Publications by authors named "Immo Burkhardt"

11 Publications

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The Termite Fungal Cultivar Combines Diverse Enzymes and Oxidative Reactions for Plant Biomass Conversion.

mBio 2021 06 15;12(3):e0355120. Epub 2021 Jun 15.

Group of Chemical Biology of Microbe-Host Interactions, Leibniz Institute for Natural Product Research and Infection Biology, Hans Knöll Institute (HKI), Jena, Germany.

Macrotermitine termites have domesticated fungi in the genus as their primary food source using predigested plant biomass. To access the full nutritional value of lignin-enriched plant biomass, the termite-fungus symbiosis requires the depolymerization of this complex phenolic polymer. While most previous work suggests that lignocellulose degradation is accomplished predominantly by the fungal cultivar, our current understanding of the underlying biomolecular mechanisms remains rudimentary. Here, we provide conclusive omics and activity-based evidence that employs not only a broad array of carbohydrate-active enzymes (CAZymes) but also a restricted set of oxidizing enzymes (manganese peroxidase, dye decolorization peroxidase, an unspecific peroxygenase, laccases, and aryl-alcohol oxidases) and Fenton chemistry for biomass degradation. We propose for the first time that induces hydroquinone-mediated Fenton chemistry (Fe + HO + H → Fe + OH + HO) using a herein newly described 2-methoxy-1,4-dihydroxybenzene (2-MHQ, compound 19)-based electron shuttle system to complement the enzymatic degradation pathways. This study provides a comprehensive depiction of how efficient biomass degradation by means of this ancient insect's agricultural symbiosis is accomplished. Fungus-growing termites have optimized the decomposition of recalcitrant plant biomass to access valuable nutrients by engaging in a tripartite symbiosis with complementary contributions from a fungal mutualist and a codiversified gut microbiome. This complex symbiotic interplay makes them one of the most successful and important decomposers for carbon cycling in Old World ecosystems. To date, most research has focused on the enzymatic contributions of microbial partners to carbohydrate decomposition. Here, we provide genomic, transcriptomic, and enzymatic evidence that also employs redox mechanisms, including diverse ligninolytic enzymes and a Fenton chemistry-based hydroquinone-catalyzed lignin degradation mechanism, to break down lignin-rich plant material. Insights into these efficient decomposition mechanisms reveal new sources of efficient ligninolytic agents applicable for energy generation from renewable sources.
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http://dx.doi.org/10.1128/mBio.03551-20DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8262964PMC
June 2021

Biochemical and Mechanistic Characterization of the Fungal Reverse 1-Dimethylallyltryptophan Synthase DMATS1.

ACS Chem Biol 2019 12 5;14(12):2922-2931. Epub 2019 Dec 5.

Kekulé Institut für Organische Chemie und Biochemie , Rheinische Friedrich Wilhelms-Universität Bonn , Gerhard-Domagk-Strasse 1 , 53121 Bonn , Germany.

Dimethylallyltryptophan synthases catalyze the regiospecific transfer of (oligo)prenylpyrophosphates to aromatic substrates like tryptophan derivatives. These reactions are key steps in many biosynthetic pathways of fungal and bacterial secondary metabolites. investigations on recombinant DMATS1 from identified the enzyme as the first selective reverse tryptophan--1 prenyltransferase of fungal origin. The enzyme was also able to catalyze the reverse -geranylation of tryptophan. DMATS1 was shown to be phylogenetically related to fungal tyrosine -prenyltransferases and fungal 7-DMATS. Like these enzymes, DMATS1 was able to convert tyrosine into its regularly -prenylated derivative. Investigation of the binding sites of DMATS1 by homology modeling and comparison to the crystal structure of 4-DMATS FgaPT2 showed an almost identical site for DMAPP binding but different residues for tryptophan coordination. Several putative active site residues were verified by site directed mutagenesis of DMATS1. Homology models of the phylogenetically related enzymes showed similar tryptophan binding residues, pointing to a unified substrate binding orientation of tryptophan and DMAPP, which is distinct from that in FgaPT2. Isotopic labeling experiments showed the reaction catalyzed by DMATS1 to be nonstereospecific. Based on these data, a detailed mechanism for DMATS1 catalysis is proposed.
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http://dx.doi.org/10.1021/acschembio.9b00828DOI Listing
December 2019

Mechanistic characterization of three sesquiterpene synthases from the termite-associated fungus Termitomyces.

Org Biomol Chem 2019 03;17(13):3348-3355

Kekulé-Institute of Organic Chemistry and Biochemistry, University of Bonn, Gerhard-Domagk-Straße 1, 53121 Bonn, Germany.

Three terpene synthases from the termite associated fungus Termitomyces were functionally characterized as (+)-intermedeol synthase, (-)-γ-cadinene synthase and (+)-germacrene D-4-ol synthase, with the germacrene D-4-ol synthase as the first reported enzyme that produces the (+)-enantiomer. The enzymatic mechanisms were thoroughly investigated by incubation with isotopically labeled precursors to follow the stereochemical courses of single reaction steps in catalysis. The role of putative active site residues was tested by site directed mutagenesis of a highly conserved tryptophan in all three enzymes and additional residues in (-)-γ-cadinene synthase that were identified by homology model analysis.
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http://dx.doi.org/10.1039/c8ob02744gDOI Listing
March 2019

The absolute configuration of isochamigrene: new insights into the cyclisation mechanism of trichodiene synthase.

Chem Commun (Camb) 2018 Apr;54(28):3540-3542

Kekulé-Institut für Organische Chemie und Biochemie, Rheinische Friedrich-Wilhelms-Universität Bonn, Gerhard-Domagk-Straße 1, 53121 Bonn, Germany.

Isochamigrene, a side product of the trichodiene synthase, which is a key enzyme from the biosynthesis of the trichothecene mycotoxins from Fusarium spp., was enantioselectively synthesised and compared to the natural product from Fusarium sporotrichioides. As a result, its absolute configuration was assigned to (S)-isochamigrene. Implications for the recently extensively discussed cyclisation mechanism towards trichodiene and its side products are discussed.
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http://dx.doi.org/10.1039/C8CC01744ADOI Listing
April 2018

The GATA-Type Transcription Factor Csm1 Regulates Conidiation and Secondary Metabolism in .

Front Microbiol 2017 26;8:1175. Epub 2017 Jun 26.

Institut für Biologie und Biotechnologie der Pflanzen, Westfälische Wilhelms-Universität MünsterMünster, Germany.

GATA-type transcription factors (TFs) such as the nitrogen regulators AreA and AreB, or the light-responsive TFs WC-1 and WC-2, play global roles in fungal growth and development. The conserved GATA TF NsdD is known as an activator of sexual development and key repressor of conidiation in , and as light-regulated repressor of macroconidia formation in In the present study, we functionally characterized the NsdD ortholog in , named Csm1. Deletion of this gene resulted in elevated microconidia formation in the wild-type (WT) and restoration of conidiation in the non-sporulating velvet mutant Δ demonstrating that Csm1 also plays a role as repressor of conidiation in . Furthermore, biosynthesis of the PKS-derived red pigments, bikaverin and fusarubins, is de-regulated under otherwise repressing conditions. Cross-species complementation of the Δ mutant with the ortholog led to full restoration of WT-like growth, conidiation and pigment formation. In contrast, the rescued only the defects in growth, the tolerance to HO and virulence, but did not restore the light-dependent differentiation when expressed in the Δ mutant. Microarray analysis comparing the expression profiles of the WT and the Δ mutant under different nitrogen conditions revealed a strong impact of this GATA TF on 19 of the 47 gene clusters in the genome of . One of the up-regulated silent gene clusters is the one containing the sesquiterpene cyclase-encoding key gene Heterologous expression of in enabled us to identify the product as the volatile bioactive compound (-)-germacrene D.
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http://dx.doi.org/10.3389/fmicb.2017.01175DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5483468PMC
June 2017

Chemical differentiation of three DMSP lyases from the marine Roseobacter group.

Org Biomol Chem 2017 May;15(20):4432-4439

Kekulé-Institut für Organische Chemie und Biochemie, Rheinische Friedrich-Wilhelms-Universität Bonn, Gerhard-Domagk-Straße 1, 53121 Bonn, Germany.

Dimethylsulfoniopropionate (DMSP) catabolism of marine bacteria plays an important role in marine and global ecology. The genome of Ruegeria pomeroyi DSS-3, a model organism from the Roseobacter group, harbours no less than three genes for different DMSP lyases (DddW, DddP and DddQ) that catalyse the degradation of DMSP to dimethyl sulfide (DMS) and acrylate. Despite their apparent similar function these enzymes show no significant overall sequence identity. In this work DddQ and DddW from R. pomeroyi and the DddP homolog from Phaeobacter inhibens DSM 17395 were functionally characterised and their substrate scope was tested using several synthetic DMSP analogues. Comparative kinetic assays revealed differences in the conversion of DMSP and its analogues in terms of selectivity and overall velocity, giving additional insights into the molecular mechanisms of DMSP lyases and into their putatively different biological functions.
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http://dx.doi.org/10.1039/c7ob00913eDOI Listing
May 2017

Comparative "Omics" of the Fusarium fujikuroi Species Complex Highlights Differences in Genetic Potential and Metabolite Synthesis.

Genome Biol Evol 2016 12 31;8(11):3574-3599. Epub 2016 Dec 31.

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

Species of the Fusarium fujikuroi species complex (FFC) cause a wide spectrum of often devastating diseases on diverse agricultural crops, including coffee, fig, mango, maize, rice, and sugarcane. Although species within the FFC are difficult to distinguish by morphology, and their genes often share 90% sequence similarity, they can differ in host plant specificity and life style. FFC species can also produce structurally diverse secondary metabolites (SMs), including the mycotoxins fumonisins, fusarins, fusaric acid, and beauvericin, and the phytohormones gibberellins, auxins, and cytokinins. The spectrum of SMs produced can differ among closely related species, suggesting that SMs might be determinants of host specificity. To date, genomes of only a limited number of FFC species have been sequenced. Here, we provide draft genome sequences of three more members of the FFC: a single isolate of F. mangiferae, the cause of mango malformation, and two isolates of F. proliferatum, one a pathogen of maize and the other an orchid endophyte. We compared these genomes to publicly available genome sequences of three other FFC species. The comparisons revealed species-specific and isolate-specific differences in the composition and expression (in vitro and in planta) of genes involved in SM production including those for phytohormome biosynthesis. Such differences have the potential to impact host specificity and, as in the case of F. proliferatum, the pathogenic versus endophytic life style.
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http://dx.doi.org/10.1093/gbe/evw259DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5203792PMC
December 2016

Gibepyrone Biosynthesis in the Rice Pathogen Fusarium fujikuroi Is Facilitated by a Small Polyketide Synthase Gene Cluster.

J Biol Chem 2016 12 17;291(53):27403-27420. Epub 2016 Nov 17.

From the Institut für Biologie und Biotechnologie der Pflanzen, Westfälische Wilhelms-Universität Münster, Schlossplatz 8, D-48143 Münster,

The 2H-pyran-2-one gibepyrone A and its oxidized derivatives gibepyrones B-F have been isolated from the rice pathogenic fungus Fusarium fujikuroi already more than 20 years ago. However, these products have not been linked to the respective biosynthetic genes, and therefore, their biosynthesis has not yet been analyzed on a molecular level. Feeding experiments with isotopically labeled precursors clearly supported a polyketide origin for the formal monoterpenoid gibepyrone A, whereas the terpenoid pathway could be excluded. Targeted gene deletion verified that the F. fujikuroi polyketide synthase PKS13, designated Gpy1, is responsible for gibepyrone A biosynthesis. Next to Gpy1, the ATP-binding cassette transporter Gpy2 is encoded by the gibepyrone gene cluster. Gpy2 was shown to have only a minor impact on the actual efflux of gibepyrone A out of the cell. Instead, we obtained evidence that Gpy2 is involved in gene regulation as it represses GPY1 gene expression. Thus, GPY1 was up-regulated and gibepyrone A production was enhanced both extra- and intracellularly in Δgpy2 mutants. Furthermore, expression of GPY genes is strictly repressed by members of the fungus-specific velvet complex, Vel1, Vel2, and Lae1, whereas Sge1, a major regulator of secondary metabolism in F. fujikuroi, affects gibepyrone biosynthesis in a positive manner. The gibepyrone A derivatives gibepyrones B and D were shown to be produced by cluster-independent P450 monooxygenases, probably to protect the fungus from the toxic product. In contrast, the formation of gibepyrones E and F from gibepyrone A is a spontaneous process and independent of enzymatic activity.
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http://dx.doi.org/10.1074/jbc.M116.753053DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5207165PMC
December 2016

Knock-down of the methyltransferase Kmt6 relieves H3K27me3 and results in induction of cryptic and otherwise silent secondary metabolite gene clusters in Fusarium fujikuroi.

Environ Microbiol 2016 11 18;18(11):4037-4054. Epub 2016 Jul 18.

Institute of Plant Biology and Biotechnology, Westfälische Wilhelms-University Münster, 48143, Münster, Germany.

Filamentous fungi produce a vast array of secondary metabolites (SMs) and some play a role in agriculture or pharmacology. Sequencing of the rice pathogen Fusarium fujikuroi revealed the presence of far more SM-encoding genes than known products. SM production is energy-consuming and thus tightly regulated, leaving the majority of SM gene clusters silent under laboratory conditions. One important regulatory layer in SM biosynthesis involves histone modifications that render the underlying genes either silent or poised for transcription. Here, we show that the majority of the putative SM gene clusters in F. fujikuroi are located within facultative heterochromatin marked by trimethylated lysine 27 on histone 3 (H3K27me3). Kmt6, the methyltransferase responsible for establishing this histone mark, appears to be essential in this fungus, and knock-down of Kmt6 in the KMT6 strain shows a drastic phenotype affecting fungal growth and development. Transcription of four so far cryptic and otherwise silent putative SM gene clusters was induced in the KMT6 strain, in which decreased expression of KMT6 is accompanied by reduced H3K27me3 levels at the respective gene loci and accumulation of novel metabolites. One of the four putative SM gene clusters, named STC5, was analysed in more detail thereby revealing a novel sesquiterpene.
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http://dx.doi.org/10.1111/1462-2920.13427DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5118082PMC
November 2016

Mechanistic Characterisation of Two Sesquiterpene Cyclases from the Plant Pathogenic Fungus Fusarium fujikuroi.

Angew Chem Int Ed Engl 2016 07 13;55(30):8748-51. Epub 2016 Jun 13.

Kekulé-Institut für Organische Chemie und Biochemie, Rheinische Friedrich-Wilhelms-Universität Bonn, Gerhard-Domagk-Straße 1, 53121, Bonn, Germany.

Two sesquiterpene cyclases from Fusarium fujikuroi were expressed in Escherichia coli and purified. The first enzyme was inactive because of a critical mutation, but activity was restored by sequence correction through site-directed mutagenesis. The mutated enzyme and two naturally functional homologues from other fusaria converted farnesyl diphosphate into guaia-6,10(14)-diene. The second enzyme produced eremophilene. The absolute configuration of guaia-6,10(14)-diene was elucidated by enantioselective synthesis, while that of eremophilene was evident from the sign of its optical rotation and is opposite to that in plants but the same as in Sorangium cellulosum. The mechanisms of both terpene cyclases were studied with various (13) C- and (2) H-labelled FPP isotopomers.
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http://dx.doi.org/10.1002/anie.201603782DOI Listing
July 2016

Two separate key enzymes and two pathway-specific transcription factors are involved in fusaric acid biosynthesis in Fusarium fujikuroi.

Environ Microbiol 2016 Mar 21;18(3):936-56. Epub 2016 Jan 21.

Institute of Plant Biology and Biotechnology, Westfälische Wilhelms-University, Schlossplatz 8, 48143, Münster, Germany.

Fusaric acid (FSA) is a mycotoxin produced by several fusaria, including the rice pathogen Fusarium fujikuroi. Genes involved in FSA biosynthesis were previously identified as a cluster containing a polyketide synthase (PKS)-encoding (FUB1) and four additional genes (FUB2-FUB5). However, the biosynthetic steps leading to FSA as well as the origin of the nitrogen atom, which is incorporated into the polyketide backbone, remained unknown. In this study, seven additional cluster genes (FUB6-FUB12) were identified via manipulation of the global regulator FfSge1. The extended FUB gene cluster encodes two Zn(II)2 Cys6 transcription factors: Fub10 positively regulates expression of all FUB genes, whereas Fub12 is involved in the formation of the two FSA derivatives, i.e. dehydrofusaric acid and fusarinolic acid, serving as a detoxification mechanism. The major facilitator superfamily transporter Fub11 functions in the export of FSA out of the cell and is essential when FSA levels become critical. Next to Fub1, a second key enzyme was identified, the non-canonical non-ribosomal peptide synthetase Fub8. Chemical analyses of generated mutant strains allowed for the identification of a triketide as PKS product and the proposition of an FSA biosynthetic pathway, thereby unravelling the unique formation of a hybrid metabolite consisting of this triketide and an amino acid moiety.
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http://dx.doi.org/10.1111/1462-2920.13150DOI Listing
March 2016