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1.
Renata Novakova Jana Bistakova Dagmar Homerova Bronislava Rezuchova Lubica Feckova Jan Kormanec 《DNA sequence》2004,15(3):188-195
We cloned a new polyketide gene cluster, aur2, in Streptomyces aureofaciens CCM3239. Sequence analysis of the 9531-bp DNA fragment revealed 10 open reading frames, majority of which showed high similarity to the previously characterized type II polyketide synthase (PKS) genes. An unusual feature of the aur2 cluster is a disconnected organization of minimal PKS genes; ACP is located apart from the genes for ketosynthases KSalpha and KSbeta. The aur2 gene cluster was disrupted in S. aureofaciens CCM3239 by a homologous recombination, replacing the four genes (aur2A, E, F, G) including ketosynthase KSalpha, with antibiotic resistance marker gene. The disruption did not affect growth and differentiation, and disrupted strain produced spores with wild-type grey-pink pigmentation. The biochromatographic analysis of the culture extracts from S. aureofaciens wild type and aur2-disrupted strains did not reveal any difference in the pattern of antibacterial compounds. 相似文献
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3.
Streptomyces arenae produces the aromatic polyketide naphthocyclinone, which exhibits activity against Gram-positive bacteria. A cosmid clone containing the putative naphthocyclinone gene cluster was isolated from a genomic library of S. arenae by hybridization with a conserved region from the actinorhodin PKS of S. coelicolor. Sequence analysis of a 5.5-kb DNA fragment, which hybridizes with the actI probe, revealed three open reading frames coding for the minimal polyketide synthase. A strong sequence similarity was found to several previously described ketosynthases, chain length factors and acyl carrier proteins from other polyketide gene clusters. An additional open reading frame downstream of the PKS genes of S. arenae showed 53% identity to act VII probably encoding an aromatase. Another open reading frame was identified in a region of 1.436 bp upstream of the PKS genes, which, however, had no similarity to known genes in the database. Approximately 8 kb upstream of the PKS genes, a DNA fragment was identified that hybridizes to an actVII--actIV specific probe coding for a cyclase and a putative regulatory protein, respectively. Disruption of the proposed naphthocyclinone gene cluster by insertion of a thiostrepton resistance gene completely abolished production of naphthocyclinones in the mutant strain, showing that indeed the naphthocyclinone gene cluster had been isolated. Heterologous expression of the minimal PKS genes in S. coelicolor CH999 in the presence of the act ketoreductase led to the production of mutactin and dehydromutactin, indicating that the S. arenae polyketide synthase forms a C-16 backbone that is subsequently dimerized to build naphthocyclinone. The functions of the proposed cyclase and aromatase were examined by coexpression with genes from different polyketide core producers. 相似文献
4.
A standard type II polyketide synthase (PKS) gene cluster was isolated while attempting to clone the biosynthetic gene for lipstatin from Streptomyces toxytricini NRRL 15,443. This result was observed using a Southern blot of a PstI-digested S. toxytricini chromosomal DNA library with a 444 bp amplified probe of a ketosynthase (KS) gene fragment. Four open reading frames [thioesterase (TE), beta-ketoacyl systhase (KAS), chain length factor (CLF), and acyl carrier protein (ACP)], were identified through the nucleotide sequence determination and analysis of a 4.5 kb cloned DNA fragment. In order to confirm the involvement of a cloned gene in lipstatin biosynthesis, a gene disruption experiment for the KS gene was performed. However, the resulting gene disruptant did not show any significant difference in lipstatin production when compared to wild-type S. toxytricini. This result suggests that lipstatin may not be synthesized by a type II PKS. 相似文献
5.
Pradimicins are potent antifungal antibiotics having an unusual dihydrobenzo[alpha]naphthacenequinone aglycone substituted with D-alanine and sugars. Pradimicins are polyketide antibiotics produced by Actinomadura hibisca P157-2. The gene cluster involved in the biosynthesis of pradimicins was cloned and sequenced. The pradimicin gene cluster was localized to a 39-kb DNA segment and its involvement in the biosynthesis of pradimicin was proven by gene inactivation of prmA and prmB (ketosynthases alpha and beta). The pradimicin gene cluster consists of 28 open reading frames (ORFs), encoding a type II polyketide synthase (PKS), the enzymes involved in sugar biosynthesis and tailoring enzymes as well as two resistance proteins. The deduced proteins showed strong similarities to the previously validated gene clusters of angucyclic polyketides such as rubromycin, griseorhodin, and fredericamycin. From the pradimicin gene cluster, prmP3 encoding a component of the acetyl-CoA carboxylase complex was disrupted. The production levels of pradimicins of the resulting mutants decreased to 62% of the level produced by the wild-type strain, which indicate that the acetyl-CoA carboxylase gene would have a significant role in the production of pradimicins through supplying the extender unit precursor, malonyl-CoA. 相似文献
6.
A hydroxylase-like gene product contributes to synthesis of a polyketide spore pigment in Streptomyces halstedii. 总被引:4,自引:3,他引:1
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G Blanco A Pereda P Brian C Mndez K F Chater J A Salas 《Journal of bacteriology》1993,175(24):8043-8048
A gene, schC, adjacent to the sch gene cluster encoding the biosynthesis of a polyketide spore pigment in Streptomyces halstedii was sequenced. Its deduced product resembled flavin adenine nucleotide-containing hydroxylases involved in the biosynthesis of polycyclic aromatic polyketide antibiotics and in catabolic pathways of aromatic compounds. When schC was disrupted, the normally green spores of S. halstedii became lilac. An schC-like gene was located in an equivalent position next to a large gene cluster (whiE) known to determine spore pigment in Streptomyces coelicolor A3(2). 相似文献
7.
Isolation and sequence analysis of polyketide synthase genes from the daunomycin-producing Streptomyces sp. strain C5. 总被引:7,自引:5,他引:2
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J Ye M L Dickens R Plater Y Li J Lawrence W R Strohl 《Journal of bacteriology》1994,176(20):6270-6280
A contiguous region of about 30 kbp of DNA putatively encoding reactions in daunomycin biosynthesis was isolated from Streptomyces sp. strain C5 DNA. The DNA sequence of an 8.1-kbp EcoRI fragment, which hybridized with actI polyketide synthase (PKS) and actIII polyketide reductase (PKR) gene probes, was determined, revealing seven complete open reading frames (ORFs), two in one cluster and five in a divergently transcribed cluster. The former two genes are likely to encode PKR and a bifunctional cyclase/dehydrase. The five latter genes encode: (i) a homolog of TcmH, an oxygenase of the tetracenomycin biosynthesis pathway; (ii) a PKS Orf1 homolog; (iii) a PKS Orf2 homolog (chain length factor); (iv) a product having moderate sequence identity with Escherichia coli beta-ketoacyl acyl carrier protein synthase III but lacking the conserved active site; and (v) a protein highly similar to several acyltransferases. The DNA within the 8.1-kbp EcoRI fragment restored daunomycin production to two dauA non-daunomycin-producing mutants of Streptomyces sp. strain C5 and restored wild-type antibiotic production to Streptomyces coelicolor B40 (act VII; nonfunctional cyclase/dehydrase), and to S. coelicolor B41 (actIII) and Streptomyces galilaeus ATCC 31671, strains defective in PKR activity. 相似文献
8.
Streptomyces curacoi produces curamycin, an antibiotic based on a modified orsellinic acid skeleton that is synthesized by the polyketide pathway. We have cloned, characterized, and partly sequenced a polyketide synthase gene cluster of S. curacoi. The sequence data reveal an organization of open reading frames that is similar to those of other polyketide synthetic clusters, although the biosynthetic products differ considerably in size and structure. We propose that one of the predicted open reading frames (curA) encodes polykeptide synthase, on the basis of its homology with other enzymes with similar functions. Expression of the cloned chromosomal fragment in the heterologous host S. lividans leads to the production of a brown pigment in large quantities. The analysis and expression of the cur genes for detailed molecular studies of the mechanism of polyketide biosynthesis is discussed. 相似文献
9.
Metsä-Ketelä M Halo L Munukka E Hakala J Mäntsälä P Ylihonko K 《Applied and environmental microbiology》2002,68(9):4472-4479
A 613-bp fragment of an essential ketosynthase gene from the biosynthetic pathway of aromatic polyketide antibiotics was sequenced from 99 actinomycetes isolated from soil. Phylogenetic analysis showed that the isolates clustered into clades that correspond to the various classes of aromatic polyketides. Additionally, sequencing of a 120-bp fragment from the gamma-variable region of 16S ribosomal DNA (rDNA) and subsequent comparative sequence analysis revealed incongruity between the ketosynthase and 16S rDNA phylogenetic trees, which strongly suggests that there has been horizontal transfer of aromatic polyketide biosynthesis genes. The results show that the ketosynthase tree could be used for DNA fingerprinting of secondary metabolites and for screening interesting aromatic polyketide biosynthesis genes. Furthermore, the movement of the ketosynthase genes suggests that traditional marker molecules like 16S rDNA give misleading information about the biosynthesis potential of aromatic polyketides, and thus only molecules that are directly involved in the biosynthesis of secondary metabolites can be used to gain information about the biodiversity of antibiotic production in different actinomycetes. 相似文献
10.
Regulation of jadomycin B production in Streptomyces venezuelae ISP5230: involvement of a repressor gene, jadR2. 总被引:1,自引:0,他引:1
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The nucleotide sequence of a region upstream of the type II polyketide synthase genes in the cluster for biosynthesis of the polyketide antibiotic jadomycin B in Streptomyces venezuelae contained an open reading frame encoding a sequence of 196 amino acids that resembeled sequences deduced for a group of repressor proteins. The strongest similarity was to EnvR of Escherichia coli, but the sequence also resembled MtrR, AcrR, TetC, and TcmR, all of which are involved in regulating resistance to antibiotics or toxic hydrophobic substances in the environment. Disruption of the nucleotide sequence of this putative S. venezuelae repressor gene (jadR2), by insertion of an apramycin resistance gene at an internal MluI site, and replacement of the chromosomal gene generated mutants that produced jadomycin B without the stress treatments (exposure to heat shock or to toxic concentrations of ethanol) required for jadomycin B production by the wild type. When cultures of the disruption mutants were ethanol stressed, they overproduced the antibiotic. From these results it was concluded that expression of the jadomycin B biosynthesis genes are negatively regulated by jadR2. 相似文献
11.
Identification of two polyketide synthase gene clusters on the linear plasmid pSLA2-L in Streptomyces rochei 总被引:4,自引:0,他引:4
The 200kb linear plasmid pSLA2-L was suggested to be involved in the production of two macrolide antibiotics, lankamycin (Lm) and lankacidin (Lc), in Streptomyces rochei 7434AN4. Hybridization experiments with the polyketide synthase (PKS) genes for erythromycin and actinorhodin identified two eryAI-homologous regions and an actI-homologous region on pSLA2-L. The nucleotide sequence of a 3.6kb SacI fragment carrying one of the eryAI-homologs revealed that it codes for part of a large protein with four domains for ketoreductase, acyl carrier protein, ketosynthase, and acyltransferase. Gene disruption confirmed that the two eryAI-homologs are parts of a large type-I PKS gene cluster for Lm. A 4.8kb DNA carrying the actI-homologous region contains four open reading frames (ORF1-ORF4) as well as an additional ORF, i.e. ORF5, which might code for a thioesterase. Deletion of the ORF2-ORF4 region showed that it is not involved in the synthesis of Lm or Lc. Thus, it was confirmed that pSLA2-L contains two PKS gene clusters for Lm and an unknown type-II polyketide. 相似文献
12.
Nonactin is the parent compound of a group of highly atypical polyketide metabolites produced by Streptomyces griseus subsp. griseus ETH A7796. In this paper we describe the isolation, sequencing, and analysis of 15? omitted?559 bp of chromosomal DNA, containing the potential nonactin biosynthesis gene cluster, from S. griseus subsp. griseus ETH A7796. Fourteen open reading frames were observed in the DNA sequence. Significantly, type II polyketide synthase (PKS) homologues were discovered in an apparent operon structure, which also contained the nonactate synthase gene (nonS), clustered with the tetranactin resistance gene. The deduced products of two of the genes (nonK and nonJ) are quite unusual ketoacyl synthase (KAS) alpha and KASbeta homologues. We speculate that nonactic acid, the polyketide precursor of nonactin, is synthesized by a type II PKS system. 相似文献
13.
Aparicio JF Colina AJ Ceballos E Martín JF 《The Journal of biological chemistry》1999,274(15):10133-10139
The biosynthetic gene cluster for the 26-membered ring of the polyene macrolide pimaricin extends for about 110 kilobase pairs of contiguous DNA in the genome of Streptomyces natalensis. Two sets of polyketide synthase (PKS) genes are separated by a group of small polyketide-functionalizing genes. Two of the polyketide synthase genes, pimS0 and pimS1, have been fully sequenced and disrupted proving the involvement of each of these genes in pimaricin biosynthesis. The pimS0 gene encodes a relatively small acetate-activating PKS (approximately 193 kDa) that appears to work as a loading protein which "presents" the starter unit to the second PKS subunit. The pimS1 gene encodes a giant multienzyme (approximately 710 kDa) harboring 15 activities responsible for the first four cycles of chain elongation in pimaricin biosynthesis, resulting in formation of the polyene chromophore. 相似文献
14.
15.
Cloning and characterization of a polyketide synthase gene from Streptomyces fradiae Tü2717, which carries the genes for biosynthesis of the angucycline antibiotic urdamycin A and a gene probably involved in its oxygenation.
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A DNA fragment was cloned as cosmid purd8, which encodes a polyketide synthase involved in the production of the angucycline antibiotic urdamycin from Streptomyces fradiae Tü2717. Deletion of the polyketide synthase genes from the chromosome abolished urdamycin production. In addition, purd8 conferred urdamycin resistance on introduction into Streptomyces lividans TK24. Sequence analysis of 5.7 kb of purd8 revealed six open reading frames transcribed in the same direction. The deduced amino acid sequences of the six open reading frames strongly resemble proteins from known type II polyketide synthase gene clusters: a ketoacyl synthase, a chain length factor, an acyl carrier protein, a ketoreductase, a cyclase, and an oxygenase. Heterologous expression of the urdamycin genes encoding a ketoacyl synthase and a chain length factor in Streptomyces glaucescens tetracenomycin C-nonproducing mutants impaired in either the TcmK ketoacyl synthase or TcmL chain length factor resulted in the production of tetracenomycin C. Heterologous expression of a putative oxygenase gene from the urdamycin gene cluster in S. glaucescens GLA.O caused production of the hybrid antibiotic 6-hydroxy tetracenomycin C. 相似文献
16.
Analysis of the nucleotide sequence of the Streptomyces glaucescens tcmI genes provides key information about the enzymology of polyketide antibiotic biosynthesis. 总被引:32,自引:2,他引:30
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Key information about the biosynthesis of polyketide metabolites has been uncovered by sequence analysis of the tetracenomycin C polyketide synthase genes (tcml) from Streptomyces glaucescens GLA.0. The sequence data revealed the presence of three complete open reading frames (ORFs). ORF1 and ORF2 appear to be translationally coupled and would encode proteins containing 426 and 405 amino acids, respectively. The two deduced proteins are homologous to known beta-ketoacyl synthases. ORF3 begins 70 nucleotides after the stop codon of ORF2 and would code for an 83 amino acid protein with a strong resemblance to known bacterial, animal and plant acyl-carrier proteins (ACP). The presence of an ACP gene within the tcm gene cluster suggests that different ACPs are used in fatty acid and polyketide biosynthesis in Streptomyces. We conclude from these data and earlier information that polyketide biosynthesis in S. glaucescens, and most likely in other bacteria, involves a multienzyme complex consisting of at least five types of enzymes: acylCoA transferases that load the acyl and 2-carboxyacyl precursors onto the ACP; a beta-ketoacyl synthase that, along with the acylated ACP, forms the poly-beta-ketoacyl intermediates; a poly-beta-ketone cyclase that forms carbocyclic structures from the latter intermediates; a beta-ketoacyl oxidoreductase that forms beta-hydroxyacyl intermediates or reduces ketone groups in fully formed polyketides; and a thioesterase that releases the assembled polyketide from the enzyme. 相似文献
17.
Song D Coughlin J Ju J Zhou X Shen B Zhao C Deng Z 《Acta biochimica et biophysica Sinica》2008,40(4):319-326
Sequence analysis of oxazolomycin (OZM) biosynthetic gene cluster from Streptomyces albus JA3453 revealed a gene, ozmH , encoding a hybrid polyketide and non-ribosomal pep-tide enzyme. Tandem ketosynthase (KS) domains (KS10–1 and KS10–2 ) were characterized and they show significant homol-ogy with known KSs. Using an alternative method that involves RecA-mediated homologous recombination, the negative selection marker sacB gene, and temperature-sensitive replications, site-directed mutagenesis of the catalytic triad amino acid cysteines were carried out in each of the tandem KS domains totest the function they play in OZM biosynthesis. HPLC-mass spectrometry analysis of the resulting mutant strains showed that KS10–2 is essential for OZM biosynthesis but KS10–1 is not indispensable and might serve as a "redundant" domain. These results confirmed the existence of an "extra domain" in complex polyketide synthase. 相似文献
18.
T A Chinenova I V Biriukova T A Voe?kova L K Emel'ianova O A Klochkova G V Sezonov N D Lomovskaia 《Genetika》1990,26(4):636-647
Data are presented on resistance of Streptomyces aureofaciens strain TB-633 FU--the producer of chlortetracycline (CTC) to autogenous antibiotics and a number of other antibiotics. It is demonstrated that resistance to CTC is specified by ctr genes of constitutive expression as well as by inducible genes. CTC and ethidium bromide may serve as efficient inductors of inducible ctr genes. The induction process is accompanied by increase in antibiotic biosynthesis level. Genes responsible for strain resistance to a number of macrolide antibiotics and thiostrepton are inducible and only function in the presence of appropriate antibiotics in the medium. The action of inducible mtr gene(s) is described in detail. The gene(s) simultaneously ensure increase in resistance to CTC and a number of macrolide antibiotics in the presence of exogenous inductors in media, such as both CTC and macrolide antibiotics. Mutants have been isolated which provide constitutive level of resistance to these antibiotics. A series of ctr and mtr mutants have increased CTC biosynthesis as compared to the initial level. Data on comparative analysis of the results obtained from hybridization of fragments of S. aureofaciens and S. rimosus DNAs to actI and actIII genes, responsible for polyketide synthases' synthesis, demonstrate that genes for CTC and OTC biosynthesis are situated on DNA fragments of similar size. This determines the strategy for cloning ctr and mtr genes as well as genes for CTC biosynthesis from S. aureofaciens. 相似文献
19.
A collection of actinomycin-producing Streptomyces strains, their variants with different levels of antibiotic biosynthesis, and recombinant strains were screened in order to select new strains that produce polyketide antibiotics. Screening with the use of the cloned act gene encoding a component of actinorhodin polyketide synthase (PKS) multienzyme complex from Streptomyces coelicolor revealed that many strains tested can synthesize polyketide antibiotics along with actinomycins. A relationship between biosynthetic pathways of actinomycins and polyketides is discussed. 相似文献