Cyclofaulknamycin with the Rare Amino Acid D-capreomycidine Isolated from a Well-Characterized Streptomyces albus Strain

Targeted genome mining is an efficient method of biosynthetic gene cluster prioritization within constantly growing genome databases. Using two capreomycidine biosynthesis genes, alphaketoglutarate-dependent arginine beta-hydroxylase and pyridoxal-phosphate-dependent aminotransferase, we identified two types of clusters: one type containing both genes involved in the biosynthesis of the abovementioned moiety, and other clusters including only arginine hydroxylase. Detailed analysis of one of the clusters, the flk cluster from Streptomyces albus, led to the identification of a cyclic peptide that contains a rare D-capreomycidine moiety for the first time. The absence of the pyridoxal-phosphate-dependent aminotransferase gene in the flk cluster is compensated by the XNR_1347 gene in the S. albus genome, whose product is responsible for biosynthesis of the abovementioned nonproteinogenic amino acid. Herein, we report the structure of cyclofaulknamycin and the characteristics of its biosynthetic gene cluster, biosynthesis and bioactivity profile

Discovery and overproduction of novel highly bioactive pamamycins through transcriptional engineering of the biosynthetic gene cluster

Background Pamamycins are a family of highly bioactive macrodiolide polyketides produced by Streptomyces alboniger as a complex mixture of derivatives with molecular weights ranging from 579 to 705 Daltons. The large derivatives are produced as a minor fraction, which has prevented their isolation and thus studies of chemical and biological properties. Results Herein, we describe the transcriptional engineering of the pamamycin biosynthetic gene cluster (pam BGC), which resulted in the shift in production profle toward high molecular weight derivatives. The pam BGC library was constructed by inserting randomized promoter sequences in front of key biosynthetic operons. The library was expressed in Streptomyces albus strain with improved resistance to pamamycins to overcome sensitivity-related host limitations. Clones with modifed pamamycin profles were selected and the properties of engineered pam BGC were studied in detail. The production level and composition of the mixture of pamamycins was found to depend on balance in expression of the corresponding biosynthetic genes. This approach enabled the isolation of known pamamycins and the discovery of three novel derivatives with molecular weights of 663 Da and higher. One of them, homopamamycin 677A, is the largest described representative of this family of natural products with an elucidated structure. The new pamamycin 663A shows extraordinary activity (IC50 2 nM) against hepatocyte cancer cells as well as strong activity (in the one-digit micromolar range) against a range of Gram-positive pathogenic bacteria. Conclusion By employing transcriptional gene cluster refactoring, we not only enhanced the production of known pamamycins but also discovered novel derivatives exhibiting promising biological activities. This approach has the potential for broader application in various biosynthetic gene clusters, creating a sustainable supply and discovery platform for bioactive natural products

Heterologe Üperproduktion von neuen Naturstoffen in Streptomyceten

Secondary metabolites, often referred to as natural products (NPs), produced by Actinobacteria (mostly by Streptomyces) are a major source for antibiotics and other drugs. The genome data of Actinobacteria revealed their great biosynthetic potential to be far from fully exploited. The major reason is that most corresponding biosynthetic gene clusters responsible for the NP production remain “silent” under laboratory conditions or their expression level is too low for the detection and isolation of new molecules. The first part of this thesis is focussing on the discovery, isolation and characterisation of the new NPs cyclofaulknamycin and aorimycins. The discovery of cyclofaulknamycin shows that even well described model organisms like Streptomyces albus J1074, which is under investigations in hundreds of laboratories worldwide, have the potential to still conceal unknown bioactive molecules. This applies even more for newly isolated bacteria, which was shown by the example of the novel NPs aorimycins. The second part focusses on known compounds with unknown pharmaceutical potential. One of the main reasons for that is a low production yield resulting in supply shortage for the profiling of compounds. Herein, a method is tested which utilised random rational designed promoters to increase the NP production level. By the use of this method new derivatives were isolated and further characterised.Sekundärmetabolite, die oft als Naturstoffe bezeichnet werden, werden oft von Actinobakterien (meist von Streptomyceten) produziert und sind eine wichtige Quelle für Antibiotika und andere Arzneimittel. Die Genomdaten von Actinobakterien haben gezeigt, dass ihr großes biosynthetisches Potenzial noch nicht ausgeschöpft ist. Der Hauptgrund dafür ist, dass die meisten für die Naturstoffproduktion verantwortlichen biosynthetischen Gencluster unter Laborbedingungen nicht aktiv sind oder ihr Expressionsniveau zu niedrig ist, um neue Moleküle nachweisen zu können. Der erste Teil dieser Arbeit befasst sich mit der Entdeckung, Isolierung und Charakterisierung der neuen Naturstoffe Cyclofaulknamycin und Aorimycin. Die Entdeckung von Cyclofaulknamycin zeigt, dass selbst gut beschriebene Modellorganismen wie Streptomyces albus J1074, die in hunderten von Laboren weltweit untersucht werden, noch unbekannte bioaktive Moleküle verbergen können. Dies gilt umso mehr für neu isolierte Bakterien, was am Beispiel der neuartigen Aorimycine gezeigt wurde. Der zweite Teil konzentriert sich auf bekannte Verbindungen mit unbekanntem pharmazeutischem Potenzial. Einer der Hauptgründe dafür ist die geringe Produktionsausbeute, die zu Engpässen bei der Profilierung von Verbindungen führt. Hier werden zufällig-rational konzipierte Promotoren eingesetzt, um die Naturstoff-Produktion zu steigern. Mit Hilfe dieser Methode wurden neue Derivate isoliert und weiter charakterisiert

Cyclofaulknamycin with the Rare Amino Acid D-capreomycidine Isolated from a Well-Characterized Strain.

Targeted genome mining is an efficient method of biosynthetic gene cluster prioritization within constantly growing genome databases. Using two capreomycidine biosynthesis genes, alpha-ketoglutarate-dependent arginine beta-hydroxylase and pyridoxal-phosphate-dependent aminotransferase, we identified two types of clusters: one type containing both genes involved in the biosynthesis of the abovementioned moiety, and other clusters including only arginine hydroxylase. Detailed analysis of one of the clusters, the flk cluster from Streptomyces albus, led to the identification of a cyclic peptide that contains a rare D-capreomycidine moiety for the first time. The absence of the pyridoxal-phosphate-dependent aminotransferase gene in the flk cluster is compensated by the XNR_1347 gene in the S. albus genome, whose product is responsible for biosynthesis of the abovementioned nonproteinogenic amino acid. Herein, we report the structure of cyclofaulknamycin and the characteristics of its biosynthetic gene cluster, biosynthesis and bioactivity profile

Discovery and overproduction of novel highly bioactive pamamycins through transcriptional engineering of the biosynthetic gene cluster

Eckert N, Rebets Y, Horbal L, et al. Discovery and overproduction of novel highly bioactive pamamycins through transcriptional engineering of the biosynthetic gene cluster. Microbial Cell Factories . 2023;22(1): 233.BACKGROUND: Pamamycins are a family of highly bioactive macrodiolide polyketides produced by Streptomyces alboniger as a complex mixture of derivatives with molecular weights ranging from 579 to 705 Daltons. The large derivatives are produced as a minor fraction, which has prevented their isolation and thus studies of chemical and biological properties.; RESULTS: Herein, we describe the transcriptional engineering of the pamamycin biosynthetic gene cluster (pam BGC), which resulted in the shift in production profile toward high molecular weight derivatives. The pam BGC library was constructed by inserting randomized promoter sequences in front of key biosynthetic operons. The library was expressed in Streptomyces albus strain with improved resistance to pamamycins to overcome sensitivity-related host limitations. Clones with modified pamamycin profiles were selected and the properties of engineered pam BGC were studied in detail. The production level and composition of the mixture of pamamycins was found to depend on balance in expression of the corresponding biosynthetic genes. This approach enabled the isolation of known pamamycins and the discovery of three novel derivatives with molecular weights of 663 Da and higher. One of them, homopamamycin 677A, is the largest described representative of this family of natural products with an elucidated structure. The new pamamycin 663A shows extraordinary activity (IC50 2nM) against hepatocyte cancer cells as well as strong activity (in the one-digit micromolar range) against a range of Gram-positive pathogenic bacteria.; CONCLUSION: By employing transcriptional gene cluster refactoring, we not only enhanced the production of known pamamycins but also discovered novel derivatives exhibiting promising biological activities. This approach has the potential for broader application in various biosynthetic gene clusters, creating a sustainable supply and discovery platform for bioactive natural products. © 2023. The Author(s)