• The diversity and antibacterial activity of culturable actinobacteria isolated from the rhizosphere soil of Deschampsia antarctica (Galindez Island, Maritime Antarctic)

      Tistechok, Stepan; Skvortsova, Maryna; Mytsyk, Yuliia; Fedorenko, Victor; Parnikoza, Ivan; Luzhetskyy, Andriy; Gromyko, Oleksandr; HIPS, Helmholtz-Institut für Pharmazeutische Forschung Saarland, Universitätscampus E8.1 66123 Saarbrücken, Germany. (Springer, 2021-09-01)
      Antarctic actinobacteria, which can be isolated from both soils and marine sediments, demonstrate a wide range of antimicrobial activities as well as significant biosynthetic potential as the producers of biologically active compounds. However, the actinobacterial diversity of the Antarctic region has not yet been sufficiently studied. The present study sought to examine the diversity and antibacterial activity of culturable actinobacteria isolated from the rhizosphere soil of Deschampsia antarctica (É. Desv.), which was collected from Galindez Island, Maritime Antarctic. Among the actinobacteria isolated using a 16S rRNA gene sequence-based phylogenetic analysis process, five genera, namely Streptomyces, Micromonospora, Umezawaea, Kribbella and Micrococcus, were identified. To the best of our knowledge, this is the first report to describe the isolation and initial characterisation of members of the genus Umezawaea from the Antarctic. The isolated actinobacteria were assayed to determine their activity against Gram-positive bacteria, Gram-negative bacteria and yeast. Among the isolated strains, only 30.2% were able to inhibit the growth of at least one of the tested pathogens. The polymerase chain reaction-based screening of the biosynthetic genes revealed the presence of type I polyketide synthases (65.1%), type II polyketide synthases (25.6%) and non-ribosomal peptide synthetases (9.3%) in the actinobacteria strains. The examination of the sensitivity/resistance to antibiotics profile of the actinobacteria strains revealed their high sensitivity in relation to the tested antibiotics. Taken together, the results showed that Antarctic actinobacteria demonstrate potential as the producers of natural bioactive compounds, which means that they represent a valuable prospect for further studies.
    • Bonsecamin: A New Cyclic Pentapeptide Discovered through Heterologous Expression of a Cryptic Gene Cluster.

      Lasch, Constanze; Stierhof, Marc; Estévez, Marta Rodríguez; Myronovskyi, Maksym; Zapp, Josef; Luzhetskyy, Andriy N; HIPS, Helmholtz-Institut für Pharmazeutische Forschung Saarland, Universitätscampus E8.1 66123 Saarbrücken, Germany. (MDPI, 2021-07-31)
      The intriguing structural complexity of molecules produced by natural organisms is uncontested. Natural scaffolds serve as an important basis for the development of molecules with broad applications, e.g., therapeutics or agrochemicals. Research in recent decades has demonstrated that by means of classic metabolite extraction from microbes only a small portion of natural products can be accessed. The use of genome mining and heterologous expression approaches represents a promising way to discover new natural compounds. In this paper we report the discovery of a novel cyclic pentapeptide called bonsecamin through the heterologous expression of a cryptic NRPS gene cluster from Streptomyces albus ssp. chlorinus NRRL B-24108 in Streptomyces albus Del14. The new compound was successfully isolated and structurally characterized using NMR. The minimal set of genes required for bonsecamin production was determined through bioinformatic analysis and gene deletion experiments. A biosynthetic route leading to the production of bonsecamin is proposed in this paper.
    • Cyclofaulknamycin with the Rare Amino Acid D-capreomycidine Isolated from a Well-Characterized Strain.

      Horbal, Liliya; Stierhof, Marc; Palusczak, Anja; Eckert, Nikolas; Zapp, Josef; Luzhetskyy, Andriy N; HIPS, Helmholtz-Institut für Pharmazeutische Forschung Saarland, Universitätscampus E8.1 66123 Saarbrücken, Germany. (MDPI, 2021-07-28)
      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 Heterologous Production of New Cyclic Depsibosamycins.

      Stierhof, Marc; Myronovskyi, Maksym; Zapp, Josef; Luzhetskyy, Andriy N; HIPS, Helmholtz-Institut für Pharmazeutische Forschung Saarland, Universitätscampus E8.1 66123 Saarbrücken, Germany. (MDPI, 2021-06-28)
      Streptomyces are producers of valuable secondary metabolites with unique scaffolds that perform a plethora of biological functions. Nonribosomal peptides are of special interest due to their variety and complexity. They are synthesized by nonribosomal peptide synthetases, large biosynthetic machineries that are encoded in the genome of many Streptomyces species. The identification of new peptides and the corresponding biosynthetic gene clusters is of major interest since knowledge can be used to facilitate combinatorial biosynthesis and chemical semisynthesis of natural products. The recently discovered bosamycins are linear octapeptides with an interesting 5-OMe tyrosine moiety and various modifications at the N-terminus. In this study, the new cyclic depsibosamycins B, C, and D from Streptomyces aurantiacus LU19075 were discovered. In comparison to the linear bosamycins B, C, and D, which were also produced by the strain, the cyclic depsibosamycins showed a side-chain-to-tail lactonization of serine and glycine, leading to a ring of four amino acids. In silico identification and heterologous expression of the depsibosamycin (dbm) gene cluster indicated that the cyclic peptides, rather than the linear derivatives, are the main products of the cluster.
    • Multiple copies of the oxytetracycline gene cluster in selected Streptomyces rimosus strains can provide significantly increased titers.

      Pikl, Špela; Carrillo Rincón, Andrés Felipe; Slemc, Lucija; Goranovič, Dušan; Avbelj, Martina; Gjuračić, Krešimir; Sucipto, Hilda; Stare, Katja; Baebler, Špela; Šala, Martin; et al. (BioMedCentral, 2021-02-17)
      Background: Natural products are a valuable source of biologically active compounds that have applications in medicine and agriculture. One disadvantage with natural products is the slow, time-consuming strain improvement regimes that are necessary to ensure sufficient quantities of target compounds for commercial production. Although great efforts have been invested in strain selection methods, many of these technologies have not been improved in decades, which might pose a serious threat to the economic and industrial viability of such important bioprocesses. Results: In recent years, introduction of extra copies of an entire biosynthetic pathway that encodes a target product in a single microbial host has become a technically feasible approach. However, this often results in minor to moderate increases in target titers. Strain stability and process reproducibility are the other critical factors in the industrial setting. Industrial Streptomyces rimosus strains for production of oxytetracycline are one of the most economically efficient strains ever developed, and thus these represent a very good industrial case. To evaluate the applicability of amplification of an entire gene cluster in a single host strain, we developed and evaluated various gene tools to introduce multiple copies of the entire oxytetracycline gene cluster into three different Streptomyces rimosus strains: wild-type, and medium and high oxytetracycline-producing strains. We evaluated the production levels of these engineered S. rimosus strains with extra copies of the oxytetracycline gene cluster and their stability, and the oxytetracycline gene cluster expression profiles; we also identified the chromosomal integration sites. Conclusions: This study shows that stable and reproducible increases in target secondary metabolite titers can be achieved in wild-type and in high oxytetracycline-producing strains, which always reflects the metabolic background of each independent S. rimosus strain. Although this approach is technically very demanding and requires systematic effort, when combined with modern strain selection methods, it might constitute a very valuable approach in industrial process development.
    • Targeted Genome Mining-From Compound Discovery to Biosynthetic Pathway Elucidation.

      Gummerlich, Nils; Rebets, Yuriy; Paulus, Constanze; Zapp, Josef; Luzhetskyy, Andriy; HIPS, Helmholtz-Institut für Pharmazeutische Forschung Saarland, Universitätscampus E8.1 66123 Saarbrücken, Germany. (MDPI, 2020-12-19)
      Natural products are an important source of novel investigational compounds in drug discovery. Especially in the field of antibiotics, Actinobacteria have been proven to be a reliable source for lead structures. The discovery of these natural products with activity- and structure-guided screenings has been impeded by the constant rediscovery of previously identified compounds. Additionally, a large discrepancy between produced natural products and biosynthetic potential in Actinobacteria, including representatives of the order Pseudonocardiales, has been revealed using genome sequencing. To turn this genomic potential into novel natural products, we used an approach including the in-silico pre-selection of unique biosynthetic gene clusters followed by their systematic heterologous expression. As a proof of concept, fifteen Saccharothrixespanaensis genomic library clones covering predicted biosynthetic gene clusters were chosen for expression in two heterologous hosts, Streptomyceslividans and Streptomycesalbus. As a result, two novel natural products, an unusual angucyclinone pentangumycin and a new type II polyketide synthase shunt product SEK90, were identified. After purification and structure elucidation, the biosynthetic pathways leading to the formation of pentangumycin and SEK90 were deduced using mutational analysis of the biosynthetic gene cluster and feeding experiments with 13C-labelled precursors.
    • Dudomycins: New Secondary Metabolites Produced After Heterologous Expression of an Nrps Cluster from ssp. Nrrl B-24108.

      Lasch, Constanze; Stierhof, Marc; Estévez, Marta Rodríguez; Myronovskyi, Maksym; Zapp, Josef; Luzhetskyy, Andriy; HIPS, Helmholtz-Institut für Pharmazeutische Forschung Saarland, Universitätscampus E8.1 66123 Saarbrücken, Germany. (MDPI, 2020-11-16)
      Since the 1950s, natural products of bacterial origin were systematically developed to be used as drugs with a wide range of medical applications. The available treatment options for many diseases are still not satisfying, wherefore, the discovery of new structures has not lost any of its importance. Beyond the great variety of already isolated and characterized metabolites, Streptomycetes still harbor uninvestigated gene clusters whose products can be accessed using heterologous expression in host organisms. This works presents the discovery of a set of structurally novel secondary metabolites, dudomycins A to D, through the expression of a cryptic NRPS cluster from Streptomyces albus ssp. Chlorinus NRRL B-24108 in the heterologous host strain Streptomyces albus Del14. A minimal set of genes, required for the production of dudomycins, was defined through gene inactivation experiments. This paper also proposes a model for dudomycin biosynthesis.
    • 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.
    • 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.
    • 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.
    • Novel Fredericamycin Variant Overproduced by a Streptomycin-resistant subsp. Strain.

      Rodríguez Estévez, Marta; Myronovskyi, Maksym; Rosenkränzer, Birgit; Paululat, Thomas; Petzke, Lutz; Ristau, Jeanette; Luzhetskyy, Andriy; HIPS, Helmholtz-Institut für Pharmazeutische Forschung Saarland, Universitätscampus E8.1 66123 Saarbrücken, Germany. (MDPI, 2020-05-28)
      Streptomycetes are an important source of natural products potentially applicable in the pharmaceutical industry. Many of these drugs are secondary metabolites whose biosynthetic genes are very often poorly expressed under laboratory cultivation conditions. In many cases, antibiotic-resistant mutants exhibit increased production of natural drugs, which facilitates the identification and isolation of new substances. In this study, we report the induction of a type II polyketide synthase gene cluster in the marine strain Streptomyces albus subsp. chlorinus through the selection of streptomycin-resistant mutants, resulting in overproduction of the novel compound fredericamycin C2 (1). Fredericamycin C2 (1) is structurally related to the potent antitumor drug lead fredericamycin A.
    • 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.
    • Baikalomycins A-C, New Aquayamycin-Type Angucyclines Isolated from Lake Baikal Derived sp. IB201691-2A.

      Voitsekhovskaia, Irina; Paulus, Constanze; Dahlem, Charlotte; Rebets, Yuriy; Nadmid, Suvd; Zapp, Josef; Axenov-Gribanov, Denis; Rückert, Christian; Timofeyev, Maxim; Kalinowski, Jörn; et al. (MDPI, 2020-05-07)
      Natural products produced by bacteria found in unusual and poorly studied ecosystems, such as Lake Baikal, represent a promising source of new valuable drug leads. Here we report the isolation of a new Streptomyces sp. strain IB201691-2A from the Lake Baikal endemic mollusk Benedictia baicalensis. In the course of an activity guided screening three new angucyclines, named baikalomycins A-C, were isolated and characterized, highlighting the potential of poorly investigated ecological niches. Besides that, the strain was found to accumulate large quantities of rabelomycin and 5-hydroxy-rabelomycin, known shunt products in angucyclines biosynthesis. Baikalomycins A-C demonstrated varying degrees of anticancer activity. Rabelomycin and 5-hydroxy-rabelomycin further demonstrated antiproliferative activities. The structure elucidation showed that baikalomycin A is a modified aquayamycin with β-d-amicetose and two additional hydroxyl groups at unusual positions (6a and 12a) of aglycone. Baikalomycins B and C have alternating second sugars attached, α-l-amicetose and α-l-aculose, respectively. The gene cluster for baikalomycins biosynthesis was identified by genome mining, cloned using a transformation-associated recombination technique and successfully expressed in S. albus J1074. It contains a typical set of genes responsible for an angucycline core assembly, all necessary genes for the deoxy sugars biosynthesis, and three genes coding for the glycosyltransferase enzymes. Heterologous expression and deletion experiments allowed to assign the function of glycosyltransferases involved in the decoration of baikalomycins aglycone.
    • 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.
    • 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.
    • Perquinolines A-C: Unprecedented Bacterial Tetrahydroisoquinolines Involving an Intriguing Biosynthesis.

      Rebets, Yuriy; Nadmid, Suvd; Paulus, Constanze; Dahlem, Charlotte; Herrmann, Jennifer; Hübner, Harald; Rückert, Christian; Kiemer, Alexandra K; Gmeiner, Peter; Kalinowski, Jörn; et al. (Wiley, 2019-08-21)
      Autophagy, a membrane-dependent catabolic process, ensures survival of aging cells and depends on the cellular energetic status. Acetyl-CoA carboxylase 1 (Acc1) connects central energy metabolism to lipid biosynthesis and is rate-limiting for the de novo synthesis of lipids. However, it is unclear how de novo lipogenesis and its metabolic consequences affect autophagic activity. Here, we show that in aging yeast, autophagy levels highly depend on the activity of Acc1. Constitutively active Acc1 (acc1S/A ) or a deletion of the Acc1 negative regulator, Snf1 (yeast AMPK), shows elevated autophagy levels, which can be reversed by the Acc1 inhibitor soraphen A. Vice versa, pharmacological inhibition of Acc1 drastically reduces cell survival and results in the accumulation of Atg8-positive structures at the vacuolar membrane, suggesting late defects in the autophagic cascade. As expected, acc1S/A cells exhibit a reduction in acetate/acetyl-CoA availability along with elevated cellular lipid content. However, concomitant administration of acetate fails to fully revert the increase in autophagy exerted by acc1S/A Instead, administration of oleate, while mimicking constitutively active Acc1 in WT cells, alleviates the vacuolar fusion defects induced by Acc1 inhibition. Our results argue for a largely lipid-dependent process of autophagy regulation downstream of Acc1. We present a versatile genetic model to investigate the complex relationship between acetate metabolism, lipid homeostasis, and autophagy and propose Acc1-dependent lipogenesis as a fundamental metabolic path downstream of Snf1 to maintain autophagy and survival during cellular aging.
    • 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.
    • 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.