Recent Submissions

  • Expanding the Scope of Detectable Microbial Natural Products by Complementary Analytical Methods and Cultivation Systems.

    Bader, Chantal D; Haack, Patrick A; Panter, Fabian; Krug, Daniel; Müller, Rolf; HIPS, Helmholtz-Institut für Pharmazeutische Forschung Saarland, Universitätscampus E8.1 66123 Saarbrücken, Germany. (American Chemical Society (ACS), 2021-01-15)
    Outer membrane vesicles (OMVs) are universally produced by prokaryotes and play important roles in symbiotic and pathogenic interactions. They often contain DNA, but a mechanism for its incorporation is lacking. Here, we show that Dinoroseobacter shibae, a dinoflagellate symbiont, constitutively secretes OMVs containing DNA. Time-lapse microscopy captured instances of multiple OMV production at the septum during cell division. DNA from the vesicle lumen was up to 22-fold enriched for the region around the terminus of replication (ter). The peak of coverage was located at dif, a conserved 28-bp palindromic sequence required for binding of the site-specific tyrosine recombinases XerC/XerD. These enzymes are activated at the last stage of cell division immediately prior to septum formation when they are bound by the divisome protein FtsK. We suggest that overreplicated regions around the terminus have been repaired by the FtsK-dif-XerC/XerD molecular machinery. The vesicle proteome was clearly dominated by outer membrane and periplasmic proteins. Some of the most abundant vesicle membrane proteins were predicted to be required for direct interaction with peptidoglycan during cell division (LysM, Tol-Pal, Spol, lytic murein transglycosylase). OMVs were 15-fold enriched for the saturated fatty acid 16:00. We hypothesize that constitutive OMV secretion in D. shibae is coupled to cell division. The footprint of the FtsK-dif-XerC/XerD molecular machinery suggests a novel potentially highly conserved route for incorporation of DNA into OMVs. Clearing the division site from small DNA fragments might be an important function of vesicles produced during exponential growth under optimal conditions.IMPORTANCE Gram-negative bacteria continually form vesicles from their outer membrane (outer membrane vesicles [OMVs]) during normal growth. OMVs frequently contain DNA, and it is unclear how DNA can be shuffled from the cytoplasm to the OMVs. We studied OMV cargo in Dinoroseobacter shibae, a symbiont of dinoflagellates, using microscopy and a multi-omics approach. We found that vesicles formed during undisturbed exponential growth contain DNA which is enriched for genes around the replication terminus, specifically, the binding site for an enzyme complex that is activated at the last stage of cell division. We suggest that the enriched genes are the result of overreplication which is repaired by their excision and excretion via membrane vesicles to clear the divisome from waste DNA.
  • Dedication: Heinz Floss and Christopher Walsh-pioneers in natural product chemical biology.

    Müller, Rolf; Wright, Gerard D; HIPS, Helmholtz-Institut für Pharmazeutische Forschung Saarland, Universitätscampus E8.1 66123 Saarbrücken, Germany. (Springer, 2019-03-01)
    [No abstract available]
  • Introduction to the special issue: "Natural Product Discovery and Development in the Genomic Era: 2019".

    Baltz, Richard H; Abe, Ikuro; Kim, Eung-Soo; Müller, Rolf; Van Lanen, Steven G; Wright, Gerry; HIPS, Helmholtz-Institut für Pharmazeutische Forschung Saarland, Universitätscampus E8.1 66123 Saarbrücken, Germany. (Springer, 2019-03-01)
    [No abstract available]
  • Ribosome-Targeting Antibiotics Impair T Cell Effector Function and Ameliorate Autoimmunity by Blocking Mitochondrial Protein Synthesis.

    Almeida, Luís; Dhillon-LaBrooy, Ayesha; Castro, Carla N; Adossa, Nigatu; Carriche, Guilhermina M; Guderian, Melanie; Lippens, Saskia; Dennerlein, Sven; Hesse, Christina; Lambrecht, Bart N; et al. (Elsevier (Cell Press), 2020-11-24)
    While antibiotics are intended to specifically target bacteria, most are known to affect host cell physiology. In addition, some antibiotic classes are reported as immunosuppressive for reasons that remain unclear. Here, we show that Linezolid, a ribosomal-targeting antibiotic (RAbo), effectively blocked the course of a T cell-mediated autoimmune disease. Linezolid and other RAbos were strong inhibitors of T helper-17 cell effector function in vitro, showing that this effect was independent of their antibiotic activity. Perturbing mitochondrial translation in differentiating T cells, either with RAbos or through the inhibition of mitochondrial elongation factor G1 (mEF-G1) progressively compromised the integrity of the electron transport chain. Ultimately, this led to deficient oxidative phosphorylation, diminishing nicotinamide adenine dinucleotide concentrations and impairing cytokine production in differentiating T cells. In accordance, mice lacking mEF-G1 in T cells were protected from experimental autoimmune encephalomyelitis, demonstrating that this pathway is crucial in maintaining T cell function and pathogenicity.
  • Angucycline-like Aromatic Polyketide from a Novel Streptomyces Species Reveals Freshwater Snail Physa acuta as Underexplored Reservoir for Antibiotic-Producing Actinomycetes.

    Safaei, Nasim; Mast, Yvonne; Steinert, Michael; Huber, Katharina; Bunk, Boyke; Wink, Joachim; HZI,Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7,38124 Braunschweig, Germany. (MDPI, 2020-12-29)
    Antibiotic producers have mainly been isolated from soil, which often has led to the rediscovery of known compounds. In this study, we identified the freshwater snail Physa acuta as an unexplored source for new antibiotic producers. The bacterial diversity associated with the snail was characterized by a metagenomic approach using cultivation-independent high-throughput sequencing. Although Actinobacteria represented only 2% of the bacterial community, the focus was laid on the isolation of the genus Streptomyces due to its potential to produce antibiotics. Three Streptomyces strains (7NS1, 7NS2 and 7NS3) were isolated from P. acuta, and the antimicrobial activity of the crude extracts were tested against a selection of Gram-positive and Gram-negative bacteria and fungi. 7NS3 showed the strongest activity against Gram-positive bacteria and, thus, was selected for genome sequencing and a phylogenomic analysis. 7NS3 represents a novel Streptomyces species, which was deposited as Streptomyces sp. DSM 110735 at the Leibniz Institute-German Collection of Microorganisms and Cell Cultures (DSMZ). Bioassay-guided high-performance liquid chromatography (HPLC) and high-resolution electrospray ionization-mass spectrometry (HR-ESI-MS) analyses of crude extract fractions resulted in the detection of four compounds, one of which matched the compound characteristics of emycin A, an angucycline-like aromatic polyketide. Genome mining studies based on the whole-genome sequence of 7NS3 resulted in the identification of a gene cluster potentially coding for emycin A biosynthesis. Our study demonstrates that freshwater snails like P. acuta can represent promising reservoirs for the isolation of new antibiotic-producing actinobacterial species.
  • Perquinolines A-C: Unprecedented Bacterial Tetrahydroisoquinolines Involving an Intriguing Biosynthesis.

    Rebets, Yuriy; Nadmid, Suvd; Paulus, Constanze; Dahlem, Charlotte; Herrmann, Jennifer; Hübner, Harald; Rückert, Christian; Kiemer, Alexandra K; Gmeiner, Peter; Kalinowski, Jörn; et al. (Wiley, 2019-08-21)
    Autophagy, a membrane-dependent catabolic process, ensures survival of aging cells and depends on the cellular energetic status. Acetyl-CoA carboxylase 1 (Acc1) connects central energy metabolism to lipid biosynthesis and is rate-limiting for the de novo synthesis of lipids. However, it is unclear how de novo lipogenesis and its metabolic consequences affect autophagic activity. Here, we show that in aging yeast, autophagy levels highly depend on the activity of Acc1. Constitutively active Acc1 (acc1S/A ) or a deletion of the Acc1 negative regulator, Snf1 (yeast AMPK), shows elevated autophagy levels, which can be reversed by the Acc1 inhibitor soraphen A. Vice versa, pharmacological inhibition of Acc1 drastically reduces cell survival and results in the accumulation of Atg8-positive structures at the vacuolar membrane, suggesting late defects in the autophagic cascade. As expected, acc1S/A cells exhibit a reduction in acetate/acetyl-CoA availability along with elevated cellular lipid content. However, concomitant administration of acetate fails to fully revert the increase in autophagy exerted by acc1S/A Instead, administration of oleate, while mimicking constitutively active Acc1 in WT cells, alleviates the vacuolar fusion defects induced by Acc1 inhibition. Our results argue for a largely lipid-dependent process of autophagy regulation downstream of Acc1. We present a versatile genetic model to investigate the complex relationship between acetate metabolism, lipid homeostasis, and autophagy and propose Acc1-dependent lipogenesis as a fundamental metabolic path downstream of Snf1 to maintain autophagy and survival during cellular aging.
  • 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.
  • Targeted Genome Mining-From Compound Discovery to Biosynthetic Pathway Elucidation.

    Gummerlich, Nils; Rebets, Yuriy; Paulus, Constanze; Zapp, Josef; Luzhetskyy, Andriy; HIPS, Helmholtz-Institut für Pharmazeutische Forschung Saarland, Universitätscampus E8.1 66123 Saarbrücken, Germany. (MDPI, 2020-12-19)
    Natural products are an important source of novel investigational compounds in drug discovery. Especially in the field of antibiotics, Actinobacteria have been proven to be a reliable source for lead structures. The discovery of these natural products with activity- and structure-guided screenings has been impeded by the constant rediscovery of previously identified compounds. Additionally, a large discrepancy between produced natural products and biosynthetic potential in Actinobacteria, including representatives of the order Pseudonocardiales, has been revealed using genome sequencing. To turn this genomic potential into novel natural products, we used an approach including the in-silico pre-selection of unique biosynthetic gene clusters followed by their systematic heterologous expression. As a proof of concept, fifteen Saccharothrixespanaensis genomic library clones covering predicted biosynthetic gene clusters were chosen for expression in two heterologous hosts, Streptomyceslividans and Streptomycesalbus. As a result, two novel natural products, an unusual angucyclinone pentangumycin and a new type II polyketide synthase shunt product SEK90, were identified. After purification and structure elucidation, the biosynthetic pathways leading to the formation of pentangumycin and SEK90 were deduced using mutational analysis of the biosynthetic gene cluster and feeding experiments with 13C-labelled precursors.
  • Supercritical Fluid Extraction Enhances Discovery of Secondary Metabolites from Myxobacteria.

    Bader, Chantal D; Neuber, Markus; Panter, Fabian; Krug, Daniel; Müller, Rolf; HIPS, Helmholtz-Institut für Pharmazeutische Forschung Saarland, Universitätscampus E8.1 66123 Saarbrücken, Germany. (American Chemical Society, 2020-11-10)
    Supercritical fluid extraction (SFE) is widely used for the isolation of natural products from plants, but its application in efforts to identify structurally and physicochemically often dissimilar microbial natural products is limited to date. In this study, we evaluated the impact of SFE on the extractability of myxobacterial secondary metabolites, aiming to improve the prospects of discovering novel natural products. We investigated the influence of different co-solvents on the extraction efficiency of secondary metabolites from three myxobacterial strains and the antimicrobial activity profiles of the corresponding extracts. For each known secondary metabolite, we found extraction conditions using SFE leading to superior yields in the extracts compared to conventional solvent extraction. Compounds with a logP higher than 3 showed the best extraction efficiency using 20% EtOAc as a co-solvent, whereas compounds with logP values lower than 3 were better extractable using more polar co-solvents such as MeOH. Extracts generated with SFE showed increased antimicrobial activities including the presence of activities not explained by known myxobacterial secondary metabolites, highlighting the advantage of SFE for bioactivity-guided isolation. Moreover, non-targeted metabolomics analysis revealed a group of chlorinated metabolites produced by the well-studied model myxobacterium Myxococcus xanthus DK1622, which were not accessible previously due to their low concentration in conventional extracts. The enriched SF extracts were used for isolation and subsequent structure elucidation of chloroxanthic acid A as the founding member of a novel secondary metabolite family. Our findings encourage the increased utilization of SFE as a part of future screening workflows of microbial natural products.
  • Corallopyronin A for short-course anti-wolbachial, macrofilaricidal treatment of filarial infections.

    Schiefer, Andrea; Hübner, Marc P; Krome, Anna; Lämmer, Christine; Ehrens, Alexandra; Aden, Tilman; Koschel, Marianne; Neufeld, Helene; Chaverra-Muñoz, Lillibeth; Jansen, Rolf; et al. (PLOS, 2020-12-07)
    Current efforts to eliminate the neglected tropical diseases onchocerciasis and lymphatic filariasis, caused by the filarial nematodes Onchocerca volvulus and Wuchereria bancrofti or Brugia spp., respectively, are hampered by lack of a short-course macrofilaricidal-adult-worm killing-treatment. Anti-wolbachial antibiotics, e.g. doxycycline, target the essential Wolbachia endosymbionts of filariae and are a safe prototype adult-worm-sterilizing and macrofilaricidal regimen, in contrast to standard treatments with ivermectin or diethylcarbamazine, which mainly target the microfilariae. However, treatment regimens of 4-5 weeks necessary for doxycycline and contraindications limit its use. Therefore, we tested the preclinical anti-Wolbachia drug candidate Corallopyronin A (CorA) for in vivo efficacy during initial and chronic filarial infections in the Litomosoides sigmodontis rodent model. CorA treatment for 14 days beginning immediately after infection cleared >90% of Wolbachia endosymbionts from filariae and prevented development into adult worms. CorA treatment of patently infected microfilaremic gerbils for 14 days with 30 mg/kg twice a day (BID) achieved a sustained reduction of >99% of Wolbachia endosymbionts from adult filariae and microfilariae, followed by complete inhibition of filarial embryogenesis resulting in clearance of microfilariae. Combined treatment of CorA and albendazole, a drug currently co-administered during mass drug administrations and previously shown to enhance efficacy of anti-Wolbachia drugs, achieved microfilarial clearance after 7 days of treatment at a lower BID dose of 10 mg/kg CorA, a Human Equivalent Dose of 1.4 mg/kg. Importantly, this combination led to a significant reduction in the adult worm burden, which has not yet been published with other anti-Wolbachia candidates tested in this model. In summary, CorA is a preclinical candidate for filariasis, which significantly reduces treatment times required to achieve sustained Wolbachia depletion, clearance of microfilariae, and inhibition of embryogenesis. In combination with albendazole, CorA is robustly macrofilaricidal after 7 days of treatment and fulfills the Target Product Profile for a macrofilaricidal drug.
  • Leben und Überleben im Boden

    Volz, Carsten; Krug, Daniel; Müller, Rolf; HIPS, Helmholtz-Institut für Pharmazeutische Forschung Saarland, Universitätscampus E8.1 66123 Saarbrücken, Germany. (Wiley-VCH, 2020-12-07)
    Myxobacteria - survivalists in soil. Myxobacteria like Myxococccus xanthus are soil-living microorganisms featuring a complex lifestyle, including movement by coordinated swarming on surfaces, predatory feeding on other microorganisms, and the formation of multicellular fruiting bodies when unfavorable environmental conditions are encountered. Bioinformatic analysis of the large myxobacterial genomes has enabled fascinating insights into the molecular basis for the biosynthesis of complex secondary metabolite structures by myxobacteria, and has set the stage for the discovery of novel natural products. Moreover, well-characterized myxobacteria like M. xanthus increasingly play a role as “biochemical factories” for the biotechnological production of bioactive molecules using synthetic biology approaches
  • The antibiotic sorangicin A inhibits promoter DNA unwinding in a rifampicin-resistant RNA polymerase.

    Lilic, Mirjana; Chen, James; Boyaci, Hande; Braffman, Nathaniel; Hubin, Elizabeth A; Herrmann, Jennifer; Müller, Rolf; Mooney, Rachel; Landick, Robert; Darst, Seth A; et al. (National Academy of Sciences, 2020-11-16)
    Rifampicin (Rif) is a first-line therapeutic used to treat the infectious disease tuberculosis (TB), which is caused by the pathogen Mycobacterium tuberculosis (Mtb). The emergence of Rif-resistant (RifR) Mtb presents a need for new antibiotics. Rif targets the enzyme RNA polymerase (RNAP). Sorangicin A (Sor) is an unrelated inhibitor that binds in the Rif-binding pocket of RNAP. Sor inhibits a subset of RifR RNAPs, including the most prevalent clinical RifR RNAP substitution found in Mtb infected patients (S456>L of the β subunit). Here, we present structural and biochemical data demonstrating that Sor inhibits the wild-type Mtb RNAP by a similar mechanism as Rif: by preventing the translocation of very short RNAs. By contrast, Sor inhibits the RifR S456L enzyme at an earlier step, preventing the transition of a partially unwound promoter DNA intermediate to the fully opened DNA and blocking the template-strand DNA from reaching the active site in the RNAP catalytic center. By defining template-strand blocking as a mechanism for inhibition, we provide a mechanistic drug target in RNAP. Our finding that Sor inhibits the wild-type and mutant RNAPs through different mechanisms prompts future considerations for designing antibiotics against resistant targets. Also, we show that Sor has a better pharmacokinetic profile than Rif, making it a suitable starting molecule to design drugs to be used for the treatment of TB patients with comorbidities who require multiple medications.
  • Neither black nor white: do altered intestinal microbiota reflect chronic liver disease severity?

    Goeser, Felix; Münch, Philipp; Lesker, Till Robin; Lutz, Philipp Ludwig; Krämer, Benjamin; Kaczmarek, Dominik J; Finnemann, Claudia; Nischalke, Hans Dieter; Geffers, Robert; Parcina, Marijo; et al. (BMJ publishing group, 2020-06-05)
    No abstract available
  • Dudomycins: New Secondary Metabolites Produced After Heterologous Expression of an Nrps Cluster from ssp. Nrrl B-24108.

    Lasch, Constanze; Stierhof, Marc; Estévez, Marta Rodríguez; Myronovskyi, Maksym; Zapp, Josef; Luzhetskyy, Andriy; HIPS, Helmholtz-Institut für Pharmazeutische Forschung Saarland, Universitätscampus E8.1 66123 Saarbrücken, Germany. (MDPI, 2020-11-16)
    Since the 1950s, natural products of bacterial origin were systematically developed to be used as drugs with a wide range of medical applications. The available treatment options for many diseases are still not satisfying, wherefore, the discovery of new structures has not lost any of its importance. Beyond the great variety of already isolated and characterized metabolites, Streptomycetes still harbor uninvestigated gene clusters whose products can be accessed using heterologous expression in host organisms. This works presents the discovery of a set of structurally novel secondary metabolites, dudomycins A to D, through the expression of a cryptic NRPS cluster from Streptomyces albus ssp. Chlorinus NRRL B-24108 in the heterologous host strain Streptomyces albus Del14. A minimal set of genes, required for the production of dudomycins, was defined through gene inactivation experiments. This paper also proposes a model for dudomycin biosynthesis.
  • Metabolic Profiling to Determine Bactericidal or Bacteriostatic Effects of New Natural Products using Isothermal Microcalorimetry.

    Cirnski, Katarina; Coetzee, Janetta; Herrmann, Jennifer; Müller, Rolf; HIPS, Helmholtz-Institut für Pharmazeutische Forschung Saarland, Universitätscampus E8.1 66123 Saarbrücken, Germany. (MyJove Corporation, 2020-10-29)
    Due to the global threat of rising antimicrobial resistance, novel antibiotics are urgently needed. We investigate natural products from Myxobacteria as an innovative source of such new compounds. One bottleneck in the process is typically the elucidation of their mode-of-action. We recently established isothermal microcalorimetry as part of a routine profiling pipeline. This technology allows for investigating the effect of antibiotic exposure on the total bacterial metabolic response, including processes that are decoupled from biomass formation. Importantly, bacteriostatic and bactericidal effects are easily distinguishable without any user intervention during the measurements. However, isothermal microcalorimetry is a rather new approach and applying this method to different bacterial species usually requires pre-evaluation of suitable measurement conditions. There are some reference thermograms available of certain bacteria, greatly facilitating interpretation of results. As the pool of reference data is steadily growing, we expect the methodology to have increasing impact in the future and expect it to allow for in-depth fingerprint analyses enabling the differentiation of antibiotic classes.
  • Special Issue: "Actinobacteria and Myxobacteria-Important Resources for Novel Antibiotics".

    Wink, Joachim; HZI,Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7,38124 Braunschweig, Germany. (MDPI, 2020-09-24)
    [[No abstract available]
  • An ambruticin-sensing complex modulates Myxococcus xanthus development and mediates myxobacterial interspecies communication.

    Marcos-Torres, Francisco Javier; Volz, Carsten; Müller, Rolf; HIPS, Helmholtz-Institut für Pharmazeutische Forschung Saarland, Universitätscampus E8.1 66123 Saarbrücken, Germany. (Nature Pulishing Group, 2020-11-04)
    Starvation induces cell aggregation in the soil bacterium Myxococcus xanthus, followed by formation of fruiting bodies packed with myxospores. Sporulation in the absence of fruiting bodies can be artificially induced by high concentrations of glycerol through unclear mechanisms. Here, we show that a compound (ambruticin VS-3) produced by a different myxobacterium, Sorangium cellulosum, affects the development of M. xanthus in a similar manner. Both glycerol (at millimolar levels) and ambruticin VS-3 (at nanomolar concentrations) inhibit M. xanthus fruiting body formation under starvation, and induce sporulation in the presence of nutrients. The response is mediated in M. xanthus by three hybrid histidine kinases (AskA, AskB, AskC) that form complexes interacting with two major developmental regulators (MrpC, FruA). In addition, AskB binds directly to the mrpC promoter in vitro. Thus, our work indicates that the AskABC-dependent regulatory pathway mediates the responses to ambruticin VS-3 and glycerol. We hypothesize that production of ambruticin VS-3 may allow S. sorangium to outcompete M. xanthus under both starvation and growth conditions in soil.
  • Non-Heme Monooxygenase ThoJ Catalyzes Thioholgamide β-Hydroxylation.

    Sikandar, Asfandyar; Lopatniuk, Maria; Luzhetskyy, Andriy; Koehnke, Jesko; HIPS, Helmholtz-Institut für Pharmazeutische Forschung Saarland, Universitätscampus E8.1 66123 Saarbrücken, Germany. (American Chemical Society (ACS), 2020-10-01)
    Thioviridamide-like compounds, including thioholgamides, are ribosomally synthesized and post-translationally modified peptide natural products with potent anticancer cell activity and an unprecedented structure. Very little is known about their biosynthesis, and we were intrigued by the β-hydroxy-N1, N3-dimethylhistidinium moiety found in these compounds. Here we report the construction of a heterologous host capable of producing thioholgamide with a 15-fold increased yield compared to the wild-type strain. A knockout of thoJ, encoding a predicted nonheme monooxygenase, shows that ThoJ is essential for thioholgamide β-hydroxylation. The crystal structure of ThoJ exhibits a typical mono/dioxygenase fold with conserved key active-site residues. Yet, ThoJ possesses a very large substrate binding pocket that appears suitable to receive a cyclic thioholgamide intermediate for hydroxylation. The improved production of the heterologous host will enable the dissection of the individual biosynthetic steps involved in biosynthesis of this exciting RiPP family.
  • Loseolamycins: A Group of New Bioactive Alkylresorcinols Produced after Heterologous Expression of a Type III PKS from micromonospora endolithica.

    Lasch, Constanze; Gummerlich, Nils; Myronovskyi, Maksym; Palusczak, Anja; Zapp, Josef; Luzhetskyy, Andriy; HIPS, Helmholtz-Institut für Pharmazeutische Forschung Saarland, Universitätscampus E8.1 66123 Saarbrücken, Germany. (MDPI, 2020-10-09)
    Natural products are a valuable source of biologically active compounds with potential applications in medicine and agriculture. Unprecedented scaffold diversity of natural products and biocatalysts from their biosynthetic pathways are of fundamental importance. Heterologous expression and refactoring of natural product biosynthetic pathways are generally regarded as a promising approach to discover new secondary metabolites of microbial origin. Here, we present the identification of a new group of alkylresorcinols after transcriptional activation and heterologous expression of the type III polyketide synthase of Micromonospora endolithica. The most abundant compounds loseolamycins A1 and A2 have been purified and their structures were elucidated by NMR. Loseolamycins contain an unusual branched hydroxylated aliphatic chain which is provided by the host metabolism and is incorporated as a starter fatty acid unit. The isolated loseolamycins show activity against gram-positive bacteria and inhibit the growth of the monocot weed Agrostis stolonifera in a germination assay. The biosynthetic pathway leading to the production of loseolamycins is proposed in this paper.
  • Isolation and identification of Streptomyces sp. Act4Zk, a good producer of Staurosporine and some derivatives.

    Khosravi Babadi, Z; Ebrahimipour, G; Wink, J; Narmani, A; Risdian, C; HZI,Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7,38124 Braunschweig, Germany. (Society for Applied Microbiology, 2020-10-15)
    In this study, strain Streptomyces sp. Act4Zk was isolated based on a method developed for the isolation of myxobacteria. Due to the low efficiency of the majority of conventional DNA extraction techniques, for molecular identification of the strain Streptomyces sp. Act4Zk, a new technique for DNA extraction of Actinobacteria was developed. In order to explore potential bioactivities of the strain, extracts of the fermented broth culture were prepared by an organic solvent (i.e. ethyl acetate) extraction method using. These ethyl acetate extracts were subjected to HPLC fractionation against standard micro-organisms, followed by LC/MS analysis. Based on morphological, physiological, biochemical and 16S rRNA gene sequence data, strain Streptomyces sp. Act4Zk is likely to be a new species of Streptomyces, close to Streptomyces genecies and Streptomyces roseolilacinus. Antimicrobial assay indicated high antifungal activity as well as antibacterial activity against Mycobacterium smegmatis and Gram-positive bacteria for the new strain. HPLC and LC/MS analyses of the extracts led to the identification of three different compounds and confirmed our hypothesis that the interesting species of the genus Streptomyces being a good producer of staurosporine and some derivatives.

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