Bacillus subtilis as heterologous host for the secretory production of the non-ribosomal cyclodepsipeptide enniatin

The heterologous expression of genes or gene clusters in microbial hosts, followed by metabolic engineering of biosynthetic pathways, is key to access industrially and pharmaceutically relevant compounds in an economically affordable and sustainable manner. Therefore, platforms need to be developed, which provide tools for the controlled synthesis of bioactive compounds. The Gram-positive bacterium Bacillus subtilis is a promising candidate for such applications, as it is generally regarded as a safe production host, its physiology is well investigated and a variety of tools is available for its genetic manipulation. Furthermore, this industrially relevant bacterium provides a high secretory potential not only for enzymes but also for primary and secondary metabolites. In this study, we present the first heterologous expression of an eukaryotic non-ribosomal peptide synthetase gene (esyn) coding for the biosynthesis of the small molecule enniatin in B. subtilis. Enniatin is a pharmaceutically used cyclodepsipeptide for treatment of topical bacterial and fungal infections. We generated various enniatin-producing B. subtilis strains, allowing for either single chromosomal or plasmid-based multi-copy expression of the esyn cluster under the control of an acetoin-inducible promoter system. Optimization of cultivation conditions, combined with modifications of the genetic background and multi-copy plasmid-based esyn expression, resulted in a secretory production of enniatin B. This work presents B. subtilis as a suitable host for the expression of heterologous eukaryotic non-ribosomal peptide synthetases (NRPS) clusters

Insect‐associated bacteria assemble the antifungal butenolide gladiofungin by non‐canonical polyketide chain termination

Genome mining of one of the protective symbionts ( Burkholderia gladioli ) of the invasive beetle Lagria villosa revealed a cryptic gene cluster that codes for the biosynthesis of a novel antifungal polyketide with a glutarimide pharmacophore. Targeted gene inactivation, metabolic profiling, and bioassays led to the discovery of the gladiofungins as previously‐overlooked components of the antimicrobial armory of the beetle symbiont, which are highly active against the entomopathogenic fungus Purpureocillium lilacinum . By mutational analyses, isotope labeling, and computational analyses of the modular polyketide synthase, we found that the rare butenolide moiety of gladiofungins derives from an unprecedented polyketide chain termination reaction involving a glycerol‐derived C3 building block. The key role of an A‐factor synthase (AfsA)‐like offloading domain was corroborated by CRISPR‐Cas‐mediated gene editing, which facilitated precise excision within a PKS domain

Multimodal Molecular Imaging and Identification of Bacterial Toxins Causing Mushroom Soft Rot and Cavity Disease

Soft rot disease of edible mushrooms leads to rapid degeneration of fungal tissue and thus severely affects farming productivity worldwide. The bacterial mushroom pathogen Burkholderia gladioli pv. agaricicola has been identified as the cause. Yet, little is known about the molecular basis of the infection, the spatial distribution and the biological role of antifungal agents and toxins involved in this infectious disease. We combine genome mining, metabolic profiling, MALDI-Imaging and UV Raman spectroscopy, to detect, identify and visualize a complex of chemical mediators and toxins produced by the pathogen during the infection process, including toxoflavin, caryoynencin, and sinapigladioside. Furthermore, targeted gene knockouts and in vitro assays link antifungal agents to prevalent symptoms of soft rot, mushroom browning, and impaired mycelium growth. Comparisons of related pathogenic, mutualistic and environmental Burkholderia spp. indicate that the arsenal of antifungal agents may have paved the way for ancestral bacteria to colonize niches where frequent, antagonistic interactions with fungi occur. Our findings not only demonstrate the power of label-free, in vivo detection of polyyne virulence factors by Raman imaging, but may also inspire new approaches to disease control. © 2021 The Authors. ChemBioChem published by Wiley-VCH Gmb

Heterologe Produktion des Polyketids 6-Desoxyerythronolid B und des nichtribosomalen Peptids Enniatin B in Bacillus subtilis

Natürliche Metabolite sind Ausgangsstoff für eine Reihe von Arzneimitteln wie zum Beispiel Antibiotika. Doch bis zur Anwendung am Menschen sind viele Analyseschritte notwendig. Da viele Naturstoffe nicht in ausreichenden Mengen zur Verfügung gestellt werden können, wird deren Funktionsanalyse und Anwendung erschwert. Für dieses Defizit sind im Wesentlichen zwei Ursachen zu nennen. Entweder ist eine Vielzahl der Produzenten nicht kultivierbar oder eine ausreichende Synthese ist unter Laborbedingungen im Ausgangsstamm nicht möglich. Aus diesem Grund sind alternative Strategien wie zum Beispiel eine heterologe Expression dieser Synthese-Cluster in geeigneten Wirten notwendig. Dies war der Ansatzpunkt für die vorliegende Arbeit. Eine besondere Bedeutung innerhalb der Naturstoffe kommt der strukturell diversen und mitunter sehr komplexen Gruppe der Polyketide und nichtribosomalen Peptide zu, die oft pharmazeutisch relevante Wirkungen aufweisen. Die für ihre Synthese verantwortlichen Enzyme (PKS und NRPS) sind häufig beachtliche Multienzymkomplexe, die durch Gencluster codiert werden, deren Größe von 10–100 kbp reichen kann. Bisher wurden für die heterologe Produktion dieser Metabolite in erster Linie Actinomyceten wie zum Beispiel Streptomyces coelicolor und Myxococcus xanthus, die selbst eine Vielzahl an Polyketiden und nichtribosomalen Peptiden synthetisieren, oder Escherichia coli genutzt. In der vorliegenden Arbeit wurde Bacillus subtilis, der bereits breite Anwendung in der industriellen Herstellung von technischen und pharmazeutischen Proteinen findet, erstmals als heterologer Wirt für die Synthese eines Polyketids (6-Desoxyerythronolid B) und eines nichtribosomalen Peptids (Enniatin B) eingesetzt. Zu diesem Zweck wurde ein Klonierungsprotokoll für die schnelle und wiederholte Genommodifizierung entwickelt. Dieses basiert auf der Kombination von transformationssteigernden Elementen (sogenannten six-sites) mit der chromosomalen Integration einer induzierbaren Kopie des Kompetenzfaktors ComS. Zur Markerentfernung wurde das Cre-lox-System implementiert. Durch die zusätzliche Deletion des Restriktions- und Modifikationssystems wurde eine weitere Voraussetzung zur chromosomalen Integration großer Gencluster geschaffen. Damit steht nun ein optimiertes Protokoll für die Konstruktion von B. subtilis-Expressionsstämmen und deren weiterer genomischer Modifizierung zur Verfügung. Als Vertreter einer komplexen Polyketidsynthase wurde die Desoxyerythronolid B-Synthase (DEBS) aus Saccharopolyspora erythraea ausgewählt. Dieser aus drei ca. 300–350 kDa großen Proteinen (DEBS1–3) bestehende Enzymkomplex ist für die Bildung des Makrolids 6-Desoxyerythronolid B (6dEB) verantwortlich, das die Vorstufe des antibiotisch wirksamen Erythromycins darstellt. Das korrespondierende Gencluster umfasst drei ca. 10 kb große Gene (eryAI–III) und konnte erfolgreich in drei Operonstrukturen im Genom von B. subtilis lokalisiert werden: als i) natürliches Operon, ii) modifiziertes Operon mit optimierten RBS und iii) drei separate Expressionskassetten. Unter fed-batch-simulierenden Bedingungen (EnBase-System) gelang dabei ein positiver Metabolitennachweis für den Stamm mit drei separaten Expressionskassetten. Um das Zellwachstum und die 6dEB-Synthese zu verbessern, wurden weiterführende Genommodifizierungen des Produktionsstammes vorgenommen, von denen sich einige positiv auf die Produktbildung auswirkten. Mit diesem Versuch wurde erstmals die prinzipielle Eignung von B. subtilis als heterologer Produzent für komplexe Polyketide erbracht. Die Enniatin-Synthetase (ESyn) aus dem filamentösen Pilz Fusarium oxysporum wurde als Beispiel einer nichtribosomalen Peptidsynthetase in die Arbeit einbezogen. Aufgrund der handhabaren Größe des esyn-Gens (10 kb) wurde die Expression auf single- und multi-copy-Level untersucht. Dabei wurde auch der Einfluss verschiedener Genommodifizierungen und Änderungen in den Wachstumsbedingungen auf die Produktbildung analysiert. Die Kultivierungsversuche inklusive Metabolitenanalyse wurden in Kooperation mit dem Institut für Biologische Chemie an der TU Berlin durchgeführt. Abschließende Konzentrationen des entsprechenden Metaboliten (Enniatin B), einem zyklischen Hexadepsipetid mit zahlreichen antiinfektiven Wirkungen, wurden auf 4,5 µg/L (single-copy) bzw. 1,2mg/L (multi-copy) beziffert. Damit konnte in B. subtilis zum ersten Mal die heterologe Produktion eines gattungsfremden nichtribosomalen Peptids demonstriert werden. Zusammengefasst beschreibt die präsentierte Arbeit die erfolgreiche Produktion eines komplexen Polyketids und eines nichtribosomalen Peptids. Obwohl weitere Untersuchungen notwendig sind, um einige unerwartete Effekte bestimmter Genommodifizierungen aufzuklären und eine weitere Steigerung in der Produktausbeute zu erreichen, konnte eindeutig belegt werden, dass sich B. subtilis als Wirt für die heterologe Produktion von Sekundärmetaboliten eignet.Secondary metabolites represent a frequently used resource for pharmaceutical drugs such as antibiotics or cytostatics. However, since most of the natural products cannot be supplied in sufficient amounts, their functional analysis as well as their application is a challenge. This is (in most cases) due to two major reasons: i) the natural producer is not cultivatable or ii) a sufficient synthesis in the natural host is impossible under laboratory conditions. Consequently, in order to improve the investigations in as well as the demand for secondary metabolites, alternative strategies are necessary. One option, the heterologous expression of the corresponding gene cluster in a suitable host-organism, served as the initial point of the present study. Within the secondary metabolites the structural diverse and quite complex groups of polyketides and non-ribosomal peptides are of particular significance since they often show a broad spectrum in bioactivities and therapeutic applications. They are produced by polyketide synthases (PKSs) and non-ribosomal peptide synthetases (NRPSs), large multi enzyme complexes which are encoded by gene clusters that can reach and exceed 10–100 kbp in length. So far heterologous production of these metabolites was predominantly achieved in actinomycetes hosts (that are themselves natural producers of a large number of polyketides and non-ribosomal peptides) or in E. coli. In the present work, the synthesis of a polyketide and a non-ribosomal peptide was successfully demonstrated in the gram-positive host-organism Bacillus subtilis for the first time. To this purpose, a cloning protocol for rapid and multiple genome modification was developed. This protocol is based on transformation enhancing elements (so called six-sites) that were combined with an inducible gene copy of the competence factor ComS for an increased natural competence. For marker removal the modified Cre-lox-system was included. Additionally, in order to allow the integration of large DNA fragments, the restriction and modification system of the host was deleted. Using this optimized cloning procedure, the investigated complex gene clusters were functionally expressed in the genome of the B. subtilis host strain. The deoxyerythronolide B synthase (DEBS) from Saccharopolyspora erythraea was chosen as a representative of a complex PKS. This multi enzyme complex consists of three approx. 300–350 kDa proteins and is responsible for the production of the macrolide 6-deoxyerythronolide B (6dEB), the precursor of the antibiotic active erythromycin. The corresponding gene cluster includes three genes of approx. 10 kb in length and was transferred to the B. subtilis host genome in three different organizations as: i) native operon, ii) modified operon with optimized ribosomal binding sites (RBS) and iii) separated cluster with three independent expression cassettes. Under fed-batch-simulating expression conditions using the EnBase-technology, successful 6dEB production was verified for the host strain expressing the DEBS genes from separate cassettes. In order to improve cell growth and 6dEB synthesis, several genome modifications were realized and some of them showed a positive effect on product formation. By this approach, the proof of principle for the successful production of a complex polyketide in B. subtilis was demonstrated. Beside DEBS, the NRPS enniatin synthetase (ESyn) from the filamentous fungi Fusarium oxysporum was also investigated. Due to the manageable size of the corresponding gene (approx. 10 kb), the expression on single-copy and multi-copy-level was realized and the influence of several genomic modifications and variations of the cultivation conditions on target gene expression was analyzed. Esyn expression studies including LC-MS analysis were carried out by our collaboration partner at the TU Berlin. Final concentrations of the metabolite enniatin B, which is a cyclic hexadepsipeptide with various antimicrobial effects, were quantified to approx. 4.5 µg/L (single-copy) and 1.2 mg/L (multi-copy), respectively. This is so far the first demonstration of the heterologous production of a non-ribosomal peptide in B. subtilis which does not originate from the genus Bacillus. In summary, the present work focused on the successful production of a complex polyketide and a fungal non-ribosomal peptide in B. subtilis. Although further investigations are required to verify some unexpected effects of certain genome-modifications and to further enhance the product yield, it is clearly demonstrated that B. subtilis is a suitable host for the heterologous production of secondary metabolites

Staphylococcal serine protease-like proteins are pacemakers of allergic airway reactions to Staphylococcus aureus.

A substantial subgroup of asthmatic patients have "nonallergic" or idiopathic asthma, which often takes a severe course and is difficult to treat. The cause might be allergic reactions to the gram-positive pathogen Staphylococcus aureus, a frequent colonizer of the upper airways. However, the driving allergens of S aureus have remained elusive

Effects of Halide Ions on the Carbamidocyclophane Biosynthesis in Nostoc sp. CAVN2

In this study, the influence of halide ions on [7.7]paracyclophane biosynthesis in the cyanobacterium Nostoc sp. CAVN2 was investigated. In contrast to KI and KF, supplementation of the culture medium with KCl or KBr resulted not only in an increase of growth but also in an up-regulation of carbamidocyclophane production. LC-MS analysis indicated the presence of chlorinated, brominated, but also non-halogenated derivatives. In addition to 22 known cylindrocyclophanes and carbamidocyclophanes, 27 putative congeners have been detected. Nine compounds, carbamidocyclophanes M−U, were isolated, and their structural elucidation by 1D and 2D NMR experiments in combination with HRMS and ECD analysis revealed that they are brominated analogues of chlorinated carbamidocyclophanes. Quantification of the carbamidocyclophanes showed that chloride is the preferably utilized halide, but incorporation is reduced in the presence of bromide. Evaluation of the antibacterial activity of 30 [7.7]paracyclophanes and related derivatives against selected pathogenic Gram-positive and Gram-negative bacteria exhibited remarkable effects especially against methicillin- and vancomycin-resistant staphylococci and Mycobacterium tuberculosis. For deeper insights into the mechanisms of biosynthesis, the carbamidocyclophane biosynthetic gene cluster in Nostoc sp. CAVN2 was studied. The gene putatively coding for the carbamoyltransferase has been identified. Based on bioinformatic analyses, a possible biosynthetic assembly is discussed