• Actinobacteria Isolated from an Underground Lake and Moonmilk Speleothem from the Biggest Conglomeratic Karstic Cave in Siberia as Sources of Novel Biologically Active Compounds.

      Axenov-Gibanov, Denis V; Voytsekhovskaya, Irina V; Tokovenko, Bogdan T; Protasov, Eugeniy S; Gamaiunov, Stanislav V; Rebets, Yuriy V; Luzhetskyy, Andriy N; Timofeyev, Maxim A; Helmholtz Institute for Pharmaceutical Research Saarland (HIPS), Saarbrucken, Germany, 3 Universität des Saarlandes–Pharmazeutische Biotechnologie, Saarbrucken, Germany. (2016)
      Actinobacteria isolated from unstudied ecosystems are one of the most interesting and promising sources of novel biologically active compounds. Cave ecosystems are unusual and rarely studied. Here, we report the isolation and characterization of ten new actinobacteria strains isolated from an ancient underground lake and moonmilk speleothem from the biggest conglomeratic karstic cave in Siberia with a focus on the biological activity of the obtained strains and the metabolite dereplication of one active strain. Streptomyces genera isolates from moonmilk speleothem demonstrated antibacterial and antifungal activities. Some of the strains were able to inhibit the growth of pathogenic Candida albicans.
    • Benzanthric Acid, a Novel Metabolite From Del14 Expressing the Nybomycin Gene Cluster.

      Rodríguez Estévez, Marta; Gummerlich, Nils; Myronovskyi, Maksym; Zapp, Josef; Luzhetskyy, Andriy; HIPS, Helmholtz-Institut für Pharmazeutische Forschung Saarland, Universitätscampus E8.1 66123 Saarbrücken, Germany. (Frontiers, 2019-01-01)
      Streptomycetes constitute a diverse bacterial group able to produce a wide variety of secondary metabolites with potential applications in the pharmacy industry. However, the genes responsible for the biosynthesis of these compounds are very frequently inactive or expressed at very low levels under standard laboratory cultivation conditions. Therefore, the activation or upregulation of secondary metabolite biosynthesis genes is a crucial step for the discovery of new bioactive natural products. We have recently reported the discovery of the biosynthetic genes for the antibiotic nybomycin (nyb genes) in Streptomyces albus subsp. chlorinus. The nyb genes were expressed in the heterologous host Streptomyces albus Del14, which produces not only nybomycin, but also a novel compound. In this study, we describe the isolation, purification, and structure elucidation of the new substance named benzanthric acid.
    • The bottromycin epimerase BotH defines a group of atypical α/β-hydrolase-fold enzymes.

      Sikandar, Asfandyar; Franz, Laura; Adam, Sebastian; Santos-Aberturas, Javier; Horbal, Liliya; Luzhetskyy, Andriy; Truman, Andrew W; Kalinina, Olga V; Koehnke, Jesko; HIPS, Helmholtz-Institut für Pharmazeutische Forschung Saarland, Universitätscampus E8.1 66123 Saarbrücken, Germany. (Springer Nature, 2020-06-29)
      d-amino acids endow peptides with diverse, desirable properties, but the post-translational and site-specific epimerization of l-amino acids into their d-counterparts is rare and chemically challenging. Bottromycins are ribosomally synthesized and post-translationally modified peptides that have overcome this challenge and feature a d-aspartate (d-Asp), which was proposed to arise spontaneously during biosynthesis. We have identified the highly unusual α/β-hydrolase (ABH) fold enzyme BotH as a peptide epimerase responsible for the post-translational epimerization of l-Asp to d-Asp during bottromycin biosynthesis. The biochemical characterization of BotH combined with the structures of BotH and the BotH–substrate complex allowed us to propose a mechanism for this reaction. Bioinformatic analyses of BotH homologs show that similar ABH enzymes are found in diverse biosynthetic gene clusters. This places BotH as the founding member of a group of atypical ABH enzymes that may be able to epimerize non-Asp stereocenters across different families of secondary metabolites.
    • Characterization of Sigma Factor Genes in streptomyces lividans TK24 Using a Genomic Library-Based Approach for Multiple Gene Deletions.

      Rebets, Yuriy; Tsolis, Konstantinos C; Guðmundsdóttir, Elísabet Eik; Koepff, Joachim; Wawiernia, Beata; Busche, Tobias; Bleidt, Arne; Horbal, Liliya; Myronovskyi, Maksym; Ahmed, Yousra; et al. (Frontiers, 2018-01-01)
      Alternative sigma factors control numerous aspects of bacterial life, including adaptation to physiological stresses, morphological development, persistence states and virulence. This is especially true for the physiologically complex actinobacteria. Here we report the development of a robust gene deletions system for Streptomyces lividans TK24 based on a BAC library combined with the λ-Red recombination technique. The developed system was validated by systematically deleting the most highly expressed genes encoding alternative sigma factors and several other regulatory genes within the chromosome of S. lividans TK24. To demonstrate the possibility of large scale genomic manipulations, the major part of the undecylprodigiosin gene cluster was deleted as well. The resulting mutant strains were characterized in terms of morphology, growth parameters, secondary metabolites production and response to thiol-oxidation and cell-wall stresses. Deletion of SLIV_12645 gene encoding S. coelicolor SigR1 ortholog has the most prominent phenotypic effect, resulted in overproduction of actinorhodin and coelichelin P1 and increased sensitivity to diamide. The secreted proteome analysis of SLIV_12645 mutant revealed SigR1 influence on trafficking of proteins involved in cell wall biogenesis and refactoring. The reported here gene deletion system will further facilitate work on S. lividans strain improvement as a host for either secondary metabolites or protein production and will contribute to basic research in streptomycetes physiology, morphological development, secondary metabolism. On the other hand, the systematic deletion of sigma factors encoding genes demonstrates the complexity and conservation of regulatory processes conducted by sigma factors in streptomycetes
    • Chromosomal position effect influences the heterologous expression of genes and biosynthetic gene clusters in Streptomyces albus J1074.

      Bilyk, Bohdan; Horbal, Liliya; Luzhetskyy, Andriy N; Helmholz-Institut für pharmazeutische Forschung , Josef-Schneider-Straße2,97080 Würzburg, Germany. (2017-01-04)
      Efforts to construct the Streptomyces host strain with enhanced yields of heterologous product have focussed mostly on engineering of primary metabolism and/or the deletion of endogenous biosynthetic gene clusters. However, other factors, such as chromosome compactization, have been shown to have a significant influence on gene expression levels in bacteria and fungi. The expression of genes and biosynthetic gene clusters may vary significantly depending on their location within the chromosome. Little is known about the position effect in actinomycetes, which are important producers of various industrially relevant bioactive molecules.
    • Cloning and Heterologous Expression of the Grecocycline Biosynthetic Gene Cluster.

      Bilyk, Oksana; Sekurova, Olga N; Zotchev, Sergey B; Luzhetskyy, Andriy N; Helmholtz Institut für Pharmazeutische Forschung Saarland, Universitätscampus E8.1, 66123 Saarbrücken, Germany. (2016)
      Transformation-associated recombination (TAR) in yeast is a rapid and inexpensive method for cloning and assembly of large DNA fragments, which relies on natural homologous recombination. Two vectors, based on p15a and F-factor replicons that can be maintained in yeast, E. coli and streptomycetes have been constructed. These vectors have been successfully employed for assembly of the grecocycline biosynthetic gene cluster from Streptomyces sp. Acta 1362. Fragments of the cluster were obtained by PCR and transformed together with the "capture" vector into the yeast cells, yielding a construct carrying the entire gene cluster. The obtained construct was heterologously expressed in S. albus J1074, yielding several grecocycline congeners. Grecocyclines have unique structural moieties such as a dissacharide side chain, an additional amino sugar at the C-5 position and a thiol group. Enzymes from this pathway may be used for the derivatization of known active angucyclines in order to improve their desired biological properties.
    • Complete Draft Genome Sequence of the Actinobacterium Nocardiopsis sinuspersici UTMC102 (DSM 45277(T)), Which Produces Serine Protease.

      Tokovenko, Bogdan; Rückert, Christian; Kalinowski, Jörn; Mohammadipanah, Fatemeh; Wink, Joachim; Rosenkränzer, Birgit; Myronovskyi, Maksym; Luzhetskyy, Andriy N; Helmholtz Institut für pharmazeutische Forschung Saarland, Universitätscampus E8.1, 66123 Saarbrücken, Germany. (2017-05-18)
      The genome sequence of alkalohalophilic actinobacterium Nocardiopsis sinuspersici UTMC102 is provided. N. sinuspersici UTMC102 produces a highly active serine alkaline protease, and contains at least 11 gene clusters encoding the biosynthesis of secondary metabolites. The N. sinuspersici UTMC102 genome was assembled into a single chromosomal scaffold.
    • Complete genome sequence of producer of the glycopeptide antibiotic Aculeximycin Kutzneria albida DSM 43870T, a representative of minor genus of Pseudonocardiaceae

      Rebets, Yuriy; Tokovenko, Bogdan; Lushchyk, Igor; Rückert, Christian; Zaburannyi, Nestor; Bechthold, Andreas; Kalinowski, Jörn; Luzhetskyy, Andriy N (2014-10-10)
      Abstract Background Kutzneria is a representative of a rarely observed genus of the family Pseudonocardiaceae. Kutzneria species were initially placed in the Streptosporangiaceae genus and later reconsidered to be an independent genus of the Pseudonocardiaceae. Kutzneria albida is one of the eight known members of the genus. This strain is a unique producer of the glycosylated polyole macrolide aculeximycin which is active against both bacteria and fungi. Kutzneria albida genome sequencing and analysis allow a deeper understanding of evolution of this genus of Pseudonocardiaceae, provide new insight in the phylogeny of the genus, as well as decipher the hidden secondary metabolic potential of these rare actinobacteria. Results To explore the biosynthetic potential of Kutzneria albida to its full extent, the complete genome was sequenced. With a size of 9,874,926 bp, coding for 8,822 genes, it stands alongside other Pseudonocardiaceae with large circular genomes. Genome analysis revealed 46 gene clusters potentially encoding secondary metabolite biosynthesis pathways. Two large genomic islands were identified, containing regions most enriched with secondary metabolism gene clusters. Large parts of this secondary metabolism “clustome” are dedicated to siderophores production. Conclusions Kutzneria albida is the first species of the genus Kutzneria with a completely sequenced genome. Genome sequencing allowed identifying the gene cluster responsible for the biosynthesis of aculeximycin, one of the largest known oligosaccharide-macrolide antibiotics. Moreover, the genome revealed 45 additional putative secondary metabolite gene clusters, suggesting a huge biosynthetic potential, which makes Kutzneria albida a very rich source of natural products. Comparison of the Kutzneria albida genome to genomes of other actinobacteria clearly shows its close relations with Pseudonocardiaceae in line with the taxonomic position of the genus.
    • Complete genome sequence of producer of the glycopeptide antibiotic Aculeximycin Kutzneria albida DSM 43870T, a representative of minor genus of Pseudonocardiaceae.

      Rebets, Yuriy; Tokovenko, Bogdan; Lushchyk, Igor; Rückert, Christian; Zaburannyi, Nestor; Bechthold, Andreas; Kalinowski, Jörn; Luzhetskyy, Andriy N; Helmholtz Institute for Pharmaceutical Research Saarland (HIPS);Saarland University, Building A4.1, 66123 Saarbruecken, Germany. (2014)
      Kutzneria is a representative of a rarely observed genus of the family Pseudonocardiaceae. Kutzneria species were initially placed in the Streptosporangiaceae genus and later reconsidered to be an independent genus of the Pseudonocardiaceae. Kutzneria albida is one of the eight known members of the genus. This strain is a unique producer of the glycosylated polyole macrolide aculeximycin which is active against both bacteria and fungi. Kutzneria albida genome sequencing and analysis allow a deeper understanding of evolution of this genus of Pseudonocardiaceae, provide new insight in the phylogeny of the genus, as well as decipher the hidden secondary metabolic potential of these rare actinobacteria.
    • Development of a Biosensor Concept to Detect the Production of Cluster-Specific Secondary Metabolites.

      Sun, Yi-Qian; Busche, Tobias; Rückert, Christian; Paulus, Constanze; Rebets, Yuriy; Novakova, Renata; Kalinowski, Jörn; Luzhetskyy, Andriy N; Kormanec, Jan; Sekurova, Olga N; et al. (ACS Publications, 2017-06-16)
      Genome mining of actinomycete bacteria aims at the discovery of novel bioactive secondary metabolites that can be developed into drugs. A new repressor-based biosensor to detect activated secondary metabolite biosynthesis gene clusters in Streptomyces was developed. Biosynthetic gene clusters for undecylprodigiosin and coelimycin in the genome of Streptomyces lividans TK24, which encoded TetR-like repressors and appeared to be almost “silent” based on the RNA-seq data, were chosen for the proof-of-principle studies. The bpsA reporter gene for indigoidine synthetase was placed under control of the promotor/operator regions presumed to be controlled by the cluster-associated TetR-like repressors. While the biosensor for undecylprodigiosin turned out to be nonfunctional, the coelimycin biosensor was shown to perform as expected, turning on biosynthesis of indigoidine in response to the concomitant production of coelimycin. The developed reporter system concept can be applied to those cryptic gene clusters that encode metabolite-sensing repressors to speed up discovery of novel bioactive compounds in Streptomyces.
    • Draft Genome Sequence of Streptomyces sp. Strain IB2014011-1, Isolated from Trichoptera sp. Larvae of Lake Baikal.

      Axenov-Gribanov, Denis V; Tokovenko, Bogdan T; Rebets, Yuriy V; Voytsekhovskaya, Irina V; Shatilina, Zhanna M; Protasov, Eugenii S; Luzhetskyy, Andriy N; Timofeyev, Maxim A; Helmholtz Institut für pharmazeutische Forschung Saarland, Universitätscampus E8.1, 66123 Saarbrücken, Germany. (2017-04-27)
      Unique ecosystems with specific environmental conditions have been proven to be a promising source for isolation of new actinobacterial strains. Ancient Lake Baikal is one of the greatest examples of an ecosystem with high species biodiversity and endemicity caused by long-lasting isolated evolution and stable environmental conditions. Herein we report the draft genome sequence of Streptomyces sp. strain IB2014011-1, which was isolated from insect Trichoptera sp. larvae collected at the bottom of Lake Baikal.
    • Dual control system - A novel scaffolding architecture of an inducible regulatory device for the precise regulation of gene expression.

      Horbal, L; Luzhetskyy, Andriy N; Helmholtz Institut für Pharmazeutische Forschung Saarland, Universitätscampus E8.1, 66123 Saarbrücken, Germany. (2016-09)
      Here, we present a novel scaffolding architecture of an inducible regulatory device. This dual control system is completely silent in the off stage and is coupled to the regulation of gene expression at both the transcriptional and translational levels. This system also functions as an AND gate. We demonstrated the effectiveness of the cumate-riboswitch dual control system for the control of pamamycin production in Streptomyces albus. Placing the cre recombinase gene under the control of this system permitted the construction of synthetic devices with non-volatile memory that sense the signal and respond by altering DNA at the chromosomal level, thereby producing changes that are heritable. In addition, we present a library of synthetic inducible promoters based on the previously described cumate switch. With only one inducer and different promoters, we demonstrate that simultaneous modulation of the expression of several genes to different levels in various operons is possible. Because all modules of the AND gates are functional in bacteria other than Streptomyces, we anticipate that these regulatory devices can be used to control gene expression in other Actinobacteria. The features described in this study make these systems promising tools for metabolic engineering and biotechnology in Actinobacteria.
    • Endophytic Streptomyces in the traditional medicinal plant Arnica montana L.: secondary metabolites and biological activity.

      Wardecki, Tina; Brötz, Elke; De Ford, Christian; von Loewenich, Friederike D; Rebets, Yuriy; Tokovenko, Bogdan; Luzhetskyy, Andriy N; Merfort, Irmgard; Helmholtz Institute for Pharmaceutical Research Saarland,Saarbrücken, Saarland 66123, Germany. (2015-08)
      Arnica montana L. is a medical plant of the Asteraceae family and grows preferably on nutrient poor soils in mountainous environments. Such surroundings are known to make plants dependent on symbiosis with other organisms. Up to now only arbuscular mycorrhizal fungi were found to act as endophytic symbiosis partners for A. montana. Here we identified five Streptomyces strains, microorganisms also known to occur as endophytes in plants and to produce a huge variety of active secondary metabolites, as inhabitants of A. montana. The secondary metabolite spectrum of these strains does not contain sesquiterpene lactones, but consists of the glutarimide antibiotics cycloheximide and actiphenol as well as the diketopiperazines cyclo-prolyl-valyl, cyclo-prolyl-isoleucyl, cyclo-prolyl-leucyl and cyclo-prolyl-phenylalanyl. Notably, genome analysis of one strain was performed and indicated a huge genome size with a high number of natural products gene clusters among which genes for cycloheximide production were detected. Only weak activity against the Gram-positive bacterium Staphylococcus aureus was revealed, but the extracts showed a marked cytotoxic activity as well as an antifungal activity against Candida parapsilosis and Fusarium verticillioides. Altogether, our results provide evidence that A. montana and its endophytic Streptomyces benefit from each other by completing their protection against competitors and pathogens and by exchanging plant growth promoting signals with nutrients.
    • Engineering of Streptomyces lividans for heterologous expression of secondary metabolite gene clusters.

      Ahmed, Yousra; Rebets, Yuriy; Estévez, Marta Rodríguez; Zapp, Josef; Myronovskyi, Maksym; Luzhetskyy, Andriy; HIPS, Helmholtz-Institut für Pharmazeutische Forschung Saarland, Universitätscampus E8.1 66123 Saarbrücken, Germany. (BioMed Central (BMC), 2020-01-09)
      BACKGROUND: Heterologous expression of secondary metabolite gene clusters is used to achieve increased production of desired compounds, activate cryptic gene clusters, manipulate clusters from genetically unamenable strains, obtain natural products from uncultivable species, create new unnatural pathways, etc. Several Streptomyces species are genetically engineered for use as hosts for heterologous expression of gene clusters. S. lividans TK24 is one of the most studied and genetically tractable actinobacteria, which remain untapped. It was therefore important to generate S. lividans chassis strains with clean metabolic backgrounds. RESULTS: In this study, we generated a set of S. lividans chassis strains by deleting endogenous gene clusters and introducing additional φC31 attB loci for site-specific integration of foreign DNA. In addition to the simplified metabolic background, the engineered S. lividans strains had better growth characteristics than the parental strain in liquid production medium. The utility of the developed strains was validated by expressing four secondary metabolite gene clusters responsible for the production of different classes of natural products. Engineered strains were found to be superior to the parental strain in production of heterologous natural products. Furthermore, S. lividans-based strains were better producers of amino acid-based natural products than other tested common hosts. Expression of a Streptomyces albus subsp. chlorinus NRRL B-24108 genomic library in the modified S. lividans ΔYA9 and S. albus Del14 strains resulted in the production of 7 potentially new compounds, only one of which was produced in both strains. CONCLUSION: The constructed S. lividans-based strains are a great complement to the panel of heterologous hosts for actinobacterial secondary metabolite gene expression. The expansion of the number of such engineered strains will contribute to an increased success rate in isolation of new natural products originating from the expression of genomic and metagenomic libraries, thus raising the chance to obtain novel biologically active compounds.
    • A gene cluster for the biosynthesis of moenomycin family antibiotics in the genome of teicoplanin producer Actinoplanes teichomyceticus.

      Horbal, Liliya; Ostash, Bohdan; Luzhetskyy, Andriy N; Walker, Suzanne; Kalinowski, Jorn; Fedorenko, Victor; Helmholtz-Institute for Pharmaceutical Research Saarland (HIPS),Saarland Universitätscampus E8.1, 66123 Saarbrücken, Germany. (2016-09)
      Moenomycins are phosphoglycolipid antibiotics notable for their extreme potency, unique mode of action, and proven record of use in animal nutrition without selection for resistant microflora. There is a keen interest in manipulation of structures of moenomycins in order to better understand their structure-activity relationships and to generate improved analogs. Only two almost identical moenomycin biosynthetic gene clusters are known, limiting our knowledge of the evolution of moenomycin pathways and our ability to genetically diversify them. Here, we report a novel gene cluster (tchm) that directs production of the phosphoglycolipid teichomycin in Actinoplanes teichomyceticus. Its overall genetic architecture is significantly different from that of the moenomycin biosynthesis (moe) gene clusters of Streptomyces ghanaensis and Streptomyces clavuligerus, featuring multiple gene rearrangements and two novel structural genes. Involvement of the tchm cluster in teichomycin biosynthesis was confirmed via heterologous co-expression of amidotransferase tchmH5 and moe genes. Our work sets the background for further engineering of moenomycins and for deeper inquiries into the evolution of this fascinating biosynthetic pathway.
    • Genome Engineering Approaches to Improve Nosokomycin A Production by Streptomyces ghanaensis B38.3

      Kuzhyk, Yuriy; Lopatniuk, Maria; Luzhetskyy, Andriy N; Fedorenko, Victor; Ostash, Bohdan; HIPS, Helmholtz-Institut für Pharmazeutische Forschung Saarland, Universitätscampus E8.1 66123 Saarbrücken, Germany. (Springer, 2018-09-25)
      Here we describe our efforts to improve the levels of phosphoglycolipid antibiotic nosokomycin A production by Streptomyces ghanaensis ATCC14672 via genome engineering approaches. Introduction of two extra copies of leucyl tRNA (UUA) gene bldA and one copy of moenomycin biosynthesis gene cluster led, on average, to threefold increase in nosokomycin A titers (up to 1.5 mg/L). Our results validate genome engineering approach as a viable strategy to improve moenomycin production.
    • Heterologous Expression of the Nybomycin Gene Cluster from the Marine StrainStreptomyces albus subsp. NRRL B-24108.

      Rodríguez Estévez, Marta; Myronovskyi, Maksym; Gummerlich, Nils; Nadmid, Suvd; Luzhetskyy, Andriy N; HIPS, Helmholtz-Institut für pharmazeutische Forschung Saarland, Universitätscampus E8.1, 66123 Saarbrücken, Germany. (MPDI, 2018-11-04)
      Streptomycetes represent an important reservoir of active secondary metabolites with potential applications in the pharmaceutical industry. The gene clusters responsible for their production are often cryptic under laboratory growth conditions. Characterization of these clusters is therefore essential for the discovery of new microbial pharmaceutical drugs. Here, we report the identification of the previously uncharacterized nybomycin gene cluster from the marine actinomycete
    • Identification and Heterologous Expression of the Albucidin Gene Cluster from the Marine Strain Subsp. NRRL B-24108.

      Myronovskyi, Maksym; Rosenkränzer, Birgit; Stierhof, Marc; Petzke, Lutz; Seiser, Tobias; Luzhetskyy, Andriy; HIPS, Helmholtz-Institut für Pharmazeutische Forschung Saarland, Universitätscampus E8.1 66123 Saarbrücken, Germany. (MDPI, 2020-02-10)
      Herbicides with new modes of action and safer toxicological and environmental profiles are needed to manage the evolution of weeds that are resistant to commercial herbicides. The unparalleled structural diversity of natural products makes these compounds a promising source for new herbicides. In 2009, a novel nucleoside phytotoxin, albucidin, with broad activity against grass and broadleaf weeds was isolated from a strain of Streptomyces albus subsp. chlorinus NRRL B-24108. Here, we report the identification and heterologous expression of the previously uncharacterized albucidin gene cluster. Through a series of gene inactivation experiments, a minimal set of albucidin biosynthetic genes was determined. Based on gene annotation and sequence homology, a model for albucidin biosynthesis was suggested. The presented results enable the construction of producer strains for a sustainable supply of albucidin for biological activity studies.
    • Identification of a Biosynthetic Gene Cluster Responsible for the Production of a New Pyrrolopyrimidine Natural Product-Huimycin.

      Shuai, Hui; Myronovskyi, Maksym; Nadmid, Suvd; Luzhetskyy, Andriy; HIPS, Helmholtz-Institut für Pharmazeutische Forschung Saarland, Universitätscampus E8.1 66123 Saarbrücken, Germany. (MDPI, 2020-07-18)
      Pyrrolopyrimidines are an important class of natural products with a broad spectrum of biological activities, including antibacterial, antifungal, antiviral, anticancer or anti-inflammatory. Here, we present the identification of a biosynthetic gene cluster from the rare actinomycete strain Kutzneria albida DSM 43870, which leads to the production of huimycin, a new member of the pyrrolopyrimidine family of compounds. The huimycin gene cluster was successfully expressed in the heterologous host strain Streptomyces albus Del14. The compound was purified, and its structure was elucidated by means of nuclear magnetic resonance spectroscopy. The minimal huimycin gene cluster was identified through sequence analysis and a series of gene deletion experiments. A model for huimycin biosynthesis is also proposed in this paper.
    • Identification of butenolide regulatory system controlling secondary metabolism in Streptomyces albus J1074.

      Ahmed, Yousra; Rebets, Yuriy; Tokovenko, Bogdan; Brötz, Elke; Luzhetskyy, Andriy N; Helmholtz-Institut für pharmazeutische Forschung Saarland,Universitätscampus E8.1, 66123 Saarbrücken, Germany. (2017-08-29)
      A large majority of genome-encrypted chemical diversity in actinobacteria remains to be discovered, which is related to the low level of secondary metabolism genes expression. Here, we report the application of a reporter-guided screening strategy to activate cryptic polycyclic tetramate macrolactam gene clusters in Streptomyces albus J1074. The analysis of the S. albus transcriptome revealed an overall low level of secondary metabolism genes transcription. Combined with transposon mutagenesis, reporter-guided screening resulted in the selection of two S. albus strains with altered secondary metabolites production. Transposon insertion in the most prominent strain, S. albus ATGSal2P2::TN14, was mapped to the XNR_3174 gene encoding an unclassified transcriptional regulator. The mutant strain was found to produce the avenolide-like compound butenolide 4. The deletion of the gene encoding a putative acyl-CoA oxidase, an orthologue of the Streptomyces avermitilis avenolide biosynthesis enzyme, in the S. albus XNR_3174 mutant caused silencing of secondary metabolism. The homologues of XNR_3174 and the butenolide biosynthesis genes were found in the genomes of multiple Streptomyces species. This result leads us to believe that the discovered regulatory elements comprise a new condition-dependent system that controls secondary metabolism in actinobacteria and can be manipulated to activate cryptic biosynthetic pathways.