Publications by authors named "Karin Kleigrewe"

26 Publications

  • Page 1 of 1

Croconaine-based nanoparticles enable efficient optoacoustic imaging of murine brain tumors.

Photoacoustics 2021 Jun 23;22:100263. Epub 2021 Mar 23.

Chair of Biological Imaging, Center for Translational Cancer Research (TranslaTUM), School of Medicine, Technical University of Munich, Munich 81675, Germany.

Contrast enhancement in optoacoustic (photoacoustic) imaging can be achieved with agents that exhibit high absorption cross-sections, high photostability, low quantum yield, low toxicity, and preferential bio-distribution and clearance profiles. Based on advantageous photophysical properties of croconaine dyes, we explored croconaine-based nanoparticles (CR780RGD-NPs) as highly efficient contrast agents for targeted optoacoustic imaging of challenging preclinical tumor targets. Initial characterization of the CR780 dye was followed by modifications using polyethylene glycol and the cancer-targeting c(RGDyC) peptide, resulting in self-assembled ultrasmall particles with long circulation time and active tumor targeting. Preferential bio-distribution was demonstrated in orthotopic mouse brain tumor models by multispectral optoacoustic tomography (MSOT) imaging and histological analysis. Our findings showcase particle accumulation in brain tumors with sustainable strong optoacoustic signals and minimal toxic side effects. This work points to CR780RGD-NPs as a promising optoacoustic contrast agent for potential use in the diagnosis and image-guided resection of brain tumors.
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http://dx.doi.org/10.1016/j.pacs.2021.100263DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8080078PMC
June 2021

Anti-inflammatory chemoprevention attenuates the phenotype in a mouse model of esophageal adenocarcinoma.

Carcinogenesis 2021 Apr 20. Epub 2021 Apr 20.

Department of Medicine II, Klinikum rechts der Isar, Technical University Munich (TUM), Ismaninger Str, München, Germany.

Barrett´s Esophagus (BE) is the main known precursor condition of Esophageal Adenocarcinoma (EAC). BE is defined by the presence of metaplasia above the normal squamous columnar junction and has mainly been attributed to gastroesophageal reflux disease (GERD) and chronic reflux esophagitis. Thus, the rising incidence of EAC in the Western world is likely mediated by chronic esophageal inflammation, secondary to GERD in combination with environmental risk factors such as a Western diet and obesity. However, (at present) risk prediction tools and endoscopic surveillance have shown limited effectiveness. Chemoprevention as an adjunctive approach remains an attractive option to reduce the incidence of neoplastic disease. Here, we investigate the feasibility of chemopreventive approaches in BE and EAC via inhibition of inflammatory signaling in a transgenic mouse model of BE and EAC (L2-IL1B mice), with accelerated tumor formation on a high fat diet (HFD). L2-IL1B mice were treated with the IL-1 receptor antagonist Anakinra and the nonsteroidal anti-inflammatory drugs (NSAIDs) aspirin or Sulindac. Interleukin-1b antagonism reduced tumor progression in L2-IL1B mice with or without a HFD, while both NSAIDs were effective chemoprevention agents in the accelerated HFD fed L2-IL1B mouse model. Sulindac treatment also resulted in a marked change in the immune profile of L2-IL-1B mice. In summary, anti-inflammatory treatment of HFD-treated L2-IL1B mice acted protectively on disease progression. These results from a mouse model of BE support results from clinical trials that suggest that anti-inflammatory medication may be effective in the chemoprevention of EAC.
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http://dx.doi.org/10.1093/carcin/bgab032DOI Listing
April 2021

Facile Synthesis of a Croconaine-Based Nanoformulation for Optoacoustic Imaging and Photothermal Therapy.

Adv Healthc Mater 2021 05 18;10(9):e2002115. Epub 2021 Mar 18.

Chair of Biological Imaging, Center for Translational Cancer Research (TranslaTUM), Technical University of Munich, Munich, 81675, Germany.

Near-infrared (NIR) light absorbing theranostic agents can integrate optoacoustic imaging and photothermal therapy for effective personalized precision medicine. However, most of these agents face the challenges of unstable optical properties, material-associated toxicity, and nonbiodegradability, all of which limit their biomedical application. Several croconaine-based organic agents able to overcome some of these limitations have been recently reported, but these suffer from complicated multistep synthesis protocols. Herein, the use of CR760, a croconaine dye with excellent optical properties, is reported for nanoparticle formulation and subsequent optoacoustic imaging and photothermal therapy. Importantly, CR760 can be conveniently prepared in a single step from commercially available materials. Furthermore, CR760 can be covalently attached, via a polyethylene glycol linker, to the α β integrin ligand c(RGDyC), resulting in self-assembled nanoparticles (NPs) with cancer-targeting capability. Such CR760RGD-NPs exhibit strong NIR absorption, high photostability, high optoacoustic generation efficiency, and active tumor-targeting, making them ideal candidates for optoacoustic imaging. Due to favorable electron transfer, CR760RGD-NPs display a 45.37% photothermal conversion efficiency thereby rendering them additionally useful for photothermal therapy. Targeted tumor elimination, biosafety, and biocompatibility are demonstrated in a 4T1 murine breast tumor model. This work points to the use of CR760RGD-NPs as a promising nanoagent for NIR-based cancer phototheranostics.
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http://dx.doi.org/10.1002/adhm.202002115DOI Listing
May 2021

Proteome activity landscapes of tumor cell lines determine drug responses.

Nat Commun 2020 07 20;11(1):3639. Epub 2020 Jul 20.

Chair of Proteomics and Bioanalytics, Technical University of Munich, Emil-Erlenmeyer-Forum 5, 85354, Freising, Germany.

Integrated analysis of genomes, transcriptomes, proteomes and drug responses of cancer cell lines (CCLs) is an emerging approach to uncover molecular mechanisms of drug action. We extend this paradigm to measuring proteome activity landscapes by acquiring and integrating quantitative data for 10,000 proteins and 55,000 phosphorylation sites (p-sites) from 125 CCLs. These data are used to contextualize proteins and p-sites and predict drug sensitivity. For example, we find that Progesterone Receptor (PGR) phosphorylation is associated with sensitivity to drugs modulating estrogen signaling such as Raloxifene. We also demonstrate that Adenylate kinase isoenzyme 1 (AK1) inactivates antimetabolites like Cytarabine. Consequently, high AK1 levels correlate with poor survival of Cytarabine-treated acute myeloid leukemia patients, qualifying AK1 as a patient stratification marker and possibly as a drug target. We provide an interactive web application termed ATLANTiC (http://atlantic.proteomics.wzw.tum.de), which enables the community to explore the thousands of novel functional associations generated by this work.
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http://dx.doi.org/10.1038/s41467-020-17336-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7371697PMC
July 2020

Complex Bacterial Consortia Reprogram the Colitogenic Activity of in a Gnotobiotic Mouse Model of Chronic, Immune-Mediated Colitis.

Front Immunol 2019 20;10:1420. Epub 2019 Jun 20.

Chair of Nutrition and Immunology, Technische Universität München, Freising, Germany.

Inflammatory bowel diseases (IBD) are associated with compositional and functional changes of the intestinal microbiota, but specific contributions of individual bacteria to chronic intestinal inflammation remain unclear. is a resident member of the human intestinal core microbiota that has been linked to the pathogenesis of IBD and induces chronic colitis in susceptible monoassociated IL-10-deficient (IL-10) mice. In this study, we characterized the colitogenic activity of as part of a simplified human microbial consortium based on seven enteric bacterial strains (SIHUMI). RNA sequencing analysis of isolated from monoassociated wild type and IL-10 mice identified 408 genes including 14 genes of the ethanolamine utilization () locus that were significantly up-regulated in response to inflammation. Despite considerable up-regulation of genes, deletion of ethanolamine utilization (Δ) had no impact on colitogenic activity in monoassociated IL-10 mice. However, replacement of the wild type bacteria by a Δ mutant in SIHUMI-colonized IL-10 mice resulted in exacerbated colitis, suggesting protective functions of ethanolamine utilization in complex bacterial communities. To better understand gene response in the presence of other microbes, we purified wild type cells from the colon content of SIHUMI-colonized wild type and IL-10 mice using immuno-magnetic separation and performed RNA sequencing. Transcriptional profiling revealed that the bacterial environment reprograms gene expression in response to inflammation, with the majority of differentially expressed genes not being shared between monocolonized and SIHUMI conditions. While in monoassociation a general bacterial stress response could be observed, expression of genes in SIHUMI-colonized mice was characterized by up-regulation of genes involved in growth and replication. Interestingly, in mice colonized with SIHUMI lacking enhanced inflammation was observed in comparison to SIHUMI-colonized mice, supporting the hypothesis that ethanolamine metabolism protects against colitis in complex consortia. In conclusion, this study demonstrates that complex bacterial consortia interactions reprogram the gene expression profile and colitogenic activity of the opportunistic pathogen toward a protective function.
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http://dx.doi.org/10.3389/fimmu.2019.01420DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6596359PMC
October 2020

Glycemic Variability Promotes Both Local Invasion and Metastatic Colonization by Pancreatic Ductal Adenocarcinoma.

Cell Mol Gastroenterol Hepatol 2018 26;6(4):429-449. Epub 2018 Jul 26.

Department of Surgery, Klinikum rechts der Isar, School of Medicine, Technical University of Munich (TUM), Munich, Germany.

Background & Aims: Although nearly half of pancreatic ductal adenocarcinoma (PDAC) patients have diabetes mellitus with episodes of hyperglycemia, its tumor microenvironment is hypoglycemic. Thus, it is crucial for PDAC cells to develop adaptive mechanisms dealing with oscillating glucose levels. So far, the biological impact of such glycemic variability on PDAC biology remains unknown.

Methods: Murine PDAC cells were cultured in low- and high-glucose medium to investigate the molecular, biochemical, and metabolic influence of glycemic variability on tumor behavior. A set of in vivo functional assays including orthotopic implantation and portal and tail vein injection were used. Results were further confirmed on tissues from PDAC patients.

Results: Glycemic variability has no significant effect on PDAC cell proliferation. Hypoglycemia is associated with local invasion and angiogenesis, whereas hyperglycemia promotes metastatic colonization. Increased metastatic colonization under hyperglycemia is due to increased expression of runt related transcription factor 3 (Runx3), which further activates expression of collagen, type VI, alpha 1 (Col6a1), forming a glycemic pro-metastatic pathway. Through epigenetic machinery, retinoic acid receptor beta (Rarb) expression fluctuates according to glycemic variability, acting as a critical sensor relaying the glycemic signal to Runx3/Col6a1. Moreover, the signal axis of Rarb/Runx3/Col6a1 is pharmaceutically accessible to a widely used antidiabetic substance, metformin, and Rar modulator. Finally, PDAC tissues from patients with diabetes show an increased expression of COL6A1.

Conclusions: Glycemic variability promotes both local invasion and metastatic colonization of PDAC. A pro-metastatic signal axis Rarb/Runx3/Col6a1 whose activity is controlled by glycemic variability is identified. The therapeutic relevance of this pathway needs to be explored in PDAC patients, especially in those with diabetes.
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http://dx.doi.org/10.1016/j.jcmgh.2018.07.003DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6154439PMC
April 2019

Fatty Acid Metabolites as Novel Regulators of Non-shivering Thermogenesis.

Handb Exp Pharmacol 2019 ;251:183-214

Molecular Nutritional Medicine, Else Kröner-Fresenius Center for Nutritional Medicine, Technical University of Munich, Freising, Germany.

Fatty acids are essential contributors to adipocyte-based non-shivering thermogenesis by acting as activators of uncoupling protein 1 and serving as fuel for mitochondrial heat production. Novel evidence suggests a contribution to this thermogenic mechanism by their conversion to bioactive compounds. Mammalian cells produce a plethora of oxylipins and endocannabinoids, some of which have been identified to affect the abundance or thermogenic activity of brown and brite adipocytes. These effectors are produced locally or at distant sites and signal toward thermogenic adipocytes via a direct interaction with these cells or indirectly via secondary mechanisms. These interactions are evoked by the activation of receptor-mediated pathways. The endogenous production of these compounds is prone to modulation by the dietary intake of the respective precursor fatty acids. The effect of nutritional interventions on uncoupling protein 1-derived thermogenesis may thus at least in part be conferred by the production of a supportive oxylipin and endocannabinoid profile. The manipulation of this system in future studies will help to elucidate the physiological potential of these compounds as novel, endogenous regulators of non-shivering thermogenesis.
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http://dx.doi.org/10.1007/164_2018_150DOI Listing
July 2019

Xanthohumol C, a minor bioactive hop compound: Production, purification strategies and antimicrobial test.

J Chromatogr B Analyt Technol Biomed Life Sci 2018 Sep 18;1095:39-49. Epub 2018 Jul 18.

Biothermodynamics, TUM School of Life and Food Sciences Weihenstephan, Technical University of Munich, Maximus-von-Imhof-Forum 2, 85354 Freising, Germany. Electronic address:

Hop has been attracting scientific attention due to its favorable bioactivity properties. It is thus desirable to relate these properties to the specific hop compounds and extract these compounds in highly purified form in order to enhance the effect. The aim of the present study is the isolation of a sufficient amount of the highly purified prenylated minor hop compound xanthohumol C (XNC) for characterizing its bioactivity. Two strategies for the production of XNC were evaluated. The first strategy involved a capture of natural XNC from a xanthohumol (XN)-enriched hop extract (XF) by countercurrent chromatography. In the second approach, a one-step semi-synthesis of XNC was performed starting from XN, which had previously been separated from a natural XN-enriched hop extract. Both methods delivered XNC in sufficient amount and purity (>95%, HPLC), whereas the second strategy was preferable in terms of purity (>99%, HPLC) as well as productivity and solvent consumption. The methods were validated by identifying and quantifying XNC using LC-MS, LC-MS/MS and H NMR analysis. The XNC obtained in this way was supplied to several bacterial, yeast and fungal cultures in order to evaluate its antimicrobial effects. For comparison, microorganisms were also treated with the natural XN-enriched hop extract, as well as the prenylated hop compound XN. While still reducing cell proliferation, XNC was found to be less effective than both XF and XN for all studied bacteria and yeasts. Furthermore, for Bacillus subtilis, a strongly pH-dependent minimal inhibition concentration was observed for all three bioactive compounds, lowest at a pH of 5 and highest at a pH of 7.
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http://dx.doi.org/10.1016/j.jchromb.2018.07.018DOI Listing
September 2018

Degradation of brown adipocyte purine nucleotides regulates uncoupling protein 1 activity.

Mol Metab 2018 02 26;8:77-85. Epub 2017 Dec 26.

Chair of Molecular Nutritional Medicine, TUM School of Life Sciences, Technical University of Munich, Germany; EKFZ - Else Kröner-Fresenius Center for Nutritional Medicine, Technical University of Munich, Freising, Germany; ZIEL - Institute for Food & Health, Technical University of Munich, Freising, Germany.

Objective: Non-shivering thermogenesis in mammalian brown adipose tissue depends on thermogenic uncoupling protein 1. Its activity is triggered by free fatty acids while purine nucleotides mediate inhibition. During activation, it is thought that free fatty acids overcome purine-mediated inhibition. We measured the cellular concentration and the release of purine nucleotide metabolites to uncover a possible role of purine nucleotide degradation in uncoupling protein 1 activation.

Methods: With mass spectrometry, purine nucleotide metabolites were quantified in cellular homogenates and supernatants of cultured primary brown adipocytes. We also determined oxygen consumption in response to a β-adrenergic agonist.

Results: Upon adrenergic activation, brown adipocytes decreased the intracellular concentration of inhibitory nucleotides (ATP, ADP, GTP and GDP) and released the respective degradation products. At the same time, an increase in cellular calcium occurred. None of these phenomena occurred in white adipocytes or myotubes. The brown adipocyte expression of enzymes implicated in purine metabolic remodeling is altered upon cold exposure. Pharmacological and genetic interference of purine metabolism altered uncoupling protein 1 mediated uncoupled respiration.

Conclusion: Adrenergic stimulation of brown adipocytes lowers the intracellular concentration of purine nucleotides, thereby contributing to uncoupling protein 1 activation.
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http://dx.doi.org/10.1016/j.molmet.2017.12.010DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5985227PMC
February 2018

Detection of the formyl radical by EPR spin-trapping and mass spectrometry.

Free Radic Biol Med 2018 02 4;116:129-133. Epub 2018 Jan 4.

Bavarian Center for Biomolecular Mass Spectrometry (BayBioMS), Technical University of Munich, Gregor-Mendel-Strasse 4, 85354 Freising, Germany. Electronic address:

For the first time we here present the unambiguous identification of the formyl radical (CHO) by EPR (Electron Paramagnetic Resonance) spectroscopy and mass spectrometry (MS) using DMPO (5,5-dimethyl-1-pyrroline N-oxide) as spin trap at ambient temperature without using any catalyst(s). The CHO was continuously generated by UV photolysis in closed anoxic environment from pure formaldehyde (HCHO) in aqueous solution. The isotropic hyperfine structure constants of CHO were determined as a = 15.72G and a = 21.27G. The signals were deconvoluted and split by simulation in their single adduct components: DMPO-CHO, DMPO-H and DMPO-OH. We verified our results at first using MNP (2-methyl-2-nitroso-propane) as spin trap with known literature data and then mass spectrometry. Similarly the MNP adduct components MNP-CHO, MNP-H as well as its own adduct, the MNP-2-methyl-2-propyl (MNP-MP) were deconvoluted. Due to the low signal intensities, we had to accumulate single measurements for both spin traps. Using MS we got the exact mass of the reduced CHO adduct independently confirming the result of EPR detection of formyl radical.
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http://dx.doi.org/10.1016/j.freeradbiomed.2018.01.002DOI Listing
February 2018

Combinatorial interaction network of abscisic acid receptors and coreceptors from .

Proc Natl Acad Sci U S A 2017 09 5;114(38):10280-10285. Epub 2017 Sep 5.

Chair of Botany, TUM School of Life Sciences Weihenstephan, Technical University of Munich, D-85354 Freising, Germany;

The phytohormone abscisic acid (ABA) is induced in response to abiotic stress to mediate plant acclimation to environmental challenge. Key players of the ABA-signaling pathway are the ABA-binding receptors (RCAR/PYR1/PYL), which, together with a plant-specific subclade of protein phosphatase 2C (PP2C), form functional holoreceptors. The genome encodes nine PP2C coreceptors and 14 different RCARs, which can be divided into three subfamilies. The presence of these gene families in higher plants points to the existence of an intriguing regulatory network and poses questions as to the functional compatibility and specificity of receptor-coreceptor interactions. Here, we analyzed all RCAR-PP2C combinations for their capacity to regulate ABA signaling by transient expression in protoplasts. Of 126 possible RCAR-PP2C pairings, 113 were found to be functional. The three subfamilies within the RCAR family showed different sensitivities to regulating the ABA response at basal ABA levels when efficiently expressed. At exogenous high ABA levels, the RCARs regulated most PP2Cs and activated the ABA response to a similar extent. The PP2C AHG1 was regulated only by RCAR1/PYL9, RCAR2/PYL7, and RCAR3/PYL8, which are characterized by a unique tyrosine residue. Site-directed mutagenesis of RCAR1 showed that its tyrosine residue is critical for AHG1 interaction and regulation. Furthermore, the PP2Cs HAI1 to HAI3 were regulated by all RCARs, and the ABA receptor RCAR4/PYL10 showed ABA-dependent PP2C regulation. The findings unravel the interaction network of possible RCAR-PP2C pairings and their different potentials to serve a rheostat function for integrating fluctuating hormone levels into the ABA-response pathway.
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http://dx.doi.org/10.1073/pnas.1706593114DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5617281PMC
September 2017

Sharing and community curation of mass spectrometry data with Global Natural Products Social Molecular Networking.

Nat Biotechnol 2016 08;34(8):828-837

Produits naturels - Synthèses - Chimie Médicinale, University of Rennes 1, Rennes Cedex, France.

The potential of the diverse chemistries present in natural products (NP) for biotechnology and medicine remains untapped because NP databases are not searchable with raw data and the NP community has no way to share data other than in published papers. Although mass spectrometry (MS) techniques are well-suited to high-throughput characterization of NP, there is a pressing need for an infrastructure to enable sharing and curation of data. We present Global Natural Products Social Molecular Networking (GNPS; http://gnps.ucsd.edu), an open-access knowledge base for community-wide organization and sharing of raw, processed or identified tandem mass (MS/MS) spectrometry data. In GNPS, crowdsourced curation of freely available community-wide reference MS libraries will underpin improved annotations. Data-driven social-networking should facilitate identification of spectra and foster collaborations. We also introduce the concept of 'living data' through continuous reanalysis of deposited data.
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http://dx.doi.org/10.1038/nbt.3597DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5321674PMC
August 2016

Unique marine derived cyanobacterial biosynthetic genes for chemical diversity.

Nat Prod Rep 2016 Feb;33(2):348-64

Center for Marine Biotechnology and Biomedicine, Scripps Institution of Oceanography, University of California San Diego, USA. and Skaggs School of Pharmacy and Pharmaceutical Sciences, University of California San Diego, USA.

Cyanobacteria are a prolific source of structurally unique and biologically active natural products that derive from intriguing biochemical pathways. Advancements in genome sequencing have accelerated the identification of unique modular biosynthetic gene clusters in cyanobacteria and reveal a wealth of unusual enzymatic reactions involved in their construction. This article examines several interesting mechanistic transformations involved in cyanobacterial secondary metabolite biosynthesis with a particular focus on marine derived modular polyketide synthases (PKS), nonribosomal peptide synthetases (NRPS) and combinations thereof to form hybrid natural products. Further, we focus on the cyanobacterial genus Moorea and the co-evolution of its enzyme cassettes that create metabolic diversity. Progress in the development of heterologous expression systems for cyanobacterial gene clusters along with chemoenzymatic synthesis makes it possible to create new analogs. Additionally, phylum-wide genome sequencing projects have enhanced the discovery rate of new natural products and their distinctive enzymatic reactions. Summarizing, cyanobacterial biosynthetic gene clusters encode for a large toolbox of novel enzymes that catalyze unique chemical reactions, some of which may be useful in synthetic biology.
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http://dx.doi.org/10.1039/c5np00097aDOI Listing
February 2016

Integrating mass spectrometry and genomics for cyanobacterial metabolite discovery.

J Ind Microbiol Biotechnol 2016 Mar 17;43(2-3):313-24. Epub 2015 Nov 17.

Center for Marine Biotechnology and Biomedicine, Scripps Institution of Oceanography, University of California, San Diego, 9500 Gilman Drive MC0212, La Jolla, CA, 92093, USA.

Filamentous marine cyanobacteria produce bioactive natural products with both potential therapeutic value and capacity to be harmful to human health. Genome sequencing has revealed that cyanobacteria have the capacity to produce many more secondary metabolites than have been characterized. The biosynthetic pathways that encode cyanobacterial natural products are mostly uncharacterized, and lack of cyanobacterial genetic tools has largely prevented their heterologous expression. Hence, a combination of cutting edge and traditional techniques has been required to elucidate their secondary metabolite biosynthetic pathways. Here, we review the discovery and refined biochemical understanding of the olefin synthase and fatty acid ACP reductase/aldehyde deformylating oxygenase pathways to hydrocarbons, and the curacin A, jamaicamide A, lyngbyabellin, columbamide, and a trans-acyltransferase macrolactone pathway encoding phormidolide. We integrate into this discussion the use of genomics, mass spectrometric networking, biochemical characterization, and isolation and structure elucidation techniques.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5065021PMC
http://dx.doi.org/10.1007/s10295-015-1705-7DOI Listing
March 2016

Genetic engineering, high resolution mass spectrometry and nuclear magnetic resonance spectroscopy elucidate the bikaverin biosynthetic pathway in Fusarium fujikuroi.

Fungal Genet Biol 2015 Nov 14;84:26-36. Epub 2015 Sep 14.

Institute of Food Chemistry, Westfälische Wilhelms-Universität Münster, Corrensstraße 45, 48149 Münster, Germany; NRW Graduate School of Chemistry, Westfälische Wilhelms-Universität Münster, Wilhelm-Klemm-Str. 10, 48149 Münster, Germany. Electronic address:

Secondary metabolites of filamentous fungi can be highly bioactive, ranging from antibiotic to cancerogenic properties. In this study we were able to identify a new, yet unknown metabolite produced by Fusarium fujikuroi, an ascomycetous rice pathogen. With the help of genomic engineering and high-performance liquid chromatography (HPLC) coupled to high resolution mass spectrometry (HRMS) followed by isolation and detailed structure elucidation, the new substance could be designated as an unknown bikaverin precursor, missing two methyl- and one hydroxy group, hence named oxo-pre-bikaverin. Though the bikaverin gene cluster has been extensively studied in the past, elucidation of the biosynthetic pathway remained elusive due to a negative feedback loop that regulates the genes within the cluster. To decipher the bikaverin biosynthetic pathway and to overcome these negative regulation circuits, the structural cluster genes BIK2 and BIK3 were overexpressed independently in the ΔΔBIK2/BIK3+OE::BIK1 mutant background by using strong constitutive promoters. Using the software tool MZmine 2, the metabolite profile of the generated mutants obtained by HPLC-HRMS was compared, revealing further intermediates.
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http://dx.doi.org/10.1016/j.fgb.2015.09.006DOI Listing
November 2015

Combining Mass Spectrometric Metabolic Profiling with Genomic Analysis: A Powerful Approach for Discovering Natural Products from Cyanobacteria.

J Nat Prod 2015 Jul 7;78(7):1671-82. Epub 2015 Jul 7.

◊Life Sciences Institute, University of Michigan, Ann Arbor, Michigan, United States.

An innovative approach was developed for the discovery of new natural products by combining mass spectrometric metabolic profiling with genomic analysis and resulted in the discovery of the columbamides, a new class of di- and trichlorinated acyl amides with cannabinomimetic activity. Three species of cultured marine cyanobacteria, Moorea producens 3L, Moorea producens JHB, and Moorea bouillonii PNG, were subjected to genome sequencing and analysis for their recognizable biosynthetic pathways, and this information was then compared with their respective metabolomes as detected by MS profiling. By genome analysis, a presumed regulatory domain was identified upstream of several previously described biosynthetic gene clusters in two of these cyanobacteria, M. producens 3L and M. producens JHB. A similar regulatory domain was identified in the M. bouillonii PNG genome, and a corresponding downstream biosynthetic gene cluster was located and carefully analyzed. Subsequently, MS-based molecular networking identified a series of candidate products, and these were isolated and their structures rigorously established. On the basis of their distinctive acyl amide structure, the most prevalent metabolite was evaluated for cannabinomimetic properties and found to be moderate affinity ligands for CB1.
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http://dx.doi.org/10.1021/acs.jnatprod.5b00301DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4681511PMC
July 2015

Genetic manipulation of the Fusarium fujikuroi fusarin gene cluster yields insight into the complex regulation and fusarin biosynthetic pathway.

Chem Biol 2013 Aug 8;20(8):1055-66. Epub 2013 Aug 8.

Institute for Biology and Biotechnology of Plants, Westfälische Wilhelms-Universität Münster, Schlossplatz 8, Münster 48143, Germany.

In this work, the biosynthesis and regulation of the polyketide synthase/nonribosomal peptide synthetase (PKS/NRPS)-derived mutagenic mycotoxin fusarin C was studied in the fungus Fusarium fujikuroi. The fusarin gene cluster consists of nine genes (fus1-fus9) that are coexpressed under high-nitrogen and acidic pH conditions. Chromatin immunoprecipitation revealed a correlation between high expression and enrichment of activating H3K9-acetylation marks under inducing conditions. We provide evidence that only four genes are sufficient for the biosynthesis. The combination of genetic engineering with nuclear magnetic resonance and mass-spectrometry-based structure elucidation allowed the discovery of the putative fusarin biosynthetic pathway. Surprisingly, we indicate that PKS/NRPS releases its product with an open ring structure, probably as an alcohol. Our data indicate that 2-pyrrolidone ring closure, oxidation at C-20, and, finally, methylation at C-20 are catalyzed by Fus2, Fus8, and Fus9, respectively.
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http://dx.doi.org/10.1016/j.chembiol.2013.07.004DOI Listing
August 2013

Deciphering the cryptic genome: genome-wide analyses of the rice pathogen Fusarium fujikuroi reveal complex regulation of secondary metabolism and novel metabolites.

PLoS Pathog 2013 27;9(6):e1003475. Epub 2013 Jun 27.

Institut für Biologie und Biotechnologie der Pflanzen, Molecular Biology and Biotechnology of Fungi, Westfälische Wilhelms-Universität Münster, Münster, Germany.

The fungus Fusarium fujikuroi causes "bakanae" disease of rice due to its ability to produce gibberellins (GAs), but it is also known for producing harmful mycotoxins. However, the genetic capacity for the whole arsenal of natural compounds and their role in the fungus' interaction with rice remained unknown. Here, we present a high-quality genome sequence of F. fujikuroi that was assembled into 12 scaffolds corresponding to the 12 chromosomes described for the fungus. We used the genome sequence along with ChIP-seq, transcriptome, proteome, and HPLC-FTMS-based metabolome analyses to identify the potential secondary metabolite biosynthetic gene clusters and to examine their regulation in response to nitrogen availability and plant signals. The results indicate that expression of most but not all gene clusters correlate with proteome and ChIP-seq data. Comparison of the F. fujikuroi genome to those of six other fusaria revealed that only a small number of gene clusters are conserved among these species, thus providing new insights into the divergence of secondary metabolism in the genus Fusarium. Noteworthy, GA biosynthetic genes are present in some related species, but GA biosynthesis is limited to F. fujikuroi, suggesting that this provides a selective advantage during infection of the preferred host plant rice. Among the genome sequences analyzed, one cluster that includes a polyketide synthase gene (PKS19) and another that includes a non-ribosomal peptide synthetase gene (NRPS31) are unique to F. fujikuroi. The metabolites derived from these clusters were identified by HPLC-FTMS-based analyses of engineered F. fujikuroi strains overexpressing cluster genes. In planta expression studies suggest a specific role for the PKS19-derived product during rice infection. Thus, our results indicate that combined comparative genomics and genome-wide experimental analyses identified novel genes and secondary metabolites that contribute to the evolutionary success of F. fujikuroi as a rice pathogen.
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http://dx.doi.org/10.1371/journal.ppat.1003475DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3694855PMC
January 2014

New approach via gene knockout and single-step chemical reaction for the synthesis of isotopically labeled fusarin c as an internal standard for the analysis of this fusarium mycotoxin in food and feed samples.

J Agric Food Chem 2012 Aug 16;60(34):8350-5. Epub 2012 Aug 16.

Institute of Food Chemistry, and ‡Molecular Biology and Biotechnology of Fungi, Westfälische Wilhelms-Universität Münster , 48149 Münster, Germany.

The gold standard for quantitation of contaminants with high-performance liquid chromatography tandem mass spectrometry (HPLC-MS/MS) is the use of isotopically labeled standards. Herein, we report a new strategy for the synthesis of isotopically labeled 21-d3-fusarin C via a genetically modified Fusarium strain, followed by a one-step derivatization reaction. Fusarin C is a Fusarium mycotoxin, which is mutagenic after metabolic activation. Its occurrence has been demonstrated recently in corn-based samples, but up to now, little is known about the contamination of other grain samples. To collect further data, the quantitation method was enhanced by application of the 21-d3-fusarin C and the use of a QTRAP 5500 mass spectrometer. This new method has a limit of detection (LOD) of 1 μg/kg, a limit of quantitation (LOQ) of 4 μg/kg, and a recovery rate of 99%. A total of 21 corn samples and 13 grain samples were analyzed, with resulting fusarin C levels varying from not detectable to 24.7 μg/kg.
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http://dx.doi.org/10.1021/jf302534xDOI Listing
August 2012

Structure elucidation of new fusarins revealing insights in the rearrangement mechanisms of the Fusarium mycotoxin fusarin C.

J Agric Food Chem 2012 May 21;60(21):5497-505. Epub 2012 May 21.

Institute of Food Chemistry, Westfälische Wilhelms-Universität Münster, Münster, Germany.

Fusarin C is a Fusarium mycotoxin that rearranges under reversed phase chromatographic conditions. In this study, the rearrangement of fusarin C was examined in detail, and the formation of fusarins under different conditions was optimized. All relevant fusarins including (10Z)-, (8Z)-, and (6Z)-fusarin C were isolated and identified by NMR. To confirm the involvement of the 2-pyrrolidone ring in the rearrangement of fusarin C, 15-methoxy-fusarin C was synthesized. For the first time, the structure of open-chain fusarin C was elucidated, and on the basis of these data, the rearrangement product of fusarin C was identified as epi-fusarin C. The results were confirmed by detailed NMR measurements and density functional theory calculations. Furthermore, a new fusarin C like metabolite, which was named dihydrofusarin C, was detected by analysis of the crude extract of fusarin C with high-performance liquid chromatography coupled to UV and Fourier transform mass spectrometry.
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http://dx.doi.org/10.1021/jf3009469DOI Listing
May 2012

Biosynthesis of fusarubins accounts for pigmentation of Fusarium fujikuroi perithecia.

Appl Environ Microbiol 2012 Jun 6;78(12):4468-80. Epub 2012 Apr 6.

Institute for Biology and Biotechnology of Plants, Westfälische Wilhelms-University, Münster, Germany.

Fusarium fujikuroi produces a variety of secondary metabolites, of which polyketides form the most diverse group. Among these are the highly pigmented naphthoquinones, which have been shown to possess different functional properties for the fungus. A group of naphthoquinones, polyketides related to fusarubin, were identified in Fusarium spp. more than 60 years ago, but neither the genes responsible for their formation nor their biological function has been discovered to date. In addition, although it is known that the sexual fruiting bodies in which the progeny of the fungus develops are darkly colored by a polyketide synthase (PKS)-derived pigment, the structure of this pigment has never been elucidated. Here we present data that link the fusarubin-type polyketides to a defined gene cluster, which we designate fsr, and demonstrate that the fusarubins are the pigments responsible for the coloration of the perithecia. We studied their regulation and the function of the single genes within the cluster by a combination of gene replacements and overexpression of the PKS-encoding gene, and we present a model for the biosynthetic pathway of the fusarubins based on these data.
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http://dx.doi.org/10.1128/AEM.00823-12DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3370568PMC
June 2012

Isolation and structure elucidation of two new cytotoxic metabolites from red yeast rice.

Nat Prod Res 2012 2;26(20):1914-21. Epub 2011 Dec 2.

Institute of Food Chemistry, Westfälische Wilhelms-Universität, Corrensstrasse 45, D-48149 Münster, Germany.

In this study, 10 already described secondary metabolites and 2 unknown metabolites were identified in an extract of Monascus purpureus by high-performance liquid chromatography-diode array detection. The unknown metabolites were isolated and their chemical structures were elucidated. The new metabolites possess the molecular formulas C(21)H(27)NO(4) and C(23)H(31)NO(4). They were named monascopyridines E and F due to their pyridine backbone. The cytotoxicity of the new compounds was studied using immortalised human kidney epithelial cells displaying IC(50) values in the micromolar range.
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http://dx.doi.org/10.1080/14786419.2011.639074DOI Listing
February 2013

A new high-performance liquid chromatography-tandem mass spectrometry method based on dispersive solid phase extraction for the determination of the mycotoxin fusarin C in corn ears and processed corn samples.

J Agric Food Chem 2011 Oct 20;59(19):10470-6. Epub 2011 Sep 20.

Institute of Food Chemistry, University of Münster, Münster, Germany.

Fusarin C is a mycotoxin that is produced by a variety of Fusarium species and is therefore a possible contaminant in food and feed. For this reason, a reliable high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) method for the determination of fusarin C in food and feed samples was developed based on dispersive solid phase extraction (DSPE). This method has a limit of detection (LOD) of 2 μg/kg, a limit of quantitation (LOQ) of 7 μg/kg, and a recovery rate of 80%. Fifty different corn samples were analyzed, and fusarin C was detected in 40 of them. The fusarin C level varied in kernels of corn ears from not detectable up to 83 mg/kg and in food samples from not detectable up to 28 μg/kg. The co-occurrence of further structural analogues of fusarin C was confirmed by high-performance liquid chromatography Fourier transformation mass spectrometry (HPLC-FTMS). In addition, the stability of fusarin C under storage conditions was evaluated.
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http://dx.doi.org/10.1021/jf2026814DOI Listing
October 2011

Investigation of the metabolism of ergot alkaloids in cell culture by fourier transformation mass spectrometry.

J Agric Food Chem 2011 Jul 23;59(14):7798-807. Epub 2011 Jun 23.

University of Münster, Wilhelm-Klemm-Strasse 10, D-48149 Münster, Germany.

Ergot alkaloids are known toxic secondary metabolites of the fungus Claviceps purpurea occurring in various grains, especially rye products. The liver is responsible for converting the ergot alkaloids into metabolites; however, the toxic impact of these end products of metabolism is still unknown. The aim of this study was to analyze the metabolism of ergot alkaloids in colon and liver cell lines (HT-29, HepG2), as well as in human primary renal cells (RPTEC). It was shown that cells in vitro are able to metabolize ergot alkaloids, forming a variety of metabolic compounds. Significant differences between the used cell types could be identified, and a suitable model system was established using HT-29 cells, performing an intensive metabolism to hydroxylated metabolites. The formed substances were analyzed by coupling of high-performance liquid chromatography with fluorescence detection and Fourier transformation mass spectrometry (HPLC-FLD-FTMS) as a powerful tool to identify known and unknown metabolites.
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http://dx.doi.org/10.1021/jf2012024DOI Listing
July 2011

FfVel1 and FfLae1, components of a velvet-like complex in Fusarium fujikuroi, affect differentiation, secondary metabolism and virulence.

Mol Microbiol 2010 Aug 21;77(4):972-94. Epub 2010 Jun 21.

Institut für Botanik, Westfälische Wilhelms-Universität Münster, Schlossgarten 3, D-48149 Münster, GermanyInstitut für Lebensmittelchemie, Westfälische Wilhelms-Universität Münster, Corrensstraße 45, D-48149 Münster, GermanyBacterial Foodborne Pathogens and Mycology Research, USDA/ARS, 1815 N University St, Peoria, IL 61604, USADepartment of Medical Microbiology and ImmunologyDepartment of Bacteriology, University of Wisconsin, 1550 Linden Dr, Madison, WI 53706-1521, USA.

Besides industrially produced gibberellins (GAs), Fusarium fujikuroi is able to produce additional secondary metabolites such as the pigments bikaverin and neurosporaxanthin and the mycotoxins fumonisins and fusarin C. The global regulation of these biosynthetic pathways is only poorly understood. Recently, the velvet complex containing VeA and several other regulatory proteins was shown to be involved in global regulation of secondary metabolism and differentiation in Aspergillus nidulans. Here, we report on the characterization of two components of the F. fujikuroi velvet-like complex, FfVel1 and FfLae1. The gene encoding this first reported LaeA orthologue outside the class of Eurotiomycetidae is upregulated in ΔFfvel1 microarray-studies and FfLae1 interacts with FfVel1 in the nucleus. Deletion of Ffvel1 and Fflae1 revealed for the first time that velvet can simultaneously act as positive (GAs, fumonisins and fusarin C) and negative (bikaverin) regulator of secondary metabolism, and that both components affect conidiation and virulence of F. fujikuroi. Furthermore, the velvet-like protein FfVel2 revealed similar functions regarding conidiation, secondary metabolism and virulence as FfVel1. Cross-genus complementation studies of velvet complex component mutants between Fusarium, Aspergillus and Penicillium support an ancient origin for this complex, which has undergone a divergence in specific functions mediating development and secondary metabolism.
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http://dx.doi.org/10.1111/j.1365-2958.2010.07263.xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2989987PMC
August 2010

Biosynthesis of the red pigment bikaverin in Fusarium fujikuroi: genes, their function and regulation.

Mol Microbiol 2009 May 14;72(4):931-46. Epub 2009 Apr 14.

Institut für Botanik, Schlossgarten 3, Westfälische Wilhelms-Universität Münster, D-48149 Münster, Germany.

Fusarium secondary metabolites are structurally diverse, have a variety of activities and are generally poorly understood biosynthetically. The F. fujikuroi polyketide synthase gene bik1 was previously shown to be responsible for formation of the mycelial pigment bikaverin. Here we present the characterization of five genes adjacent to bik1 as encoding a putative FAD-dependent monooxygenase (bik2), an O-methyltransferase (bik3), an NmrA-like protein (bik4), a Zn(II)2Cys6 transcription factor (bik5) and an MFS transporter (bik6). Deletion of each gene resulted in total loss or significant reduction of bikaverin synthesis. Expression studies revealed that all bik genes are repressed by high amounts of nitrogen in an AreA-independent manner and are subject to a time- and pH-dependent regulation. Deletion of the pH regulatory gene pacC resulted in partial derepression while complementation with a dominant active allele resulted in repression of bik genes at acidic ambient pH. Transcription of all bik genes in strains lacking bik1, bik2 or bik3 was essentially eliminated, while transcription of some bik genes was detected in strains lacking bik4, bik5 or bik6. Thus, bikaverin synthesis is regulated by a complex regulatory network. Understanding how different factors influence the synthesis of this model secondary metabolite will aid understanding secondary metabolism in general.
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http://dx.doi.org/10.1111/j.1365-2958.2009.06695.xDOI Listing
May 2009