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Preparation of genetically manipulated producers of hybrid manumycins
Krýslová, Dita ; Petříčková, Kateřina (advisor) ; Doubravová, Linda (referee)
Streptomyces are one of the most prolific producers of various secondary metabolites. Manumycin antibiotics represent an important class of these compounds. They belong to a big class of polyketide metabolites. While their antibiotic effect is not very significant, their other biological properties have a big potential in the treatment of inflammations, tumors, etc. They are characterized by two short polyketide chains, which are attached to a central subunit. At the end of the lower polyketide chain, a C5N cyclic unit is frequently attached. This thesis originates from the colabomycin E, an antibiotic, which was discovered by our team. This antibiotic is a new member of manumycin-type metabolites and is produced by Streptomyces aureus SOK1/5-04 strain. Previous studies on the function of individual genes in the biosynthetic gene cluster of colabomycin E inspired us to consider editing of the current biological activity of colabomycin E by replacement of the C5N unit with another, structurally similar bioactive subunit. Due to high structural similarity, we have selected 4,7- dihydroxycoumarin unit of novobiocin, an aminocoumarin-type metabolite produced also by Streptomyces. The 4,7-dihydroxycoumarin unit is pharmacophore with a cancerostatic activity. We expected that the cancerostatic activity...
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Mutational analysis of manumycin antibiotics biosynthetic routes
Kolek, Jan ; Petříček, Miroslav (advisor) ; Zikánová, Blanka (referee)
Mutational analysis of manumycin antibiotics biosynthetic routes The manumycin antibiotics are secondary metabolites, which come from a big group of polyketide metabolites. They are produced by bacteria from genus Streptomyces. Manumycin antibiotics are characterized by two linear polyketide chains, which are connected to a central mC7N moiety. The lower chain is often terminated by the C5N moiety. Manumycin metabolites show many biological activities. They have antimicrobial activity, especially against gram-positive bacteria. Next, they posses antifungal, insecticidal or antiinflamatory activities. Manumycins are also potentional anticancer agents. In order to prepare these compounds by the fermentation, the detailed knowledge of their biosynthetic routes is required. Mutational analysis is based on techniques of genetic engineering. Mutational analysis is a useful pool for analysis of biosynthetic pathways of secondary metabolites and the genes, which are involved in these pathways. This knowledge is essential for application of combinatorial biosynthesis for the design of new metabolites. The new hybrid compound could be used in future as new antibiotics or anticancer drugs. Keywords: manumycin, polyketide, streptomyces, asukamycin
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The effect of polyketide antibiotics on signalling and functional activity of human monocytic cell line
Kopecká, Kristýna ; Stříž, Ilja (advisor) ; Kovářová, Jaromíra (referee)
Anti-inflammatory cytokines have an important role in the development of inflammatory reactions. If an acute inflammation turns into chronical it is very often a pathological phenomenon. Chronicle inflammations accompany a whole number of serious diseases with an unclear prognosis, such as some of the autoimmune diseases. Usually, the cause of these diseases is not quite clear and the treatment is mainly symptomatic with an effort to suppress the immunity system. For this purpose we use various immunosuppressant drugs, and biological treatment is used, too. Another possibility is to use bioactive secondary metabolites produced by various microorganisms. In this group there are for example macrolides antibiotics, and a big potential is also seen in the recently discovered polyketides. The objective of this work is to test the newly acquired secondary metabolites that were isolated in the Laboratory of Molecular Biology of Actinomycetes at the Czech Academy of Sciences. Tested were manumycin A, manumycin B, colabomycin E, asukamycin A, asukamycin D, β-rubromycin, deoxynybomycin. As comparative substances were used the macrolides antibiotics clarithromycin and azithromycin dehydrate, all of them commercial pharmaceuticals. These substances were tested on the monocytic line THP-1. Cells were stimulated...
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Analysis of the biosynthetic gene cluster encoding biosynthesis of the manumycin antibiotic U-62162, and the ways of its modification.
Urbanová, Daniela ; Petříček, Miroslav (advisor) ; Schierová, Michaela (referee)
Streptomyces is the largest antibiotic-producing genus in the microbial world. Manumycin-type antibiotics are a small group of its metabolites. Their antibiotic activities are not very important but they show biological properties which can be potencially used e. g. to treat inflammation, cancer or Alzheimer's disease. The structure of manumycin compounds is formed by a central unit with connected upper and lower polyketide chain. The lower chain is mostly terminated by so called C5N unit. The substance U-62162 produced by the strain Streptomyces verdensis differs significantly from the other members of the manumycin-type metabolites in the structure of the lower chain which is fully saturated and lacking the C5N unit. The U-62162 biosynthetic gene cluster was sequenced and functions of identified open reading frames were deduced. Heterologous expressions of the cluster showed some genes reguired for the biosynthesis of the upper chain to be encoded on a different part of the chromosome. The insertional inactivation of the vrdER gene confirmed the enoylreductase to be responsible for the saturation of the lower chain. DSBA oxidoreductase, which gene is located at the edge of the cluster, is probably not involved in the biosynthesis. The insertion of genes for the biosynthesis of the C5N unit did...
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Lessons from nature - preparation of hybrid bioactive compounds
Vobruba, Šimon ; Janata, Jiří (advisor) ; Zikánová, Blanka (referee)
Secondary metabolites are biologically active compounds produced mainly by microorganisms. They are not essential for survival of producing strains, however, they significantly affect their physiology and ecology. They are frequently used in pharmacology, biology and chemistry. The present work describes the current state of knowledge concerning origin and evolution of secondary metabolites. The secondary metabolites biosynthetic genes are usually organised in clusters. The basic mechanisms of secondary metabolite gene clusters modification are gene mutations or intragenic rearrangements. These mechanisms are typically involved in natural evolution of gene clusters coding for secondary metabolites with modular type of biosynthesis. The subclusters of different origin can also fuse to form a new hybrid compound biosynthetic gene cluster. Similar evolutionary event probably occurred also in case of biosynthesis of two model groups of natural compounds - lincosamides and pyrrolobenzodiazepines. Analogous approaches are used in genetic engineering to construct producers of new more efficient bioactive compounds. Examples of such genetic modifications of gene clusters involved in the biosynthesis of compounds from nonribosomal peptides, polyketides and lincosamides groups are described. Possible future...
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The effect of polyketide antibiotics on signalling and functional activity of human monocytic cell line
Kopecká, Kristýna ; Stříž, Ilja (advisor) ; Kovářová, Jaromíra (referee)
Anti-inflammatory cytokines have an important role in the development of inflammatory reactions. If an acute inflammation turns into chronical it is very often a pathological phenomenon. Chronicle inflammations accompany a whole number of serious diseases with an unclear prognosis, such as some of the autoimmune diseases. Usually, the cause of these diseases is not quite clear and the treatment is mainly symptomatic with an effort to suppress the immunity system. For this purpose we use various immunosuppressant drugs, and biological treatment is used, too. Another possibility is to use bioactive secondary metabolites produced by various microorganisms. In this group there are for example macrolides antibiotics, and a big potential is also seen in the recently discovered polyketides. The objective of this work is to test the newly acquired secondary metabolites that were isolated in the Laboratory of Molecular Biology of Actinomycetes at the Czech Academy of Sciences. Tested were manumycin A, manumycin B, colabomycin E, asukamycin A, asukamycin D, β-rubromycin, deoxynybomycin. As comparative substances were used the macrolides antibiotics clarithromycin and azithromycin dehydrate, all of them commercial pharmaceuticals. These substances were tested on the monocytic line THP-1. Cells were stimulated...
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Identification and activation of a cryptic biosynthetic gene cluster for manumycin-type metabolites in Saccharothrix espanaensis DSM44229
Zelenka, Tomáš ; Petříčková, Kateřina (advisor) ; Beranová, Jana (referee)
1 Abstract: Secondary metabolism of Gram-positive soil bacteria from the genus Streptomyces is a inestimable source of natural products including manumycins, which belong to a polyketide group. These products possess weak antimicrobial, but important antiinflammatory, and antitumor activities. Streptomyces sp. offers broad amounts of yet undiscovered antibiotics, potentially utilizable in clinical medicine. This fact makes out of these organisms a promising solution to our present problem with rising antibiotic resistance among microorganisms. Two main ways are applied in this research: There are efforts of prepairing new derivates based on known products and creating various modifications in their structure. Next, new producers are discovered by "genome mining" methods, activation of silent gene clusters, followed by improvements of antibiotic production. One of those silent clusters was found in the Saccharothrix espanaensis DSM44229 strain. The genetic information has been transferred to a heterologous host in order to characterize its product. Cluster activation and production of novel manumycin-type metabolites occurred in the host after the transfer.
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Analysis of the biosynthetic gene cluster encoding biosynthesis of the manumycin antibiotic U-62162, and the ways of its modification.
Urbanová, Daniela ; Petříček, Miroslav (advisor) ; Schierová, Michaela (referee)
Streptomyces is the largest antibiotic-producing genus in the microbial world. Manumycin-type antibiotics are a small group of its metabolites. Their antibiotic activities are not very important but they show biological properties which can be potencially used e. g. to treat inflammation, cancer or Alzheimer's disease. The structure of manumycin compounds is formed by a central unit with connected upper and lower polyketide chain. The lower chain is mostly terminated by so called C5N unit. The substance U-62162 produced by the strain Streptomyces verdensis differs significantly from the other members of the manumycin-type metabolites in the structure of the lower chain which is fully saturated and lacking the C5N unit. The U-62162 biosynthetic gene cluster was sequenced and functions of identified open reading frames were deduced. Heterologous expressions of the cluster showed some genes reguired for the biosynthesis of the upper chain to be encoded on a different part of the chromosome. The insertional inactivation of the vrdER gene confirmed the enoylreductase to be responsible for the saturation of the lower chain. DSBA oxidoreductase, which gene is located at the edge of the cluster, is probably not involved in the biosynthesis. The insertion of genes for the biosynthesis of the C5N unit did...
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Lessons from nature - preparation of hybrid bioactive compounds
Vobruba, Šimon ; Janata, Jiří (advisor) ; Zikánová, Blanka (referee)
Secondary metabolites are biologically active compounds produced mainly by microorganisms. They are not essential for survival of producing strains, however, they significantly affect their physiology and ecology. They are frequently used in pharmacology, biology and chemistry. The present work describes the current state of knowledge concerning origin and evolution of secondary metabolites. The secondary metabolites biosynthetic genes are usually organised in clusters. The basic mechanisms of secondary metabolite gene clusters modification are gene mutations or intragenic rearrangements. These mechanisms are typically involved in natural evolution of gene clusters coding for secondary metabolites with modular type of biosynthesis. The subclusters of different origin can also fuse to form a new hybrid compound biosynthetic gene cluster. Similar evolutionary event probably occurred also in case of biosynthesis of two model groups of natural compounds - lincosamides and pyrrolobenzodiazepines. Analogous approaches are used in genetic engineering to construct producers of new more efficient bioactive compounds. Examples of such genetic modifications of gene clusters involved in the biosynthesis of compounds from nonribosomal peptides, polyketides and lincosamides groups are described. Possible future...
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Biosynthesis of lower polyketide chains in manumycin antibiotics - the length-affecting factors
Kolek, Jan ; Petříček, Miroslav (advisor) ; Seydlová, Gabriela (referee)
Manumycin antibiotics represent an important class of secondary metabolites produced by Streptomyces bacteria. They belong to a big class of polyketide metabolites and posses significant antimicrobial, anti-inflammatory, antitumor, and many other biological activities. They are characterized by two short polyketide chains, which are attached to a central subunit. Polyketide chains are synthesized by enzymes of the iterative type II polyketide-synthase. Mechanism of regulation of the polyketide chains length has not been known yet. Understanding mechanism can lead to biosynthesis of novel manumycin antibiotics with predetermined chain lengths what may improve their biological activities in favour of a practical use of these compounds. We prepared a mutant strain of asukamycin producer Streptomyces nodosus ssp. asukaensis with deletion of genes coding for type I/II β-ketoacylsynthase and protein AsuC14, which is a potential factor affecting lower polyketide chain length, for the identification of the chain length factor in manumycin antibiotics producers. Next, the genes for type I/II β-ketoacylsynthase and potential chain length-affecting factor C14 from strains producing manumycins with variable length of the lower polyketide chains were expressed in this mutant strain. Our results demonstrate...
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