1. |
Katsuyama K,
Iwata N,
Shimazu A,
( 1992 ) Purification and primary structure of proteinous alpha-amylase inhibitor from Streptomyces chartreusis. PMID : 1369094 : Abstract >>
A new polypeptide inhibitor, AI-409, that inhibits human salivary alpha-amylase, was purified from a fermentation broth of Streptomyces chartreusis strain No. 409. This protein consists of a single-chain polypeptide of 78 amino acid residues, and includes two disulfide bridges. The primary structure of AI-409 and the locations of the disulfide bridges were identified by enzymatic digestion and the automatic Edman technique. Enzymatic digestion was done with trypsin, carboxypeptidase Y, and chymotrypsin. One of the disulfide bridges was between Cys(10) and Cys(26), and the other between Cys(44) and Cys(71).
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2. |
Kaneko S,
Kuno A,
Matsuo N,
( 2000 ) Purification, characterization and gene cloning of two alpha-L-arabinofuranosidases from streptomyces chartreusis GS901. PMID : 10657233 : PMC : PMC1220816 Abstract >>
alpha-L-Arabinofuranosidases I and II were purified from the culture filtrate of Streptomyces chartreusis GS901 and were found to have molecular masses of 80 and 37 kDa and pI values of 6.6 and 7.5 respectively. Both enzymes demonstrated slight reactivity towards arabinoxylan and arabinogalactan as substrates but did not hydrolyse gum arabic or arabinoxylo-oligosaccharides. alpha-L-Arabinofuranosidase I hydrolysed all of the alpha-linkage types that normally occur between two alpha-L-arabinofuranosyl residues, with the following decreasing order of reactivity being observed for the respective disaccharide linkages: alpha-(1-->2) alpha-(1-->3) alpha-(1-->5). This enzyme cleaved the (1-->3) linkages of the arabinosyl side-chains of methyl 3, 5-di-O-alpha-L-arabinofuranosyl-alpha-L-arabinofuranoside in preference to the (1-->5) linkages. alpha-L-Arabinofuranosidase I hydrolysed approx. 30% of the arabinan but hydrolysed hardly any linear arabinan. In contrast, alpha-L-Arabinofuranosidase II hydrolysed only (1-->5)-arabinofuranobioside among the regioisomeric methyl arabinobiosides and did not hydrolyse the arabinotrioside. Linear 1-->5-linked arabinan was a good substrate for this enzyme, but it hydrolysed hardly any of the arabinan. Synergism between the two enzymes was observed in the conversion of arabinan and debranched arabinan into arabinose. Complete amino acid sequencing of alpha-L-arabinofuranosidase I indicated that the enzyme consists of a central catalytic domain that belongs to family 51 of the glycoside hydrolases and additionally that unknown functional domains exist in the N-terminal and C-terminal regions. The amino acid sequence of alpha-L-arabinofuranosidase II indicated that this enzyme belongs to family 43 of the glycoside hydrolase family and, as this is the first report of an exo-1, 5-alpha-L-arabinofuranosidase, it represents a novel type of enzyme.
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3. |
Xu Z,
Jakobi K,
Welzel K,
Hertweck C,
( 2005 ) Biosynthesis of the antitumor agent chartreusin involves the oxidative rearrangement of an anthracyclic polyketide. PMID : 15911378 : DOI : 10.1016/j.chembiol.2005.04.017 Abstract >>
Chartreusin is a potent antitumor agent with a mixed polyketide-carbohydrate structure produced by Streptomyces chartreusis. Three type II polyketide synthase (PKS) gene clusters were identified from an S. chartreusis HKI-249 genomic cosmid library, one of which encodes chartreusin (cha) biosynthesis, as confirmed by heterologous expression of the entire cha gene cluster in Streptomyces albus. Molecular analysis of the approximately 37 kb locus and structure elucidation of a linear pathway intermediate from an engineered mutant reveal that the unusual bis-lactone aglycone chartarin is derived from an anthracycline-type polyketide. A revised biosynthetic model involving an oxidative rearrangement is presented.
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4. |
Hsu JS,
Yang YB,
Deng CH,
Wei CL,
Liaw SH,
Tsai YC,
( 2004 ) Family shuffling of expandase genes to enhance substrate specificity for penicillin G. PMID : 15466573 : DOI : 10.1128/AEM.70.10.6257-6263.2004 PMC : PMC522083 Abstract >>
Deacetoxycephalosporin C synthase (expandase) from Streptomyces clavuligerus, encoded by cefE, is an important industrial enzyme for the production of 7-aminodeacetoxycephalosporanic acid from penicillin G. To improve the substrate specificity for penicillin G, eight cefE-homologous genes were directly evolved by using the DNA shuffling technique. After the first round of shuffling and screening, using an Escherichia coli ESS bioassay, four chimeras with higher activity were subjected to a second round. Subsequently, 20 clones were found with significantly enhanced activity. The kinetic parameters of two isolates that lack substrate inhibition showed 8.5- and 118-fold increases in the k(cat)/K(m) ratio compared to the S. clavuligerus expandase. The evolved enzyme with the 118-fold increase is the most active obtained to date anywhere. Our shuffling results also indicate the remarkable plasticity of the expandase, suggesting that more-active chimeras might be achievable with further rounds.
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5. |
Chen W,
Qu D,
Zhai L,
Tao M,
Wang Y,
Lin S,
Price NP,
Deng Z,
( 2010 ) Characterization of the tunicamycin gene cluster unveiling unique steps involved in its biosynthesis. PMID : 21153459 : DOI : 10.1007/s13238-010-0127-6 PMC : PMC4875072 Abstract >>
Tunicamycin, a potent reversible translocase I inhibitor, is produced by several Actinomycetes species. The tunicamycin structure is highly unusual, and contains an 11-carbon dialdose sugar and an �\, �]-1?,11'-glycosidic linkage. Here we report the identification of a gene cluster essential for tunicamycin biosynthesis by high-throughput heterologous expression (HHE) strategy combined with a bioassay. Introduction of the genes into heterologous non-producing Streptomyces hosts results in production of tunicamycin by these strains, demonstrating the role of the genes for the biosynthesis of tunicamycins. Gene disruption experiments coupled with bioinformatic analysis revealed that the tunicamycin gene cluster is minimally composed of 12 genes (tunA-tunL). Amongst these is a putative radical SAM enzyme (Tun B) with a potentially unique role in biosynthetic carbon-carbon bond formation. Hence, a seven-step novel pathway is proposed for tunicamycin biosynthesis. Moreover, two gene clusters for the potential biosynthesis of tunicamycin-like antibiotics were also identified in Streptomyces clavuligerus ATCC 27064 and Actinosynnema mirums DSM 43827. These data provide clarification of the novel mechanisms for tunicamycin biosynthesis, and for the generation of new-designer tunicamycin analogs with selective/enhanced bioactivity via combinatorial biosynthesis strategies.
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6. |
Wu Q,
Liang J,
Lin S,
Zhou X,
Bai L,
Deng Z,
Wang Z,
( 2011 ) Characterization of the biosynthesis gene cluster for the pyrrole polyether antibiotic calcimycin (A23187) in Streptomyces chartreusis NRRL 3882. PMID : 21173184 : DOI : 10.1128/AAC.01130-10 PMC : PMC3067094 Abstract >>
The pyrrole polyether antibiotic calcimycin (A23187) is a rare ionophore that is specific for divalent cations. It is widely used as a biochemical and pharmacological tool because of its multiple, unique biological effects. Here we report on the cloning, sequencing, and mutational analysis of the 64-kb biosynthetic gene cluster from Streptomyces chartreusis NRRL 3882. Gene replacements confirmed the identity of the gene cluster, and in silico analysis of the DNA sequence revealed 27 potential genes, including 3 genes for the biosynthesis of the �\-ketopyrrole moiety, 5 genes that encode modular type I polyketide synthases for the biosynthesis of the spiroketal ring, 4 genes for the biosynthesis of 3-hydroxyanthranilic acid, an N-methyltransferase tailoring gene, a resistance gene, a type II thioesterase gene, 3 regulatory genes, 4 genes with other functions, and 5 genes of unknown function. We propose a pathway for the biosynthesis of calcimycin and assign the genes to the biosynthesis steps. Our findings set the stage for producing much desired calcimycin derivatives using genetic modification instead of chemical synthesis.
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7. |
Gao P,
Huang Y,
( 2009 ) Detection, distribution, and organohalogen compound discovery implications of the reduced flavin adenine dinucleotide-dependent halogenase gene in major filamentous actinomycete taxonomic groups. PMID : 19447951 : DOI : 10.1128/AEM.02958-08 PMC : PMC2708417 Abstract >>
Halogenases have been shown to play a significant role in biosynthesis and introducing the bioactivity of many halogenated secondary metabolites. In this study, 54 reduced flavin adenine dinucleotide (FADH(2))-dependent halogenase gene-positive strains were identified after the PCR screening of a large collection of 228 reference strains encompassing all major families and genera of filamentous actinomycetes. The wide distribution of this gene was observed to extend to some rare lineages with higher occurrences and large sequence diversity. Subsequent phylogenetic analyses revealed that strains containing highly homologous halogenases tended to produce halometabolites with similar structures, and halogenase genes are likely to propagate by horizontal gene transfer as well as vertical inheritance within actinomycetes. Higher percentages of halogenase gene-positive strains than those of halogenase gene-negative ones contained polyketide synthase genes and/or nonribosomal peptide synthetase genes or displayed antimicrobial activities in the tests applied, indicating their genetic and physiological potentials for producing secondary metabolites. The robustness of this halogenase gene screening strategy for the discovery of particular biosynthetic gene clusters in rare actinomycetes besides streptomycetes was further supported by genome-walking analysis. The described distribution and phylogenetic implications of the FADH(2)-dependent halogenase gene present a guide for strain selection in the search for novel organohalogen compounds from actinomycetes.
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8. |
Everest GJ,
Cook AE,
Kirby BM,
Meyers PR,
( 2011 ) Evaluation of the use of recN sequence analysis in the phylogeny of the genus Amycolatopsis. PMID : 21671192 : DOI : 10.1007/s10482-011-9604-z Abstract >>
Partial recN gene sequences (>1 kb) were obtained from 35 type strains of the genus Amycolatopsis. Phylogenetic trees were constructed to determine the effectiveness of using this gene to predict taxonomic relationships within the genus. The use of recN sequence analysis as an alternative to DNA-DNA hybridization (DDH) for distinguishing closely related species was also assessed. The recN based phylogeny mostly confirmed the conventional 16S rRNA and gyrB gene-based phylogenies and thus provides further support for these phylogenetic groupings. As is the case for the gyrB gene, pairwise recN sequence similarities cannot be used to predict the DNA relatedness between type strains but the recN genetic distance can be used as a means to assess quickly whether an isolate is likely to represent a new species in the genus Amycolatopsis. A recN genetic distance of >0.04 between two Amycolatopsis strains is proposed to provide a good indication that they belong to different species (and that polyphasic taxonomic characterization of the unknown strain is worth undertaking).
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9. |
Hu Y,
Sun F,
Liu W,
( 2018 ) The heat shock protein 70 gene as a new alternative molecular marker for the taxonomic identification of Streptomyces strains. PMID : 30203150 : DOI : 10.1186/s13568-018-0674-4 PMC : PMC6134474 Abstract >>
With the developments in taxonomy, the classically used highly conserved 16S rRNA molecular marker has shown some disadvantages among closely related species. For further taxonomic studies of the prokaryotes, specific PCR primers were designed from two conserved regions in the amino acid sequences of the 70-kDa heat shock protein sourced from 20 different genera in actinomycetes. These were used for the amplification of the hsp70 genes in 16 Streptomyces strains. Then, we investigated the phylogenetic relationships among these Streptomyces strains and compared the tree topology based on the hsp70 gene with those based on the previously used markers (16S rRNA and gyrB). To our knowledge, this is the first use of the hsp70 gene as a molecular marker for the taxonomic identification of Streptomyces.
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10. |
Wang YS,
Zhang B,
Zhu J,
Yang CL,
Guo Y,
Liu CL,
Liu F,
Huang H,
Zhao S,
Liang Y,
Jiao RH,
Tan RX,
Ge HM,
( 2018 ) Molecular Basis for the Final Oxidative Rearrangement Steps in Chartreusin Biosynthesis. PMID : 30067334 : DOI : 10.1021/jacs.8b06623 Abstract >>
Oxidative rearrangements play key roles in introducing structural complexity and biological activities of natural products biosynthesized by type II polyketide synthases (PKSs). Chartreusin (1) is a potent antitumor polyketide that contains a unique rearranged pentacyclic aromatic bilactone aglycone derived from a type II PKS. Herein, we report an unprecedented dioxygenase, ChaP, that catalyzes the final �\-pyrone ring formation in 1 biosynthesis using flavin-activated oxygen as an oxidant. The X-ray crystal structures of ChaP and two homologues, docking studies, and site-directed mutagenesis provided insights into the molecular basis of the oxidative rearrangement that involves two successive C-C bond cleavage steps followed by lactonization. ChaP is the first example of a dioxygenase that requires a flavin-activated oxygen as a substrate despite lacking flavin binding sites, and represents a new class in the vicinal oxygen chelate enzyme superfamily.
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