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Gamle, Denmark

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.

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.

Pesnot T.,University of East Anglia | Palcic M.M.,Carlsberg Research Center | Wagner G.K.,University of East Anglia | Wagner G.K.,Kings 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.

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).

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.

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