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Glycopeptide antibiotics (GPA), which include vancomycin and teicoplanin, are important lastresort antibiotics used to treat multidrug-resistant Gram-positive bacterial infections. Whilst second generation GPAs – generated through chemical modification of natural GPAs – have proven successful, the emergence of GPA resistance has underlined the need to develop new members of this compound class. Significant recent advances have been made in GPA research, including gaining an in-depth understanding of their biosynthesis, improving titre in production strains, developing new derivatives via novel chemical modifications, and identifying a new mode of action for structurally diverse Type V GPAs. Taken together, these advances demonstrate significant untapped potential for the further development of GPAs to tackle the growing threat of multidrug resistant bacteria.

An ever-increasing demand for novel antimicrobials to treat life-threatening infections caused by the global spread of multidrug-resistant bacterial pathogens stands in stark contrast to the current level of investment in their development, particularly in the fields of natural-product-derived and synthetic small molecules. New agents displaying innovative chemistry and modes of action are desperately needed worldwide to tackle the public health menace posed by antimicrobial resistance. Here, our consortium presents a strategic blueprint to substantially improve our ability to discover and develop new antibiotics. We propose both short-term and long-term solutions to overcome the most urgent limitations in the various sectors of research and funding, aiming to bridge the gap between academic, industrial and political stakeholders, and to unite interdisciplinary expertise in order to efficiently fuel the translational pipeline for the benefit of future generations.

By culturing microorganisms under standard laboratory conditions, most biosynthetic gene clusters (BGCs) are not expressed, and thus, the products are not produced. To explore this biosynthetic potential, we developed a novel “semi-targeted” approach focusing on activating “silent” BGCs by concurrently introducing a group of regulator genes into streptomycetes of the Tübingen strain collection. We constructed integrative plasmids containing two classes of regulatory genes under the control of the constitutive promoter ermE*p (cluster situated regulators (CSR) and Streptomyces antibiotic regulatory proteins (SARPs)). These plasmids were introduced into Streptomyces sp. TÜ17, Streptomyces sp. TÜ10 and Streptomyces sp. TÜ102. Introduction of the CSRs-plasmid into strain S. sp. TÜ17 activated the production of mayamycin A. By using the individual regulator genes, we proved that Aur1P, was responsible for the activation. In strain S. sp. TÜ102, the introduction of the SARP-plasmid triggered the production of a chartreusin-like compound. Insertion of the CSRs-plasmid into strain S. sp. TÜ10 resulted in activating the warkmycin-BGC. In both recombinants, activation of the BGCs was only possible through the simultaneous expression of aur1PR3 and griR in S. sp. TÜ102 and aur1P and pntR in of S. sp. TÜ10.

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We report the draft genome sequence of Microbispora sp. strain ATCC-PTA-5024, a soil isolate that produces NAI-107, a new lantibiotic with the potential to treat life-threatening infections caused by multidrug-resistant Gram-positive pathogens. The draft genome of strain Microbispora sp. ATCC-PTA-5024 consists of 8,543,819 bp, with a 71.2% G+C content and 7,860 proteincoding genes.

A wide variety of enzymatic pathways that produce specialized metabolites in bacteria, fungi and plants are known to be encoded in biosynthetic gene clusters. Information about these clusters, pathways and metabolites is currently dispersed throughout the literature, making it difficult to exploit. To facilitate consistent and systematic deposition and retrieval of data on biosynthetic gene clusters, we propose the Minimum Information about a Biosynthetic Gene cluster (MIBiG) data standard.

Zinc is a crucial trace element for bacteria and plays an essential role in several physiological processes, including DNA replication and metabolism. Bacteria have evolved various mechanisms to acquire zinc from the environment, such as the production of zincophores. One example is the zincophore [S,S]-EDDS produced by Amycolatopsis japonicum. [S,S]-EDDS is an isomer of ethylenediamine tetraacetate (EDTA), which is widely used in industry. However, unlike EDTA, [S,S]-EDDS is biodegradable, making it a promising alternative with a favorable environmental profile. Besides its application in various industrial processes, [S,S]- EDDS is also interesting for agriculture. These two aspects were investigated in the present work. (i) [S,S]-EDDS has demonstrated efficacy as a Fe2+ and Zn2+ fertilizer, but its short degradation time limits its long-term impact. To overcome this limitation, a promising approach is to use the [S,S]-EDDS producer A. japonicum as a biofertilizer. Continuous production of [S,S]-EDDS would avoid the need for multiple applications of [S,S]-EDDS. The experiments carried out with Phaseolus vulgaris cv. Black pole in calcareous soil showed that the use of A. japonicum as a biofertilizer, however, did not lead to any improvement in plant growth or microelement concentration. (ii) For the use of [S,S]-EDDS as a chelating agent in various industrial applications, the yield of [S,S]-EDDS in A. japonicum needs to be improved. A prerequisite for this is the understanding of the biosynthesis of [S,S]-EDDS. Using a wide range of methods (biochemistry, genetics, and labeling studies) it was confirmed that L-aspartic acid and oxaloacetic acid are precursors for [S,S]-EDDS biosynthesis. In addition, ((S)-2-amino-2-carboxyethyl)-L-aspartic acid (ACEAA) was identified for the first time as a biosynthetic intermediate. Furthermore, the results strongly indicated that the previously proposed precursor, L-diaminopropionic acid (L-DAPP), is not involved in the biosynthesis, suggesting a novel biosynthetic pathway for [S,S]-EDDS. Genetic strategies were used to redirect the metabolic flux towards the desired metabolites to further optimize [S,S]-EDDS yields. To prioritize target genes for metabolic engineering, the bioinformatic tool Secondary Metabolite Transcriptomic Pipeline (Sema-Trap) was used for RNA-Seq-based transcriptome analysis. This identified bldCAj, lacIAj and gltsAj as target genes for engineering in A. japonicum. Overexpression of these genes resulted in a 3-fold increase in [S,S]-EDDS production compared to A. japonicum wild type. Taken together, these findings provide the potential for further progress in optimizing [S,S]-EDDS production. Die Dissertation ist gesperrt bis zum 20. Juni 2025 !

Although they are only needed in small amounts, trace elements, e.g. zinc or iron, are still essential for all living organisms. To facilitate the uptake of those trace elements, bacteria are able to produce so-called metallophores, which are usually low molecular weight compounds. These naturally produced chelators (ion complexing agents) are secreted to extracellularly bind hardly soluble metal ions. When metal ions are complexed by metallophores, their solubility increases and thus their uptake improves. However, not only bacteria rely on chelators to increase the solubility and bioavailability of metal ions. The synthetic chelator ethylenediaminetetraacetic acid (EDTA) e.g. is one of the most abundantly used commercial complexing agents. Its field of application ranges from an ingredient in various washing agents, cosmetics or fertilizers to a use as additive in foods and medicinal products. Due to a combination of both, the abundant industrial usage as well as a poor biodegradability, the synthetic chelator EDTA has accumulated in rivers and lakes in the last decades. Since this poses a putative environmental threat, there is an ongoing effort to replace EDTA with a sustainable alternative with a higher biodegradability, while having similar chelating capacities. One of the compounds that is considered as a potential substitute is ethylenediaminedisuccinc acid (EDDS). EDDS is a structural isomer of EDTA and is naturally produced by various species of actinomycete bacteria of the genus Amycolatopsis, e.g. by Amycolatopsis japonicum. Although both compounds share similar chelating capacities, the [S,S]-isomer of EDDS is extremely-well biodegradable compared to EDTA, making it an efficient as well as a sustainable alternative to EDTA. Since the biosynthesis of [S,S]-EDDS in A. japonicum is zinc regulated, the metallophore is presumably used as zincophore in order to facilitate the uptake of zinc. However, even trace amounts of zinc, which occur ubiquitously in glass or steel fermenters, are able to inhibit the production of [S,S]-EDDS. Aiming at an economical biotechnological production of this compound, the production of [S,S]-EDDS in A. japonicum was optimized by metabolic engineering. Since both the biosynthesis genes as well as the molecular mechanisms of the zinc regulation had already been elucidated in this strain, the first step of the optimization process focused on the exchange of the native promoter of the [S,S]-EDDS biosynthesis genes with a strong, constitutive one. Hence, the zinc inhibition by the so called Zur-regulator was abolished and the productivity increased. Furthermore, additional copies of the biosynthesis genes were introduced and the supply of the precursor O-phosphoserine was optimized. This optimization process resulted in an increase of [S,S]-EDDS production in A. japonicum from 0.3 g/L to 3.0 g/L when minimal medium was used in small scale cultivation. Moreover, when tested in complex medium in bioreactors, the newly generated [S,S]-EDDS-producer strain reached a production titer of 9.8 g/L. Another focus of this work lies on investigating the distribution of the potential to produce [S,S]-EDDS or EDDS-like compounds in actinomycetes and their functional role in the producer strains. A MultiGene-Blast analysis searching for EDDS-like biosynthesis proteins in actinomycetes genomes led to the discovery of a new biosynthesis gene cluster, which is present is various species from different actinomycete genera. Based on the high similarity of the genes in this cluster to the known genes of the EDDS biosynthesis as well as to another known gene of the viomycin-biosynthesis, the metallophore ethylendiaminesuccinic acid hydroxyarginine (EDHA) could be assigned as the putative gene cluster product. Although the compound EDHA had already been discovered, the corresponding gene cluster has not been described so far. Bioinformatic analysis of two EDHA cluster containing strains, Streptomyces scabies and Streptomyces sp. MA5143a, revealed a putative DNA binding motif of an IdeR-regulator in this gene cluster. These IdeR regulators are known to be iron dependent, DNA binding proteins, which regulate the expression of siderophore biosynthesis clusters in Gram-positive bacteria. Transcription of the EDHA biosynthesis genes in those two strains was analyzed by reverse transcriptase and polymerase chain reaction (RT-PCR) and was shown to be iron repressed. Additionally, in both strains iron inhibited EDHA production could be detected via high performance liquid chromatography-electrospray ionization-mass spectrometry (HPLC-ESI-MS). These experiments resulted in the conclusion, that the EDDS-like metallophore EDHA in contrast to EDDS presumably functions as siderophore to facilitate the iron uptake in the strains S. scabies und Streptomyces sp. MA5143a.

The microbial secondary metabolism is a rich source for valuable products that have found their way into various clinical and industrial applications. A particularly productive bacterial genus for the discovery of natural products is Amycolatopsis. The most frequently reported type of secondary metabolites produced by this genus, are glycopeptide antibiotics like balhimycin or the medically relevant vancomycin. In contrast to most other members of the Amycolatopsis genus, Amycolatopsis japonicum was never described to produce any product with antibacterial activity. This strain however is known to synthesize the chelating agent ethylenediamine-disuccinate ([S,S]-EDDS), a biodegradable EDTA isomer in response to zinc deficiency. This zinc responsive repression of [S,S]-EDDS production indicates that it contributes to zinc uptake and that it belongs to the rarely described physiological group of the zincophores. Combining excellent chelating properties with the accessibility to biodegradation, [S,S]-EDDS is considered as a sustainable chelating agent, possessing the potential to replace EDTA and other environmentally threatening chelating agents in various applications. In this study, two distinct molecular genetic strategies were developed and implemented to activate the biosynthesis of the glycopeptide antibiotic ristomycin A or to identify the [S,S]-EDDS biosynthetic genes in Amycolatopsis japonicum, respectively. Genetic evaluation of the Amycolatopsis antibiotic biosynthetic potential indicated that A. japonicum might has the capability to produce a glycopeptide antibiotic. Since the biosynthesis of the predicted glycopeptide was not inducible by variations in culture conditions, a molecular genetic approach was employed to activate its production. Heterologous expression of the characterized pathway specific activator Bbr, naturally inducing the balhimycin biosynthesis in A. balhimycina, also induced the synthesis of a bioactive substance by A. japonicum. The bioactivity could be assigned to the production of ristomycin A, a highly glycosylated peptide antibiotic which is used as compound in diagnostic kits to detect widespread hereditary coagulation disorders. Full sequencing of the A. japonicum genome and its computational analysis led to the identification of the corresponding biosynthetic gene cluster which is directing the biosynthesis of ristomycin A. Such computational genome analyses by various bioinformatic tools are nowadays standardized applied strategies to identify secondary metabolite gene clusters. These approaches however failed in the identification of the [S,S]-EDDS biosynthetic genes. This required the development of a new approach which relies on the assumption that the zinc repressed biosynthesis of [S,S]-EDDS is regulated by a zinc responsive regulatory element. Therefore, the major zinc responsive transcriptional regulator of A. japonicum (Zur) was characterized in detail. Zur regulates the expression of the high affinity zinc uptake system ZnuABC by binding to a specific DNA binding sequence. The screening of the A. japonicum genome for further Zur regulated genes by using this deduced Zur binding sequence led to the identification of the operon aesA-D. Extensive transcriptional analyses and band shift assays revealed that aesA-D is zinc responsively regulated by Zur and involved in [S,S]-EDDS biosynthesis, as shown by inactivation studies. The [S,S]-EDDS biosynthesis was uncoupled from zinc repression by deleting zur. This mutant sets the stage to establish a sustainable [S,S]-EDDS production process without limits formerly imposed by zinc repression. The strategy to awake predicted silent gene clusters by using a characterized regulator as well as the strategy to identify new biosynthetic genes by characterizing an environmental signal-sensing regulator enabled the isolation of novel biosynthetic pathways in A. japonicum. Both approaches follow the joint concept to exploit knowledge of regulatory pathways and have the prospect to be generally applicable in order to guide future detection of new natural products., Der mikrobielle Sekundärmetabolismus ist eine reichhaltige Quelle für Naturstoffe, von denen viele klinische beziehungsweise industrielle Anwendung gefunden haben. Die Gattung Amycolatopsis ist für die Synthese vieler Naturstoffe bekannt. Beispielsweise werden viele Glykopeptid-Antibiotika, wie das klinisch relevante Vancomycin oder das Balhimycin, von Stämmen dieser Gattung produziert. Im Gegensatz dazu wurde der Stamm Amycolatopsis japonicum nie als Produzent einer biologisch aktiven Substanz beschrieben. Dieser Stamm produziert jedoch unter Zinkmangelbedingungen das EDTA-Isomer Ethylendiamindisuccinat ([S,S]-EDDS). Diese zinkabhängige [S,S]-EDDS Produktion lässt darauf schließen, dass [S,S]-EDDS ein Zinkophor ist, das an der Zinkaufnahme beteiligt ist. [S,S]-EDDS weist Komplexbildungseigenschaften auf, die mit denen von EDTA vergleichbar sind. Im Gegensatz zu EDTA ist [S,S]-EDDS jedoch biologisch abbaubar. Die weite industrielle Anwendung von EDTA in Kombination mit dessen Unzugänglichkeit für biologische Abbauprozesse führt zu einer umweltgefährdenden EDTA-Persistenz in aquatischen Lebensräumen. Der Naturstoff [S,S]-EDDS ist deshalb ein nachhaltiger EDTA Ersatz mit einem verbesserten ökologischen Fingerabdruck. In dieser Arbeit wurden zwei molekulargenetische Strategien entwickelt, um die Biosynthese des Glykopeptid-Antibiotikums Ristomycin A zu aktivieren und um die [S,S]-EDDS-Biosynthese-Gene in A. japonicum zu identifizieren. Untersuchungen des genetischen Potenzials der Gattung Amycolatopsis ließen vermuten, dass auch A. japonicum die Fähigkeit besitzt, ein Glykopeptid-Antibiotikum zu synthetisieren. Um dieses nicht exprimierte, sogenannte „stille Gencluster“ zu aktivieren, wurde ein molekulargenetischer Ansatz verwendet, bei dem der Biosynthese-spezifische Aktivator Bbr heterolog in A. japonicum exprimiert wurde. Bbr reguliert die Balhimycin-Biosynthese in Amycolatopsis balhimycina. In A. japonicum induzierte dessen Expression die Produktion von Ristomycin A, was durch HPLC-DAD, MS, MS/MS, HR-MS, und NMR-Analysen bestätigt werden konnte. Ristomycn A ist ein vielfach glykosyliertes Heptapeptid, das als Hauptwirkstoff in Diagnoseverfahren zur Bestimmung von angeborenen und weitverbreiteten Blutgerinnungsstörungen verwendet wird. Die Sequenzierung des A. japonicum Genoms und dessen computergestützte Auswertung führten zur Identifizierung des Biosynthese-Genclusters, das für die Synthese von Ristomycin A verantwortlich ist. Solche computergestützten Genomanalysen mittels verschiedenster bioinformatischen Plattformen werden heutzutage standardmäßig zur Identifizierung von Sekundärmetabolit-Gencluster angewandt, die bekannten Synthesemechanismen zugeordnet werden können. Allerdings konnten die [S,S]-EDDS-Biosynthese-Gene mit diesen Tools nicht entdeckt werden, was auf einen bislang nicht bekannten Biosynthesemechanismus hindeutet. Um diesen zu identifizieren, wurde ein neuer Ansatz entwickelt, der auf der Annahme beruht, dass die Zink-reprimierte [S,S]-EDDS-Biosynthese durch einen Zink-sensitiven Regulator gewährleistet wird. Die bakterielle Zink-Homöostase wird meistens durch den globalen Zink-spezifische Transkriptionsregulator Zur reguliert. Das Zur Protein von A. japonicum wurde identifiziert und detailliert charakterisiert. Es konnten gezeigt werden, dass ZurAj die Transkription des hoch affinen Zinkaufnahmesystems ZnuABCAj durch seine Zink-abhängige Bindung an spezifische DNA Bindesequenzen reguliert. Diese Zur-Bindesequenzen wurden verwendet, um das A. japonicum Genom nach weiteren, ZurAj regulierten, Genen zu durchsuchen. Dies führte zur Auffindung des aesA-D Operons. Umfangreiche Transkriptions-Untersuchungen ergaben, dass aesA-D Zink-abhängig von ZurAj reguliert wird. Die Beteiligung von aesA-D an der [S,S]-EDDS konnte durch Inaktivierungsversuche nachgewiesen werden. Zusätzlich führte die Deletion des Zinkregulators ZurAj (A. japonicum Δzur) dazu, dass auch in Gegenwart von hohen Zink-Konzentrationen [S,S]-EDDS in hohen Mengen produziert wird. A. japonicum Δzur ist eine erfolgversprechende Ausgangsbasis, um einen nachhaltigen und wirtschaftlich verwertbaren [S,S]-EDDS Produktionsprozess zu entwickeln, der keiner Limitierung durch negative Einflüsse von Zink unterliegt. Die Strategie, ein vorhergesagtes, stilles Gencluster durch die Expression eines spezifischen Regulators zu aktivieren, sowie auch die Strategie, neue Biosynthese-Gene durch die Charakterisierung eines globalen Regulators, der spezifische Umweltsignale wahrnimmt, zu identifizieren, ermöglichte die Charakterisierung neuer Naturstoffsynthesewege in A. japonicum. Beide Ansätze nutzen Erkenntnisse über regulatorische Mechanismen und besitzen das Potenzial zukünftig angewendet zu werden, um neue Naturstoffe und neue Synthesewege zu identifizieren.