Isolation and characterization of novel natural compounds from Myxobacteria

Microbial secondary metabolites are known to be an excellent source for novel therapeutic agents. Among other microorganisms, myxobacteria are continuously providing new biologically active natural compounds with unique structures. Here, chemical screening of these gram-negative bacteria has resulted in the identification of two new structural classes of natural products along with a new derivative of a sponge-derived natural product. In this thesis, isolation, structural elucidation, and biological activity of these new secondary metabolites are presented. New catecholate-type siderophores, hyalachelins A-C, were isolated from the strain belonging to the underexplored species Hyalangium minutum. Their complete 3D structure was obtained by combining the spectroscopic data and quantum mechanical calculations. Iron binding activity of hyalachelins was determined by CAS assay. Moreover, novel polyketides, named cystochromones, were isolated from Cystobacter sp. Cystochromones bear a chromone ring that is substituted by a long aliphatic chain on position C-5 which is not preceded among natural chromones. Additionally, a biosynthetic pathway was proposed on the basis of the results of the feeding experiments. Furthermore, a new derivative of the marine sponge-derived peptide microsclerodermin was isolated from the terrestrial myxobacterium. This result represents the rare example of isolation of same compounds from terrestrial and marine sources.Mikrobielle Naturstoffe sind bekanntermaßen eine ergiebige Quelle für neue therapeutische Wirkstoffe. Aus Myxobakterien werden fortwährend neue biologisch aktive Naturstoffe mit einzigartigen Strukturen isoliert. Ein chemisches screening dieser Gram-negativen Bakterien resultierte in der Identifizierung von zwei strukturell neuen Klassen von Sekundärmetaboliten und einem neuen Derivat eines bereits bekannten Naturstoffes. In der vorliegenden Arbeit werden die Isolierung, Strukturaufklärung und die biologischen Aktivitäten dieser Substanzen diskutiert. Die Hyacheline A-C, neue Siderophore vom Catecholat-Typ, wurden aus einem Stamm der wenig erforschten myxobakteriellen Spezies Hyalangium minutum isoliert. Die dreidimensionale Struktur der Hyacheline wurde mittels Kombination von spektroskopischen Daten mit quantenmechanischen Berechnungen aufgeklärt, sowie ihr Eisen-Bindungsverhalten anhand von CAS Assays bestimmt. Die Cystochromone wurden aus Extrakten von Cystobacter sp. isoliert. Die chromonartigen Polyketide tragen an Position C-5 des Chromonsystems einen langkettigen aliphatischen Rest. Diese Substitution ist von natürlichen Chromonen bisher nicht bekannt. Auf Basis von Fütterungsexperimenten konnte ein Biosyntheseweg für die Cystochromone vorgeschlagen werden. Weiterhin wurde ein neues Derivat der Mikrosklerodermine aus dem Extrakt eines terrestrischen Myxobakteriums isoliert. Diese Naturstoffe waren bisher aus Meeresschwämmen bekannt und stellen ein Beispiel des selten beschriebenen Falles eines gemeinsamen oder ähnlichen Sekundärmetabolismus von marinen und terrestrischen Mikroorganismen dar

Heterologous Expression of the Nybomycin Gene Cluster from the Marine Strain Streptomyces albus subsp. chlorinus NRRL B-24108

Streptomycetes represent an important reservoir of active secondary metabolites with potential applications in the pharmaceutical industry. The gene clusters responsible for their production are often cryptic under laboratory growth conditions. Characterization of these clusters is therefore essential for the discovery of new microbial pharmaceutical drugs. Here, we report the identification of the previously uncharacterized nybomycin gene cluster from the marine actinomycete Streptomyces albus subsp. chlorinus through its heterologous expression. Nybomycin has previously been reported to act against quinolone-resistant Staphylococcus aureus strains harboring a mutated gyrA gene but not against those with intact gyrA. The nybomycin-resistant mutants generated from quinolone-resistant mutants have been reported to be caused by a back-mutation in the gyrA gene that restores susceptibility to quinolones. On the basis of gene function assignment from bioinformatics analysis, we suggest a model for nybomycin biosynthesis

Identification of a Biosynthetic Gene Cluster Responsible for the Production of a New Pyrrolopyrimidine Natural Product-Huimycin.

Pyrrolopyrimidines are an important class of natural products with a broad spectrum of biological activities, including antibacterial, antifungal, antiviral, anticancer or anti-inflammatory. Here, we present the identification of a biosynthetic gene cluster from the rare actinomycete strain Kutzneria albida DSM 43870, which leads to the production of huimycin, a new member of the pyrrolopyrimidine family of compounds. The huimycin gene cluster was successfully expressed in the heterologous host strain Streptomyces albus Del14. The compound was purified, and its structure was elucidated by means of nuclear magnetic resonance spectroscopy. The minimal huimycin gene cluster was identified through sequence analysis and a series of gene deletion experiments. A model for huimycin biosynthesis is also proposed in this paper

Heterologous Expression of the Nybomycin Gene Cluster from the Marine Strain <i>Streptomyces albus</i> subsp. <i>chlorinus</i> NRRL B-24108

Streptomycetes represent an important reservoir of active secondary metabolites with potential applications in the pharmaceutical industry. The gene clusters responsible for their production are often cryptic under laboratory growth conditions. Characterization of these clusters is therefore essential for the discovery of new microbial pharmaceutical drugs. Here, we report the identification of the previously uncharacterized nybomycin gene cluster from the marine actinomycete Streptomyces albus subsp. chlorinus through its heterologous expression. Nybomycin has previously been reported to act against quinolone-resistant Staphylococcus aureus strains harboring a mutated gyrA gene but not against those with intact gyrA. The nybomycin-resistant mutants generated from quinolone-resistant mutants have been reported to be caused by a back-mutation in the gyrA gene that restores susceptibility to quinolones. On the basis of gene function assignment from bioinformatics analysis, we suggest a model for nybomycin biosynthesis

Cystochromones, Unusual Chromone-Containing Polyketides from the Myxobacterium <i>Cystobacter</i> sp. MCy9104

Seven new chromone-containing polyketides, termed cystochromones A–G, were isolated from the myxobacterial strain <i>Cystobacter</i> sp. MCy9104. Their structures were elucidated using comprehensive NMR spectroscopy and HR-MS/MS. Cystochromones bear a pentadecyl moiety unusually attached at C-5 of the chromone ring. Moreover, isotope-labeled substrate feeding experiments and NMR analysis suggested a hybrid iso-fatty acid and polyketide synthase biosynthetic pathway for these secondary metabolites

Microsclerodermins from Terrestrial Myxobacteria: An Intriguing Biosynthesis Likely Connected to a Sponge Symbiont

The microsclerodermins are unusual peptide natural products exhibiting potent antifungal activity reported from marine sponges of the genera Microscleroderma and Theonella. We here describe a variety of microbial producers of microsclerodermins and pedeins among myxobacteria along with the isolation of several new derivatives. A retrobiosynthetic approach led to the identification of microsclerodermin biosynthetic gene clusters in genomes of Sorangium and Jahnella species, allowing for the first time insights into the intriguing hybrid PKS/NRPS machinery required for microsclerodermin formation. This study reveals the biosynthesis of a “marine natural product” in a terrestrial myxobacterium where even the identical structure is available from both sources. Thus, the newly identified terrestrial producers provide access to additional chemical diversity; moreover, they are clearly more amenable to production optimization and genetic modification than the original source from the marine habitat. As sponge metagenome data strongly suggest the presence of associated myxobacteria, our findings underpin the recent notion that many previously described “sponge metabolites” might in fact originate from such microbial symbionts

Hyalachelins A–C, Unusual Siderophores Isolated from the Terrestrial Myxobacterium <i>Hyalangium minutum</i>

Three new siderophores, termed hyalachelins A–C (<b>1</b>–<b>3</b>), were isolated from the terrestrial myxobacterium <i>Hyalangium minutum</i>. Their structures were determined by 2D NMR and HR-MS/MS experiments, and their stereochemical configuration was established by a combination of NMR data, quantum mechanical calculations, and circular dichroism experiments. Hyalachelins are unusual catecholate-type siderophores that bear a 3,7,8-trihydroxy-1-oxo-1,2,3,4-tetra­hydro­isoquinoline-3-carbox­ylic acid. Their iron chelating activities were evaluated in a CAS assay showing EC₅₀ values of ∼30 μM

Perquinolines A-C: Unprecedented Bacterial Tetrahydroisoquinolines Involving an Intriguing Biosynthesis

Rebets Y, Nadmid S, Paulus C, et al. Perquinolines A-C: Unprecedented Bacterial Tetrahydroisoquinolines Involving an Intriguing Biosynthesis. Angewandte Chemie International Edition. 2019;58(37):12930-12934.Metabolic profiling of Streptomyces sp. IB2014/016-6 led to the identification of three new tetrahydroisoquinoline natural products, perquinolines A-C (1-3). Labelled precursor feeding studies and the cloning of the pqr biosynthetic gene cluster revealed that 1-3 are assembled by the action of several unusual enzymes. The biosynthesis starts with the condensation of succinyl-CoA and l-phenylalanine catalyzed by the amino-7-oxononanoate synthase-like enzyme PqrA, representing rare chemistry in natural product assembly. The second condensation and cyclization events are conducted by PqrG, an enzyme resembling an acyl-CoA ligase. Last, ATP-grasp RimK-type ligase PqrI completes the biosynthesis by transferring a gamma-aminobutyric acid or beta-alanine moiety. The discovered pathway represents a new route for assembling the tetrahydroisoquinoline cores of natural products

Discovery of the first small-molecule CsrA-RNA interaction inhibitors using biophysical screening technologies.

CsrA is a global post-transcriptional regulator protein affecting mRNA translation and/or stability. Widespread among bacteria, it is essential for their full virulence and thus represents a promising anti-infective drug target. Therefore, we aimed at the discovery of CsrA-RNA interaction inhibitors. Results & methodology: We followed two strategies: a screening of small molecules (A) and an RNA ligand-based approach (B). Using surface plasmon resonance-based binding and fluorescence polarization-based competition assays, (A) yielded seven small-molecule inhibitors, among them MM14 (IC50 of 4 µM). (B) resulted in RNA-based inhibitor GGARNA (IC50 of 113 µM)

Baikalomycins A-C, New Aquayamycin-Type Angucyclines Isolated from Lake Baikal Derived sp. IB201691-2A.

Natural products produced by bacteria found in unusual and poorly studied ecosystems, such as Lake Baikal, represent a promising source of new valuable drug leads. Here we report the isolation of a new Streptomyces sp. strain IB201691-2A from the Lake Baikal endemic mollusk Benedictia baicalensis. In the course of an activity guided screening three new angucyclines, named baikalomycins A-C, were isolated and characterized, highlighting the potential of poorly investigated ecological niches. Besides that, the strain was found to accumulate large quantities of rabelomycin and 5-hydroxy-rabelomycin, known shunt products in angucyclines biosynthesis. Baikalomycins A-C demonstrated varying degrees of anticancer activity. Rabelomycin and 5-hydroxy-rabelomycin further demonstrated antiproliferative activities. The structure elucidation showed that baikalomycin A is a modified aquayamycin with β-d-amicetose and two additional hydroxyl groups at unusual positions (6a and 12a) of aglycone. Baikalomycins B and C have alternating second sugars attached, α-l-amicetose and α-l-aculose, respectively. The gene cluster for baikalomycins biosynthesis was identified by genome mining, cloned using a transformation-associated recombination technique and successfully expressed in S. albus J1074. It contains a typical set of genes responsible for an angucycline core assembly, all necessary genes for the deoxy sugars biosynthesis, and three genes coding for the glycosyltransferase enzymes. Heterologous expression and deletion experiments allowed to assign the function of glycosyltransferases involved in the decoration of baikalomycins aglycone