Your browser doesn't support javascript.
loading
Show: 20 | 50 | 100
Results 1 - 20 de 60
Filter
Add more filters










Publication year range
1.
Biochim Biophys Acta ; 1860(3): 465-75, 2016 Mar.
Article in English | MEDLINE | ID: mdl-26475642

ABSTRACT

BACKGROUND: OleP is a cyt P450 from Streptomyces antibioticus carrying out epoxigenation of the antibiotic oleandomycin during its biosynthesis. The timing of its reaction has not been fully clarified, doubts remain regarding its substrate and catalytic mechanism. METHODS: The crystal structure of OleP in complex with clotrimazole, an inhibitor of P450s used in therapy, was solved and the complex formation dynamics was characterized by equilibrium and kinetic binding studies and compared to ketoconazole, another azole differing for the N1-substituent. RESULTS: Clotrimazole coordinates the heme and occupies the active site. Most of the residues interacting with clotrimazole are conserved and involved in substrate binding in MycG, the P450 epoxigenase with the highest homology with OleP. Kinetic characterization of inhibitor binding revealed OleP to follow a simple bimolecular reaction, without detectable intermediates. CONCLUSIONS: Clotrimazole-bound OleP adopts an open form, held by a π-π stacking chain that fastens helices F and G and the FG loop. Affinity is affected by the interactions of the N1 substituent within the active site, given the one order of magnitude difference of the off-rate constants between clotrimazole and ketoconazole. Based on structural similarities with MycG, we propose a binding mode for both oleandomycin intermediates, that are the candidate substrates of OleP. GENERAL SIGNIFICANCE: Among P450 epoxigenases OleP is the only one that introduces an epoxide on a non-activated C­C bond. The data here presented are necessary to understand the rare chemistry carried out by OleP, to engineer it and to design more selective and potent P450-targeted drugs.


Subject(s)
Anti-Bacterial Agents/biosynthesis , Clotrimazole/chemistry , Cytochrome P-450 Enzyme System/chemistry , Oleandomycin/biosynthesis , Oxidoreductases/chemistry , Streptomyces antibioticus/enzymology , Catalytic Domain , Crystallography , Cytochrome P-450 Enzyme System/physiology , Oxidoreductases/physiology , Protein Structure, Secondary , Structure-Activity Relationship
2.
J Bacteriol ; 183(18): 5358-63, 2001 Sep.
Article in English | MEDLINE | ID: mdl-11514520

ABSTRACT

Oleandomycin, a macrolide antibiotic produced by Streptomyces antibioticus, contains two sugars attached to the aglycon: L-oleandrose and D-desosamine. oleY codes for a methyltransferase involved in the biosynthesis of L-oleandrose. This gene was overexpressed in Escherichia coli to form inclusion bodies and in Streptomyces lividans, producing a soluble protein. S. lividans overexpressing oleY was used as a biotransformation host, and it converted the precursor L-olivosyl-erythronolide B into its 3-O-methylated derivative, L-oleandrosyl-erythronolide B. Two other monoglycosylated derivatives were also substrates for the OleY methyltransferase: L-rhamnosyl- and L-mycarosyl-erythronolide B. OleY methyltransferase was purified yielding a 43-kDa single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The native enzyme showed a molecular mass of 87 kDa by gel filtration chromatography, indicating that the enzyme acts as a dimer. It showed a narrow pH range for optimal activity, and its activity was clearly stimulated by the presence of several divalent cations, being maximal with Co(2+). The S. antibioticus OleG2 glycosyltransferase is proposed to transfer L-olivose to the oleandolide aglycon, which is then converted into L-oleandrose by the OleY methyltransferase. This represents an alternative route for L-oleandrose biosynthesis from that in the avermectin producer Streptomyces avermitilis, in which L-oleandrose is transferred to the aglycon by a glycosyltransferase.


Subject(s)
Anti-Bacterial Agents/biosynthesis , Deoxy Sugars , Methyltransferases/metabolism , Oleandomycin/biosynthesis , Streptomyces antibioticus/enzymology , Escherichia coli/enzymology , Escherichia coli/genetics , Hexoses/metabolism , Methyltransferases/genetics , Methyltransferases/isolation & purification , Streptomyces/enzymology , Streptomyces/genetics , Streptomyces antibioticus/genetics , Streptomyces antibioticus/growth & development , Substrate Specificity
3.
J Antibiot (Tokyo) ; 53(5): 502-8, 2000 May.
Article in English | MEDLINE | ID: mdl-10908114

ABSTRACT

The gene cluster encoding the deoxyoleandolide polyketide synthase (OlePKS) was isolated from the oleandomycin producing strain Streptomnyces antibioticus. Sequencing of the first two genes encoding OlePKS, together with the previously identified third gene revealed an overall genetic and protein architecture similar to that of the erythromycin gene cluster encoding the 6-deoxyerythronolide B synthase (DEBS) from Saccharopolyspora erythraea. When the entire OlePKS (10,487 amino acids) was expressed in the heterologous host Streptomyces lividans, it produced 8,8a-deoxyoleandolide, an aglycone precursor of oleandomycin. The role of the P-450 monooxygenase, OleP, in oleandomycin biosynthesis was also examined in vivo by co-expression with DEBS in S. lividans. The production of 8,8a-dihydroxy-6-deoxyerythronolide B and other derivatives indicates that OleP is involved in the epoxidation pathway of oleandomycin biosynthesis. Since there are currently no genetic systems available for manipulation of the natural oleandomycin producing strain, the heterologous expression system reported here provides a useful tool for studying this important macrolide antibiotic.


Subject(s)
Anti-Bacterial Agents/biosynthesis , Multienzyme Complexes/genetics , NADH, NADPH Oxidoreductases/genetics , Oleandomycin/biosynthesis , Base Sequence , Cloning, Molecular , DNA Primers , Epoxy Compounds/metabolism , Genes, Bacterial , Multienzyme Complexes/metabolism , Multigene Family , NADH, NADPH Oxidoreductases/metabolism , NADPH-Ferrihemoprotein Reductase , Streptomyces/metabolism
4.
Antimicrob Agents Chemother ; 44(5): 1266-75, 2000 May.
Article in English | MEDLINE | ID: mdl-10770761

ABSTRACT

A 9.8-kb DNA region from the oleandomycin gene cluster in Streptomyces antibioticus was cloned. Sequence analysis revealed the presence of 8 open reading frames encoding different enzyme activities involved in the biosynthesis of one of the two 2, 6-deoxysugars attached to the oleandomycin aglycone: L-oleandrose (the oleW, oleV, oleL, and oleU genes) and D-desosamine (the oleNI and oleT genes), or of both (the oleS and oleE genes). A Streptomyces albus strain harboring the oleG2 glycosyltransferase gene integrated into the chromosome was constructed. This strain was transformed with two different plasmid constructs (pOLV and pOLE) containing a set of genes proposed to be required for the biosynthesis of dTDP-L-olivose and dTDP-L-oleandrose, respectively. Incubation of these recombinant strains with the erythromycin aglycon (erythronolide B) gave rise to two new glycosylated compounds, identified as L-3-O-olivosyl- and L-3-O-oleandrosyl-erythronolide B, indicating that pOLV and pOLE encode all enzyme activities required for the biosynthesis of these two 2,6-dideoxysugars. A pathway is proposed for the biosynthesis of these two deoxysugars in S. antibioticus.


Subject(s)
Bacterial Proteins/genetics , Deoxy Sugars/biosynthesis , Hexoses/biosynthesis , Streptomyces antibioticus/genetics , Amino Acid Sequence , Amino Sugars/biosynthesis , Amino Sugars/genetics , Bacterial Proteins/metabolism , Cloning, Molecular , Gene Expression Regulation, Bacterial , Glycosyltransferases/biosynthesis , Glycosyltransferases/genetics , Molecular Sequence Data , Multigene Family , Oleandomycin/biosynthesis , Sequence Homology, Amino Acid , Streptomyces antibioticus/metabolism
5.
Chem Biol ; 7(2): 77-84, 2000 Feb.
Article in English | MEDLINE | ID: mdl-10662693

ABSTRACT

BACKGROUND: Recently developed tools for the genetic manipulation of modular polyketide synthases (PKSs) have advanced the development of combinatorial biosynthesis technologies for drug discovery. Although many of the current techniques involve engineering individual domains or modules of the PKS, few experiments have addressed the ability to combine entire protein subunits from different modular PKSs to create hybrid polyketide pathways. We investigated this possibility by in vivo assembly of heterologous PKS complexes using natural and altered subunits from related macrolide PKSs. RESULTS: The pikAI and pikAII genes encoding subunits 1 and 2 (modules 1-4) of the picromycin PKS (PikPKS) and the eryAIII gene encoding subunit 3 (modules 5-6) of the 6-deoxyerythronolide B synthase (DEBS) were cloned in two compatible Streptomyces expression vectors. A strain of Streptomyces lividans co-transformed with the two vectors produced the hybrid macrolactone 3-hydroxynarbonolide. Co-expression of the same pik genes with the gene for subunit 3 of the oleandomycin PKS (OlePKS) was also successful. A series of hybrid polyketide pathways was then constructed by combining PikPKS subunits 1 and 2 with modified DEBS3 subunits containing engineered domains in modules 5 or 6. We also report the effect of junction location in a set of DEBS-PikPKS fusions. CONCLUSIONS: We show that natural as well as engineered protein subunits from heterologous modular PKSs can be functionally assembled to create hybrid polyketide pathways. This work represents a new strategy that complements earlier domain engineering approaches for combinatorial biosynthesis in which complete modules or PKS protein subunits, in addition to individual enzymatic domains, are used as building blocks for PKS engineering.


Subject(s)
Anti-Bacterial Agents/biosynthesis , Macrolides , Multienzyme Complexes/biosynthesis , Multienzyme Complexes/genetics , Anti-Bacterial Agents/chemistry , Erythromycin/biosynthesis , Genes, Bacterial/genetics , Genetic Engineering , Genetic Vectors/chemical synthesis , Multienzyme Complexes/chemistry , Multigene Family/genetics , Oleandomycin/biosynthesis , Protein Structure, Quaternary , Saccharopolyspora/enzymology , Streptomyces/enzymology , Streptomyces antibioticus/enzymology
6.
Mol Microbiol ; 34(5): 1039-48, 1999 Dec.
Article in English | MEDLINE | ID: mdl-10594828

ABSTRACT

Two glycosyltransferase genes, oleG1 and oleG2, and a putative isomerase gene, oleP1, have previously been identified in the oleandomycin biosynthetic gene cluster of Streptomyces antibioticus. In order to identify which of these two glycosyltransferases encodes the desosaminyltransferase and which the oleandrosyltransferase, interspecies complementation has been carried out, using two mutant strains of Saccharopolyspora erythraea, one strain carrying an internal deletion in the eryCIII (desosaminyltransferase) gene and the other an internal deletion in the eryBV (mycarosyltransferase) gene. Expression of the oleG1 gene in the eryCIII deletion mutant restored the production of erythromycin A (although at a low level), demonstrating that oleG1 encodes the desosaminyltransferase required for the biosynthesis of oleandomycin and indicating that, as in erythromycin biosynthesis, the neutral sugar is transferred before the aminosugar onto the macrocyclic ring. Significantly, when an intact oleG2 gene (presumed to encode the oleandrosyltransferase) was expressed in the eryBV deletion mutant, antibiotic activity was also restored and, in addition to erythromycin A, new bioactive compounds were produced with a good yield. The neutral sugar residue present in these compounds was identified as L-rhamnose attached at position C-3 of an erythronolide B or a 6-deoxyerythronolide B lactone ring, thus indicating a relaxed specificity of the oleandrosyltransferase, OleG2, for both the activated sugar and the macrolactone substrate. The oleP1 gene located immediately upstream of oleG1 was likewise introduced into an eryCII deletion mutant of Sac. erythraea, and production of erythromycin A was again restored, demonstrating that the function of OleP1 is identical to that of EryCII in the biosynthesis of dTDP-D-desosamine, which we have previously proposed to be a dTDP-4-keto-6-deoxy-D-glucose 3, 4-isomerase.


Subject(s)
Erythromycin/analogs & derivatives , Genes, Bacterial , Oleandomycin/biosynthesis , Saccharopolyspora/genetics , Saccharopolyspora/metabolism , Streptomyces antibioticus/genetics , Chromatography, High Pressure Liquid , Chromatography, Thin Layer , DNA, Bacterial/genetics , Erythromycin/biosynthesis , Genetic Complementation Test , Glycosyltransferases/genetics , Glycosyltransferases/metabolism , Isomerases/genetics , Isomerases/metabolism , Magnetic Resonance Spectroscopy , Mass Spectrometry , Multigene Family , Plasmids/genetics , Saccharopolyspora/growth & development , Streptomyces antibioticus/growth & development , Streptomyces antibioticus/metabolism , Transformation, Bacterial
7.
Mol Gen Genet ; 259(3): 299-308, 1998 Aug.
Article in English | MEDLINE | ID: mdl-9749673

ABSTRACT

A 6-kb region from the chromosome of Streptomyces antibioticus, an oleandomycin producer, was cloned and sequenced. This region was located between the 3' end of the gene encoding the third subunit of the oleandomycin type I polyketide synthase and the oleP and oleB genes, which encode a cytochrome P450 monooxygenase and an oleandomycin resistance gene, respectively. Analysis of the nucleotide sequence revealed the presence of five genes encoding a cytochrome P450-like protein (oleP1), two glycosyltransferases (oleG1 and oleG2) involved in the transfer of the two 6-deoxysugars (L-oleandrose and D-desosamine) to the oleandomycin macrolactone ring, a methyltransferase (oleM1), and a gene (oleY) of unknown function. Insertional inactivation of this region by gene disruption generated an oleandomycin non-producing mutant which accumulated a compound that, according to mass spectrometry analysis, could correspond to the oleandomycin macrolactone ring (oleandolide), suggesting that the mutation affects oleandrosyl glycosyltransferase.


Subject(s)
Anti-Bacterial Agents/biosynthesis , Chromosomes, Bacterial/genetics , Glycosyltransferases/genetics , Glycosyltransferases/metabolism , Oleandomycin/biosynthesis , Streptomyces antibioticus/genetics , Streptomyces antibioticus/metabolism , Amino Acid Sequence , Base Sequence , Cloning, Molecular , DNA, Bacterial/genetics , Genes, Bacterial , Glycosylation , Methyltransferases/genetics , Methyltransferases/metabolism , Molecular Sequence Data , Mutagenesis, Insertional , Sequence Homology, Amino Acid , Streptomyces antibioticus/enzymology
8.
FEMS Microbiol Lett ; 147(1): 29-36, 1997 Feb 01.
Article in English | MEDLINE | ID: mdl-9037760

ABSTRACT

A chromosomal DNA fragment from Streptomyces antibioticus ATCC11891 was isolated by its ability to stimulate actinorhodin and undecylprodigiosin biosynthesis in Streptomyces lividans TK21. This fragment includes two open reading frames, whose deduced translated products resemble enzymes involved in sulfur metabolism (ORF1) and LysR-type transcriptional regulators (ORF2). The cloning of the promoter region of ORF2 (abaB) in high copy number led to overproduction of both antibiotics suggesting that this phenotype might well be due to titration by this region of one or more proteins. Southern blot analysis revealed that abaB gene is highly conserved among all streptomycetes tested.


Subject(s)
Genes, Fungal/genetics , Promoter Regions, Genetic/physiology , Streptomyces/genetics , Streptomyces/metabolism , Anti-Bacterial Agents/biosynthesis , Base Sequence , Blotting, Southern , Chromatography, High Pressure Liquid , Cloning, Molecular , DNA, Fungal/genetics , Gene Dosage , Gene Expression Regulation, Fungal/genetics , Molecular Sequence Data , Oleandomycin/biosynthesis , Open Reading Frames/genetics , Plasmids , Restriction Mapping , Sequence Analysis, DNA , Sequence Homology, Amino Acid , Transcription, Genetic/genetics
10.
Antibiot Khimioter ; 40(11-12): 10-5, 1995.
Article in Russian | MEDLINE | ID: mdl-8830632

ABSTRACT

The data on the study of the electrocoagulation treatment of highly concentrated sewage in antibiotic production are presented. Aluminium plates were used as the electrodes. The influence of the anode current density, pH and temperature on the process of the electrocoagulation treatment of the sewage was investigated. The electrocoagulation treatment of the sewage in production of tetracycline, oleandomycin and benzyl-penicillin was shown to be efficient.


Subject(s)
Anti-Bacterial Agents/biosynthesis , Waste Disposal, Fluid/methods , Aluminum/analysis , Electrocoagulation , Electrodes , Hydrogen-Ion Concentration , Oleandomycin/biosynthesis , Penicillin G/metabolism , Solubility , Temperature , Tetracycline/biosynthesis
11.
J Biol Chem ; 270(31): 18234-9, 1995 Aug 04.
Article in English | MEDLINE | ID: mdl-7629141

ABSTRACT

The oleandomycin (OM) producer, Streptomyces antibioticus, possesses a mechanism involving two enzymes for the intracellular inactivation and extracellular reactivation of the antibiotic. Inactivation takes place by transfer of a glucose molecule from a donor (UDP-glucose) to OM, a process catalyzed by an intracellular glucosyltransferase. Glucosyltransferase activity is detectable in cell-free extracts concurrent with biosynthesis of OM. The enzyme has been purified 1,097-fold as a monomer, with a molecular mass of 57.1 kDa by a four-step procedure using three chromatographic columns. The reaction operates via a compulsory-order mechanism. This has been shown by steady-state kinetic studies using either OM or an alternative substrate (rosaramycin) and dead-end inhibitors, and isotopic exchange reactions at equilibrium. OM binds first to the enzyme, followed by UDP-glucose. A ternary complex is thus formed prior to transfer of glucose. UDP is then released, followed by the glycosylated oleandomycin (GS-OM).


Subject(s)
Glucosyltransferases/isolation & purification , Glucosyltransferases/metabolism , Oleandomycin/biosynthesis , Streptomyces antibioticus/metabolism , Erythromycin/pharmacology , Glycosylation , Kinetics , Streptomyces antibioticus/enzymology , Uridine Diphosphate Glucose/pharmacology
12.
FEMS Microbiol Lett ; 127(1-2): 117-20, 1995 Mar 15.
Article in English | MEDLINE | ID: mdl-7737473

ABSTRACT

A cosmid clone from an oleandomycin producer, Streptomyces antibioticus, contains a large open reading frame encoding a type I polyketide synthase subunit and an oleandomycin resistance gene (oleB). Sequencing of a 1.4-kb DNA fragment adjacent to oleB revealed the existence of an open reading frame (oleP) encoding a protein similar to several cytochrome P450 monooxygenases from different sources, including the products of the eryF and eryK genes from Saccharopolyspora erythraea that participate in erythromycin biosynthesis. The oleP gene was expressed in Escherichia coli as a fusion protein to a maltose-binding protein. Using polyclonal antibodies against this fusion protein it was observed that the synthesis of the cytochrome P450 was in parallel to that of oleandomycin. The cytochrome P450 encoded by the oleP gene could be responsible for the epoxidation of carbon 8 of the oleandomycin lactone ring.


Subject(s)
Cytochrome P-450 Enzyme System/genetics , Genes, Bacterial , Oleandomycin/biosynthesis , Oxygenases/genetics , Streptomyces antibioticus/genetics , Streptomyces antibioticus/metabolism , Amino Acid Sequence , Bacterial Proteins/biosynthesis , Bacterial Proteins/genetics , Bacterial Proteins/immunology , Drug Resistance, Microbial/genetics , Escherichia coli/genetics , Kinetics , Molecular Sequence Data , Multienzyme Complexes/genetics , Open Reading Frames , Sequence Homology, Amino Acid
13.
Science ; 265(5171): 509-12, 1994 Jul 22.
Article in English | MEDLINE | ID: mdl-8036492

ABSTRACT

Macrocyclic polyketides have been subjects of great interest in synthetic and biosynthetic chemistry because of their structural complexity and medicinal activities. With expression of the entire 6-deoxyerythronolide B synthase (DEBS) (10,283 amino acids) in a heterologous host, substantial quantities of 6-deoxyerythronolide B (6dEB), the aglycone of the macrolide antibiotic erythromycin, and 8,8a-deoxyoleandolide, a 14-membered lactone ring identical to 6dEB except for a methyl group side chain in place of an ethyl unit, were synthesized in Streptomyces coelicolor. The biosynthetic strategy utilizes a genetic approach that facilitates rapid structural manipulation of DEBS or other modular polyketide synthases (PKSs), including those found in actinomycetes with poorly developed genetic methods. From a technological viewpoint, this approach should allow the rational design of biosynthetic products and may eventually lead to the generation of diverse polyketide libraries by means of combinatorial cloning of naturally occurring and mutant PKS modules.


Subject(s)
Erythromycin/analogs & derivatives , Multienzyme Complexes/genetics , Oleandomycin/analogs & derivatives , Acyl Coenzyme A/metabolism , Base Sequence , Binding Sites , Cloning, Molecular , Drug Design , Erythromycin/biosynthesis , Erythromycin/isolation & purification , Escherichia coli/genetics , Genes, Bacterial , Genetic Engineering , Genetic Vectors , Molecular Sequence Data , Multienzyme Complexes/chemistry , Multienzyme Complexes/metabolism , Multigene Family , Mutation , Oleandomycin/biosynthesis , Oleandomycin/isolation & purification , Recombinant Proteins/metabolism , Streptomyces/enzymology , Streptomyces/genetics , Structure-Activity Relationship
14.
Eur J Biochem ; 222(1): 129-35, 1994 May 15.
Article in English | MEDLINE | ID: mdl-8200337

ABSTRACT

Cell-free extracts from the oleandomycin producer, Streptomyces antibioticus, possess an intracellular glycosyltransferase capable of inactivating oleandomycin by glycosylation of the 2'-hydroxyl group in the desosamine moiety of the molecule [Vilches, C., Hernández, C., Méndez, C. & Salas, J. A. (1992) J. Bacteriol. 174, 161-165]. Using a four-step purification procedure, we have purified an enzyme activity from the culture supernatants from this organism which is able to release glucose from the inactive glycosylated molecule thus reactivating the antibiotic activity. This enzyme activity appeared in the culture supernatants immediately before oleandomycin is detected. The enzyme (molecular mass 87 kDa) showed a high degree of substrate specificity, not acting on other glycosylated macrolides such as methymycin, lankamycin and rosaramicin which are substrates for the glycosyltransferase. A second activity was detected corresponding to a 34-kDa polypeptide which probably originates from proteolytic cleavage of the larger polypeptide. The 87-kDa polypeptide possibly catalyses the last biosynthetic step in oleandomycin biosynthesis by S. antibioticus.


Subject(s)
Enzymes/isolation & purification , Oleandomycin/metabolism , Streptomyces antibioticus/enzymology , Biotransformation , Chromatography, Ion Exchange , Electrophoresis, Paper , Electrophoresis, Polyacrylamide Gel , Enzyme Activation , Enzymes/metabolism , Glycosylation , Kinetics , Oleandomycin/biosynthesis , Substrate Specificity
15.
Microbiologia ; 10(1-2): 37-48, 1994.
Article in English | MEDLINE | ID: mdl-7946126

ABSTRACT

Resistance to macrolides in producing organisms can be achieved by target site modification, intracellular inactivation of the antibiotic or active efflux mechanisms for the excretion of the antibiotic. The oleandomycin producer, Streptomyces antibioticus, possesses oleandomycin-sensitive ribosomes all along the cell cycle. However, it contains an intracellular glycosyltransferase capable of inactivating oleandomycin in the presence of UDP-glucose as cofactor. The correspondent gene (oleD) has been cloned and sequenced and the glycosyltransferase purified. Two other genes (oleB and oleC) that confer oleandomycin resistance have been cloned and characterized and both encode ABC (ATP-Binding Cassette) transporters. These may constitute the excretion mechanism throughout which the glycosylated oleandomycin is excreted. A second enzyme activity has been purified from culture supernatants of the oleandomycin producer that releases the glucose from the inactive glycosylated oleandomycin generating active antibiotic. This enzyme would probably catalyse the last step in the biosynthesis of oleandomycin.


Subject(s)
Drug Resistance, Microbial/genetics , Oleandomycin/metabolism , Streptomyces antibioticus/genetics , Amino Acid Sequence , Biological Transport, Active , Cell Membrane Permeability , Glycosylation , Methylation , Molecular Sequence Data , Molecular Structure , Oleandomycin/biosynthesis , RNA, Ribosomal/genetics , Streptomyces antibioticus/enzymology , Streptomyces antibioticus/metabolism
16.
Mol Gen Genet ; 242(3): 358-62, 1994 Feb.
Article in English | MEDLINE | ID: mdl-8107683

ABSTRACT

A gene (ORFB) from Streptomyces antibioticus (an oleandomycin producer) encoding a large, multifunctional polyketide synthase (PKS) was cloned and sequenced. Its product shows an internal duplication and a close similarity to the third subunit of the PKS involved in erythromycin biosynthesis by Saccharopolyspora erythraea, showing the equivalent nine active site domains in the same order along the polypeptide. An unusual feature of this ORF is the GC content of most of the sequence, which is surprisingly low, for a Streptomyces gene; the large number of codons with T in the third position is particularly striking. The last 800 bp of the gene stand out as being normal in their GC content, this region corresponding almost exactly to the thioesterase domain of the gene and suggesting that this domain was a late addition to the PKS. Based on the high degree of similarity between the ORFB product and the third subunit of the erythromycin PKS and the occurrence nearby of a gene conferring oleandomycin resistance, it is possible that this gene might be involved in the biosynthesis of the oleandomycin lactone ring.


Subject(s)
Multienzyme Complexes/genetics , Streptomyces antibioticus/enzymology , Amino Acid Sequence , Cloning, Molecular , Genes, Bacterial , Molecular Sequence Data , Multienzyme Complexes/classification , Oleandomycin/biosynthesis , Restriction Mapping , Sequence Alignment , Streptomyces antibioticus/genetics
17.
Mikrobiol Zh (1978) ; 53(5): 11-5, 1991.
Article in Russian | MEDLINE | ID: mdl-1791777

ABSTRACT

Strains, producers of oleandomycin, with different level of antibiotic-formation have been studied for their resistance to their own antibiotic. The obtained highly active strain possesses double resistance to oleandomycin and 50% higher activity. Identity of oleandomycin phosphate substances synthesized by initial and produced highly active strains is shown by the HELC method.


Subject(s)
Oleandomycin/antagonists & inhibitors , Oleandomycin/biosynthesis , Streptomyces antibioticus/drug effects , Streptomyces antibioticus/metabolism , Chromatography, High Pressure Liquid , Dose-Response Relationship, Drug , Drug Resistance, Microbial , Oleandomycin/analysis , Time Factors
18.
J Gen Microbiol ; 136(8): 1447-54, 1990 Aug.
Article in English | MEDLINE | ID: mdl-2262785

ABSTRACT

The influence of different nutritional compounds on oleandomycin biosynthesis by Streptomyces antibioticus was studied, resulting in the design of a chemically defined medium for production of the antibiotic. Of the variety of carbon and nitrogen compounds tested, fructose and aspartic acid (carbon and nitrogen sources, respectively) supported the highest oleandomycin titres. Addition of propionate but not acetate, both precursors of the skeleton of the macrolide lactone ring, stimulated the biosynthesis of the antibiotic. Oleandomycin biosynthesis was repressed by glucose but not by phosphate. S. antibioticus develops oleandomycin resistance shortly before the antibiotic begins to be synthesized, showing a triphasic pattern of resistance: spores and producing mycelium are resistant, while non-producing mycelium is sensitive.


Subject(s)
Culture Media , Oleandomycin/biosynthesis , Streptomyces antibioticus/metabolism , Amino Acids/pharmacology , Carbohydrates/pharmacology , Carbon/pharmacology , Carboxylic Acids/pharmacology , Drug Resistance, Microbial , Nitrogen/pharmacology , Phosphates/pharmacology , Streptomyces antibioticus/drug effects
20.
Antibiot Khimioter ; 35(6): 7-12, 1990 Jun.
Article in Russian | MEDLINE | ID: mdl-2169231

ABSTRACT

A low productive laboratory strain of S. antibioticus and a strain with an increased productivity of oleandomycin derived from it were studied comparatively with using restriction analysis and blotting hybridization. Amplification, site specific integration and segregation of the DNA sequence 32.0 kb in size were detected in the strains. The chromosomes of the laboratory strain contained one copy of the amplifying sequence AUD. After uniting of the end sequences AUD appeared to be capable of segregating from the chromosomes and its one copy per five genomes was present in the form of an extrachromosomal genetic element eSA1. The genome of the strain with increased productivity of oleandomycin contained in its chromosomes sequence ADS-Sa1 amplified to 150 copies and the eSA1 extrachromosomal genetic element in the form of mono-, di- and trimeric structures in the quantity of approximately one copy per genome. The BamHIB fragment of the eSA1 DNA 4 kb in size was identified. The fragment was able to participate in segregation or integration of eSA1 from or into the chromosomes since its subfragments were flanking AUD and ADS-SA1 in the chromosomes. The BamHIB fragment was hybridizing with a number of fragments of the chromosomal DNA of S. antibioticus, S. erythraeus. S. lividans and other strains of streptomycetes. It probably contained an IS-like element or a dispersed genetic element of another class. The DNA sequence of the eSA1 genetic element contained regions homologous to the sequence of the Erm E gene in S. erythraeus NRRL 2338.


Subject(s)
DNA, Bacterial/genetics , Nucleic Acid Hybridization/physiology , Repetitive Sequences, Nucleic Acid/physiology , Streptomyces antibioticus/genetics , Streptomyces/genetics , Blotting, Southern/methods , Culture Media , DNA Restriction Enzymes/pharmacology , In Vitro Techniques , Models, Genetic , Nucleic Acid Hybridization/drug effects , Oleandomycin/biosynthesis , Repetitive Sequences, Nucleic Acid/drug effects , Streptomyces antibioticus/metabolism
SELECTION OF CITATIONS
SEARCH DETAIL
...