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ABSTRACT: The five-membered aromatic nitrogen heterocyclic pyrrole ring is a building block for a wide variety of natural products. Aiming at generating new pyrrole-containing derivatives as well as to identify new candidates that may be of value in designing new anticancer, antiviral, and/or antimicrobial agents, we employed a strategy on pyrrole-containing compound mutasynthesis using the pyrrole-containing calcimycin biosynthetic gene cluster. We blocked the biosynthesis of the calcimycin precursor, 3-hydroxy anthranilic acid, by deletion of calB1-3 and found that two intermediates containing the pyrrole and the spiroketal moiety were accumulated in the culture. We then fed the mutant using the structurally similar compound of 3-hydroxy anthranilic acid. At least four additional new pyrrole spiroketal derivatives were obtained. The structures of the intermediates and the new pyrrole spiroketal derivatives were identified using LC-MS and NMR. One of them shows enhanced antibacterial activity. Our work shows a new way of pyrrole derivative biosynthetic mutasynthesis.
Applied Microbiology and Biotechnology 05/2013; · 3.42 Impact Factor
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ABSTRACT: The identity and reactivity of the intermediates in agglomerin biosynthesis were established and the respective roles of the acetyltransferase Agg4 and the eliminating enzyme Agg5 identified. It is proposed that enzymes homologous to Agg4 and Agg5 carry out the dehydration steps in all spirotetronate biosynthetic pathways. If this proves correct, it may assist engineering of these pathways.
Angewandte Chemie International Edition 04/2013; · 13.45 Impact Factor
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ABSTRACT: Calcimycin is a rare divalent cation specific ionophore antibiotic that has many biochemical and pharmaceutical applications. We have recently cloned and sequenced the Streptomyces chartreusis calcimycin biosynthesis gene cluster as well as identified the genes required for the synthesis of the polyketide backbone of calcimycin. Additional modifying or decorating enzymes are required to convert the polyketide backbone into the biologically active calcimycin. Using targeted mutagenesis of Streptomyces we were able to show that calM from the calcimycin biosynthesis gene cluster is required for calcimycin production. Inactivating calM by PCR targeting, caused high level accumulation of N-demethyl calcimycin. CalM in the presence of S-adenosyl-L-methionine converted N-demethyl calcimycin to calcimycin in vitro. The enzyme was determined to have a kinetic parameter of Km 276 μM, kcat 1.26 min(-1) and kcat/ Km 76.2 M(-1) s(-1). These results proved that CalM is a N-methyltransferase that is required for calcimycin biosynthesis, and they set the stage for generating much desired novel calcimycin derivatives by rational genetic and chemical engineering.
Biochimie 04/2013; · 3.02 Impact Factor
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ABSTRACT: New assembly line, new compound: SIA7248, a new symmetric macrolide, was isolated from a marine-derived Streptomyces strain. Bioinformatic analyses of the identified biosynthetic gene cluster (sia) for SIA7248 suggested a polyketide biosynthesis utilizing an iteratively trans-acting ketoreductase (KR). We characterized SiaM as a trans-KR to catalyse reductions of various β-ketoacyl-thioesters with D-stereospecificity.
ChemBioChem 03/2013; · 3.94 Impact Factor
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ABSTRACT: Strain 268506(T) was isolated from a root of Avicennia marina collected at mangrove forest in Wengchang, Hainan province, China. The 16S rRNA gene sequence of strain 268506(T) showed the highest similarity with Micromonospora equina Y22(T) (98.8 %) and Micromonospora olivasterospora DSM 43868(T) (98.7 %). In addition, gyrB gene phylogeny clearly showed strain 268506(T) should be assigned to the genus Micromonospora but different from any established Micromonospora species. The predominant menaquinones are MK-9(H(8)) and MK-9(H(6)). The major fatty acids are iso-C(16:0), iso-C(15:0) and anteiso-C(17:0). The characteristic whole-cell sugars are xylose, mannose and arabinose. The cell wall contains meso-DAP and glycine. Phosphatidylinositol, diphosphatidylglycerol and phosphatidylethanolamine are the characteristic polar lipids. The DNA G+C content is 70.3 mol%. Some physiological and biochemical properties combined with low DNA-DNA relatedness indicated that the novel strain could be readily distinguished from the closest phylogenetic relatives. On the basis of these phenotypic and genotypic data, strain 268506(T) represents a novel species of the genus Micromonospora, for which the name Micromonospora avicenniae sp. nov. is proposed. The type strain is 268506(T) ( = CCTCC AA 2012010(T) = DSM 45758(T)).
Antonie van Leeuwenhoek 02/2013; · 2.09 Impact Factor
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ABSTRACT: DNA phosphorothioate modification (DNA sulfur modification, a non-bridging oxygen swapped with a sulfur) exists in diverse bacteria. Salmonella enterica serovar Cerro 87 is one of the bacteria that harbor the DNA sulfur modification. The modification is carried out by the products of a four-membered gene cluster, dptBCDE. Transformation of Escherichia coli DH10B with the dptBCDE gene cluster endows the strain with DNA sulfur modification capability. Deletion of dptC abolished the modification. Here, we studied the function of dptC in DNA sulfur modification.
Six cysteine residues in dptC were mutated individually within the dptBCDE gene cluster. Mutants were then tested for DNA sulfur modification.
Among the 6 cysteine mutations (C39, C146, C262, C273, C280, and C283), 5 abolished DNA modification except for C39, suggesting that C146, C262, C273, C280, and C283 are essential for DNA sulfur modification. Sequence alignment shows that these five cysteine residues are conserved among different strains.
Mutation at anyone of C146, C262, C273, C280 and C283 of dptC abolished DNA modification. Our results shed light on further study of DNA sulfur modification biochemical pathway.
ACTA MICROBIOLOGICA SINICA 02/2013; 53(2):204-9.
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ABSTRACT: Blasticidin S is a peptidyl-nucleoside antibiotic produced by Streptomyces griseochromogenes that exhibits strong fungicidal activity. To circumvent an effective DNA uptake barrier system in the native producer and investigate its biosynthesis in vivo, the blasticidin S biosynthetic gene cluster (bls) was engrafted to the chromosome of Streptomyces lividans. However, the resulting mutant LL2 produced the inactive deaminohydroxyblasticidin S instead of blasticidin S. Subsequently, a blasticidin S deaminase (SLBSD) was identified in S. lividans and shown to govern this in vivo conversion. Purified SLBSD was found to be capable of transforming blasticidin S to deaminohydroxyblasticidin S in vitro. It also catalyzed deamination of the cytosine moiety of cytosylglucuronic acid, an intermediate in blasticidin S biosynthesis. Disruption of SLBSD in S. lividans LL2 led to successful production of active blasticidin S in the resultant mutant, S. lividans WJ2. To demonstrate the easy manipulation of the blasticidin S biosynthetic gene cluster, blsE, F and L, encoding a predicted radical SAM protein, an unknown protein and a guanidino methyltransferase, were individually inactivated to access their role in blasticidin S biosynthesis.
Applied and environmental microbiology 02/2013; · 3.69 Impact Factor
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ABSTRACT: Alteration of sugar moieties of natural products often leads to novel antibiotics with different chemical and physical properties. fscMI is a putative glycosyltransferase (GT) in a gene cluster for the production of candicidin, a polyene macrolide antibiotic, produced by Streptomyces sp. FR-008. In this report, we established an in vivo biochemical detection system by inactivating fscMI and the DH11 domain of polyketide synthase (PKS) through double homologous recombination to unveil the interaction between polyene GTs and their substrates. We found that homologous GT genes including amphDI, nysDI and pimK can catalyze the conversion of candicidin aglycone into candicidin/FR-008-III in fscMI mutant, suggesting that homologous polyene GTs show some tolerance toward aglycones and that it is possible to create new polyene analogues with altered aglycones through genetic engineering. Inactivation of the DH11 domain of PKS led to novel polyene derivatives with mycosamine added to the altered polyketide backbones, further confirming the loose substrate specificity of polyene GTs. Furthermore, mutation of Ser346, Ser361, His362 or Cys387 of FscMI by site-directed mutagenesis significantly reduced its catalytic activity. Further analysis suggested that Ser361 and Cys387 are likely the critical donor interacting residues that could affect the activity of GT FscMI. To our knowledge, this is the first report of the critical residues in a polyene GT.
Molecular BioSystems 01/2013; · 3.53 Impact Factor
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ABSTRACT: The orthologous penR and pntR genes from the pentalenolactone biosynthetic gene clusters of Streptomyces exfoliatus UC5319 and S. arenae TÜ469, respectively, were predicted to encode MarR/SlyA family transcriptional regulators, responsible for regulation of the biosynthesis of the sesquiterpenoid antibiotic pentalenolactone. The intrinsic target DNA sequences and small molecule ligands of purified recombinant PenR and PntR were identified by Electrophoretic Mobility Shift Assays. PenR bound to DNA from both the penR-gapN and penM-penH intergenic regions while PntR bound only the corresponding pntR-gapR intergenic region. The targets of PenR and PntR were shown to be limited to conserved 37-bp DNA segments. Pentalenolactone and two late-stage biosynthetic intermediates, pentalenolactones D and F, act as ligands of both PenR and PntR, resulting in release of these proteins from their target DNA. The production of pentalenolactones was significantly decreased in the penR deletion mutant S. exfoliatus ΔpenR ZD27 but could be restored by complementation with either penR or pntR. Reverse transcription PCR (RT-PCR) established that transcription of pentalenolactone biosynthetic and resistance genes decreased while that of the penR gene itself increased in the penR deletion mutant S. exfoliatus ZD27 compared with the wild-type strain. The PenR protein thus serves as a positive regulator of pentalenolactone biosynthesis and self-resistance while acting as an autorepressor of penR.
Journal of bacteriology 01/2013; · 3.94 Impact Factor
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Fei Xu,
Dekun Kong,
Xinyi He,
Zhang Zhang,
Mo Han,
Xinqiang Xie,
Peng Wang,
Hairong Cheng,
Meifeng Tao,
Liping Zhang, Zixin Deng,
Shuangjun Lin
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ABSTRACT: Streptonigrin (STN, 1) is a highly functionalized aminoquinone alkaloid with broad and potent antitumor activity. Here, we reported the biosynthetic gene cluster of STN identified by genome scanning of a STN producer Streptomyces flocculus CGMCC4.1223. This cluster consists of 48 genes determined by a series of gene inactivations. Based on the structures of intermediates and shunt products accumulated from five specific gene inactivation mutants and feeding experiments, the biosynthetic pathway was proposed and the sequence of tailoring steps was preliminarily determined. In this pathway, a cryptic methylation of lavendamycin was genetically and biochemically characterized to be catalyzed by a leucine carboxyl methyltransferase StnF2. A [2Fe-2S](2+) cluster-containing aromatic ring dioxygenase StnB1/B2 system was biochemically characterized to catalyze a regiospecific cleavage of the N-C8' bond of indole ring of methyl ester of lavendamycin. This work provides opportunities to illuminate the enzymology of novel reactions involved in this pathway and to create, using genetic and chemo-enzymatic methods, new streptonigrinoid analogues as potential therapeutic agents.
Journal of the American Chemical Society 01/2013; · 9.91 Impact Factor
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ABSTRACT: Quinoxaline-2-carboxylic acid (QXC) and 3-hydroxyquinaldic acid (HQA) feature in quinomycin family and confer anticancer activity. In light of the significant potency against cancer, the biosynthetic gene clusters have been reported from many different Streptomyces strains, and the biosynthetic pathway were proposed mainly based on the in vivo feeding experiment with isotope labeled putative intermediates. Herein we report another gene cluster from Streptomyces griseovariabilis subsp. bandungensis subsp. nov responsible for the biosynthesis of echinomycin (a member of quinomycin family, also named quinomycin A) and presented in vitro evidence to corroborate the previous hypothesis on QXC biosynthesis, showing that only with the assistance of a MbtH-like protein Qui5, did the didomain NRPS protein (Qui18) perform the loading of a L-tryptophan onto its own PCP domain. Particularly, it was found that Qui5 and Qui18 subunits form a functional tetramer through size exclusion chromatography. The subsequent hydroxylation on β-carbon of the loaded L-tryptophan proved in vitro to be completed by cytochrome P450-dependent hydroxylase Qui15. Importantly, only the Qui18 loaded L-tryptophan can be hydroxylated by Qui15 and the enzyme was inactive on free L-tryptophan. Additionally, the chemically synthesized (2S,3S) β-hydroxytryptophan was detected to be converted by the tryptophan 2,3-dioxygenase Qui17 through LC-MS, which enriched our previous knowledge that tryptophan 2,3-dioxygenase nearly exclusively acted on L-tryptophan and 6-fluoro-tryptophan.
PLoS ONE 01/2013; 8(2):e56772. · 4.09 Impact Factor
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ABSTRACT: OBJECTIVE To investigate function of transporter genes fscTI and fscTII in the biosynthetic gene cluster of candicidin/FR-008.
We constructed a plasmid pJTU4137 for disruption of transporter genes fscTI and fscTII by conjugation and homologous recombinant. The transporter genes were also PCR amplified and cloned into the high-copy plasmid pJTU1278 for overexpression in strain ZYJ-6 derived from Streptomyces sp. FR-008.
The disruption mutant LX10 was unable to produce candicidin and its analogues. Overexpression of FscTI and FscTII in ZYJ-6 caused a 1.5-fold increase in FR-008-III production compared with the control.
We confirmed that fscTI and fscTII are function as ATP dependent ATP binding cassetle (ABC) transporters in the biosynthetic gene cluster of FR-008. Furthermore, a positive example was provided for improving antibiotic production in other polyene producing strains based on the results that overexpression of fscTI and fscTI increased candicidin production.
ACTA MICROBIOLOGICA SINICA 12/2012; 52(12):1458-66.
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Peiji Zhao,
Linquan Bai,
Juan Ma,
Ying Zeng,
Lei Li,
Yirong Zhang,
Chunhua Lu,
Huanqin Dai,
Zhaoxian Wu,
Yaoyao Li,
Xuan Wu,
Gang Chen,
Xiaojiang Hao,
Yuemao Shen, Zixin Deng,
Heinz G Floss
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ABSTRACT: SecReT4 (http://db-mml.sjtu.edu.cn/SecReT4/) is an integrated database providing comprehensive information of type IV secretion systems (T4SSs) in bacteria. T4SSs are versatile assemblages that promote genetic exchange and/or effector translocation with consequent impacts on pathogenesis and genome plasticity. T4SSs have been implicated in conjugation, DNA uptake and release and effector translocation. The effectors injected into eukaryotic target cells can lead to alteration of host cellular processes during infection. SecReT4 offers a unique, highly organized, readily exploreable archive of known and putative T4SSs and cognate effectors in bacteria. It currently contains details of 10 752 core components mapping to 808 T4SSs and 1884 T4SS effectors found in representatives of 289 bacterial species, as well as a collection of more than 900 directly related references. A broad range of similarity search, sequence alignment, phylogenetic, primer design and other functional analysis tools are readily accessible via SecReT4. We propose that SecReT4 will facilitate efficient investigation of large numbers of these systems, recognition of diverse patterns of sequence-, gene- and/or functional conservation and an improved understanding of the biological roles and significance of these versatile molecular machines. SecReT4 will be regularly updated to ensure its ongoing maximum utility to the research community.
Nucleic Acids Research 11/2012; · 8.03 Impact Factor
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ABSTRACT: A novel endophytic actinomycete strain 274745T was isolated from a root of Sonneratia apetala collected at a mangrove forest in Sanya, Hainan province, China. The 16S rRNA gene sequence of strain 274745T showed the highest similarity to Micromonospora pattaloongensis TJ2-2T (98.3%). The phylogenetic analysis based on the gyrB gene also supported the close relationship of these two strains. The predominant menaquinone was MK-10(H8) and the major fatty acids are iso-C15:0, C17:0 and anteiso-C15:0. The characteristic whole-cell sugars were xylose and mannose. The cell wall contained meso-DAP and glycine. The polar lipid profile mainly comprised phosphatidylethanolamine, phosphatidylinositol and diphosphatidylglycerol. The DNA G+C content is 71.6 mol%. Furthermore, a combination of DNA-DNA relatedness and some physiological and biochemical properties indicated that the novel strain could be readily distinguished from the closest phylogenetic relatives. On the basis of these phenotypic and genotypic data, strain 274745T represents a novel species of the genus Micromonospora, for which the name Micromonospora sonneratiae sp. nov. is proposed. The type strain is 274745T (=CCTCC AA2012003T=DSM 45704T).
INTERNATIONAL JOURNAL OF SYSTEMATIC AND EVOLUTIONARY MICROBIOLOGY 11/2012; · 2.11 Impact Factor
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ABSTRACT: A polyene macrolide antibiotic tetramycin biosynthetic gene cluster was identified by genome mining and isolated from Streptomyces hygrospinosus var. beijingensis. Genetic and in silico analyses gave insights into the mechanism of biosynthesis of tetramycin, and a model of the tetramycin biosynthetic pathway is proposed. Inactivation of a cytochrome P450 monooxygenase gene, tetrK, resulted in the production of a tetramycin B precursor: tetramycin A, which lacks a hydroxy group in its polyol region. TetrK was subsequently overexpressed heterologously in E. coli with a His(6) tag, and purified TetrK efficiently hydroxylated tetramycin A to afford tetramycin B. Kinetic studies revealed no inhibition of TetrK by substrate or product. Surprisingly, sequence-alignment analysis showed that TetrK, as a hydroxylase, has much higher homology with epoxidase PimD than with hydroxylases NysL and AmphL. The 3D structure of TetrK was then constructed by homology modeling with PimD as reference. Although TetrK and PimD catalyzed different chemical reactions, homology modeling indicated that they might share the same catalytic sites, despite also possessing some different sites correlated with substrate binding and substrate specificity. These findings offer good prospects for the production of improved antifungal polyene analogues.
ChemBioChem 09/2012; 13(15):2234-42. · 3.94 Impact Factor
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ABSTRACT: Phosphorothioation, with sulfur replacing a nonbridging oxygen of phosphate, has surfaced in bacterial DNA electrophoresis. To understand structural characteristics of the thio-substituted DNA, we have investigated the correlation between the relative energy of phosphate/phosphorothioate linkage and the backbone torsions. The relative energies (R.E.) computed by the quantum mechanical method, the PBE1PBE(CPCM, solvent=water)//PBE1PBE/6-31+G(2df) level of theory, were used to construct energy-scoring functions against backbone torsion variables, resulting in the squared correlation coefficients r(2) of 0.90-0.95. Then, the DNA energy alteration by phosphorothioation is estimated with the relative energy difference (ΔR.E.) between phosphate and phosphorothioate of the phosphate linkages in the DNA crystallographic database (NDB). As a result, Rp-phosphorothioation shifts the relative energy of B-helical structures by 2.7 ± 3.4 kcal/mol, destabilizing about 95% linkages, while Sp-phosphorothioation by -1.4 ± 2.4 kcal/mol, stabilizing over 84% linkages in the data sets. The B-helical destabilization is likely caused by the steric effect between the sulfur atom of Rp-phosphorothioate and the neighboring C-H groups of deoxyribose on the groove wall in B-helix. The unfavorable interaction may be magnified by the increasing rigidness of P-O-involving backbone torsions α and ζ upon the nonbridging phosphorothioations. Since B-helix is the most prevalent DNA double-helical structure and Rp-phosphorothioation is the exclusive configuration in bacteria thio-DNA found to date, the observed stereospecificity-destabilization correlation may reflect a structure-function relationship of biological DNA-phosphorothiation.
The Journal of Physical Chemistry B 08/2012; 116(35):10639-48. · 3.70 Impact Factor
