1. |
Yanai K,
Murakami T,
Bibb M,
( 2006 ) Amplification of the entire kanamycin biosynthetic gene cluster during empirical strain improvement of Streptomyces kanamyceticus. PMID : 16766657 : DOI : 10.1073/pnas.0603251103 PMC : PMC1475798 Abstract >>
Streptomyces kanamyceticus 12-6 is a derivative of the wild-type strain developed for industrial kanamycin (Km) production. Southern analysis and DNA sequencing revealed amplification of a large genomic segment including the entire Km biosynthetic gene cluster in the chromosome of strain 12-6. At 145 kb, the amplifiable unit of DNA (AUD) is the largest AUD reported in Streptomyces. Striking repetitive DNA sequences belonging to the clustered regularly interspaced short palindromic repeats family were found in the AUD and may play a role in its amplification. Strain 12-6 contains a mixture of different chromosomes with varying numbers of AUDs, sometimes exceeding 36 copies and producing an amplified region >5.7 Mb. The level of Km production depended on the copy number of the Km biosynthetic gene cluster, suggesting that DNA amplification occurred during strain improvement as a consequence of selection for increased Km resistance. Amplification of DNA segments including entire antibiotic biosynthetic gene clusters might be a common mechanism leading to increased antibiotic production in industrial strains.
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2. |
Kharel MK,
Subba B,
Basnet DB,
Woo JS,
Lee HC,
Liou K,
Sohng JK,
( 2004 ) A gene cluster for biosynthesis of kanamycin from Streptomyces kanamyceticus: comparison with gentamicin biosynthetic gene cluster. PMID : 15313224 : DOI : 10.1016/j.abb.2004.06.009 Abstract >>
Gene clusters for the biosynthesis of kanamycin (Km) and gentamicin (Gm) were isolated from the genomic libraries of Streptomyces kanamyceticus and Micromonospora echinospora, respectively. The sequencing of the 47 kb-region of S. kanamyceticus genomic DNA revealed 40 putative open reading frames (ORFs) encoding Km biosynthetic proteins, regulatory proteins, and resistance and transport proteins. Similarly, the sequencing of 32.6 kb genomic DNA of M. echinospora revealed a Gm biosynthetic gene cluster flanked by resistant genes. Biosynthetic pathways for the formation of Km were proposed by the comparative study of biosynthetic genes. Out of 12 putative Km biosynthetic genes, kanA was expressed in Escherichia coli and determined its function as a 2-deoxy-scyllo-inosose synthase. Furthermore, the acetylations of aminoglycoside-aminocyclitols (AmAcs) by Km acetyltransferase (KanM) were also demonstrated. The acetylated derivatives completely lost their antibacterial activities against Bacillus subtilis. The comparative genetic studies of Gm, Km, tobramycin (Tm), and butirosin (Bn) reveal their similar biosynthetic routes and provide a framework for the further biosynthetic studies.
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3. |
Yanai K,
Murakami T,
( 2004 ) The kanamycin biosynthetic gene cluster from Streptomyces kanamyceticus. PMID : 15303497 : DOI : 10.7164/antibiotics.57.351 Abstract >>
N/A
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4. |
Mo S,
Kim DH,
Lee JH,
Park JW,
Basnet DB,
Ban YH,
Yoo YJ,
Chen SW,
Park SR,
Choi EA,
Kim E,
Jin YY,
Lee SK,
Park JY,
Liu Y,
Lee MO,
Lee KS,
Kim SJ,
Kim D,
Park BC,
Lee SG,
Kwon HJ,
Suh JW,
Moore BS,
Lim SK,
Yoon YJ,
( 2011 ) Biosynthesis of the allylmalonyl-CoA extender unit for the FK506 polyketide synthase proceeds through a dedicated polyketide synthase and facilitates the mutasynthesis of analogues. PMID : 21175203 : DOI : 10.1021/ja108399b PMC : PMC3030623 Abstract >>
The allyl moiety of the immunosuppressive agent FK506 is structurally unique among polyketides and critical for its potent biological activity. Here, we detail the biosynthetic pathway to allylmalonyl-coenzyme A (CoA), from which the FK506 allyl group is derived, based on a comprehensive chemical, biochemical, and genetic interrogation of three FK506 gene clusters. A discrete polyketide synthase (PKS) with noncanonical domain architecture presumably in coordination with the fatty acid synthase pathway of the host catalyzes a multistep enzymatic reaction to allylmalonyl-CoA via trans-2-pentenyl-acyl carrier protein. Characterization of this discrete pathway facilitated the engineered biosynthesis of novel allyl group-modified FK506 analogues, 36-fluoro-FK520 and 36-methyl-FK506, the latter of which exhibits improved neurite outgrowth activity. This unique feature of FK506 biosynthesis, in which a dedicated PKS provides an atypical extender unit for the main modular PKS, illuminates a new strategy for the combinatorial biosynthesis of designer macrolide scaffolds as well as FK506 analogues.
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5. |
Jnawali HN,
Subba B,
Liou K,
Sohng JK,
( 2009 ) Functional characterization of kanB by complementing in engineered Streptomyces fradiae Deltaneo6::tsr. PMID : 19219581 : DOI : 10.1007/s10529-009-9937-0 Abstract >>
A putative aminotransferase gene, kanB, lies in the biosynthetic gene cluster of Streptomyces kanamyceticus ATCC 12853 and has 66% identity with neo6 in neomycin biosynthesis. Streptomyces fradiae Deltaneo6::tsr was generated by disrupting neo6 in the neomycin producer Streptomyces fradiae. Neomycin production was completely abolished in the disruptant mutant but was restored through self-complementation of neo6. S. fradiae HN4 was generated through complementation with kanB in Streptomyces fradiae neo6::tsr. Based on metabolite analysis by ESI/MS and LC/MS, neomycin production was restored in Streptomyces fradiae HN4. Thus, like neo6, kanB also functions as a 2-deoxy-scyllo-inosose aminotransferase that has dual functions in the formation of 2-deoxy-scyllo-inosose (DOS).
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6. |
Guo Y,
Zheng W,
Rong X,
Huang Y,
( 2008 ) A multilocus phylogeny of the Streptomyces griseus 16S rRNA gene clade: use of multilocus sequence analysis for streptomycete systematics. PMID : 18175701 : DOI : 10.1099/ijs.0.65224-0 Abstract >>
Streptomycetes are a complex group of actinomycetes that produce diverse bioactive metabolites of commercial significance. Systematics can provide a useful framework for identifying species that may produce novel metabolites. However, previously proposed approaches to the systematics of Streptomyces have suffered from either poor interlaboratory comparability or insufficient resolution. In particular, the Streptomyces griseus 16S rRNA gene clade is the most challenging and least defined group within the genus Streptomyces in terms of phylogeny. Here we report the results of a multilocus sequence analysis scheme developed to address the phylogeny of this clade. Sequence fragments of six housekeeping genes, atpD, gyrB, recA, rpoB, trpB and 16S rRNA, were obtained for 53 reference strains that represent 45 valid species and subspecies. Analysis of each individual locus confirmed the suitability of loci and the congruence of single-gene trees for concatenation. Concatenated trees of three, four, five and all six genes were constructed, and the stability of the topology and discriminatory power of each tree were analysed. It can be concluded from the results that phylogenetic analysis based on multilocus sequences is more accurate and robust for species delineation within Streptomyces. A multilocus phylogeny of six genes proved to be optimal for elucidating the interspecies relationships within the S. griseus 16S rRNA gene clade. Our multilocus sequence analysis scheme provides a valuable tool that can be applied to other Streptomyces clades for refining the systematic framework of this genus.
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7. |
Pet?í?ková K,
Chro?áková A,
Zelenka T,
Chrudimský T,
Pospíšil S,
Pet?í?ek M,
Krištůfek V,
( 2015 ) Evolution of cyclizing 5-aminolevulinate synthases in the biosynthesis of actinomycete secondary metabolites: outcomes for genetic screening techniques. PMID : 26300877 : DOI : 10.3389/fmicb.2015.00814 PMC : PMC4525017 Abstract >>
A combined approach, comprising PCR screening and genome mining, was used to unravel the diversity and phylogeny of genes encoding 5-aminolevulinic acid synthases (ALASs, hemA gene products) in streptomycetes-related strains. In actinomycetes, these genes were believed to be directly connected with the production of secondary metabolites carrying the C5N unit, 2-amino-3-hydroxycyclopent-2-enone, with biological activities making them attractive for future use in medicine and agriculture. Unlike "classical" primary metabolism ALAS, the C5N unit-forming cyclizing ALAS (cALAS) catalyses intramolecular cyclization of nascent 5-aminolevulinate. Specific amino acid sequence changes can be traced by comparison of "classical" ALASs against cALASs. PCR screening revealed 226 hemA gene-carrying strains from 1,500 tested, with 87% putatively encoding cALAS. Phylogenetic analysis of the hemA homologs revealed strain clustering according to putative type of metabolic product, which could be used to select producers of specific C5N compound classes. Supporting information was acquired through analysis of actinomycete genomic sequence data available in GenBank and further genetic or metabolic characterization of selected strains. Comparison of 16S rRNA taxonomic identification and BOX-PCR profiles provided evidence for numerous horizontal gene transfers of biosynthetic genes or gene clusters within actinomycete populations and even from non-actinomycete organisms. Our results underline the importance of environmental and evolutionary data in the design of efficient techniques for identification of novel producers.
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8. |
Rodríguez-Mata M,
Frank A,
Wells E,
Leipold F,
Turner NJ,
Hart S,
Turkenburg JP,
Grogan G,
( 2013 ) Structure and activity of NADPH-dependent reductase Q1EQE0 from Streptomyces kanamyceticus, which catalyses the R-selective reduction of an imine substrate. PMID : 23813853 : DOI : 10.1002/cbic.201300321 Abstract >>
NADPH-dependent oxidoreductase Q1EQE0 from Streptomyces kanamyceticus catalyzes the asymmetric reduction of the prochiral monocyclic imine 2-methyl-1-pyrroline to the chiral amine (R)-2-methylpyrrolidine with >99% ee, and is thus of interest as a potential biocatalyst for the production of optically active amines. The structures of Q1EQE0 in native form, and in complex with the nicotinamide cofactor NADPH have been solved and refined to a resolution of 2.7 ?. Q1EQE0 functions as a dimer in which the monomer consists of an N-terminal Rossman-fold motif attached to a helical C-terminal domain through a helix of 28 amino acids. The dimer is formed through reciprocal domain sharing in which the C-terminal domains are swapped, with a substrate-binding cleft formed between the N-terminal subunit of monomer A and the C-terminal subunit of monomer B. The structure is related to those of known �]-hydroxyacid dehydrogenases, except that the essential lysine, which serves as an acid/base in the (de)protonation of the nascent alcohol in those enzymes, is replaced by an aspartate residue, Asp187 in Q1EQE0. Mutation of Asp187 to either asparagine or alanine resulted in an inactive enzyme.
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9. |
Sucipto H,
Kudo F,
Eguchi T,
( 2012 ) The last step of kanamycin biosynthesis: unique deamination reaction catalyzed by the �\-ketoglutarate-dependent nonheme iron dioxygenase KanJ and the NADPH-dependent reductase KanK. PMID : 22374809 : DOI : 10.1002/anie.201108122 Abstract >>
Mystery solved: using heterologous expression, the activities of two enzymes exclusively belonging to the kanamycin biosynthetic pathway have been identified in vitro. A distinctive reaction mechanism to produce kanamycin is proposed and the previously unknown catalytic deamination activity of KanJ dioxygenase is uncovered.
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10. |
Park JW,
Park SR,
Nepal KK,
Han AR,
Ban YH,
Yoo YJ,
Kim EJ,
Kim EM,
Kim D,
Sohng JK,
Yoon YJ,
( 2011 ) Discovery of parallel pathways of kanamycin biosynthesis allows antibiotic manipulation. PMID : 21983602 : DOI : 10.1038/nchembio.671 Abstract >>
Kanamycin is one of the most widely used antibiotics, yet its biosynthetic pathway remains unclear. Current proposals suggest that the kanamycin biosynthetic products are linearly related via single enzymatic transformations. To explore this system, we have reconstructed the entire biosynthetic pathway through the heterologous expression of combinations of putative biosynthetic genes from Streptomyces kanamyceticus in the non-aminoglycoside-producing Streptomyces venezuelae. Unexpectedly, we discovered that the biosynthetic pathway contains an early branch point, governed by the substrate promiscuity of a glycosyltransferase, that leads to the formation of two parallel pathways in which early intermediates are further modified. Glycosyltransferase exchange can alter flux through these two parallel pathways, and the addition of other biosynthetic enzymes can be used to synthesize known and new highly active antibiotics. These results complete our understanding of kanamycin biosynthesis and demonstrate the potential of pathway engineering for direct in vivo production of clinically useful antibiotics and more robust aminoglycosides.
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11. |
Murakami T,
Nojiri C,
Toyama H,
Hayashi E,
Katumata K,
Anzai H,
Matsuhashi Y,
Yamada Y,
Nagaoka K,
( 1983 ) Cloning of antibiotic-resistance genes in Streptomyces. PMID : 6315667 : DOI : 10.7164/antibiotics.36.1305 Abstract >>
Antibiotic-resistance genes were shotgun cloned from antibiotic-producing Streptomyces sp. using pock-forming plasmids (pSF689 and pSF765), as cloning vectors. Streptomyces chartreusis SF1623 and S. lividans 66 were used as host strains. The ribostamycin (RSM) resistance gene was cloned from S. ribosidificus SF733 DNA (on a 2.3 Md PstI fragment) into both S. chartreusis SF1623 and S. lividans 66, using pSF689 as vector. Kanamycin (KM), novobiocin (NB), destomycin (DM) and racemomycin (RM) resistance genes were cloned from S. kanamyceticus M1164, S. spheroides M1469, S. rimofaciens M1470 and S. lavendulae A249 genomic DNA into S. lividans 66, using pSF765 as vector. Furthermore two types of KM resistance determinants derived from S. kanamyceticus M1164 were cloned using S. lividans 66, the pSF689 vector. The RSM resistance gene showed no homology to plasmid pSF733 of S. ribosidificus SF733, but hybridized to PstI or BclI digested total DNA of S. ribosidificus SF733.
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12. |
Matsuhashi Y,
Murakami T,
Nojiri C,
Toyama H,
Anzai H,
Nagaoka K,
( 1985 ) Mechanisms of aminoglycoside-resistance of Streptomyces harboring resistant genes obtained from antibiotic-producers. PMID : 3838980 : DOI : 10.7164/antibiotics.38.279 Abstract >>
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