Zwingenberg, Germany
Zwingenberg, Germany
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Suggested is a method for enhancing the expression of taste related receptor genes encompassing the following steps:(i) providing a culture of mammalian cells, the genome of said cells comprising at least one sweet receptor domain;(ii) designing at least one type of single-guide RNA (sgRNA), the 10 to 30 nt guide sequence of said sgRNA being complementary to stretches within the non-coding and/or putative regulatory region upstream of the translation start codon of at least one sweet receptor gene;(iii) preparing a vector comprising an expression cassette encompassing at least one optionally modified CRISPR-Cas9, preferably CRISPR-dCas9VP64, and at least one optionally modified sg-RNA optionally containing aptamer structures for binding activator proteins;(iv) transfecting said culture of mammalian cells with said vector to target the genome for the presence of a DNA sequence that is complementary to the 10 to 30 nt guide sequence of said sgRNA; and(v) measuring the transcriptional enhancement of the sweet receptor mRNA by quantitative RT-PCR.

Riedel K.,BRAIN AG | Sombroek D.,BRAIN AG | Fiedler B.,BRAIN AG | Siems K.,AnalytiCon Discovery GmbH | Krohn M.,BRAIN AG
Natural Product Reports | Year: 2017

Covering: 2000 to 2016 On the molecular level humans sense food by a variety of specialized tissues which express sensory receptors to handle nutritive value. In general, this means the interplay of gustatory, olfactory, trigeminal and haptic sensation is translated into perception and leads, in terms of taste, to descriptions like sweet, bitter, salty, sour and umami. Further perceptions include astringent, cool, hot, prickle, lingering, kokumi and fatty to name predominant characterizations. It is still not fully understood how this plethora of impressions can be perceived by quite a limited number of receptors obviously being the initial compilers to judge palatability. However, since the discovery of mammalian taste receptors (TASRs) almost 30 years ago the use of taste receptors in cell-based screening campaigns is advancing in industrial approaches. The article will highlight the impacts and the limits of cell-based guided identification of taste modulators for food applications with an emphasis on sweet, bitter and savory taste as well as implications emerging from natural products. © 2017 The Royal Society of Chemistry.

Jankowitsch F.,Mannheim University of Applied Sciences | Schwarz J.,Mannheim University of Applied Sciences | Ruckert C.,Bielefeld University | Gust B.,University of Tübingen | And 5 more authors.
Journal of Bacteriology | Year: 2012

Streptomyces davawensis JCM 4913 synthesizes the antibiotic roseoflavin, a structural riboflavin (vitamin B2) analog. Here, we report the 9,466,619-bp linear chromosome of S. davawensis JCM 4913 and a 89,331-bp linear plasmid. The sequence has an average G+C content of 70.58% and contains six rRNA operons (16S-23S-5S) and 69 tRNA genes. The 8,616 predicted proteincoding sequences include 32 clusters coding for secondary metabolites, several of which are unique to S. davawensis. The chromosome contains long terminal inverted repeats of 33,255 bp each and atypical telomeres. Sequence analysis with regard to riboflavin biosynthesis revealed three different patterns of gene organization in Streptomyces species. Heterologous expression of a set of genes present on a subgenomic fragment of S. davawensis resulted in the production of roseoflavin by the host Streptomyces coelicolor M1152. Phylogenetic analysis revealed that S. davawensis is a close relative of Streptomyces cinnabarinus, and much to our surprise, we found that the latter bacterium is a roseoflavin producer as well. © 2012, American Society for Microbiology.

Kueper T.,Symrise GmbH and Co KG | Krohn M.,BRAIN AG | Haustedt L.O.,AnalytiCon Discovery GmbH | Hatt H.,Ruhr University Bochum | And 2 more authors.
Experimental Dermatology | Year: 2010

During the past years the topic sensitive skin became one of the most important fields in dermatology. The tremendous interest is based on several studies showing that about 50% of the population declares to have sensitive skin. The human thermoreceptor hTRPV1 was previously identified to contribute to this skin condition while facilitating neurogenic inflammation leading to hyperalgesia. Furthermore, skin sensitivity towards capsaicin, a natural activator of TRPV1, was shown to correlate with sensitive skin. In a screening campaign based on recombinant HEK293-cells stably transfected with hTRPV1, the selective antagonist trans-4-tert-butylcyclohexanol was identified. This antagonist is able to inhibit capsaicin-induced hTRPV1 activation with an IC50 value of 34±5μm tested in HEK293-cells as well as in electrophysiological recordings performed in oocytes expressing hTRPV1. Strikingly, in a clinical study with 30 women using topical treatment with o/w emulsions containing 31.6 ppm capsaicin, we were able to show that 0.4% of this inhibitor significantly reduces capsaicin-induced burning (P<0.0001) in vivo. Thus trans-4-tert-butylcyclohexanol has the potential as a novel bioactive for the treatment of sensitive skin. © 2010 John Wiley & Sons A/S.

Liebeton K.,BRAIN AG | Lengefeld J.,ETH Zurich | Eck J.,BRAIN AG
Journal of Biotechnology | Year: 2014

Bacillus subtilis is a commonly used host for the heterologous expression of genes in academia and industry. Many factors are known to influence the expression yield in this organism e.g. the complementarity between the Shine-Dalgarno sequence (SD) and the 16S-rRNA or secondary structures in the translation initiation region of the transcript. In this study, we analysed the impact of the nucleotide composition between the SD sequence and the start codon (the spacer sequence) on the expression yield. We demonstrated that a polyadenylate-moiety spacer sequence moderately increases the expression level of laccase CotA from B. subtilis. By screening a library of artificially generated spacer variants, we identified clones with greatly increased expression levels of two model enzymes, the laccase CotA from B. subtilis (11 fold) and the metagenome derived protease H149 (30 fold). Furthermore, we demonstrated that the effect of the spacer sequence is specific to the gene of interest. These results prove the high impact of the spacer sequence on the expression yield in B. subtilis. © 2014 Elsevier B.V.

Rehdorf J.,University of Greifswald | Rehdorf J.,BRAIN AG | Behrens G.A.,University of Greifswald | Nguyen G.-S.,University of Greifswald | And 2 more authors.
Applied Microbiology and Biotechnology | Year: 2012

An esterase from Pseudomonas putida JD1 (PPE) was successfully cloned, actively expressed in Escherichia coli, and characterized. It was discovered that PPE is more active towards short-chain esters, hydrolyzed δ-valerolactone, and ε-caprolactone and was most active at 37°C and pH 8. After purification to homogeneity by Ni-NTA-assisted affinity chromatography, the kinetic parameters K M and k cat were determined for p-nitrophenyl acetate and butyrate, respectively, showing better catalytic efficiency for hydrolysis of the acetate residue. Investigation of the protein sequence revealed not only the classical catalytic triad for carboxylesterases, additionally the interesting GGG(A)X-motif, which is associated to activity towards tertiary alcohols, was found. Indeed, enzymatic activity was shown for a set of different tertiary alcohols with enantioselectivities up to E∈=∈20, suggesting PPE to be a promising biocatalyst. In addition, PPE also hydrolyzed 4-hydroxyphenyl acetate, the product of a Baeyer-Villiger monooxygenase-catalyzed oxidation of 4-hydroxyacetophenone with a specific activity of 34.36 U/mg suggesting a physiological role in P. putida JD1. © 2011 Springer-Verlag.

Ertongur-Fauth T.,BRAIN AG | Hochheimer A.,BRAIN AG | Hochheimer A.,Amgen Research GmbH | Buescher J.M.,BRAIN AG | And 2 more authors.
Experimental Dermatology | Year: 2014

Sweating is an important physiological process to regulate body temperature in humans, and various disorders are associated with dysregulated sweat formation. Primary sweat secretion in human eccrine sweat glands involves Ca2+-activated Cl- channels (CaCC). Recently, members of the TMEM16 family were identified as CaCCs in various secretory epithelia; however, their molecular identity in sweat glands remained elusive. Here, we investigated the function of TMEM16A in sweat glands. Gene expression analysis revealed that TMEM16A is expressed in human NCL-SG3 sweat gland cells as well as in isolated human eccrine sweat gland biopsy samples. Sweat gland cells express several previously described TMEM16A splice variants, as well as one novel splice variant, TMEM16A(acΔe3) lacking the TMEM16A-dimerization domain. Chloride flux assays using halide-sensitive YFP revealed that TMEM16A is functionally involved in Ca2+-dependent Cl- secretion in NCL-SG3 cells. Recombinant expression in NCL-SG3 cells showed that TMEM16A(acΔe3) is forming a functional CaCC, with basal and Ca2+-activated Cl- permeability distinct from canonical TMEM16A(ac). Our results suggest that various TMEM16A isoforms contribute to sweat gland-specific Cl- secretion providing opportunities to develop sweat gland-specific therapeutics for treatment of sweating disorders. © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Hochheimer A.,BRAIN AG | Hochheimer A.,Amgen Research GmbH | Krohn M.,BRAIN AG | Rudert K.,BRAIN AG | And 4 more authors.
Chemical Senses | Year: 2014

Investigating molecular mechanisms underlying human taste sensation requires functionally dedicated and at the same time proliferating human taste cells. Here, we isolated viable human fungiform taste papillae cells from biopsy samples, adenovirally transduced proliferation promoting genes, and obtained stably proliferating cell lines. Analysis of gene expression of 1 human taste cell line termed HTC-8 revealed that these cells express 13 TAS2R bitter taste receptor genes, CD36, OXTR encoding oxytocin receptor, as well as genes implicated with signal transduction and cell fate control. Bitter tastants triggered functionally distinct signaling pathways in HTC-8 cells. Salicin elicited phospholipase C-dependent calcium signaling and no cell depolarization. In contrast, stimulation with saccharin, aristolochic acid, or phenylthiocarbamide triggered cell depolarization and phospholipase C-independent calcium influx. Simultaneous stimulation with salicin and saccharin revealed that saccharin can enhance the phospholipase C-dependent response to salicin indicating crosstalk of signaling pathways. Our results show that HTC-8 cells are programmed to bitter taste reception but are also responsive to fatty acids, oxytocin, and somatosensory stimuli, whereas HTC-8 cells are insensitive to compounds representing other basic taste qualities. © The Author 2014. Published by Oxford University Press.

Wippo C.J.,Ludwig Maximilians University of Munich | Israel L.,Ludwig Maximilians University of Munich | Watanabe S.,University of Massachusetts Medical School | Hochheimer A.,Ludwig Maximilians University of Munich | And 3 more authors.
EMBO Journal | Year: 2011

Nucleosomes impede access to DNA. Therefore, nucleosome positioning is fundamental to genome regulation. Nevertheless, the molecular nucleosome positioning mechanisms are poorly understood. This is partly because in vitro reconstitution of in vivo-like nucleosome positions from purified components is mostly lacking, barring biochemical studies. Using a yeast extract in vitro reconstitution system that generates in vivo-like nucleosome patterns at S. cerevisiae loci, we find that the RSC chromatin remodelling enzyme is necessary for nucleosome positioning. This was previously suggested by genome-wide in vivo studies and is confirmed here in vivo for individual loci. Beyond the limitations of conditional mutants, we show biochemically that RSC functions directly, can be sufficient, but mostly relies on other factors to properly position nucleosomes. Strikingly, RSC could not be replaced by either the closely related SWI/SNF or the Isw2 remodelling enzyme. Thus, we pinpoint that nucleosome positioning specifically depends on the unique properties of the RSC complex. © 2011 European Molecular Biology Organization. All Rights Reserved.

The present invention provides a polynucleotide or a pair of polynucleotides encoding an enzyme having nitrile hydratase (NHase) [E.C.] activity. Furthermore, a vector and a host comprising the disclosed polynucleotide or pair of polynucleotides and methods for the production of the same are provided. Moreover, the invention relates to a pair of polypeptides or a fusion protein having NHase activity, an antibody specifically binding to the pair of polypeptides or fusion protein, a primer or probe, which specifically hybridizes under stringent conditions to the disclosed polynucleotide or either one of the pair of polynucleotides, a composition comprising the polynucleotide or pair of polynucleotides, the pair of polypeptides or fusion protein, the antibody and/or one or more primers or probes of the invention and a method for the production of amides comprising the enantioselective conversion of nitriles.

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