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  • 1
    Electronic Resource
    Electronic Resource
    Engineering specificity of starter unit selection by the erythromycin-producing polyketide synthase (2002)
    Oxford, UK : Blackwell Science Ltd.
    Molecular microbiology 43 (2002), S. 0 
    Details
    ISSN: 1365-2958
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: Chain initiation on many modular polyketide synthases is mediated by acyl transfer from the CoA ester of a dicarboxylic acid, followed by decarboxylation in situ by KSQ, a ketosynthase-like decarboxylase domain. Consistent with this, the acyltransferase (AT) domains of all KSQ-containing loading modules are shown here to contain a key arginine residue at their active site. Site-specific replacement of this arginine residue in the oleandomycin (ole) loading AT domain effectively abolished AT activity, consistent with its importance for catalysis. Substitution of the ole PKS loading module, or of the tylosin PKS loading module, for the erythromycin (ery) loading module gave polyketide products almost wholly either acetate derived or propionate derived, respectively, instead of the mixture found normally. An authentic extension module AT domain, rap AT2 from the rapamycin PKS, functioned appropriately when engineered in the place of the ole loading AT domain, and gave rise to substantial amounts of C13-methylerythromycins, as predicted. The role of direct acylation of the ketosynthase domain of ex-tension module 1 in chain initiation was confirmed by demonstrating that a mutant of the triketide synthase DEBS1-TE, in which the 4′-phosphopante-theine attachment site for starter acyl groups was specifically removed, produced triketide lactone pro-ducts in detectable amounts.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1046/j.1365-2958.2002.02815.x
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  • 2
    Electronic Resource
    Electronic Resource
    Parallel pathways for oxidation of 14-membered polyketide macrolactones in Saccharopolyspora erythraea (2002)
    Oxford, UK : Blackwell Science Ltd.
    Molecular microbiology 44 (2002), S. 0 
    Details
    ISSN: 1365-2958
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: The glycosyltransferases OleG1 and OleG2 and the cytochrome P450 oxidase OleP from the oleandomycin biosynthetic gene cluster of Streptomyces antibioticus have been expressed, either separately or from artificial gene cassettes, in strains of Saccharopolyspora erythraea blocked in erythromycin biosynthesis, to investigate their potential for the production of diverse novel macrolides from erythronolide precursors. OleP was found to oxidize 6-deoxyerythronolide B, but not erythronolide B. However, OleP did oxidize derivatives of erythronolide B in which a neutral sugar is attached at C-3. The oxidized products 3-O-mycarosyl-8a-hydroxyerythronolide B, 3-O-mycarosyl-8,8a-epoxyerythronolide B, 6-deoxy-8-hydroxyerythronolide B and the olefin 6-deoxy-8,8a-dehydroerythronolide B were all isolated and their structures determined. When oleP and the mycarosyltransferase eryBV were co-expressed in a gene cassette, 3-O-mycarosyl-6-deoxy-8,8a-dihydroxyerythronolide B was directly obtained. When oleG2 was co-expressed in a gene cassette together with oleP, 6-deoxyerythronolide B was converted into a mixture of 3-O-rhamnosyl-6-deoxy-8,8a-dehydroerythronolide B and 3-O-rhamnosyl-6-deoxy-8,8a-dihydroxyerythronolide B, confirming previous reports that OleG2 can transfer rhamnose, and confirming that oxidation by OleP and attachment of the neutral sugar to the aglycone can occur in either order. Similarly, four different 3-O-mycarosylerythronolides were found to be substrates for the desosaminyltransferase OleG1. These results provide additional insight into the nature of the intermediates in OleP-mediated oxidation, and suggest that oleandomycin biosynthesis might follow parallel pathways in which epoxidation either precedes or follows attachment of the neutral sugar.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1046/j.1365-2958.2002.02910.x
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  • 3
    Electronic Resource
    Electronic Resource
    Streptomyces antibioticus contains at least three oleandomycin-resistance determinants, one of which shows similarity with proteins of the ABC-transporter superfamily (1993)
    Oxford, UK : Blackwell Publishing Ltd
    Molecular microbiology 8 (1993), S. 0 
    Details
    ISSN: 1365-2958
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: Three different DNA fragments of an oleandomycin producer, Streptomyces antibioticus, conferring oleandomycin resistance were cloned in plasmid pIJ702 and expressed in Streptomyces lividans and in Streptomyces albus. These oleandomycin resistance determinants were designated as oleA (pOR400), oleB (pOR501) and oleC (pOR800). oleA and oleC are closely linked in the chromosome as they were both obtained together in two cosmid clones that were isolated from a genomic library. Sequencing of the oleC resistance determinant revealed four complete open reading frames (ORFs) and the C-terminal end of a fifth. The functions of orf1 and orf2 are unknown since they did not show significant similarity with other sequences in the data bases. The orf3 gene product has similarity with some proteins involved in iron and vitamin B12 uptake in bacteria. The orf4 gene product had a hydrophilic profile and showed important similarity with proteins containing typical ATP-binding domains characteristic of the ABC-transporter superfamily and involved in membrane transport and, particularly, with several genes conferring resistance to various macrolide antibiotics and anticancer drugs. The last gene, orf5, is translationally coupled to orf4 and codes for a hydrophobic polypeptide containing several trans-membrane domains characteristic of integral membrane proteins. Subcloning and deletion experiments limited the resistance determinant to a 0.9kb Pst1-Sph1 fragment and only orf4 is included in this fragment. These results suggest that resistance to oleandomycin conferred by oleC (orf4) is probably due to an efflux transport system of the ABC-transporter superfamily.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1111/j.1365-2958.1993.tb01601.x
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  • 4
    Electronic Resource
    Electronic Resource
    A second ABC transporter is involved in oleandomycin resistance and its secretion by Streptomyces antibioticus (1995)
    Oxford, UK : Blackwell Publishing Ltd
    Molecular microbiology 16 (1995), S. 0 
    Details
    ISSN: 1365-2958
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: A 3.2 kb Sstl-Sphl DNA fragment of Streptomyces antibioticus, an oleandomycin producer, conferring resistance to oleandomycin was sequenced and found to contain an open reading frame of 1710 bp (oleB). Its deduced gene product (OleB) showed a high degree of similarity with other proteins belonging to the ABC-transporter superfamily including the gene product of another oleandomycin-resistance gene (OleC). The OleB protein contains two ATP-binding domains, each of approximately 200 amino acids in length, and no hydrophobic transmembrane regions. Functional analysis of the oleB gene was carried out by deleting specific regions of the gene and assaying for oleandomycin resistance. These experiments showed that either the first or the second half of the gene containing only one ATP-binding domain was sufficient to confer resistance to oleandomycin. The gene oleB was expressed in Escherichia coli fused to a maltose-binding protein (MBP) using the pMal-c2 vector. The MBP-OleB hybrid protein was purified by affinity chromatography on an amylose resin and polyclonal antibodies were raised against the fusion protein. These were used to monitor the biosynthesis and physical location of OleB during growth. By Western analysis, the OleB protein was detected both in the soluble and in the membrane fraction and its synthesis paralleled oleandomycin biosynthesis. It was also shown that a Streptomyces albus strain, containing both a glycosyltransferase (OleD) able to inactivate oleandomycin and the OleB protein, was capable of glycosylating oleandomycin and secreting the inactive glycosylated molecule. It is proposed that OleB constitutes the secretion system by which oleandomycin or its inactive glycosylated form could be secreted by S. antibioticus.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1111/j.1365-2958.1995.tb02305.x
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  • 5
    Electronic Resource
    Electronic Resource
    Two glycosyltransferases and a glycosidase are involved in oleandomycin modification during its biosynthesis by Streptomyces antibioticus (1998)
    Oxford BSL : Blackwell Science Ltd
    Molecular microbiology 28 (1998), S. 0 
    Details
    ISSN: 1365-2958
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: A 5.2 kb region from the oleandomycin gene cluster in Streptomyces antibioticus located between the oleandomycin polyketide synthase gene and sugar biosynthetic genes was cloned. Sequence analysis revealed the presence of three open reading frames (designated oleI, oleN2 and oleR). The oleI gene product resembled glycosyltransferases involved in macrolide inactivation including the oleD product, a previously described glycosyltransferase from S. antibioticus. The oleN2 gene product showed similarities with different aminotransferases involved in the biosynthesis of 6-deoxyhexoses. The oleR gene product was similar to several glucosidases from different origins. The oleI, oleR and oleD genes were expressed in Streptomyces lividans. OleI and OleD intracellular proteins were partially purified by affinity chromatography in an UDP-glucuronic acid agarose column and OleR was detected as a major band from the culture supernatant. OleI and OleD showed oleandomycin glycosylating activity but they differ in the pattern of substrate specificity: OleI being much more specific for oleandomycin. OleR showed glycosidase activity converting glycosylated oleandomycin into active oleandomycin. A model is proposed integrating these and previously reported results for intracellular inactivation, secretion and extracellular reactivation of oleandomycin.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1046/j.1365-2958.1998.00880.x
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  • 6
    Electronic Resource
    Electronic Resource
    Synthesis of ribosomal proteins during growth of Streptomyces coelicolor (1994)
    Oxford, UK : Blackwell Publishing Ltd
    Molecular microbiology 12 (1994), S. 0 
    Details
    ISSN: 1365-2958
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: Changes in expression of ribosomal protein genes during growth and stationary phase of Streptomyces coelicolor A3(2) in liquid medium were studied. Proteins being synthesized were pulse-labelled with [35 S]-methionine, separated by two-dimensional poly-acrylamide gel electrophoresis, and quantified using the Bioimage computer software. Most of the ribosomal proteins were synthesized throughout the life cycle. Exceptions were two proteins whose synthesis drastically decreased at the approach of stationary phase. These two proteins were identified in purified ribosomes as homologues of Escherichia coli ribosomal proteins L10 and L7/L12, using antibodies raised against fusion proteins between these ribosomal proteins and Escherichia coliβ-galactosldase. The genes (rplJ and rplL) encoding the L10 and L7/L12 proteins were contained in a 1.2 kb BamHl fragment that was cloned and sequenced. The linkage and order of the genes coincide with other L10-L7/L12 operons. However, L11 and L1 genes were not present immediately upstream of the L10 gene, as is the case for E. coli and other bacteria. Instead, two open reading frames of unknown function were found immediately upstream of the L10 gene, in an adjacent 1.9 kb BamHl fragment.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1111/j.1365-2958.1994.tb01027.x
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  • 7
    Electronic Resource
    Electronic Resource
    Deciphering the late steps in the biosynthesis of the anti-tumour indolocarbazole staurosporine: sugar donor substrate flexibility of the StaG glycosyltransferase (2005)
    Oxford, UK : Blackwell Science Ltd
    Molecular microbiology 58 (2005), S. 0 
    Details
    ISSN: 1365-2958
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: The indolocarbazole staurosporine is a potent inhibitor of a variety of protein kinases. It contains a sugar moiety attached through C-N linkages to both indole nitrogen atoms of the indolocarbazole core. Staurosporine biosynthesis was reconstituted in vivo in a heterologous host Streptomyces albus by using two different plasmids: the ‘aglycone vector’ expressing a set of genes involved in indolocarbazole biosynthesis together with staG (encoding a glycosyltransferase) and/or staN (coding for a P450 oxygenase), and the ‘sugar vector’ expressing a set of genes responsible for the biosynthesis of the sugar moiety. Attachment of the sugar to the two indole nitrogens of the indolocarbazole core was dependent on the combined action of StaG and StaN. When StaN was absent, the sugar was attached only to one of the nitrogen atoms, through an N-glycosidic linkage, as in the indolocarbazole rebeccamycin. The StaG glycosyltransferase showed flexibility with respect to the sugar donor. When the ‘sugar vector’ was substituted by constructs directing the biosynthesis of l-rhamnose, l-digitoxose, l-olivose and d-olivose, respectively, StaG and StaN were able to transfer and attach all of these sugars to the indolocarbazole aglycone.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1111/j.1365-2958.2005.04777.x
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  • 8
    Electronic Resource
    Electronic Resource
    Tailoring modification of deoxysugars during biosynthesis of the antitumour drug chromomycin A3 by Streptomyces griseus ssp. griseus (2004)
    Oxford, UK : Blackwell Science Ltd
    Molecular microbiology 53 (2004), S. 0 
    Details
    ISSN: 1365-2958
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: Chromomycin A3 is a member of the aureolic acid group family of antitumour drugs. Three tailoring modification steps occur during its biosynthesis affecting the sugar moieties: two O-acetylations and one O-methylation. The 4-O-methylation in the 4-O-methyl-D-oliose moiety of the disaccharide chain is catalysed by the cmmMIII gene product. Inactivation of this gene generated a chromomycin-non-producing mutant that accumulated three unmethylated derivatives containing all sugars but differing in the acylation pattern. Two of these compounds were shown to be substrates of the methyltransferase as determined by their bioconversion into chromomycin A2 and A3 after feeding these compounds to a Streptomyces albus strain expressing the cmmMIII gene. The same single membrane-bound enzyme, encoded by the cmmA gene, is responsible for both acetyl transfer reactions, which convert a relatively inactive compound into the bioactive chromomycin A3. Insertional inactivation of this gene resulted in a mutant accumulating a dideacetylated chromomycin A3 derivative. This compound, lacking both acetyl groups, was converted in a two-step reaction via the 4E-monoacetylated intermediate into chromomycin A3 when fed to cultures of S. albus expressing the cmmA gene. This acetylation step would occur as the last step in chromomycin biosynthesis, being a very important event for self-protection of the producing organism. It would convert a molecule with low biological activity into an active one, in a reaction catalysed by an enzyme that is predicted to be located in the cell membrane.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1111/j.1365-2958.2004.04166.x
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  • 9
    Electronic Resource
    Electronic Resource
    Biosynthesis of the angiogenesis inhibitor borrelidin by Streptomyces parvulus Tü4055: insights into nitrile formation (2004)
    Oxford, UK : Blackwell Science Ltd
    Molecular microbiology 52 (2004), S. 0 
    Details
    ISSN: 1365-2958
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: The 18-membered polyketide macrolide borrelidin exhibits a number of important biological activities, including potent angiogenesis inhibition. This has prompted two recent total syntheses as well as the cloning of the biosynthetic gene cluster from Streptomyces parvulus Tü4055. Borrelidin possesses some unusual structural characteristics, including a cyclopentane carboxylic acid moiety at C17 and a nitrile moiety at C12 of the macrocyclic ring. Nitrile groups are relatively rare in nature, and little is known of their biosynthesis during secondary metabolism. The nitrile group of borrelidin is shown here to arise from the methyl group of a methylmalonyl-CoA extender unit incorporated during polyketide chain extension. Insertional inactivation of two genes in the borrelidin gene cluster, borI (coding for a cytochrome P450 monooxygenase) and borJ (coding for an aminotransferase), generated borrelidin non-producing mutants. These mutants accumulated different compounds lacking the C12 nitrile moiety, with the product of the borI-minus mutant (12-desnitrile-12-methyl-borrelidin) possessing a methyl group and that of the borJ-minus mutant (12-desnitrile-12-carboxyl-borrelidin) a carboxyl group at C12. The former but not the latter was converted into borrelidin when biotransformed by an S. parvulus mutant that is deficient in the biosynthesis of the borrelidin starter unit. This suggests that 12-desnitrile-12-methyl-borrelidin is a competent biosynthetic intermediate, whereas the carboxylated derivative is a shunt metabolite. Bioconversion of 12-desnitrile-12-methyl-borrelidin into borrelidin was also achieved in a heterologous system co-expressing borI and borJ in Streptomyces albus J1074. This bioconversion was more efficient when borK, which is believed to encode a dehydrogenase, was simultaneously expressed with borI and borJ. On the basis of these findings, a pathway is proposed for the formation of the nitrile moiety during borrelidin biosynthesis.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1111/j.1365-2958.2004.04090.x
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  • 10
    Electronic Resource
    Electronic Resource
    ABC transporters in antibiotic-producing actinomycetes (1998)
    Oxford, UK : Blackwell Publishing Ltd
    FEMS microbiology letters 158 (1998), S. 0 
    Details
    ISSN: 1574-6968
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology
    Notes: Many antibiotic-producing actinomycetes posses at least one ABC (ATP-binding cassette) transporter which forms part of the antibiotic biosynthetic pathway and in most cases confers resistance to the drug in an heterologous host. Three types of antibiotic ABC transporters have been so far described in producer organisms. In Type I two genes are involved, one encoding a hydrophilic ATP-binding protein with one nucleotide-binding domain and the other encoding a hydrophobic membrane protein. In Type II transporters only a gene encoding the hydrophilic ATP-binding protein with two nucleotide-binding domains is present and no gene encoding a hydrophobic membrane protein has been found. In Type III only one gene is involved which encodes both the hydrophilic and hydrophobic components. Possibly these ABC transporters are responsible for secretion of the antibiotics outside the cells. A comparative analysis of the ATP-binding components of the different antibiotic ABC transporters and analysis of the amino acid distances between the so-called Walker motifs suggests that the three types of transporters have probably evolved from a common ancestor containing a single nucleotide-binding domain.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1111/j.1574-6968.1998.tb12792.x
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