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group leader: Luzhetskyy

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  • A set of synthetic versatile genetic control elements for the efficient expression of genes in Actinobacteria.

    Horbal, Lilya; Siegl, Theresa; Luzhetskyy, Andriy; HIPS, Helmholtz-Institut für pharmazeutische Forschung Saarland, Universitätscampus 8.1, 66123 Saarbrücken, Germany. (2018-01-11)
    The design and engineering of secondary metabolite gene clusters that are characterized by complicated genetic organization, require the development of collections of well-characterized genetic control elements that can be reused reliably. Although a few intrinsic terminators and RBSs are used routinely, their translation and termination efficiencies have not been systematically studied in Actinobacteria. Here, we analyzed the influence of the regions surrounding RBSs on gene expression in these bacteria. We demonstrated that inappropriate RBSs can reduce the expression efficiency of a gene to zero. We developed a genetic device - an in vivo RBS-selector - that allows selection of an optimal RBS for any gene of interest, enabling rational control of the protein expression level. In addition, a genetic tool that provides the opportunity for measurement of termination efficiency was developed. Using this tool, we found strong terminators that lead to a 17-100-fold reduction in downstream expression and are characterized by sufficient sequence diversity to reduce homologous recombination when used with other elements. For the first time, a C-terminal degradation tag was employed for the control of protein stability in Streptomyces. Finally, we describe a collection of regulatory elements that can be used to control metabolic pathways in Actinobacteria.
  • Identification of butenolide regulatory system controlling secondary metabolism in Streptomyces albus J1074.

    Ahmed, Yousra; Rebets, Yuriy; Tokovenko, Bogdan; Brötz, Elke; Luzhetskyy, Andriy; 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.
  • Chromosomal position effect influences the heterologous expression of genes and biosynthetic gene clusters in Streptomyces albus J1074.

    Bilyk, Bohdan; Horbal, Liliya; Luzhetskyy, Andriy; 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.
  • 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 (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.
  • New natural products identified by combined genomics-metabolomics profiling of marine Streptomyces sp. MP131-18.

    Paulus, Constanze; Rebets, Yuriy; Tokovenko, Bogdan; Nadmid, Suvd; Terekhova, Larisa P; Myronovskyi, Maksym; Zotchev, Sergey B; Rückert, Christian; Braig, Simone; Zahler, Stefan; Kalinowski, Jörn; Luzhetskyy, Andriy; Helmholtz-Institut für Pharmazeutische Forschung Saarland, Universitätscampus E8.1, 66123 Saarbrücken, Germany. (2017-02-10)
    Marine actinobacteria are drawing more and more attention as a promising source of new natural products. Here we report isolation, genome sequencing and metabolic profiling of new strain Streptomyces sp. MP131-18 isolated from marine sediment sample collected in the Trondheim Fjord, Norway. The 16S rRNA and multilocus phylogenetic analysis showed that MP131-18 belongs to the genus Streptomyces. The genome of MP131-18 isolate was sequenced, and 36 gene clusters involved in the biosynthesis of 18 different types of secondary metabolites were predicted using antiSMASH analysis. The combined genomics-metabolics profiling of the strain led to the identification of several new biologically active compounds. As a result, the family of bisindole pyrroles spiroindimicins was extended with two new members, spiroindimicins E and F. Furthermore, prediction of the biosynthetic pathway for unusual α-pyrone lagunapyrone isolated from MP131-18 resulted in foresight and identification of two new compounds of this family - lagunapyrones D and E. The diversity of identified and predicted compounds from Streptomyces sp. MP131-18 demonstrates that marine-derived actinomycetes are not only a promising source of new natural products, but also represent a valuable pool of genes for combinatorial biosynthesis of secondary metabolites.
  • 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.
  • Properties of Streptomyces albus J1074 mutant deficient in tRNA(Leu)UAA gene bldA.

    Koshla, Oksana; Lopatniuk, Maria; Rokytskyy, Ihor; Yushchuk, Oleksandr; Dacyuk, Yuriy; Fedorenko, Victor; Luzhetskyy, Andriy; Ostash, Bohdan; Helmholtz Institut für phsarmazeutische Forschung Saarland (HIPS), Universitätscampus E8.1, 66123 Saarbrücken, Germany. (2017-05-20)
    Streptomyces albus J1074 is one of the most popular and convenient hosts for heterologous expression of gene clusters directing the biosynthesis of various natural metabolic products, such as antibiotics. This fuels interest in elucidation of genetic mechanisms that may limit secondary metabolism in J1074. Here, we report the generation and initial study of J1074 mutant, deficient in gene bldA for tRNA(Leu)UAA, the only tRNA capable of decoding rare leucyl TTA codon in Streptomyces. The bldA deletion in J1074 resulted in a highly conditional Bld phenotype, with depleted formation of aerial hyphae on certain solid media. In addition, bldA mutant of J1074 was unable to produce endogenous antibacterial compounds and two heterologous antibiotics, moenomycin and aranciamycin, whose biosynthesis is directed by TTA-containing genes. We have employed a new TTA codon-specific β-galactosidase reporter system to provide genetic evidence that J1074 bldA mutant is impaired in translation of TTA. In addition, we have discussed the possible reasons for differences in the phenotypes of bldA mutants described here and in previous studies, providing knowledge to study bldA-based regulation of antibiotic biosynthesis.
  • 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; 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.
  • Insights into naturally minimised Streptomyces albus J1074 genome

    Zaburannyi, Nestor; Rabyk, Mariia; Ostash, Bohdan; Fedorenko, Victor; Luzhetskyy, Andriy (2014-02-05)
    Abstract Background The Streptomyces albus J1074 strain is one of the most widely used chassis for the heterologous production of bioactive natural products. The fast growth and an efficient genetic system make this strain an attractive model for expressing cryptic biosynthetic pathways to aid drug discovery. Results To improve its capabilities for the heterologous expression of biosynthetic gene clusters, the complete genomic sequence of S. albus J1074 was obtained. With a size of 6,841,649 bp, coding for 5,832 genes, its genome is the smallest within the genus streptomycetes. Genome analysis revealed a strong tendency to reduce the number of genetic duplicates. The whole transcriptomes were sequenced at different time points to identify the early metabolic switch from the exponential to the stationary phase in S. albus J1074. Conclusions S. albus J1074 carries the smallest genome among the completely sequenced species of the genus Streptomyces. The detailed genome and transcriptome analysis discloses its capability to serve as a premium host for the heterologous production of natural products. Moreover, the genome revealed 22 additional putative secondary metabolite gene clusters that reinforce the strain’s potential for natural product synthesis.
  • A gene cluster for the biosynthesis of moenomycin family antibiotics in the genome of teicoplanin producer Actinoplanes teichomyceticus.

    Horbal, Liliya; Ostash, Bohdan; Luzhetskyy, Andriy; 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.
  • 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.
  • Dual control system - A novel scaffolding architecture of an inducible regulatory device for the precise regulation of gene expression.

    Horbal, L; Luzhetskyy, A; 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.
  • 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.
  • 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; 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.
  • Testing the utility of site-specific recombinases for manipulations of genome of moenomycin producer Streptomyces ghanaensis ATCC14672.

    Lopatniuk, M; Ostash, B; Makitrynskyy, R; Walker, S; Luzhetskyy, A; Fedorenko, V; Helmholtz Institute for Pharmaceutical Research Saarland,Saarbrücken, Saarland 66123, Germany. (2015-11)
    Streptomyces ghanaensis ATCC14672 is the producer of phosphoglycolipid antibiotics moenomycins that for almost 40 years were used worldwide as an animal feed additive. As the use of moenomycins narrows down (due to bans in the EU and some other countries), it opens the opportunity to develop much-needed antibiotics against Gram-positive human pathogens, such as cocci. It is desirable to develop ATCC14672 strains accumulating only certain members of moenomycin family which would facilitate their purification, analysis and/or chemical modification. Here we tested site-specific recombinases (SSRs) as a tool to manipulate the genome of ATCC14672 and to achieve aforementioned goals. We show that of three SSRs tested - Cre, Dre, and Flp - the first two efficiently catalyzed recombination reactions, while Flp showed no activity in ATCC14672 cells. Cre recombinase can be reused at least three times to modify ATCC14672 genome without detrimental effects, such as large-scale inversions or deletions. Properties of the generated strains and SSRs are discussed.
  • 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; 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.
  • SimReg1 is a master switch for biosynthesis and export of simocyclinone D8 and its precursors.

    Horbal, Liliya; Rebets, Yuriy; Rabyk, Mariya; Makitrynskyy, Roman; Luzhetskyy, Andriy; Fedorenko, Victor; Bechthold, Andreas (2012)
    Analysis of the simocyclinone biosynthesis (sim) gene cluster of Streptomyces antibioticus Tü6040 led to the identification of a putative pathway specific regulatory gene simReg1. In silico analysis places the SimReg1 protein in the OmpR-PhoB subfamily of response regulators. Gene replacement of simReg1 from the S. antibioticus chromosome completely abolishes simocyclinone production indicating that SimReg1 is a key regulator of simocyclinone biosynthesis. Results of the DNA-shift assays and reporter gene expression analysis are consistent with the idea that SimReg1 activates transcription of simocyclinone biosynthesis, transporter genes, regulatory gene simReg3 and his own transcription. The presence of extracts (simocyclinone) from S. antibioticus Tü6040 × pSSimR1-1 could dissociate SimReg1 from promoter regions. A preliminary model for regulation of simocyclinone biosynthesis and export is discussed.
  • Tracking down biotransformation to the genetic level: identification of a highly flexible glycosyltransferase from Saccharothrix espanaensis.

    Strobel, Tina; Schmidt, Yvonne; Linnenbrink, Anton; Luzhetskyy, Andriy; Luzhetska, Marta; Taguchi, Takaaki; Brötz, Elke; Paululat, Thomas; Stasevych, Maryna; Stanko, Oleg; Novikov, Volodymyr; Bechthold, Andreas; Albert Ludwigs University of Freiburg, Department of Pharmaceutical Biology and Biotechnology, Freiburg, Germany. (2013-09)
    Saccharothrix espanaensis is a member of the order Actinomycetales. The genome of the strain has been sequenced recently, revealing 106 glycosyltransferase genes. In this paper, we report the detection of a glycosyltransferase from Saccharothrix espanaensis which is able to rhamnosylate different phenolic compounds targeting different positions of the molecules. The gene encoding the flexible glycosyltransferase is not located close to a natural product biosynthetic gene cluster. Therefore, the native function of this enzyme might be not the biosynthesis of a secondary metabolite but the glycosylation of internal and external natural products as part of a defense mechanism.