• Fatty acid elongation in non-alcoholic steatohepatitis and hepatocellular carcinoma.

      Kessler, Sonja M; Simon, Yvette; Gemperlein, Katja; Gianmoena, Kathrin; Cadenas, Cristina; Zimmer, Vincent; Pokorny, Juliane; Barghash, Ahmad; Helms, Volkhard; van Rooijen, Nico; et al. (2014)
      Non-alcoholic steatohepatitis (NASH) represents a risk factor for the development of hepatocellular carcinoma (HCC) and is characterized by quantitative and qualitative changes in hepatic lipids. Since elongation of fatty acids from C16 to C18 has recently been reported to promote both hepatic lipid accumulation and inflammation we aimed to investigate whether a frequently used mouse NASH model reflects this clinically relevant feature and whether C16 to C18 elongation can be observed in HCC development. Feeding mice a methionine and choline deficient diet to model NASH not only increased total hepatic fatty acids and cholesterol, but also distinctly elevated the C18/C16 ratio, which was not changed in a model of simple steatosis (ob/ob mice). Depletion of Kupffer cells abrogated both quantitative and qualitative methionine-and-choline deficient (MCD)-induced alterations in hepatic lipids. Interestingly, mimicking inflammatory events in early hepatocarcinogenesis by diethylnitrosamine-induced carcinogenesis (48 h) increased hepatic lipids and the C18/C16 ratio. Analyses of human liver samples from patients with NASH or NASH-related HCC showed an elevated expression of the elongase ELOVL6, which is responsible for the elongation of C16 fatty acids. Taken together, our findings suggest a detrimental role of an altered fatty acid pattern in the progression of NASH-related liver disease.
    • Fatty acid-related phylogeny of myxobacteria as an approach to discover polyunsaturated omega-3/6 Fatty acids.

      Garcia, Ronald; Pistorius, Dominik; Stadler, Marc; Müller, Rolf (2011-04)
      In an analysis of 47 aerobic myxobacterial strains, representing 19 genera in suborders Cystobacterineae, Nannocystineae, Sorangiineae, and a novel isolate, "Aetherobacter" SBSr008, an enormously diverse array of fatty acids (FAs) was found. The distribution of straight-chain fatty acids (SCFAs) and branched-chain fatty acids (BCFAs) supports the reported clustering of strains in the phylogenetic tree based on 16S rRNA genes. This finding additionally allows the prediction and assignment of the novel isolate SBSr008 into its corresponding taxon. Sorangiineae predominantly contains larger amounts of SCFA (57 to 84%) than BCFA. On the other hand, Cystobacterineae exhibit significant BCFA content (53 to 90%), with the exception of the genus Stigmatella. In Nannocystineae, the ratio of BCFA and SCFA seems dependent on the taxonomic clade. Myxobacteria could also be identified and classified by using their specific and predominant FAs as biomarkers. Nannocystineae is remarkably unique among the suborders for its absence of hydroxy FAs. After the identification of arachidonic (AA) FA in Phaselicystidaceae, eight additional polyunsaturated fatty acids (PUFAs) belonging to the omega-6 and omega-3 families were discovered. Here we present a comprehensive report of FAs found in aerobic myxobacteria. Gliding bacteria belonging to Flexibacter and Herpetosiphon were chosen for comparative analysis to determine their FA profiles in relation to the myxobacteria.
    • First Bispecific Inhibitors of the Epidermal Growth Factor Receptor Kinase and the NF-κB Activity As Novel Anticancer Agents.

      Hamed, Mostafa M; Darwish, Sarah S; Herrmann, Jennifer; Abadi, Ashraf H; Engel, Matthias; HIPS, Helmholtz-Institut für pharmazeutische Forschung Saarland, Universitätscampus E8.1,66123 Saarbrücken, Germany. (2017-04-13)
      The activation of the NF-κB transcription factor is a major adaptive response induced upon treatment with EGFR kinase inhibitors, leading to the emergence of resistance in nonsmall cell lung cancer and other tumor types. To suppress this survival mechanism, we developed new thiourea quinazoline derivatives that are dual inhibitors of both EGFR kinase and the NF-κB activity. Optimization of the hit compound, identified in a NF-κB reporter gene assay, led to compound 9b, exhibiting a cellular IC50 for NF-κB inhibition of 0.3 μM while retaining a potent EGFR kinase inhibition (IC50 = 60 nM). The dual inhibitors showed a higher potency than gefitinib to inhibit cell growth of EGFR-overexpressing tumor cell lines in vitro and in a xenograft model in vivo, while no signs of toxicity were observed. An investigation of the molecular mechanism of NF-κB suppression revealed that the dual inhibitors depleted the transcriptional coactivator CREB-binding protein from the NF-κB complex in the nucleus.
    • A fluorescence anisotropy assay to discover and characterize ligands targeting the maytansine site of tubulin.

      Menchon, Grégory; Prota, Andrea E; Lucena-Agell, Daniel; Bucher, Pascal; Jansen, Rolf; Irschik, Herbert; Müller, Rolf; Paterson, Ian; Díaz, J Fernando; Altmann, Karl-Heinz; et al. (2018-05-29)
      Microtubule-targeting agents (MTAs) like taxol and vinblastine are among the most successful chemotherapeutic drugs against cancer. Here, we describe a fluorescence anisotropy-based assay that specifically probes for ligands targeting the recently discovered maytansine site of tubulin. Using this assay, we have determined the dissociation constants of known maytansine site ligands, including the pharmacologically active degradation product of the clinical antibody-drug conjugate trastuzumab emtansine. In addition, we discovered that the two natural products spongistatin-1 and disorazole Z with established cellular potency bind to the maytansine site on β-tubulin. The high-resolution crystal structures of spongistatin-1 and disorazole Z in complex with tubulin allowed the definition of an additional sub-site adjacent to the pocket shared by all maytansine-site ligands, which could be exploitable as a distinct, separate target site for small molecules. Our study provides a basis for the discovery and development of next-generation MTAs for the treatment of cancer.
    • Foxp3+ regulatory T cells are required for recovery from severe sepsis.

      Kühlhorn, Franziska; Rath, Matthias; Schmoeckel, Katrin; Cziupka, Katharina; Nguyen, Huu Hung; Hildebrandt, Petra; Hünig, Thomas; Sparwasser, Tim; Huehn, Jochen; Pötschke, Christian; et al. (2013)
      The role of regulatory T cells (Tregs) in bacterial sepsis remains controversial because antibody-mediated depletion experiments gave conflicting results. We employed DEREG mice (DEpletion of REGulatory T cells) and a caecal ligation and puncture model to elucidate the role of CD4(+)Foxp3(+) Tregs in sepsis. In DEREG mice natural Tregs can be visualized easily and selectively depleted by diphtheria toxin because the animals express the diphtheria toxin receptor and enhanced green fluorescent protein as a fusion protein under the control of the foxp3 locus. We confirmed rapid Treg-activation and an increased ratio of Tregs to Teffs in sepsis. Nevertheless, 24 h after sepsis induction, Treg-depleted and control mice showed equally strong inflammation, immune cell immigration into the peritoneum and bacterial dissemination. During the first 36 h of disease survival was not influenced by Treg-depletion. Later, however, only Treg-competent animals recovered from the insult. We conclude that the suppressive capacity of Tregs is not sufficient to control overwhelming inflammation and early mortality, but is a prerequisite for the recovery from severe sepsis.
    • Future Directions of Marine Myxobacterial Natural Product Discovery Inferred from Metagenomics.

      Garcia, Ronald; La Clair, James J; Müller, Rolf; HIPS, Helmholtz-Institut für pharmazeutische Forschung Saarland, Universitätscampus 8.1, 66123 Saarbrücken, Germany. (2018-08-29)
      Over the last two decades, halophilic (organisms that thrive at high salt concentrations) and halotolerant (organisms that have adapted to high salt concentrations) myxobacteria emerged as an important source of structurally diverse secondary metabolites from the marine environment. This review explores the advance of metagenomics analysis and 16S rRNA gene phylogeny of the cultured and uncultured myxobacteria from marine and other salt-environments up to July 2018. The diversity of novel groups of myxobacteria in these environments appears unprecedented, especially in the
    • Genetic engineering and heterologous expression of the disorazol biosynthetic gene cluster via Red/ET recombineering.

      Tu, Qiang; Herrmann, Jennifer; Hu, Shengbiao; Raju, Ritesh; Bian, Xiaoying; Zhang, Youming; Müller, Rolf; Helmholtz-Institute for Pharmaceutical Research Saarland (HIPS),Saarland 9 University, 66123 Saarbrücken, Germany. (2016)
      Disorazol, a macrocyclic polykitide produced by the myxobacterium Sorangium cellulosum So ce12 and it is reported to have potential cytotoxic activity towards several cancer cell lines, including multi-drug resistant cells. The disorazol biosynthetic gene cluster (dis) from Sorangium cellulosum (So ce12) was identified by transposon mutagenesis and cloned in a bacterial artificial chromosome (BAC) library. The 58-kb dis core gene cluster was reconstituted from BACs via Red/ET recombineering and expressed in Myxococcus xanthus DK1622. For the first time ever, a myxobacterial trans-AT polyketide synthase has been expressed heterologously in this study. Expression in M. xanthus allowed us to optimize the yield of several biosynthetic products using promoter engineering. The insertion of an artificial synthetic promoter upstream of the disD gene encoding a discrete acyl transferase (AT), together with an oxidoreductase (Or), resulted in 7-fold increase in disorazol production. The successful reconstitution and expression of the genetic sequences encoding for these promising cytotoxic compounds will allow combinatorial biosynthesis to generate novel disorazol derivatives for further bioactivity evaluation.
    • Genome Analysis of the Fruiting Body-Forming Myxobacterium Chondromyces crocatus Reveals High Potential for Natural Product Biosynthesis.

      Zaburannyi, Nestor; Bunk, Boyke; Maier, Josef; Overmann, Jörg; Müller, Rolf; Helmholtz-Institute for Pharmaceutical Research Saarland (HIPS), Saarland University, Campus C2.3, D-66123 Saarbrücken, Germany. (2016)
      Here, we report the complete genome sequence of the type strain of the myxobacterial genus Chondromyces, Chondromyces crocatus Cm c5. It presents one of the largest prokaryotic genomes featuring a single circular chromosome and no plasmids. Analysis revealed an enlarged set of tRNA genes, along with reduced pressure on preferred codon usage compared to that of other bacterial genomes. The large coding capacity and the plethora of encoded secondary metabolite biosynthetic gene clusters are in line with the capability of Cm c5 to produce an arsenal of antibacterial, antifungal, and cytotoxic compounds. Known pathways of the ajudazol, chondramide, chondrochloren, crocacin, crocapeptin, and thuggacin compound families are complemented by many more natural compound biosynthetic gene clusters in the chromosome. Whole-genome comparison of the fruiting-body-forming type strain (Cm c5, DSM 14714) to an accustomed laboratory strain which has lost this ability (nonfruiting phenotype, Cm c5 fr-) revealed genetic changes in three loci. In addition to the low synteny found with the closest sequenced representative of the same family, Sorangium cellulosum, extensive genetic information duplication and broad application of eukaryotic-type signal transduction systems are hallmarks of this 11.3-Mbp prokaryotic genome.
    • The glucocorticoid-induced leucine zipper mediates statin-induced muscle damage.

      Hoppstädter, Jessica; Valbuena Perez, Jenny Vanessa; Linnenberger, Rebecca; Dahlem, Charlotte; Legroux, Thierry M; Hecksteden, Anne; Tse, William K F; Flamini, Sara; Andreas, Anastasia; Herrmann, Jennifer; et al. (Wiley, 2020-02-06)
      Statins, the most prescribed class of drugs for the treatment of hypercholesterolemia, can cause muscle-related adverse effects. It has been shown that the glucocorticoid-induced leucine zipper (GILZ) plays a key role in the anti-myogenic action of dexamethasone. In the present study, we aimed to evaluate the role of GILZ in statin-induced myopathy. Statins induced GILZ expression in C2C12 cells, primary murine myoblasts/myotubes, primary human myoblasts, and in vivo in zebrafish embryos and human quadriceps femoris muscle. Gilz induction was mediated by FOXO3 activation and binding to the Gilz promoter, and could be reversed by the addition of geranylgeranyl, but not farnesyl, pyrophosphate. Atorvastatin decreased Akt phosphorylation and increased cleaved caspase-3 levels in myoblasts. This effect was reversed in myoblasts from GILZ knockout mice. Similarly, myofibers isolated from knockout animals were more resistant toward statin-induced cell death than their wild-type counterparts. Statins also impaired myoblast differentiation, and this effect was accompanied by GILZ induction. The in vivo relevance of our findings was supported by the observation that gilz overexpression in zebrafish embryos led to impaired embryonic muscle development. Taken together, our data point toward GILZ as an essential mediator of the molecular mechanisms leading to statin-induced muscle damage.
    • Growth Medium-Dependent Glycine Incorporation into the Peptidoglycan of Caulobacter crescentus.

      Takacs, Constantin N; Hocking, Jason; Cabeen, Matthew T; Bui, Nhat Khai; Poggio, Sebastian; Vollmer, Waldemar; Jacobs-Wagner, Christine; Department of Molecular, Cellular and Developmental Biology, Yale University, New Haven, Connecticut, United States of America. (2013)
      The peptidoglycan (PG) is a macromolecular component of the bacterial cell wall that maintains the shape and integrity of the cell. The PG of , unlike that of many other Gram-negative bacteria, has repeatedly been shown to contain significant amounts of glycine. This compositional peculiarity has been deemed an intrinsic characteristic of this species. By performing a comprehensive qualitative and quantitative analysis of the PG by high-performance liquid chromatography (HPLC) and mass spectrometry (MS), we show here that glycine incorporation into the PG depends on the presence of exogenous glycine in the growth medium. High levels of glycine were detected at the fifth position of the peptide side chains of PG isolated from cells grown in the complex laboratory medium PYE or in defined medium (M2G) supplemented with casamino acids or glycine alone. In contrast, glycine incorporation was undetectable when cells were grown in M2G medium lacking glycine. Remarkably, glycine incorporation into peptidoglycan occurred even in the presence of low millimolar to sub-millimolar concentrations of free glycine. High glycine content in the PG had no obvious effects on growth rates, mode of PG incorporation or cell morphology. Hence, the PG is able to retain its physiological functions in cell growth and morphogenesis despite significant alterations in its composition, in what we deem to be unprecedented plasticity.
    • Heterologous expression of the atypical tetracycline chelocardin reveals the full set of genes required for its biosynthesis.

      Lukežič, Tadeja; Pikl, Špela; Zaburannyi, Nestor; Remškar, Maja; Petković, Hrvoje; Müller, Rolf; HIPS, Helmholtz-Institut für Pharmazeutische Forschung Saarland, Universitätscampus E8.1 66123 Saarbrücken, Germany. (BMC (part of Springer), 2020-12-19)
      Background: Chelocardin (CHD) exhibits a broad-spectrum antibiotic activity and showed promising results in a small phase II clinical study conducted on patients with urinary tract infections. Importantly, CHD was shown to be active also against tetracycline-resistant Gram-negative pathogens, which is gaining even more importance in today’s antibiotic crisis. We have demonstrated that modifications of CHD through genetic engineering of its producer, the actinomycete Amycolatopsis sulphurea, are not only possible but yielded even more potent antibiotics than CHD itself, like 2-carboxamido-2-deacetyl-chelocardin (CD-CHD), which is currently in preclinical evaluation. A. sulphurea is difficult to genetically manipulate and therefore manipulation of the chd biosynthetic gene cluster in a genetically amenable heterologous host would be of high importance for further drug-discovery efforts. Results: We report heterologous expression of the CHD biosynthetic gene cluster in the model organism Streptomyces albus del14 strain. Unexpectedly, we found that the originally defined CHD gene cluster fails to provide all genes required for CHD formation, including an additional cyclase and two regulatory genes. Overexpression of the putative pathway-specific streptomyces antibiotic regulatory protein chdB in A. sulphurea resulted in an increase of both, CHD and CD-CHD production. Applying a metabolic-engineering approach, it was also possible to generate the potent CHD analogue, CD-CHD in S. albus. Finally, an additional yield increase was achieved in S. albus del14 by in-trans overexpression of the chdR exporter gene, which provides resistance to CHD and CDCHD. Conclusions: We identified previously unknown genes in the CHD cluster, which were shown to be essential for chelocardin biosynthesis by expression of the full biosynthetic gene cluster in S. albus as heterologous host. When comparing to oxytetracycline biosynthesis, we observed that the CHD gene cluster contains additional enzymes not found in gene clusters encoding the biosynthesis of typical tetracyclines (such as oxytetracycline). This finding probably explains the different chemistries and modes of action, which make CHD/CD-CHD valuable lead structures for clinical candidates. Even though the CHD genes are derived from a rare actinomycete A. sulphurea, the yield of CHD in the heterologous host was very good. The corrected nucleotide sequence of the CHD gene cluster now contains allgene products required for the production of CHD in a genetically amenable heterologous host, thus opening new possibilities towards production of novel and potent tetracycline analogues with a new mode of action.
    • A high-affinity fluorescence probe for copper(II) ions and its application in fluorescence lifetime correlation spectroscopy.

      Grüter, Andreas; Hoffmann, Michael; Müller, Rolf; Wohland, Thorsten; Jung, Gregor; HIPS, Helmholtz-Institut für Pharmazeutische Forschung Saarland, Universitätscampus E8.1 66123 Saarbrücken, Germany. (Springer, 2019-04-26)
      Copper is one of the most important transition metals in many organisms where it catalyzes a manifold of different processes. As a result of copper's redox activity, organisms have to avoid unbound ions, and a dysfunctional copper homeostasis may lead to multifarious pathological processes in cells with very severe ramifications for the affected organisms. In many neurodegenerative diseases, however, the exact role of copper ions is still not completely clarified. In this work, a high-affinity and highly selective copper probe molecule, based on the naturally occurring tetrapeptide DAHK is synthesized. The sensor (log KD = - 12.8 ± 0.1) is tagged with a fluorescent BODIPY dye whose fluorescence lifetime distinctly decreases from 5.8 ns ± 0.2 ns to 0.4 ns ± 0.1 ns on binding to copper(II) cations. It is shown by using fluorescence lifetime correlation spectroscopy that the concentration of both probe and probe-copper complex can be simultaneously measured even at nanomolar concentration levels. This work presents a possible starting point for a new type of probe and method for future in vivo studies to further reveal the exact role of copper ions in organisms. Graphical abstract.
    • Homologous bd oxidases share the same architecture but differ in mechanism.

      Theßeling, Alexander; Rasmussen, Tim; Burschel, Sabrina; Wohlwend, Daniel; Kägi, Jan; Müller, Rolf; Böttcher, Bettina; Friedrich, Thorsten; HIPS, Helmholtz-Institut für Pharmazeutische Forschung Saarland, Universitätscampus E8.1 66123 Saarbrücken, Germany. (Nature Publication group, 2019-11-13)
      Cytochrome bd oxidases are terminal reductases of bacterial and archaeal respiratory chains. The enzyme couples the oxidation of ubiquinol or menaquinol with the reduction of dioxygen to water, thus contributing to the generation of the protonmotive force. Here, we determine the structure of the Escherichia coli bd oxidase treated with the specific inhibitor aurachin by cryo-electron microscopy (cryo-EM). The major subunits CydA and CydB are related by a pseudo two fold symmetry. The heme b and d cofactors are found in CydA, while ubiquinone-8 is bound at the homologous positions in CydB to stabilize its structure. The architecture of the E. coli enzyme is highly similar to that of Geobacillus thermodenitrificans, however, the positions of heme b595 and d are interchanged, and a common oxygen channel is blocked by a fourth subunit and substituted by a more narrow, alternative channel. Thus, with the same overall fold, the homologous enzymes exhibit a different mechanism.
    • How to Study the Metabolism of New Psychoactive Substances for the Purpose of Toxicological Screenings-A Follow-Up Study Comparing Pooled Human Liver S9, HepaRG Cells, and Zebrafish Larvae.

      Wagmann, Lea; Frankenfeld, Fabian; Park, Yu Mi; Herrmann, Jennifer; Fischmann, Svenja; Westphal, Folker; Müller, Rolf; Flockerzi, Veit; Meyer, Markus R; HIPS, Helmholtz-Institut für Pharmazeutische Forschung Saarland, Universitätscampus E8.1 66123 Saarbrücken, Germany. (Frontiers, 2020-07-17)
      The new psychoactive substances (NPS) market continues to be very dynamic. A large number of compounds belonging to diverse chemical groups continue to emerge. This makes their detection in biological samples challenging for clinical and forensic toxicologists. Knowledge of the metabolic fate of NPS is crucial for developing comprehensive screening procedures. As human studies are not feasible due to ethical concerns, the current study aimed to compare the NPS' metabolic pattern in incubations with pooled human liver S9 fraction (pHLS9), human liver HepaRG cells, and zebrafish larvae. The latter model was recently shown to be a promising preclinical surrogate for human hepatic metabolism of a synthetic cannabinoid. However, studies concerning other NPS classes are still missing and therefore an amphetamine-based N-methoxybenzyl (NBOMe) compound, a synthetic cathinone, a pyrrolidinophenone analog, a lysergamide, as well as another synthetic cannabinoid were included in the current study. Liquid chromatography coupled to Orbitrap-based high-resolution tandem mass spectrometry was used to analyze metabolic data. Zebrafish larvae were found to produce the highest number of phase I but also phase II metabolites (79 metabolites in total), followed by HepaRG cells (66 metabolites). Incubations with pHLS9 produced the least metabolites (57 metabolites). Furthermore, the involvement of monooxygenases and esterases in the metabolic phase I transformations of 4F-MDMB-BINACA was elucidated using single-enzyme incubations. Several cytochrome P450 (CYP) isozymes were shown to contribute, and CYP3A5 was involved in all CYP-catalyzed reactions, while amide and ester hydrolysis were catalyzed by the human carboxylesterase (hCES) isoforms hCES1b and/or hCES1c. Finally, metabolites were compared to those present in human biosamples if data were available. Overall, the metabolic patterns in HepaRG cells provided the worst overlap with that in human biosamples. Zebrafish larvae experiments agreed best with data found in human plasma and urine analysis. The current study underlines the potential of zebrafish larvae as a tool for elucidating the toxicokinetics of NPS in the future.
    • Human microbial metabolite mimicry as a strategy to expand the chemical space of potential drugs.

      Li, Hao; Ranhotra, Harmit S; Mani, Sridhar; Dvořák, Zdeněk; Sokol, Harry; Müller, Rolf; HIRI, Helmholtz-Institut für RNA-basierte Infektionsforschung, Josef-Shneider Strasse 2, 97080 Würzburg, Germany. (Elsevier, 2020-06-17)
      he concept of small-molecule mimicry even of weak microbial metabolites present in rodents and humans, as a means to expand drug repertoires, is new. Hitherto, there are few proof-of-concept papers demonstrating utility of this concept. More recently, papers demonstrating mimicry of intestinal microbial metabolites could expand the drug repertoire for diseases such as inflammatory bowel disease (IBD). We opine that, as more functional metabolite-receptor pairings are discovered, small-molecule metabolite mimicry could be a significant effort in drug discovery.
    • Inactivation of SACE_3446, a TetR family transcriptional regulator, stimulates erythromycin production in Saccharopolyspora erythraea.

      Wu, Hang; Wang, Yansheng; Yuan, Li; Mao, Yongrong; Wang, Weiwei; Zhu, Lin; Wu, Panpan; Fu, Chengzhang; Müller, Rolf; Weaver, David T; et al. (2016-03)
      Erythromycin A is a widely used antibiotic produced by Saccharopolyspora erythraea; however, its biosynthetic cluster lacks a regulatory gene, limiting the yield enhancement via regulation engineering of S. erythraea. Herein, six TetR family transcriptional regulators (TFRs) belonging to three genomic context types were individually inactivated in S. erythraea A226, and one of them, SACE_3446, was proved to play a negative role in regulating erythromycin biosynthesis. EMSA and qRT-PCR analysis revealed that SACE_3446 covering intact N-terminal DNA binding domain specifically bound to the promoter regions of erythromycin biosynthetic gene eryAI, the resistant gene ermE and the adjacent gene SACE_3447 (encoding a long-chain fatty-acid CoA ligase), and repressed their transcription. Furthermore, we explored the interaction relationships of SACE_3446 and previously identified TFRs (SACE_3986 and SACE_7301) associated with erythromycin production. Given demonstrated relatively independent regulation mode of SACE_3446 and SACE_3986 in erythromycin biosynthesis, we individually and concomitantly inactivated them in an industrial S. erythraea WB. Compared with WB, the WBΔ3446 and WBΔ3446Δ3986 mutants respectively displayed 36% and 65% yield enhancement of erythromycin A, following significantly elevated transcription of eryAI and ermE. When cultured in a 5 L fermentor, erythromycin A of WBΔ3446 and WBΔ3446Δ3986 successively reached 4095 mg/L and 4670 mg/L with 23% and 41% production improvement relative to WB. The strategy reported here will be useful to improve antibiotics production in other industrial actinomycete.
    • Insights into the complex biosynthesis of the leupyrrins in Sorangium cellulosum So ce690.

      Kopp, Maren; Irschik, Herbert; Gemperlein, Katja; Buntin, Kathrin; Meiser, Peter; Weissman, Kira J; Bode, Helge B; Müller, Rolf; Helmholtz Institute for Pharmaceutical Research, Helmholtz Center for Infection Research and Department of Pharmaceutical Biotechnology, Saarland University, PO Box 151150, 66041 Saarbrücken, Germany. (2011-05)
      The anti-fungal leupyrrins are secondary metabolites produced by several strains of the myxobacterium Sorangium cellulosum. These intriguing compounds incorporate an atypically substituted γ-butyrolactone ring, as well as pyrrole and oxazolinone functionalities, which are located within an unusual asymmetrical macrodiolide. Previous feeding studies revealed that this novel structure arises from the homologation of four distinct structural units, nonribosomally-derived peptide, polyketide, isoprenoid and a dicarboxylic acid, coupled with modification of the various building blocks. Here we have attempted to reconcile the biosynthetic pathway proposed on the basis of the feeding studies with the underlying enzymatic machinery in the S. cellulosum strain So ce690. Gene products can be assigned to many of the suggested steps, but inspection of the gene set provokes the reconsideration of several key transformations. We support our analysis by the reconstitution in vitro of the biosynthesis of the pyrrole carboxylic starter unit along with gene inactivation. In addition, this study reveals that a significant proportion of the genes for leupyrrin biosynthesis are located outside the core cluster, a 'split' organization which is increasingly characteristic of the myxobacteria. Finally, we report the generation of four novel deshydroxy leupyrrin analogues by genetic engineering of the pathway.
    • Integrating Culture-based Antibiotic Resistance Profiles with Whole-genome Sequencing Data for 11,087 Clinical Isolates.

      Galata, Valentina; Laczny, Cédric C; Backes, Christina; Hemmrich-Stanisak, Georg; Schmolke, Susanne; Franke, Andre; Meese, Eckart; Herrmann, Mathias; von Müller, Lutz; Plum, Achim; et al. (Elsevier, 2019-05-14)
      Emergingantibiotic resistanceis a major global health threat. The analysis of nucleic acidsequences linked to susceptibility phenotypes facilitates the study of genetic antibiotic resistancedeterminants to inform molecular diagnostics and drug development. We collected genetic data(11,087 newly-sequenced whole genomes) and culture-based resistance profiles (10,991 out of the11,087 isolates comprehensively tested against 22 antibiotics in total) of clinical isolates including18 main species spanning a time period of 30 years. Species and drug specific resistance patternswere observed including increased resistance rates forAcinetobacter baumanniito carbapenemsand forEscherichia colito fluoroquinolones. Species-levelpan-genomeswere constructed to reflectthe genetic repertoire of the respective species, including conserved essential genes and known resis-tance factors. Integrating phenotypes and genotypes through species-level pan-genomes allowed toinfer gene–drug resistance associations using statistical testing. The isolate collection and the analysis results have been integrated into GEAR-base, a resource available for academic research use free of charge athttps://gear-base.com
    • Interrogation of Streptomyces avermitilis for efficient production of avermectins

      Chen, Jinsong; Liu, Mei; Liu, Xueting; Miao, Jin; Fu, Chengzhang; Gao, Heyong; Müller, Rolf; Zhang, Qing; Zhang, Lixin; Helmholtz-Institute for Pharmaceutical Research Saarland (HIPS),Saarland Universitätscampus E8.1, 66123 Saarbrücken, Germany. (2016-03)
    • Investigation of cytochromes P450 in myxobacteria: excavation of cytochromes P450 from the genome of Sorangium cellulosum So ce56.

      Khatri, Yogan; Hannemann, Frank; Perlova, Olena; Müller, Rolf; Bernhardt, Rita (2011-06-06)
      The exploitation of cytochromes P450 for novel biotechnological application and for the investigation of their physiological function is of great scientific interest in this post genomic era, where an extraordinary biodiversity of P450 genes has been derived from all forms of life. The study of P450s in the myxobacterium Sorangium cellulosum strain So ce56, the producer of novel secondary metabolites of pharmaceutical interest is the research topic, in which we were engaged since the beginning of its genome sequencing project. We herein disclosed the cytochrome P450 complements (CYPomes) of spore-forming myxobacterial species, Stigmatella aurantiaca DW4/3-1, Haliangium ochraceum DSM 14365 and Myxococcus xanthus DK1622, and their potential pharmaceutical significance has been discussed.