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group leader: Luzhetskyy

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  • Non-Heme Monooxygenase ThoJ Catalyzes Thioholgamide β-Hydroxylation.

    Sikandar, Asfandyar; Lopatniuk, Maria; Luzhetskyy, Andriy; Koehnke, Jesko; HIPS, Helmholtz-Institut für Pharmazeutische Forschung Saarland, Universitätscampus E8.1 66123 Saarbrücken, Germany. (American Chemical Society (ACS), 2020-10-01)
    Thioviridamide-like compounds, including thioholgamides, are ribosomally synthesized and post-translationally modified peptide natural products with potent anticancer cell activity and an unprecedented structure. Very little is known about their biosynthesis, and we were intrigued by the β-hydroxy-N1, N3-dimethylhistidinium moiety found in these compounds. Here we report the construction of a heterologous host capable of producing thioholgamide with a 15-fold increased yield compared to the wild-type strain. A knockout of thoJ, encoding a predicted nonheme monooxygenase, shows that ThoJ is essential for thioholgamide β-hydroxylation. The crystal structure of ThoJ exhibits a typical mono/dioxygenase fold with conserved key active-site residues. Yet, ThoJ possesses a very large substrate binding pocket that appears suitable to receive a cyclic thioholgamide intermediate for hydroxylation. The improved production of the heterologous host will enable the dissection of the individual biosynthetic steps involved in biosynthesis of this exciting RiPP family.
  • Loseolamycins: A Group of New Bioactive Alkylresorcinols Produced after Heterologous Expression of a Type III PKS from micromonospora endolithica.

    Lasch, Constanze; Gummerlich, Nils; Myronovskyi, Maksym; Palusczak, Anja; Zapp, Josef; Luzhetskyy, Andriy; HIPS, Helmholtz-Institut für Pharmazeutische Forschung Saarland, Universitätscampus E8.1 66123 Saarbrücken, Germany. (MDPI, 2020-10-09)
    Natural products are a valuable source of biologically active compounds with potential applications in medicine and agriculture. Unprecedented scaffold diversity of natural products and biocatalysts from their biosynthetic pathways are of fundamental importance. Heterologous expression and refactoring of natural product biosynthetic pathways are generally regarded as a promising approach to discover new secondary metabolites of microbial origin. Here, we present the identification of a new group of alkylresorcinols after transcriptional activation and heterologous expression of the type III polyketide synthase of Micromonospora endolithica. The most abundant compounds loseolamycins A1 and A2 have been purified and their structures were elucidated by NMR. Loseolamycins contain an unusual branched hydroxylated aliphatic chain which is provided by the host metabolism and is incorporated as a starter fatty acid unit. The isolated loseolamycins show activity against gram-positive bacteria and inhibit the growth of the monocot weed Agrostis stolonifera in a germination assay. The biosynthetic pathway leading to the production of loseolamycins is proposed in this paper.
  • Engineering Corynebacterium glutamicum with a comprehensive genomic library and phage-based vectors.

    Marques, Filipe; Luzhetskyy, Andriy; Mendes, Marta V; HIPS, Helmholtz-Institut für Pharmazeutische Forschung Saarland, Universitätscampus E8.1 66123 Saarbrücken, Germany. (Elsevier, 2020-08-20)
    The Gram-positive bacterium Corynebacterium glutamicum sustains the industrial production of chiral molecules such as L-amino acids. Through heterologous gene expression, C. glutamicum is becoming a sustainable source of small organic molecules and added-value chemicals. The current methods to implement heterologous genes in C. glutamicum rely on replicative vectors requiring lasting selection or chromosomal integration using homologous recombination. Here, we present a set of dedicated and transversal tools for genome editing and gene delivery into C. glutamicum. We generated a cosmid-based library suitable for efficient double allelic exchange, covering more than 94% of the chromosome with an average 5.1x coverage. We employed the library and an iterative marker excision system to generate the carotenoid-free C. glutamicum BT1-C31-Albino (BCA) host, featuring the attachment sites for actinophages ϕC31 and ϕBT1 for one-step chromosomal integration. As a proof-of-principle, we employed a ϕC31-based integration and a Cre system for the markerless expression of the type III polyketide synthase RppA, and a ϕBT1-based integration system for the expression of the phosphopantetheinylation-dependent non-ribosomal peptide synthetase BpsA in the C. glutamicum BCA host. The developed genomic library and microbial host, and the characterized molecular tools will contribute to the study of the physiology and the rise of C. glutamicum as a leading host for drug discovery.
  • 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.
  • 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.
  • Thioholgamide A, a New Anti-Proliferative Anti-Tumor Agent, Modulates Macrophage Polarization and Metabolism.

    Dahlem, Charlotte; Siow, Wei Xiong; Lopatniuk, Maria; Tse, William K F; Kessler, Sonja M; Kirsch, Susanne H; Hoppstädter, Jessica; Vollmar, Angelika M; Müller, Rolf; Luzhetskyy, Andriy; et al. (MDPI, 2020-05-19)
    Natural products represent powerful tools searching for novel anticancer drugs. Thioholgamide A (thioA) is a ribosomally synthesized and post-translationally modified peptide, which has been identified as a product of Streptomyces sp. MUSC 136T. In this study, we provide a comprehensive biological profile of thioA, elucidating its effects on different hallmarks of cancer in tumor cells as well as in macrophages as crucial players of the tumor microenvironment. In 2D and 3D in vitro cell culture models thioA showed potent anti-proliferative activities in cancer cells at nanomolar concentrations. Anti-proliferative actions were confirmed in vivo in zebrafish embryos. Cytotoxicity was only induced at several-fold higher concentrations, as assessed by live-cell microscopy and biochemical analyses. ThioA exhibited a potent modulation of cell metabolism by inhibiting oxidative phosphorylation, as determined in a live-cell metabolic assay platform. The metabolic modulation caused a repolarization of in vitro differentiated and polarized tumor-promoting human monocyte-derived macrophages: ThioA-treated macrophages showed an altered morphology and a modulated expression of genes and surface markers. Taken together, the metabolic regulator thioA revealed low activities in non-tumorigenic cells and an interesting anti-cancer profile by orchestrating different hallmarks of cancer, both in tumor cells as well as in macrophages as part of the tumor microenvironment.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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
  • 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.
  • Monitoring Protein Secretion in Using Fluorescent Proteins.

    Hamed, Mohamed Belal; Vrancken, Kristof; Bilyk, Bohdan; Koepff, Joachim; Novakova, Renata; van Mellaert, Lieve; Oldiges, Marco; Luzhetskyy, Andriy N; Kormanec, Jan; Anné, Jozef; et al. (2018-12-07)
    Fluorescent proteins are a major cell biology tool to analyze protein sub-cellular topology. Here we have applied this technology to study protein secretion in the Gram-positive bacterium Streptomyces lividans TK24, a widely used host for heterologous protein secretion biotechnology. Green and monomeric red fluorescent proteins were fused behind Sec (SPSec) or Tat (SPTat) signal peptides to direct them through the respective export pathway. Significant secretion of fluorescent eGFP and mRFP was observed exclusively through the Tat and Sec pathways, respectively. Plasmid over-expression was compared to a chromosomally integrated spSec-mRFP gene to allow monitoring secretion under high and low level synthesis in various media. Fluorimetric detection of SPSec-mRFP recorded folded states, while immuno-staining detected even non-folded topological intermediates. Secretion of SPSec-mRFP is unexpectedly complex, is regulated independently of cell growth phase and is influenced by the growth regime. At low level synthesis, highly efficient secretion occurs until it is turned off and secretory preforms accumulate. At high level synthesis, the secretory pathway overflows and proteins are driven to folding and subsequent degradation. High-level synthesis of heterologous secretory proteins, whether secretion competent or not, has a drastic effect on the endogenous secretome, depending on their secretion efficiency. These findings lay the foundations of dissecting how protein targeting and secretion are regulated by the interplay between the metabolome, secretion factors and stress responses in the S. lividans model.
  • Post-translational Serine/Threonine Phosphorylation and Lysine Acetylation: A Novel Regulatory Aspect of the Global Nitrogen Response Regulator GlnR in S. coelicolor M145.

    Amin, Rafat; Franz-Wachtel, Mirita; Tiffert, Yvonne; Heberer, Martin; Meky, Mohamed; Ahmed, Yousra; Matthews, Arne; Krysenko, Sergii; Jakobi, Marco; Hinder, Markus; et al. (Frontiers, 2016-01-01)
    Soil-dwelling Streptomyces bacteria such as S.coelicolor have to constantly adapt to the nitrogen (N) availability in their habitat. Thus, strict transcriptional and post-translational control of the N-assimilation is fundamental for survival of this species. GlnR is a global response regulator that controls transcription of the genes related to the N-assimilation in S. coelicolor and other members of the Actinomycetales. GlnR represents an atypical orphan response regulator that is not activated by the phosphorylation of the conserved aspartate residue (Asp 50). We have applied transcriptional analysis, LC-MS/MS analysis and electrophoretic mobility shift assays (EMSAs) to understand the regulation of GlnR in S. coelicolor M145. The expression of glnR and GlnR-target genes was revisited under four different N-defined conditions and a complex N-rich condition. Although, the expression of selected GlnR-target genes was strongly responsive to changing N-concentrations, the glnR expression itself was independent of the N-availability. Using LC-MS/MSanalysis we demonstrated that GlnR was post-translationally modified. The post-translational modifications of GlnR comprise phosphorylation of the serine/threonine residues and acetylation of lysine residues. In the complex N-rich medium GlnR was phosphorylated on six serine/threonine residues and acetylated on one lysine residue. Under defined N-excess conditions only two phosphorylated residues were detected whereas under defined N-limiting conditions no phosphorylation was observed. GlnR phosphorylation is thus clearly correlated with N-rich conditions. Furthermore, GlnR was acetylated on four lysine residues independently of the N-concentration in the defined media and on only one lysine residue in the complex N-rich medium. Using EMSAs we demonstrated that phosphorylation inhibited the binding of GlnR to its targets genes, whereas acetylation had little influence on the formation of GlnR-DNA complex. This study clearly demonstrated that GlnR DNA-binding affinity is modulated by post-translational modifications in response to changing N-conditions in order to elicit a proper transcriptional response to the latter.
  • 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
  • New Alpiniamides From sp. IB2014/011-12 Assembled by an Unusual Hybrid Non-ribosomal Peptide Synthetase -AT Polyketide Synthase Enzyme.

    Paulus, Constanze; Rebets, Yuriy; Zapp, Josef; Rückert, Christian; Kalinowski, Jörn; Luzhetskyy, Andriy N; HIPS, Helmholtz-Institut füt Pharmazeutische Forschung Saarland, Universitätscampus E8.1 66123 Saarbrücken, Germany. (2018-01-01)
    he environment of Lake Baikal is a well-known source of microbial diversity. The strain Streptomyces sp. IB2014/011-12, isolated from samples collected at Lake Baikal, was found to exhibit potent activity against Gram-positive bacteria. Here, we report isolation and characterization of linear polyketide alpiniamide A (1) and its new derivatives B–D (2–5). The structures of alpiniamides A–D were established and their relative configuration was determined by combination of partial Murata’s method and ROESY experiment. The absolute configuration of alpiniamide A was established through Mosher’s method. The gene cluster, responsible for the biosynthesis of alpiniamides (alp) has been identified by genome mining and gene deletion experiments. The successful expression of the cloned alp gene cluster in a heterologous host supports these findings. Analysis of the architecture of the alp gene cluster and the feeding of labeled precursors elucidated the alpiniamide biosynthetic pathway. The biosynthesis of alpiniamides is an example of a rather simple polyketide assembly line generating unusual chemical diversity through the combination of domain/module skipping and double bond migration events.
  • Multi-Omics and Targeted Approaches to Determine the Role of Cellular Proteases in Protein Secretion.

    Busche, Tobias; Tsolis, Konstantinos C; Koepff, Joachim; Rebets, Yuriy; Rückert, Christian; Hamed, Mohamed B; Bleidt, Arne; Wiechert, Wolfgang; Lopatniuk, Mariia; Yousra, Ahmed; et al. (2018-01-01)
    Gram-positive
  • A set of synthetic versatile genetic control elements for the efficient expression of genes in Actinobacteria.

    Horbal, Lilya; Siegl, Theresa; Luzhetskyy, Andriy N; 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 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.
  • 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.

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