell surface flocculins and mannan carbohydrates. In the present study, the crystal structure of the soluble 25-kDa lectin domain of flocculin 1 from brewer's yeast (Lg-Flo1p) was resolved to 2.5 Å, and its binding specificity towards oligosaccharides was investigated by fluorescence spectroscopy. Lg-Flo1p displays broad specificity towards sugars and has a 14-fold higher affinity for mannose 1-phosphate and glucose 1-phosphate compared to their unphosphorylated counterparts. Based on the results of a structural analysis, we propose that this higher affinity is the result of a charge interaction with a lysine residue in a carbohydrate-binding loop region, NAKAL, unique to NewFlo type flocculins. This raises the possibility of a unique mechanism of flocculation in NewFlo type yeast, which recognizes phosphorylated cell surface mannans. © 2013 The Authors Journal compilation © 2013 FEBS.


Garcia Sanchez R.,Lund University | Karhumaa K.,Lund University | Karhumaa K.,Technical University of Denmark | Fonseca C.,New University of Lisbon | And 10 more authors.
Biotechnology for Biofuels | Year: 2010

Background. Cost-effective fermentation of lignocellulosic hydrolysate to ethanol by Saccharomyces cerevisiae requires efficient mixed sugar utilization. Notably, the rate and yield of xylose and arabinose co-fermentation to ethanol must be enhanced. Results. Evolutionary engineering was used to improve the simultaneous conversion of xylose and arabinose to ethanol in a recombinant industrial Saccharomyces cerevisiae strain carrying the heterologous genes for xylose and arabinose utilization pathways integrated in the genome. The evolved strain TMB3130 displayed an increased consumption rate of xylose and arabinose under aerobic and anaerobic conditions. Improved anaerobic ethanol production was achieved at the expense of xylitol and glycerol but arabinose was almost stoichiometrically converted to arabitol. Further characterization of the strain indicated that the selection pressure during prolonged continuous culture in xylose and arabinose medium resulted in the improved transport of xylose and arabinose as well as increased levels of the enzymes from the introduced fungal xylose pathway. No mutation was found in any of the genes from the pentose converting pathways. Conclusion. To the best of our knowledge, this is the first report that characterizes the molecular mechanisms for improved mixed-pentose utilization obtained by evolutionary engineering of a recombinant S. cerevisiae strain. Increased transport of pentoses and increased activities of xylose converting enzymes contributed to the improved phenotype. © 2010 Sanchez et al; licensee BioMed Central Ltd.


Falk J.,Carlsberg Research Center | Munne-Bosch S.,University of Barcelona
Journal of Experimental Botany | Year: 2010

Tocopherols and tocotrienols, collectively known as tocochromanols, are lipid-soluble molecules that belong to the group of vitamin E compounds and are essential in the human diet. Not surprisingly, most of what is known about the biological functions of tocochromanols comes from studies of mammalian systems, yet they have been shown to be synthesized only by photosynthetic organisms. The last decade has seen a radical change in the appreciation of the biological role of tocochromanols in plants thanks to a detailed characterization of mutant and transgenic plants, including several Arabidopsis thaliana mutants, the sucrose export defective1 (sxd1) maize mutant, and some transgenic potato and tobacco lines altered in tocochromanol biosynthesis. Recent findings indicate that tocopherols may play important roles in plants beyond their antioxidant function in photosynthetic membranes. Plants deficient in tocopherols show alterations in germination and export of photoassimilates, and growth, leaf senescence, and plant responses to abiotic stresses, thus suggesting that tocopherols may influence a number of physiological processes in plants. Thus, in this review not only the antioxidant function of tocochromanols in plants, but also these new emerging possible roles will be considered. Particular attention will be paid to specific roles attributed to different tocopherol homologues (particularly α-and γ-tocopherol) and the possible functions of tocotrienols, which in contrast to tocopherols are only present in a range of unrelated plant groups and are almost exclusively found in seeds and fruits. © 2010 The Author(s).


Rudder S.,Teagasc | Rudder S.,University College Dublin | Doohan F.,University College Dublin | Creevey C.J.,Teagasc | And 5 more authors.
BMC Genomics | Year: 2014

Background: Recently it has been shown that Ensifer adhaerens can be used as a plant transformation technology, transferring genes into several plant genomes when equipped with a Ti plasmid. For this study, we have sequenced the genome of Ensifer adhaerens OV14 (OV14) and compared it with those of Agrobacterium tumefaciens C58 (C58) and Sinorhizobium meliloti 1021 (1021); the latter of which has also demonstrated a capacity to genetically transform crop genomes, albeit at significantly reduced frequencies.Results: The 7.7 Mb OV14 genome comprises two chromosomes and two plasmids. All protein coding regions in the OV14 genome were functionally grouped based on an eggNOG database. No genes homologous to the A. tumefaciens Ti plasmid vir genes appeared to be present in the OV14 genome. Unexpectedly, OV14 and 1021 were found to possess homologs to chromosomal based genes cited as essential to A. tumefaciens T-DNA transfer. Of significance, genes that are non-essential but exert a positive influence on virulence and the ability to genetically transform host genomes were identified in OV14 but were absent from the 1021 genome.Conclusions: This study reveals the presence of homologs to chromosomally based Agrobacterium genes that support T-DNA transfer within the genome of OV14 and other alphaproteobacteria. The sequencing and analysis of the OV14 genome increases our understanding of T-DNA transfer by non-Agrobacterium species and creates a platform for the continued improvement of Ensifer-mediated transformation (EMT). © 2014 Rudder et al.; licensee BioMed Central Ltd.


Holler J.G.,Copenhagen University | Christensen S.B.,Copenhagen University | Slotved H.-C.,Statens Serum Institute | Rasmussen H.B.,Copenhagen University | And 4 more authors.
Journal of Antimicrobial Chemotherapy | Year: 2012

Objectives: To isolate a plant-derived compound with efflux inhibitory activity towards the NorA transporter of Staphylococcus aureus. Methods: Bioassay-guided isolation was used, with inhibition of ethidium bromide efflux via NorA as a guide. Characterization of activity was carried out using MIC determination and potentiation studies of a fluoroquinolone antibiotic in combination with the isolated compound. Everted membrane vesicles of Escherichia coli cells enriched with NorA were prepared to study efflux inhibitory activity in an isolated manner. Results: The ethanolic extract of Persea lingue was subjected to bioassay-guided fractionation and led to the isolation of the known compound kaempferol-3-O-α-l-(2,4-bis-E-p-coumaroyl)rhamnoside (compound 1). Evaluation of the dose-response relationship of compound 1 showed that ethidium bromide efflux was inhibited, with an IC 50 value of 2 μM. The positive control, reserpine, was found to have an IC 50 value of 9 μM. Compound 1 also inhibited NorA in enriched everted membrane vesicles of E. coli. Potentiation studies revealed that compound 1 at 1.56 mg/L synergistically increased the antimicrobial activity of ciprofloxacin 8-fold against a NorA overexpresser, and the synergistic activity was exerted at a fourth of the concentration necessary for reserpine. Compound 1 was not found to exert a synergistic effect on ciprofloxacin against a norA deletion mutant. The 2,3-coumaroyl isomer of compound 1 has been shown previously not to cause acute toxicity in mice at 20 mg/kg/day. Conclusions: Our results show that compound 1 acts through inhibition of the NorA efflux pump. Combination of compound 1 with subinhibitory concentrations of ciprofloxacin renders a wild-type more susceptible and a NorA overexpresser S. aureus susceptible. © The Author 2012. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved.


Beeren S.R.,Carlsberg Laboratory | Christensen C.E.,Carlsberg Laboratory | Tanaka H.,Carlsberg Laboratory | Jensen M.G.,Carlsberg Research Center | And 2 more authors.
Carbohydrate Polymers | Year: 2014

β-Glucans from cereals are β(1-3)(1-4)-mixed linkage linear homopolysaccharides of d-glucopyranosyl residues, recently recognised as functional components of foods with benefits in maintaining the health of the digestive tract not least through a prebiotic effect. Here we describe the development of methodology to facilitate the study of β-glucans as prebiotics. Relatively short β-glucan fragments (DP 6-50) were produced by partial hydrolysis of β-glucan fibres with Lichenase then functionalised at their reducing end with a tetramethylrhodamine dye. Their enzymatic break down by human colon microbiota in an in vitro fermentation model was examined. Digestion products were isolated by virtue of their fluorescence labels, identified and characterised using capillary electrophoresis and mass spectrometry. Complete digestion of the labelled substrates was indicated, as fluorescently labelled glucose was obtained as the final product. Furthermore, a pathway of enzymatic breakdown was proposed on the basis of a time course experiment; initial fast hydrolysis with an endo-1,3(4)-β-glucanase was followed by slow degradation with an exo-1,4-β-glucanase and finally slow action of an exo-1,3-β-glucanase. © 2014 Elsevier Ltd. All rights reserved.


Dasgupta J.,University of Southern California | Dasgupta J.,St Xaviers College | Bienkowska-Haba M.,Louisiana State University Health Sciences Center | Ortega M.E.,University of Southern California | And 7 more authors.
Journal of Biological Chemistry | Year: 2011

High risk human papillomavirus types 16 (HPV16) and 18 (HPV18) can cause cervical cancer. Efficient infection by HPV16 and HPV18 pseudovirions requires interactions of particles with cell-surface receptor heparan sulfate oligosaccharide. To understand the virus-receptor interactions for HPV infection, we determined the crystal structures of HPV16 and HPV18 capsids bound to the oligosaccharide receptor fragment using oligomeric heparin. The HPV-heparin structures revealed multiple binding sites for the highly negatively charged oligosaccharide fragment on the capsid surface, which is different from previously reported virus-receptor interactions in which a single type of binding pocket is present for a particular receptor. We performed structure-guided mutagenesis to generate mutant viruses, and cell binding and infectivity assays demonstrated the functional role of viral residues involved in heparin binding. These results provide a basis for understanding virus-heparan sulfate receptor interactions critical for HPV infection and for the potential development of inhibitors against HPV infection. © 2011 by The American Society for Biochemistry and Molecular Biology, Inc.

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