The Experts below are selected from a list of 360 Experts worldwide ranked by ideXlab platform
Koji Ichinose - One of the best experts on this subject based on the ideXlab platform.
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biosynthetic conclusions from the functional dissection of oxygenases for biosynthesis of actinorhodin and related streptomyces antibiotics
Chemistry & Biology, 2013Co-Authors: Takaaki Taguchi, Mikko Metsaketela, Susumu Okamoto, Masaki Yabe, Hitomi Odaki, Miki Shinozaki, Takao Arai, Koji IchinoseAbstract:Summary Actinorhodin (ACT) produced by Streptomyces coelicolor A3(2) belongs to the benzoisochromanequinone (BIQ) class of antibiotics. ActVA-ORF5, a flavin-dependent monooxygenase (FMO) essential for ACT biosynthesis, forms a two-component enzyme system in combination with a flavin:NADH oxidoreductase, ActVB. The genes for homologous two-component FMOs are found in the biosynthetic gene clusters for two other BIQs, granaticin (GRA) and medermycin (MED), and a closely related antibiotic, alnumycin (ALN). Our functional analysis of these FMOs (ActVA-ORF5, Gra-ORF21, Med-ORF7, and AlnT) in S. coelicolor unambiguously demonstrated that ActVA-ORF5 and Gra-ORF21 are bifunctional and capable of both p -quinone formation at C-6 in the central ring and C-8 hydroxylation in the lateral ring, whereas Med-ORF7 catalyzes only p -quinone formation. No p -quinone formation on a BIQ substrate was observed for AlnT, which is involved in lateral p -quinone formation in ALN.
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identification of the actinorhodin monomer and its related compound from a deletion mutant of the actva orf4 gene of streptomyces coelicolor a3 2
Bioorganic & Medicinal Chemistry Letters, 2012Co-Authors: Takaaki Taguchi, Susumu Okamoto, Takayuki Ebihara, Atsushi Furukawa, Yumiko Hidaka, Ryuji Ariga, Koji IchinoseAbstract:An oxygenated derivative of dihydrokalafungin (DHK) was isolated from a deletion mutant of the actVA-ORF4 gene involved in the biosynthesis of a dimeric benzoisochromanequinone (BIQ) antibiotic, actinorhodin (ACT), in Streptomyces coelicolor A3(2). Spectroscopic analysis elucidated its structure as 8-hydroxy-DHK, corresponding to the monomeric unit of ACT. Further metabolite analysis identified its related compound, clearly derived from the reduction of 8-hydroxy-DHK. The structures of these metabolites indicate the essential role of ActVA-ORF4 in ACT biosynthesis, specifically in dimerization of a BIQ intermediate via C-C bond formation.
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epoxyquinone formation catalyzed by a two component flavin dependent monooxygenase involved in biosynthesis of the antibiotic actinorhodin
ChemBioChem, 2011Co-Authors: Takaaki Taguchi, Susumu Okamoto, Kimiko Hasegawa, Koji IchinoseAbstract:The biosynthetic gene cluster of the aromatic polyketide antibiotic actinorhodin (ACT) in Streptomyces coelicolor A3(2) carries a pair of genes, actVA-ORF5 and actVB, that encode a two-component flavin-dependent monooxygenase (FMO). Our previous studies have demonstrated that the ActVA-ORF5/ActVB system functions as a quinone-forming C-6 oxygenase in ACT biosynthesis. Furthermore, we found that this enzyme system exhibits an additional oxygenation activity with dihydrokalafungin (DHK), a proposed intermediate in the ACT biosynthetic pathway, and generates two reaction products. These compounds were revealed to be monooxygenated derivatives of kalafungin, which is spontaneously formed through oxidative lactonization of DHK. Their absolute structures were elucidated from their NMR spectroscopic data and by computer modeling and X-ray crystallography as (5S,14R)-epoxykalafungin and (5R,14S)-epoxykalafungin, demonstrating an additional epoxyquinone-forming activity of the ActVA-ORF5/ActVB system in vitro.
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biosynthesis of actinorhodin and related antibiotics discovery of alternative routes for quinone formation encoded in the act gene cluster
Chemistry & Biology, 2009Co-Authors: Susumu Okamoto, Takaaki Taguchi, Kozo Ochi, Koji IchinoseAbstract:All known benzoisochromanequinone (BIQ) biosynthetic gene clusters carry a set of genes encoding a two-component monooxygenase homologous to the ActVA-ORF5/ActVB system for actinorhodin biosynthesis in Streptomyces coelicolor A3(2). Here, we conducted molecular genetic and biochemical studies of this enzyme system. Inactivation of actVA-ORF5 yielded a shunt product, actinoperylone (ACPL), apparently derived from 6-deoxy-dihydrokalafungin. Similarly, deletion of actVB resulted in accumulation of ACPL, indicating a critical role for the monooxygenase system in C-6 oxygenation, a biosynthetic step common to all BIQ biosyntheses. Furthermore, in vitro, we showed a quinone-forming activity of the ActVA-ORF5/ActVB system in addition to that of a known C-6 monooxygenase, ActVA-ORF6, by using emodinanthrone as a model substrate. Our results demonstrate that the act gene cluster encodes two alternative routes for quinone formation by C-6 oxygenation in BIQ biosynthesis.
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actinoperylone a novel perylenequinone type shunt product from a deletion mutant of the actva orf5 and orf6 genes for actinorhodin biosynthesis in streptomyces coelicolor a3 2
Tetrahedron Letters, 2008Co-Authors: Takaaki Taguchi, Yutaka Ebizuka, Kozo Ochi, Susumu Okamoto, Takayuki Itoh, Koji IchinoseAbstract:A novel shunt product, actinoperylone, has been isolated from a deletion mutant of the actVA-ORF5 and ORF6 genes involved in the biosynthesis of a benzoisochromanequinone (BIQ) antibiotic actinorhodin (ACT) in Streptomyces coelicolor A3(2). Spectroscopic analysis revealed its perylenequinone-type skeleton with the four chiral centers, obviously derived from the dimerization of an ACT intermediate. The structure of actinoperylone indicates the essential role of ActVA-ORF5 in the oxygen introduction at C-6, which is common to the formation of BIQ chromophore. The present results also agree with the distribution of the actVA-ORF5 homologues in all known BIQ biosynthetic clusters in streptomycetes.
David A Hopwood - One of the best experts on this subject based on the ideXlab platform.
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A putative twocomponent regulatory system involved in secondary metabolism in Streptomyces spp
2016Co-Authors: Hiroshi Ishizuka, David A Hopwood, Sueharu Horinouchi, Helen M. Kieser, Teruhiko BeppuAbstract:A DNA fragment stimulating actinorhodin, undecylprodigiosin, and A-factor production in Streptomyces lividans 66 was cloned from Streptomyces coelicolor A3(2). Nucleotide sequencing revealed the presence of an open reading frame of 225 codons, named afsQl, that showed great similarity in amino acid sequence to the response regulators of typical prokaryotic two-component regulatory systems responsible for adaptive responses. The termination codon, TGA, of afsQl overlapped the initiation codon, GTG, of a second open reading frame, afsQ2, of 535 codons. The afsQ2 gene product showed homology with the sensory histidine protein kinases of two-component systems. In agreement with the assumption that the AfsQl and AfsQ2 proteins comprise an aspartate-histidine phosphotransfer system, an amino acid replacement from Asp to Glu at residue 52 of AfsQl, generated by site-directed mutagenesis, resulted in loss of the protein's ability to stimulate antibiotic production in S. lividans. Primer extension experiments indicated that transcription of the afsQl and afsQ2 genes initiates at the translational start codon (GTG) of the afsQl gene. The afsQ1 and afsQ2 genes were physically mapped at a chromosomal position near the actinorhodin biosynthetic gene cluster (act) by hybridization to Southern blots of restriction fragments separated by pulsed-field gel electrophoresis. Disruption of either afsQl or afsQ2 on the S. coelicolor chromosome by use of phage 4C31KC515 led to no detectable change in secondary metabolite formation or morphogenesis. The afsQl gene on pU922 suppresse
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functional complementation of pyran ring formation in actinorhodin biosynthesis in streptomyces coelicolor a3 2 by ketoreductase genes for granaticin biosynthesis
Journal of Bacteriology, 2001Co-Authors: Koji Ichinose, Takaaki Taguchi, Yutaka Ebizuka, David J Bedford, David A HopwoodAbstract:A mutation in actVI-ORF1, which controls C-3 reduction in actinorhodin biosynthesis by Streptomyces coelicolor, was complemented by gra-ORF5 and -ORF6 from the granaticin biosynthetic gene cluster of Streptomyces violaceoruber Tu22. It is hypothesized that, while gra-ORF5 alone is a ketoreductase for C-9, gra-ORF6 gives the enzyme regiospecificity also for C-3.
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identification of a novel shunt product produced by a disruptant of the actvi orfa gene involved in the biosynthesis of actinorhodin in streptomyces coelicolor a3 2
Tetrahedron Letters, 2000Co-Authors: Takaaki Taguchi, Yutaka Ebizuka, David A Hopwood, Koji IchinoseAbstract:Abstract NMR and MS spectroscopy elucidated the structure of a novel shunt product isolated from a disruptant of the act VI-ORFA gene involved in the biosynthesis of actinorhodin (ACT) in Streptomyces coelicolor A3(2). The product, 1,4-naphthoquinone-8-hydroxy-3-[(3 S )-acetoxybutyric acid], was found without complete abolition of ACT production, implying that the ORFA product might stabilise the multicomponent, type II PKS complex and perhaps assist the chemically spontaneous dehydration of a hemiketal intermediate for efficient pyran ring formation.
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proof that the actvi genetic region of streptomyces coelicolor a3 2 is involved in stereospecific pyran ring formation in the biosynthesis of actinorhodin
Bioorganic & Medicinal Chemistry Letters, 1999Co-Authors: Koji Ichinose, Chandres Surti, Takaaki Taguchi, Kevin I Bookermilburm, Yutaka Ebizuka, Richard G Stephenson, Francisco Malpartida, David A HopwoodAbstract:Pyran ring formation in the biosynthesis of actinorhodin in Streptomyces coelicolor A3(2) was studied using the act cluster deficient strain, CH999, carrying pRM5-based plasmids harbouring combinations of the actVI genes. The strain, CH999/pIJ5660 (pRM5 + actVI-ORF1), produced a chiral intermediate, (S)-DNPA, suggesting that the actVI-ORF1 product is a reductase determining the C-3 stereochemical centre.
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the granaticin biosynthetic gene cluster of streptomyces violaceoruber tu22 sequence analysis and expression in a heterologous host
Chemistry & Biology, 1998Co-Authors: Koji Ichinose, Heinz G. Floss, Maureen J Bibb, David J Bedford, Diethild Tornus, Andreas Bechthold, Peter W Revill, David A HopwoodAbstract:Abstract Introduction: The granaticins are members of the benzoisochromanequinone class of aromatic polyketides, the best known member of which is actinorhodin made by Streptomyces coelicolor A3(2). Genetic analysis of this class of compounds has played a major role in the development of hypotheses about the way in which aromatic polyketide synthases (PKSs) control product structure. Although the granaticin nascent polyketide is identical to that of actinorhodin, post-PKS steps involve different pyran-ring stereochemistry and glycosylation. Comparison of the complete gene clusters for the two metabolites is therefore of great interest. Results: The entire granaticin gene cluster (the gra cluster) from Streptomyces violaceoruber Tu22 was cloned on either of two overlapping cosmids and expressed in the heterologous host, Streptomyces coelicolor A3(2), strain CH999. Chemical analysis of the recombinant strains demonstrated production of granaticin, granaticin B, dihydrogranaticin and dihydrogranaticin B, which are the four known metabolites of S. violaceoruber . Analysis of the complete 39,250 base pair sequence of the insert of one of the cosmids, pOJ466-22-24, revealed 37 complete open reading frames (ORFs), 15 of which resemble ORFs from the act (actinorhodin) gene cluster of S. coelicolor A3(2). Among the rest, nine resemble ORFs potentially involved in deoxysugar metabolism from Streptomyces spp. and other bacteria, and six resemble regulatory ORFs. Conclusions: On the basis of these resemblances, putative functional assignments of the products of most of the newly discovered ORFs were made, including those of genes involved in the PKS and tailoring steps in the biosynthesis of the granaticin aglycone, steps in the deoxy sugar pathway, and putative regulatory and export functions.
Mervyn J. Bibb - One of the best experts on this subject based on the ideXlab platform.
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Synthetic RNA silencing of actinorhodin biosynthesis
2016Co-Authors: Gabriel C. Uguru, Mervyn J. Bibb, Shan Goh, Liam Good, James E. M. StachAbstract:We demonstrate the first application of synthetic RNA gene silencers in Streptomyces coelicolor A3(2). Peptide nucleic acid and expressed antisense RNA silencers successfully inhibited actinorhodin production. Synthetic RNA silencing was target-specific and is a new tool for gene regulation and metabolic engineering studies in Streptomyces
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a system for the targeted amplification of bacterial gene clusters multiplies antibiotic yield in streptomyces coelicolor
Proceedings of the National Academy of Sciences of the United States of America, 2011Co-Authors: Takeshi Murakami, Mervyn J. Bibb, Jan Burian, Koji Yanai, Charles J ThompsonAbstract:Gene clusters found in bacterial species classified as Streptomyces encode the majority of known antibiotics as well as many pharmaceutically active compounds. A site-specific recombination system similar to those that mediate plasmid conjugation was engineered to catalyze tandem amplification of one of these gene clusters in a heterologous Streptomyces species. Three genetic elements were known to be required for DNA amplification in S. kanamyceticus: the oriT-like recombination sites RsA and RsB, and ZouA, a site-specific relaxase similar to TraA proteins that catalyze plasmid transfer. We inserted RsA and RsB sequences into the S. coelicolor genome flanking a cluster of 22 genes (act) responsible for biosynthesis of the polyketide antibiotic actinorhodin. Recombination between RsA and RsB generated zouA-dependent DNA amplification resulting in 4–12 tandem copies of the act gene cluster averaging nine repeats per genome. This resulted in a 20-fold increase in actinorhodin production compared with the parental strain. To determine whether the recombination event required taxon-specific genetic effectors or generalized bacterial recombination (recA), it was also analyzed in the heterologous host Escherichia coli. zouA was expressed under the control of an inducible promoter in wild-type and recA mutant strains. A plasmid was constructed with recombination sites RsA and RsB bordering a drug resistance marker. Induction of zouA expression generated hybrid RsB/RsA sites, evidence of site-specific recombination that occurred independently of recA. ZouA-mediated DNA amplification promises to be a valuable tool for increasing the activities of commercially important biosynthetic, degradative, and photosynthetic pathways in a wide variety of organisms.
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Engineering Streptomyces coelicolor for heterologous expression of secondary metabolite gene clusters
Microbial biotechnology, 2010Co-Authors: Juan Pablo Gomez-escribano, Mervyn J. BibbAbstract:We have constructed derivatives of Streptomyces coelicolor M145 as hosts for the heterologous expression of secondary metabolite gene clusters. To remove potentially competitive sinks of carbon and nitrogen, and to provide a host devoid of antibiotic activity, we deleted four endogenous secondary metabolite gene clusters from S. coelicolor M145 – those for actinorhodin, prodiginine, CPK and CDA biosynthesis. We then introduced point mutations into rpoB and rpsL to pleiotropically increase the level of secondary metabolite production. Introduction of the native actinorhodin gene cluster and of gene clusters for the heterologous production of chloramphenicol and congocidine revealed dramatic increases in antibiotic production compared with the parental strain. In addition to lacking antibacterial activity, the engineered strains possess relatively simple extracellular metabolite profiles. When combined with liquid chromatography and mass spectrometry, we believe that these genetically engineered strains will markedly facilitate the discovery of new compounds by heterologous expression of cloned gene clusters, particularly the numerous cryptic secondary metabolic gene clusters that are prevalent within actinomycete genome sequences.
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the role of absc a novel regulatory gene for secondary metabolism in zinc dependent antibiotic production in streptomyces coelicolor a3 2
Molecular Microbiology, 2009Co-Authors: Andrew Hesketh, Holger Kock, Saraspadee Mootien, Mervyn J. BibbAbstract:The availability of zinc was shown to have a marked influence on the biosynthesis of actinorhodin in Streptomyces coelicolor A3(2). Production of actinorhodin and undecylprodigiosin was abolished when a novel pleiotropic regulatory gene, absC, was deleted, but only when zinc concentrations were low. AbsC was shown to control expression of the gene cluster encoding production of coelibactin, an uncharacterized non-ribosomally synthesized peptide with predicted siderophore-like activity, and the observed defect in antibiotic production was found to result from elevated expression of this gene cluster. Promoter regions in the coelibactin cluster contain predicted binding motifs for the zinc-responsive regulator Zur, and dual regulation of coelibactin expression by zur and absC was demonstrated using strains engineered to contain deletions in either or both of these genes. An AbsC binding site was identified in a divergent promoter region within the coelibactin biosynthetic gene cluster, adjacent to a putative Zur binding site. Repression of the coelibactin gene cluster by both AbsC and Zur appears to be required to maintain appropriate intracellular levels of zinc in S. coelicolor.
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the global role of ppgpp synthesis in morphological differentiation and antibiotic production in streptomyces coelicolor a3 2
Genome Biology, 2007Co-Authors: Andrew Hesketh, Wenqiong Joan Chen, Jamie Ryding, Sherman Chang, Mervyn J. BibbAbstract:Background: Regulation of production of the translational apparatus via the stringent factor ppGpp in response to amino acid starvation is conserved in many bacteria. However, in addition to this core function, it is clear that ppGpp also exhibits genus-specific regulatory effects. In this study we used Affymetrix GeneChips to more fully characterize the regulatory influence of ppGpp synthesis on the biology of Streptomyces coelicolor A3(2), with emphasis on the control of antibiotic biosynthesis and morphological differentiation. Results: Induction of ppGpp synthesis repressed transcription of the major sigma factor hrdB, genes with functions associated with active growth, and six of the thirteen conservons present in the S. coelicolor genome. Genes induced following ppGpp synthesis included the alternative sigma factor SCO4005, many for production of the antibiotics CDA and actinorhodin, the regulatory genes SCO4198 and SCO4336, and two alternative ribosomal proteins. Induction of the CDA and actinorhodin clusters was accompanied by an increase in transcription of the pathway regulators cdaR and actII-ORF4, respectively. Comparison of transcriptome profiles of a relA null strain, M570, incapable of ppGpp synthesis with its parent M600 suggested the occurrence of metabolic stress in the mutant. The failure of M570 to sporulate was associated with a stalling between production of the surfactant peptide SapB, and of the hydrophobins: it overproduced SapB but failed to express the chaplin and rodlin genes. Conclusion: In S. coelicolor, ppGpp synthesis influences the expression of several genomic elements that are particularly characteristic of streptomycete biology, notably antibiotic gene clusters, conservons, and morphogenetic proteins.
Meifeng Tao - One of the best experts on this subject based on the ideXlab platform.
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engineering the erythromycin producing strain saccharopolyspora erythraea hoe107 for the heterologous production of polyketide antibiotics
Frontiers in Microbiology, 2020Co-Authors: Qingshan Long, Zixin Deng, Zhilong Zhao, Lu Chen, Jiali Hong, Kai Liu, Yemin Wang, Xiuhua Pang, Meifeng TaoAbstract:Bacteria of the genus Saccharopolyspora produce important polyketide antibiotics, including erythromycin A (Sac. erythraea) and spinosad (Sac. spinosa). We herein report the development of an industrial erythromycin-producing strain, Sac. erythraea HOE107, into a host for the heterologous expression of polyketide biosynthetic gene clusters (BGCs) from other Saccharopolyspora species and related actinomycetes. To facilitate the integration of natural product BGCs and auxiliary genes beneficial for the production of natural products, the erythromycin polyketide synthase (ery) genes were replaced with two bacterial attB genomic integration sites associated with bacteriophages ϕC31 and ϕBT1. We also established a highly efficient conjugation protocol for the introduction of large bacterial artificial chromosome (BAC) clones into Sac. erythraea strains. Based on this optimized protocol, an arrayed BAC library was effectively transferred into Sac. erythraea. The large spinosad gene cluster from Sac. spinosa and the actinorhodin gene cluster from Streptomyces coelicolor were successfully expressed in the ery deletion mutant. Deletion of the endogenous giant polyketide synthase genes pkeA1-pkeA4, the product of which is not known, and the flaviolin gene cluster (rpp) from the bacterium increased the heterologous production of spinosad and actinorhodin. Furthermore, integration of pJTU6728 carrying additional beneficial genes dramatically improved the yield of actinorhodin in the engineered Sac. erythraea strains. Our study demonstrated that the engineered Sac. erythraea strains SLQ185, LJ161, and LJ162 are good hosts for the expression of heterologous antibiotics and should aid in expression-based genome-mining approaches for the discovery of new and cryptic antibiotics from Streptomyces and rare actinomycetes.
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engineering the erythromycin producing strain saccharopolyspora erythraea hoe107 for the heterologous production of polyketide antibiotics
Frontiers in Microbiology, 2020Co-Authors: Qingshan Long, Zixin Deng, Zhilong Zhao, Lu Chen, Jiali Hong, Kai Liu, Yemin Wang, Xiuhua Pang, Meifeng TaoAbstract:Bacteria of the genus Saccharopolyspora produce important polyketide antibiotics, including erythromycin A (Sac. erythraea) and spinosad (Sac. spinosa). We herein report the development of an early industrial erythromycin-producing strain, Sac. erythraea HOE107, into a host for the heterologous expression of polyketide biosynthetic gene clusters (BGCs) from other Saccharopolyspora species and related actinomycetes. To facilitate the integration of natural product BGCs and auxiliary genes beneficial for the production of natural products, the erythromycin polyketide synthase (ery) genes were replaced with two bacterial attB genomic integration sites associated with bacteriophages phiC31 and phiBT1. We also established a highly efficient conjugation protocol for the introduction of large bacterial artificial chromosome (BAC) clones into Sac. erythraea strains. Based on this optimized protocol, an arrayed BAC library was effectively transferred into Sac. erythraea. The large spinosad gene cluster from Sac. spinosa and the actinorhodin gene cluster from Streptomyces coelicolor were successfully expressed in the ery deletion mutant. Deletion of the endogenous giant polyketide synthase genes pkeA1-pkeA4, the product of which is not known, and the flaviolin gene cluster (rpp) from the bacterium increased the heterologous production of spinosad and actinorhodin. Furthermore, integration of pJTU6728 carrying additional beneficial genes dramatically improved the yield of actinorhodin in the engineered Sac. erythraea strains. Our study demonstrated that the engineered Sac. erythraea strains SLQ185, LJ161, and LJ162 are good hosts for the expression of heterologous antibiotics and should aid in expression-based genome-mining approaches for the discovery of new and cryptic antibiotics from Streptomyces and rare actinomycetes.
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nsdb a tpr like domain containing protein negatively affecting production of antibiotics in streptomyces coelicolor a3 2
Acta Microbiologica Sinica, 2007Co-Authors: Li Zhang, Keith F Chater, Chunhua Zhao, Meifeng TaoAbstract:Tetratricopeptide repeat (TPR) domains usually mediate protein-protein interactions. NsdA, one of the 70 proteins containing TPR-like domains in Streptomyces coelicolor A3 (2), was previously found to negatively control sporulation and antibiotic production. Here we show that elimination of SCO7252, which encodes another of these proteins, also caused overproduction of two antibiotics, actinorhodin and CDA, but did not affect morphological differentiation. Disruption of SCO1593, encoding another of the family, had no obvious phenotypic effects. In surface-grown cultures, expression of SCO7252, which was named nsdB, was initiated at about 30 h, like that of nsdA. Analysis in silico of the 70 predicted TPR-like-containing proteins of S. coelicolor showed that 32 of them contained only TPR-like domains, and 25 of the remainder contained additional DNA-binding domains, implying that they might control gene expression directly.
Takaaki Taguchi - One of the best experts on this subject based on the ideXlab platform.
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biosynthetic conclusions from the functional dissection of oxygenases for biosynthesis of actinorhodin and related streptomyces antibiotics
Chemistry & Biology, 2013Co-Authors: Takaaki Taguchi, Mikko Metsaketela, Susumu Okamoto, Masaki Yabe, Hitomi Odaki, Miki Shinozaki, Takao Arai, Koji IchinoseAbstract:Summary Actinorhodin (ACT) produced by Streptomyces coelicolor A3(2) belongs to the benzoisochromanequinone (BIQ) class of antibiotics. ActVA-ORF5, a flavin-dependent monooxygenase (FMO) essential for ACT biosynthesis, forms a two-component enzyme system in combination with a flavin:NADH oxidoreductase, ActVB. The genes for homologous two-component FMOs are found in the biosynthetic gene clusters for two other BIQs, granaticin (GRA) and medermycin (MED), and a closely related antibiotic, alnumycin (ALN). Our functional analysis of these FMOs (ActVA-ORF5, Gra-ORF21, Med-ORF7, and AlnT) in S. coelicolor unambiguously demonstrated that ActVA-ORF5 and Gra-ORF21 are bifunctional and capable of both p -quinone formation at C-6 in the central ring and C-8 hydroxylation in the lateral ring, whereas Med-ORF7 catalyzes only p -quinone formation. No p -quinone formation on a BIQ substrate was observed for AlnT, which is involved in lateral p -quinone formation in ALN.
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identification of the actinorhodin monomer and its related compound from a deletion mutant of the actva orf4 gene of streptomyces coelicolor a3 2
Bioorganic & Medicinal Chemistry Letters, 2012Co-Authors: Takaaki Taguchi, Susumu Okamoto, Takayuki Ebihara, Atsushi Furukawa, Yumiko Hidaka, Ryuji Ariga, Koji IchinoseAbstract:An oxygenated derivative of dihydrokalafungin (DHK) was isolated from a deletion mutant of the actVA-ORF4 gene involved in the biosynthesis of a dimeric benzoisochromanequinone (BIQ) antibiotic, actinorhodin (ACT), in Streptomyces coelicolor A3(2). Spectroscopic analysis elucidated its structure as 8-hydroxy-DHK, corresponding to the monomeric unit of ACT. Further metabolite analysis identified its related compound, clearly derived from the reduction of 8-hydroxy-DHK. The structures of these metabolites indicate the essential role of ActVA-ORF4 in ACT biosynthesis, specifically in dimerization of a BIQ intermediate via C-C bond formation.
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epoxyquinone formation catalyzed by a two component flavin dependent monooxygenase involved in biosynthesis of the antibiotic actinorhodin
ChemBioChem, 2011Co-Authors: Takaaki Taguchi, Susumu Okamoto, Kimiko Hasegawa, Koji IchinoseAbstract:The biosynthetic gene cluster of the aromatic polyketide antibiotic actinorhodin (ACT) in Streptomyces coelicolor A3(2) carries a pair of genes, actVA-ORF5 and actVB, that encode a two-component flavin-dependent monooxygenase (FMO). Our previous studies have demonstrated that the ActVA-ORF5/ActVB system functions as a quinone-forming C-6 oxygenase in ACT biosynthesis. Furthermore, we found that this enzyme system exhibits an additional oxygenation activity with dihydrokalafungin (DHK), a proposed intermediate in the ACT biosynthetic pathway, and generates two reaction products. These compounds were revealed to be monooxygenated derivatives of kalafungin, which is spontaneously formed through oxidative lactonization of DHK. Their absolute structures were elucidated from their NMR spectroscopic data and by computer modeling and X-ray crystallography as (5S,14R)-epoxykalafungin and (5R,14S)-epoxykalafungin, demonstrating an additional epoxyquinone-forming activity of the ActVA-ORF5/ActVB system in vitro.
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biosynthesis of actinorhodin and related antibiotics discovery of alternative routes for quinone formation encoded in the act gene cluster
Chemistry & Biology, 2009Co-Authors: Susumu Okamoto, Takaaki Taguchi, Kozo Ochi, Koji IchinoseAbstract:All known benzoisochromanequinone (BIQ) biosynthetic gene clusters carry a set of genes encoding a two-component monooxygenase homologous to the ActVA-ORF5/ActVB system for actinorhodin biosynthesis in Streptomyces coelicolor A3(2). Here, we conducted molecular genetic and biochemical studies of this enzyme system. Inactivation of actVA-ORF5 yielded a shunt product, actinoperylone (ACPL), apparently derived from 6-deoxy-dihydrokalafungin. Similarly, deletion of actVB resulted in accumulation of ACPL, indicating a critical role for the monooxygenase system in C-6 oxygenation, a biosynthetic step common to all BIQ biosyntheses. Furthermore, in vitro, we showed a quinone-forming activity of the ActVA-ORF5/ActVB system in addition to that of a known C-6 monooxygenase, ActVA-ORF6, by using emodinanthrone as a model substrate. Our results demonstrate that the act gene cluster encodes two alternative routes for quinone formation by C-6 oxygenation in BIQ biosynthesis.
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actinoperylone a novel perylenequinone type shunt product from a deletion mutant of the actva orf5 and orf6 genes for actinorhodin biosynthesis in streptomyces coelicolor a3 2
Tetrahedron Letters, 2008Co-Authors: Takaaki Taguchi, Yutaka Ebizuka, Kozo Ochi, Susumu Okamoto, Takayuki Itoh, Koji IchinoseAbstract:A novel shunt product, actinoperylone, has been isolated from a deletion mutant of the actVA-ORF5 and ORF6 genes involved in the biosynthesis of a benzoisochromanequinone (BIQ) antibiotic actinorhodin (ACT) in Streptomyces coelicolor A3(2). Spectroscopic analysis revealed its perylenequinone-type skeleton with the four chiral centers, obviously derived from the dimerization of an ACT intermediate. The structure of actinoperylone indicates the essential role of ActVA-ORF5 in the oxygen introduction at C-6, which is common to the formation of BIQ chromophore. The present results also agree with the distribution of the actVA-ORF5 homologues in all known BIQ biosynthetic clusters in streptomycetes.
