Recent Submissions

  • Characterization of the Stereoselective P450 Enzyme BotCYP Enables the Biosynthesis of the Bottromycin Core Scaffold.

    Adam, Sebastian; Franz, Laura; Milhim, Mohammed; Bernhardt, Rita; Kalinina, Olga V; Koehnke, Jesko; HIPS, Helmholtz-Institut für Pharmazeutische Forschung Saarland, Universitätscampus E8.1 66123 Saarbrücken, Germany. (American Chemical Society, 2020-11-28)
    Bottromycins are ribosomally synthesized and post-translationally modified peptide natural product antibiotics that are effective against high-priority human pathogens such as methicillin-resistant Staphylococcus aureus. The total synthesis of bottromycins involves at least 17 steps, with a poor overall yield. Here, we report the characterization of the cytochrome P450 enzyme BotCYP from a bottromycin biosynthetic gene cluster. We determined the structure of a close BotCYP homolog and used our data to conduct the first large-scale survey of P450 enzymes associated with RiPP biosynthetic gene clusters. We demonstrate that BotCYP converts a C-terminal thiazoline to a thiazole via an oxidative decarboxylation reaction and provides stereochemical resolution for the pathway. Our data enable the two-pot in vitro production of the bottromycin core scaffold and may allow the rapid generation of bottromycin analogues for compound development.
  • Synergizing the potential of bacterial genomics and metabolomics to find novel antibiotics.

    Panter, Fabian; Bader, Chantal D; Müller, Rolf; HIPS, Helmholtz-Institut für Pharmazeutische Forschung Saarland, Universitätscampus E8.1 66123 Saarbrücken, Germany. (Royal Society of Chemistry, 2021-03-29)
    Antibiotic development based on natural products has faced a long lasting decline since the 1970s, while both the speed and the extent of antimicrobial resistance (AMR) development have been severely underestimated. The discovery of antimicrobial natural products of bacterial and fungal origin featuring new chemistry and previously unknown mode of actions is increasingly challenged by rediscovery issues. Natural products that are abundantly produced by the corresponding wild type organisms often featuring strong UV signals have been extensively characterized, especially the ones produced by extensively screened microbial genera such as streptomycetes. Purely synthetic chemistry approaches aiming to replace the declining supply from natural products as starting materials to develop novel antibiotics largely failed to provide significant numbers of antibiotic drug leads. To cope with this fundamental issue, microbial natural products science is being transformed from a 'grind-and-find' study to an integrated approach based on bacterial genomics and metabolomics. Novel technologies in instrumental analytics are increasingly employed to lower detection limits and expand the space of detectable substance classes, while broadening the scope of accessible and potentially bioactive natural products. Furthermore, the almost exponential increase in publicly available bacterial genome data has shown that the biosynthetic potential of the investigated strains by far exceeds the amount of detected metabolites. This can be judged by the discrepancy between the number of biosynthetic gene clusters (BGC) encoded in the genome of each microbial strain and the number of secondary metabolites actually detected, even when considering the increased sensitivity provided by novel analytical instrumentation. In silico annotation tools for biosynthetic gene cluster classification and analysis allow fast prioritization in BGC-to-compound workflows, which is highly important to be able to process the enormous underlying data volumes. BGC prioritization is currently accompanied by novel molecular biology-based approaches to access the so-called orphan BGCs not yet correlated with a secondary metabolite. Integration of metabolomics, in silico genomics and molecular biology approaches into the mainstream of natural product research will critically influence future success and impact the natural product field in pharmaceutical, nutritional and agrochemical applications and especially in anti-infective research.
  • Human IFITM3 restricts chikungunya virus and Mayaro virus infection and is susceptible to virus-mediated counteraction.

    Franz, Sergej; Pott, Fabian; Zillinger, Thomas; Schüler, Christiane; Dapa, Sandra; Fischer, Carlo; Passos, Vânia; Stenzel, Saskia; Chen, Fangfang; Döhner, Katinka; et al. (Life Science Alliance, 2021-06-02)
    Interferon-induced transmembrane (IFITM) proteins restrict membrane fusion and virion internalization of several enveloped viruses. The role of IFITM proteins during alphaviral infection of human cells and viral counteraction strategies are insufficiently understood. Here, we characterized the impact of human IFITMs on the entry and spread of chikungunya virus and Mayaro virus and provide first evidence for a CHIKV-mediated antagonism of IFITMs. IFITM1, 2, and 3 restricted infection at the level of alphavirus glycoprotein-mediated entry, both in the context of direct infection and cell-to-cell transmission. Relocalization of normally endosomal IFITM3 to the plasma membrane resulted in loss of antiviral activity. rs12252-C, a naturally occurring variant of IFITM3 that may associate with severe influenza in humans, restricted CHIKV, MAYV, and influenza A virus infection as efficiently as wild-type IFITM3 Antivirally active IFITM variants displayed reduced cell surface levels in CHIKV-infected cells involving a posttranscriptional process mediated by one or several nonstructural protein(s) of CHIKV. Finally, IFITM3-imposed reduction of specific infectivity of nascent particles provides a rationale for the necessity of a virus-encoded counteraction strategy against this restriction factor.
  • Structure-Activity Relationship and Mode-of-Action Studies Highlight 1-(4-Biphenylylmethyl)-1H-imidazole-Derived Small Molecules as Potent CYP121 Inhibitors.

    Walter, Isabell; Adam, Sebastian; Gentilini, Maria Virginia; Kany, Andreas M; Brengel, Christian; Thomann, Andreas; Sparwasser, Tim; Köhnke, Jesko; Hartmann, Rolf W; HIPS, Helmholtz-Institut für Pharmazeutische Forschung Saarland, Universitätscampus E8.1 66123 Saarbrücken, Germany.; TWINCORE, Zentrum für experimentelle und klinische Infektionsforschung GmbH,Feodor-Lynen Str. 7, 30625 Hannover, Germany. (Wiley-VCH, 2021-05-19)
    CYP121 of Mycobacterium tuberculosis (Mtb) is an essential target for the development of novel potent drugs against tuberculosis (TB). Besides known antifungal azoles, further compounds of the azole class were recently identified as CYP121 inhibitors with antimycobacterial activity. Herein, we report the screening of a similarity-oriented library based on the former hit compound, the evaluation of affinity toward CYP121, and activity against M. bovis BCG. The results enabled a comprehensive SAR study, which was extended through the synthesis of promising compounds and led to the identification of favorable features for affinity and/or activity and hit compounds with 2.7-fold improved potency. Mode of action studies show that the hit compounds inhibit substrate conversion and highlighted CYP121 as the main antimycobacterial target of our compounds. Exemplified complex crystal structures of CYP121 with three inhibitors reveal a common binding site. Engaging in both hydrophobic interactions as well as hydrogen bonding to the sixth iron ligand, our compounds block a solvent channel leading to the active site heme. Additionally, we report the first CYP inhibitors that are able to reduce the intracellular replication of M. bovis BCG in macrophages, emphasizing their potential as future drug candidates against TB.
  • Leader peptide exchange to produce hybrid, new-to-nature ribosomal natural products.

    Franz, Laura; Koehnke, Jesko; HIPS, Helmholtz-Institut für Pharmazeutische Forschung Saarland, Universitätscampus E8.1 66123 Saarbrücken, Germany. (RSC, 2021-05-24)
    Ribosomal natural products contain exquisite post-translational peptide modifications that are installed by a range of pathway-specific enzymes. We present proof of principle for a Sortase A-based approach that enables peptide modification by enzymes from unrelated pathways. This allowed the one-pot synthesis of a new-to-nature, hybrid ribosomal natural product.
  • Structure and biosynthesis of sorangipyranone - a new γ-dihydropyrone from the myxobacterial strain MSr12020.

    Okoth, Dorothy A; Hug, Joachim J; Mándi, Attila; Kurtán, Tibor; Garcia, Ronald; Müller, Rolf; HIPS, Helmholtz-Institut für Pharmazeutische Forschung Saarland, Universitätscampus E8.1 66123 Saarbrücken, Germany. (Oxford Academic, 2021-05-18)
    Sorangipyranone was isolated as a novel natural product featuring a unique 2,3-dihydro-γ-4H-pyrone scaffold from cultures of the myxobacterial strain MSr12020. We report here the full structure elucidation of sorangipyranone by spectroscopic techniques including 2D NMR and high-resolution mass spectrometry together with the analysis of the biosynthetic pathway. Determination of the absolute configuration was performed by time-dependent density functional theory-electronic circular dichroism calculations and determination of the applicability of the Snatzke's helicity rule, to correlate the high-wavelength n→π* electronic circular dichroism (ECD) transition and the absolute configuration of the 2,3-dihydro-4H-γ-pyrone, was done by the analysis of low-energy conformers and the Kohn-Sham orbitals. Sorangipyranone outlines a new class of a γ-dihydropyrone-containing natural product comprised of malonyl-CoA-derived building blocks and features a unique polyketide scaffold. In silico analysis of the genome sequence of the myxobacterial strain MSr12020 complemented with feeding experiments employing stable isotope-labeled precursors allowed the identification and annotation of a candidate biosynthetic gene cluster that encodes a modular polyketide synthase assembly line. A model for the biosynthetic pathway leading to the formation of the γ-dihydropyrone scaffold is presented in this study.
  • An extended catalogue of tandem alternative splice sites in human tissue transcriptomes.

    Mironov, Aleksei; Denisov, Stepan; Gress, Alexander; Kalinina, Olga V; Pervouchine, Dmitri D; HIPS, Helmholtz-Institut für Pharmazeutische Forschung Saarland, Universitätscampus E8.1 66123 Saarbrücken, Germany. (PLOS, 2021-04-07)
    Tandem alternative splice sites (TASS) is a special class of alternative splicing events that are characterized by a close tandem arrangement of splice sites. Most TASS lack functional characterization and are believed to arise from splicing noise. Based on the RNA-seq data from the Genotype Tissue Expression project, we present an extended catalogue of TASS in healthy human tissues and analyze their tissue-specific expression. The expression of TASS is usually dominated by one major splice site (maSS), while the expression of minor splice sites (miSS) is at least an order of magnitude lower. Among 46k miSS with sufficient read support, 9k (20%) are significantly expressed above the expected noise level, and among them 2.5k are expressed tissue-specifically. We found significant correlations between tissue-specific expression of RNA-binding proteins (RBP), tissue-specific expression of miSS, and miSS response to RBP inactivation by shRNA. In combination with RBP profiling by eCLIP, this allowed prediction of novel cases of tissue-specific splicing regulation including a miSS in QKI mRNA that is likely regulated by PTBP1. The analysis of human primary cell transcriptomes suggested that both tissue-specific and cell-type-specific factors contribute to the regulation of miSS expression. More than 20% of tissue-specific miSS affect structured protein regions and may adjust protein-protein interactions or modify the stability of the protein core. The significantly expressed miSS evolve under the same selection pressure as maSS, while other miSS lack signatures of evolutionary selection and conservation. Using mixture models, we estimated that not more than 15% of maSS and not more than 54% of tissue-specific miSS are noisy, while the proportion of noisy splice sites among non-significantly expressed miSS is above 63%.
  • Search for the Active Ingredients from a 2-Aminothiazole DMSO Stock Solution with Antimalarial Activity.

    Ropponen, Henni-Karoliina; Bader, Chantal D; Diamanti, Eleonora; Illarionov, Boris; Rottmann, Matthias; Fischer, Markus; Witschel, Matthias; Müller, Rolf; Hirsch, Anna K H; HIPS, Helmholtz-Institut für Pharmazeutische Forschung Saarland, Universitätscampus E8.1 66123 Saarbrücken, Germany. (Wiley-VCH, 2021-04-12)
    Chemical decomposition of DMSO stock solutions is a common incident that can mislead biological screening campaigns. Here, we share our case study of 2-aminothiazole 1, originating from an antimalarial class that undergoes chemical decomposition in DMSO at room temperature. As previously measured biological activities observed against Plasmodium falciparum NF54 and for the target enzyme PfIspE were not reproducible for a fresh batch, we tackled the challenge to understand where the activity originated from. Solvent- and temperature-dependent studies using HRMS and NMR spectroscopy to monitor the decomposition led to the isolation and in vitro evaluation of several fractions against PfIspE. After four days of decomposition, we successfully isolated the oxygenated and dimerised compounds using SFC purification and correlated the observed activities to them. Due to the unstable nature of the two isolates, it is likely that they undergo further decomposition contributing to the overall instability of the compound.
  • miRMaster 2.0: multi-species non-coding RNA sequencing analyses at scale.

    Fehlmann, Tobias; Kern, Fabian; Laham, Omar; Backes, Christina; Solomon, Jeffrey; Hirsch, Pascal; Volz, Carsten; Müller, Rolf; Keller, Andreas; HIPS, Helmholtz-Institut für Pharmazeutische Forschung Saarland, Universitätscampus E8.1 66123 Saarbrücken, Germany. (Oxford University Press, 2021-04-19)
    Analyzing all features of small non-coding RNA sequencing data can be demanding and challenging. To facilitate this process, we developed miRMaster. After the analysis of over 125 000 human samples and 1.5 trillion human small RNA reads over 4 years, we present miRMaster 2 with a wide range of updates and new features. We extended our reference data sets so that miRMaster 2 now supports the analysis of eight species (e.g. human, mouse, chicken, dog, cow) and 10 non-coding RNA classes (e.g. microRNAs, piRNAs, tRNAs, rRNAs, circRNAs). We also incorporated new downstream analysis modules such as batch effect analysis or sample embeddings using UMAP, and updated annotation data bases included by default (miRBase, Ensembl, GtRNAdb). To accommodate the increasing popularity of single cell small-RNA sequencing data, we incorporated a module for unique molecular identifier (UMI) processing. Further, the output tables and graphics have been improved based on user feedback and new output formats that emerged in the community are now supported (e.g. miRGFF3). Finally, we integrated differential expression analysis with the miRNA enrichment analysis tool miEAA. miRMaster is freely available at https://www.ccb.uni-saarland.de/mirmaster2.
  • Baikalomycins A-C, New Aquayamycin-Type Angucyclines Isolated from Lake Baikal Derived sp. IB201691-2A.

    Voitsekhovskaia, Irina; Paulus, Constanze; Dahlem, Charlotte; Rebets, Yuriy; Nadmid, Suvd; Zapp, Josef; Axenov-Gribanov, Denis; Rückert, Christian; Timofeyev, Maxim; Kalinowski, Jörn; et al. (MDPI, 2020-05-07)
    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.
  • Diversity of Isolated from Date Palms Rhizosphere and Saline Environments: Isolation, Identification and Biological Activity Evaluation.

    Messaoudi, Omar; Wink, Joachim; Bendahou, Mourad; HZI,Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7,38124 Braunschweig, Germany. (MDPI, 2020-11-25)
    The diversity of cultural Actinobacteria in two types of Algerian Sahara environments, including saline environments and date palms rhizosphere, was investigated. In this study, a total of 40 strains of actinomycetes was isolated from different soil samples, using a rehydration and centrifugation method. Molecular identification, based on 16S rRNA gene sequence analysis, revealed that these isolates were affiliated to six clusters corresponding to eight genera, including Streptomyces, Nocardiopsis, Saccharopolyspora, Actinomadura, Actinocorallia, Micromonospora, Couchioplanes, and Planomonospora. A taxonomic analysis, based on the morphological, physiological, biochemical, and molecular investigation, of selected strains, which belong to the rare Actinobacteria, was undertaken. Four strains (CG3, A111, A93, and A79) were found to form distinct phyletic lines and represent new actinobacterial taxa. An assessment of antimicrobial proprieties of the 40 obtained actinomycetes strains, showed moderate to strong antimicrobial activities against fungi and bacteria. This study demonstrated the richness of Algerian Sahara with rare Actinobacteria, which can provide novel bioactive metabolites, to solving some of the most challenging problems of the day, such as multi-drug resistance.
  • In vivo and in vitro reconstitution of unique key steps in cystobactamid antibiotic biosynthesis.

    Groß, Sebastian; Schnell, Bastien; Haack, Patrick A; Auerbach, David; Müller, Rolf; HIPS, Helmholtz-Institut für Pharmazeutische Forschung Saarland, Universitätscampus E8.1 66123 Saarbrücken, Germany. (Nature Research, 2021-03-16)
    Cystobactamids are myxobacteria-derived topoisomerase inhibitors with potent anti-Gram-negative activity. They are formed by a non-ribosomal peptide synthetase (NRPS) and consist of tailored para-aminobenzoic acids, connected by a unique α-methoxy-L-isoasparagine or a β-methoxy-L-asparagine linker moiety. We describe the heterologous expression of the cystobactamid biosynthetic gene cluster (BGC) in Myxococcus xanthus. Targeted gene deletions produce several unnatural cystobactamids. Using in vitro experiments, we reconstitute the key biosynthetic steps of linker formation and shuttling via CysB to the NRPS. The biosynthetic logic involves a previously uncharacterized bifunctional domain found in the stand-alone NRPS module CysH, albicidin biosynthesis and numerous BGCs of unknown natural products. This domain performs either an aminomutase (AM) or an amide dehydratase (DH) type of reaction, depending on the activity of CysJ which hydroxylates CysH-bound L-asparagine. Furthermore, CysQ O-methylates hydroxyl-L-(iso)asparagine only in the presence of the AMDH domain. Taken together, these findings provide direct evidence for unique steps in cystobactamid biosynthesis.
  • Amycolatomycins A and B, Cyclic Hexapeptides Isolated from an amycolatopsis sp. 195334CR.

    Primahana, Gian; Risdian, Chandra; Mozef, Tjandrawati; Wink, Joachim; Surup, Frank; Stadler, Marc; HZI,Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7,38124 Braunschweig, Germany. (MDPI, 2021-03-05)
    The rare actinobacterium Amycolatopsis sp. strain 195334CR was found to produce previously undescribed cyclic hexapeptides, which we named amycolatomycin A and B (1 and 2). Their planar structures were determined by high-resolution mass spectrometry as well as extensive 1D and 2D NMR spectroscopy, while the absolute stereochemistry of its amino acids were determined by Marfey's method. Moreover, 1 and 2 differ by the incorporation of l-Ile and l-allo-Ile, respectively, whose FDVA (Nα-(2,4-Dinitro-5-fluorphenyl)-L-valinamide) derivatives were separated on a C4 column. Their hallmark in common is a unique 2,6-dichloro-tryptophan amino acid unit. Amycolatomycin A (1) exhibited weak activity against Bacillus subtilis DSM 10 (minimum inhibitory concentration (MIC) = 33.4 µg/mL).
  • Streptomyces bathyalis sp. nov., an actinobacterium isolated from the sponge in a deep sea.

    Risdian, Chandra; Landwehr, Wiebke; Rohde, Manfred; Schumann, Peter; Hahnke, Richard L; Spröer, Cathrin; Bunk, Boyke; Kämpfer, Peter; Schupp, Peter J; Wink, Joachim; et al. (Springer, 2021-02-17)
    A novel actinobacterium, designated ASO4wetT, was isolated from the unidentified sponge (SO4) in the deep sea collected of the North Atlantic Ocean. Study of 16S rRNA gene sequences indicated that strain ASO4wetT is a member of the genus Streptomyces and showed the closest similarities to Streptomyces karpasiensis K413T (98.87 %), Streptomyces glycovorans YIM M 10366T (98.38 %), and Streptomyces abyssalis YIM M 10400T (97.53 %). Strain ASO4wetT contained MK-9(H8) as the predominant menaquinone and the major fatty acids are iso-C16:0, anteiso-C15:0, and iso-C15:0. Polyphasic taxonomy was carried out between strain ASO4wetT and its phylogenetically closely related Streptomyces strains, which further elucidated their relatedness and revealed that strain ASO4wetT could be distinguished from currently known Streptomyces species. Strain ASO4wetT clearly represents a novel species in genus Streptomyces. We propose the name Streptomyces bathyalis sp. nov., with the type strain ASO4wetT (= DSM 106605T = NCCB 100657T). Analysis of the whole-genome sequence of S. bathyalis revealed that genome size is 7,377,472 bp with 6332 coding sequences.
  • Kibdelosporangium persicum sp. nov., a new member of the Actinomycetes from a hot desert in Iran.

    Safaei, Nasim; Nouioui, Imen; Mast, Yvonne; Zaburannyi, Nestor; Rohde, Manfred; Schumann, Peter; Müller, Rolf; Wink, Joachim; HZI,Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7,38124 Braunschweig, Germany.;HIPS, Helmholtz-Institut für Pharmazeutische Forschung Saarland, Universitätscampus E8.1 66123 Saarbrücken, Germany. (Microbiology Society, 2021-01-11)
    Isolate 4NS15T was isolated from a neglected arid habitat in Kerman, Iran. The strain showed 16S rRNA gene sequence similarity values of 98.9 % to the type strains of Kibdelosporangium aridum subsp. aridum, Kibdelosporangium phytohabitans and Kibdelosporangium philippinense and 98.6 % to the type strain K. aridum subsp. largum, respectively. Genome-based phylogenetic analysis revealed that isolate 4NS15T is closely related to Kibdelosporangium aridum subsp. aridum DSM 43828T. The digital DNA-DNA hybridization value between the genome sequences of 4NS15T and strain DSM 43828T is 29.8 %. Strain 4NS15T produces long chains of spores without a sporangium-like structure which can be distinguished from other Kibdelosporangium species. Isolate 4NS15T has a genome size of 10.35 Mbp with a G+C content of 68.1 mol%. Whole-cell hydrolysates of isolate 4NS15T are rich in meso-diaminopimelic acid and cell-wall sugars such as arabinose, galactose, glucose and ribose. Major fatty acids (>10 %) are C16 : 0, iso-C16 : 0 and iso-C15 : 0. The phospholipid profile contains diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylhydroxyethanolamine, aminolipid and glycoaminolipid. The predominant menaquinone is MK-9(H4). Based on its phenotypic and genotypic characteristics, isolate 4NS15T (NCCB 100701=CIP 111705=DSM 110728) merits recognition as representing a novel species of the genus Kibdelosporangium, for which the name Kibdelosporangium persicum sp. nov. is proposed.
  • Bottromycins - biosynthesis, synthesis and activity.

    Franz, Laura; Kazmaier, Uli; Truman, Andrew W; Koehnke, Jesko; HIPS, Helmholtz-Institut für Pharmazeutische Forschung Saarland, Universitätscampus E8.1 66123 Saarbrücken, Germany. (Royal Chemistry Society, 2021-02-23)
  • Multiple copies of the oxytetracycline gene cluster in selected Streptomyces rimosus strains can provide significantly increased titers.

    Pikl, Špela; Carrillo Rincón, Andrés Felipe; Slemc, Lucija; Goranovič, Dušan; Avbelj, Martina; Gjuračić, Krešimir; Sucipto, Hilda; Stare, Katja; Baebler, Špela; Šala, Martin; et al. (BioMedCentral, 2021-02-17)
    Background: Natural products are a valuable source of biologically active compounds that have applications in medicine and agriculture. One disadvantage with natural products is the slow, time-consuming strain improvement regimes that are necessary to ensure sufficient quantities of target compounds for commercial production. Although great efforts have been invested in strain selection methods, many of these technologies have not been improved in decades, which might pose a serious threat to the economic and industrial viability of such important bioprocesses. Results: In recent years, introduction of extra copies of an entire biosynthetic pathway that encodes a target product in a single microbial host has become a technically feasible approach. However, this often results in minor to moderate increases in target titers. Strain stability and process reproducibility are the other critical factors in the industrial setting. Industrial Streptomyces rimosus strains for production of oxytetracycline are one of the most economically efficient strains ever developed, and thus these represent a very good industrial case. To evaluate the applicability of amplification of an entire gene cluster in a single host strain, we developed and evaluated various gene tools to introduce multiple copies of the entire oxytetracycline gene cluster into three different Streptomyces rimosus strains: wild-type, and medium and high oxytetracycline-producing strains. We evaluated the production levels of these engineered S. rimosus strains with extra copies of the oxytetracycline gene cluster and their stability, and the oxytetracycline gene cluster expression profiles; we also identified the chromosomal integration sites. Conclusions: This study shows that stable and reproducible increases in target secondary metabolite titers can be achieved in wild-type and in high oxytetracycline-producing strains, which always reflects the metabolic background of each independent S. rimosus strain. Although this approach is technically very demanding and requires systematic effort, when combined with modern strain selection methods, it might constitute a very valuable approach in industrial process development.
  • Natural products in drug discovery: advances and opportunities.

    Atanasov, Atanas G; Zotchev, Sergey B; Dirsch, Verena M; Supuran, Claudiu T; HZI,Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7,38124 Braunschweig, Germany.; HIPS, Helmholtz-Institut für Pharmazeutische Forschung Saarland, Universitätscampus E8.1 66123 Saarbrücken, Germany. (Springer Nature, 2021-01-28)
    Natural products and their structural analogues have historically made a major contribution to pharmacotherapy, especially for cancer and infectious diseases. Nevertheless, natural products also present challenges for drug discovery, such as technical barriers to screening, isolation, characterization and optimization, which contributed to a decline in their pursuit by the pharmaceutical industry from the 1990s onwards. In recent years, several technological and scientific developments - including improved analytical tools, genome mining and engineering strategies, and microbial culturing advances - are addressing such challenges and opening up new opportunities. Consequently, interest in natural products as drug leads is being revitalized, particularly for tackling antimicrobial resistance. Here, we summarize recent technological developments that are enabling natural product-based drug discovery, highlight selected applications and discuss key opportunities.
  • Enhancing glycan stabilityviasite-selective fluorination: modulating substrate orientation by molecular design

    Axer, Alexander; Jumde, Ravindra P.; Adam, Sebastian; Faust, Andreas; Schäfers, Michael; Fobker, Manfred; Koehnke, Jesko; Hirsch, Anna K.H.; Gilmour, Ryan; HIPS, Helmholtz-Institut für Pharmazeutische Forschung Saarland, Universitätscampus E8.1 66123 Saarbrücken, Germany. (Royal Chemistry Society (RCS), 2021-01-28)
    ingle site OH → F substitution at the termini of maltotetraose leads to significantly improved hydrolytic stability towards α-amylase and α-glucosidase relative to the natural compound. To explore the effect of molecular editing, selectively modified oligosaccharides were preparedviaa convergent α-selective strategy. Incubation experiments in purified α-amylase and α-glucosidase, and in human and murine blood serum, provide insight into the influence of fluorine on the hydrolytic stability of these clinically important scaffolds. Enhancements ofca. 1 order of magnitude result from these subtle single point mutations. Modification at the monosaccharide furthest from the probable enzymatic cleavage termini leads to the greatest improvement in stability. In the case of α-amylase, docking studies revealed that retentive C2-fluorination at the reducing end inverts the orientation in which the substrate is bound. A co-crystal structure of human α-amylase revealed maltose units bound at the active-site. In view of the evolving popularity of C(sp3)-F bioisosteres in medicinal chemistry, and the importance of maltodextrins in bacterial imaging, this discovery begins to reconcile the information-rich nature of carbohydrates with their intrinsic hydrolytic vulnerabilities. © The Royal Society of Chemistry 2020.
  • 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.

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