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1. Ikeno  S, Aoki  D, Sato  K, Hamada  M, Hori  M, Tsuchiya  KS,     ( 2002 )

kasT gene of Streptomyces kasugaensis M338-M1 encodes a DNA-binding protein which binds to intergenic region of kasU-kasJ in the kasugamycin biosynthesis gene cluster.

The Journal of antibiotics 55 (12)
PMID : 12617515  :   DOI  :   10.7164/antibiotics.55.1053    
Abstract >>
We previously reported that a 4.2 kb SacI-EcoRI DNA region from Streptomyces kasugaensis M338-M1, a kasugamycin (KSM) producer, included KSM transporter genes (kasKLM). As an extension of that study, a 3.7 kb Psti-SacI DNA region, located at 1.5 approximately 5.2 kb upstream of kasK, was cloned and sequenced, revealing three complete open reading frames, designated kasT, kasU and kasJ. The kasJ gene encodes a protein (KasJ) with a conserved dinucleotide (FAD)-binding motif Homology search for KasJ showed its similarity to NADH: N-amidino-scyllo-inosamine oxidoreductase (StsB) which is involved in biosynthesis of the streptidine moiety of streptomycin (SM) in S. griseus. The kasT gene encodes a DNA-binding protein (KasT), including a helix-turn-helix motif near the center of the sequence. This protein is similar in structure to a pathway-specific activator protein (StrR) that plays a role in regulating the SM biosynthesis gene cluster of S. griseus. A fusion protein (Trx-KasT) clearly showed DNA binding activity with the intergenic region of kasU-kasJ, suggesting that KasT is a pathway-specific regulator of the KSM biosynthesis gene cluster.
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2. Kojima  I, Kasuga  K, Kobayashi  M, Fukasawa  A, Mizuno  S, Arisawa  A, Akagawa  H,     ( 2002 )

The rpoZ gene, encoding the RNA polymerase omega subunit, is required for antibiotic production and morphological differentiation in Streptomyces kasugaensis.

Journal of bacteriology 184 (23)
PMID : 12426327  :   DOI  :   10.1128/jb.184.23.6417-6423.2002     PMC  :   PMC135429    
Abstract >>
The occurrence of pleiotropic mutants that are defective in both antibiotic production and aerial mycelium formation is peculiar to streptomycetes. Pleiotropic mutant KSB was isolated from wild-type Streptomyces kasugaensis A1R6, which produces kasugamycin, an antifungal aminoglycoside antibiotic. A 9.3-kb DNA fragment was cloned from the chromosomal DNA of strain A1R6 by complementary restoration of kasugamycin production and aerial hypha formation to mutant KSB. Complementation experiments with deletion plasmids and subsequent DNA analysis indicated that orf5, encoding 90 amino acids, was responsible for the restoration. A protein homology search revealed that orf5 was a homolog of rpoZ, the gene that is known to encode RNA polymerase subunit omega (omega), thus leading to the conclusion that orf5 was rpoZ in S. kasugaensis. The pleiotropy of mutant KSB was attributed to a 2-bp frameshift deletion in the rpoZ region of mutant KSB, which probably resulted in a truncated, incomplete omega of 47 amino acids. Furthermore, rpoZ-disrupted mutant R6D4 obtained from strain A1R6 by insertion of Tn5 aphII into the middle of the rpoZ-coding region produced neither kasugamycin nor aerial mycelia, similar to mutant KSB. When rpoZ of S. kasugaensis and Streptomyces coelicolor, whose deduced products differed in the sixth amino acid residue, were introduced into mutant R6D4 via a plasmid, both transformants produced kasugamycin and aerial hyphae without significant differences. This study established that rpoZ is required for kasugamycin production and aerial mycelium formation in S. kasugaensis and responsible for pleiotropy.
KeywordMeSH Terms
DNA-Binding Proteins
3. Hamada  M, Kinoshita  N, Ohishi  Y, Yamane  Y, Ikeno  S,     ( 2000 )

ABC transporter genes, kasKLM, responsible for self-resistance of a kasugamycin producer strain.

The Journal of antibiotics 53 (4)
PMID : 10866219  :   DOI  :   10.7164/antibiotics.53.373    
Abstract >>
We previously reported that a 7.6-kb DNA fragment from Streptomyces kasugaensis M338-M1, a kasugamycin (KSM) producer, included KSM acetyltransferase gene (kac338) and some other genes possibly involved in KSM biosynthesis. As an extension of that study, a 10-kb SacI-KpnI DNA fragment, located approximately 5-15-kb upstream of kac33, was cloned and a 4.2-kb SacI-EcoRI fragment therefrom was sequenced, revealing one incomplete (designated ORF J) and three complete open reading frames (designated kasK, kasL and kasM). The coding frames of kasK, L and M overlap one another with terminator/initiator ATGA sequence. RT-PCR analysis of a DNA region including kasKLM indicated the presence of one transcript that is long enough to span the three genes. The kasK gene potentially encodes an ATP-binding protein of the ATP-binding cassette (ABC) transporter superfamily. Homology search for the deduced KasK protein shows similarity to other ABC transporters involved in self-resistance of a mithramycin and possibly doxorubicin producer strain. The kasL and kasM genes encode different integral membrane proteins, both having six putative transmembrane helices. An expression plasmid for kasKLM (pTV-KLM) was constructed and these genes were expressed in E. coli JM 109, which had been sensitive to KSM. The transformant acquired resistance to KSM, suggesting that KasK, L and M proteins as a set in S. kasugaensis M338-M1 pump out KSM to protect the producer from its toxic metabolite.
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4. Ikeno  S, Aoki  D, Hamada  M, Hori  M, Tsuchiya  KS,     ( 2006 )

DNA sequencing and transcriptional analysis of the kasugamycin biosynthetic gene cluster from Streptomyces kasugaensis M338-M1.

The Journal of antibiotics 59 (1)
PMID : 16568715  :   DOI  :   10.1038/ja.2006.4    
Abstract >>
Streptomyces kasugaensis M338-M1 produces the aminoglycoside antibiotic kasugamycin (KSM). We previously cloned, sequenced and characterized the KSM acetyltransferase, transporter, and some of the biosynthetic genes from this strain. To identify other potential genes in a chromosome walk experiment, a 6.8-kb EcoRI-PstI region immediately downstream from the KSM transporter genes was sequenced. Five open reading frames (designated as kasN, kasO, kasP, kasQ, kasR) and the 5' region of kasA were found in this region. The genes are apparently co-transcribed as bicistrons, all of which are co-directional except for the kasPQ transcript. Homology analysis of the deduced products of kasN, kasP, kasQ and kasR revealed similarities with known enzymes: KasN, D-amino acid oxidase from Pseudomonas aeruginosa (35% identity); KasP, F420-dependent H4MPT reductase from Streptomyces lavendulae (33% identity); KasQ, UDP-N-acetylglucosamine 2-epimerase from Streptomyces verticillus (45% identity); and KasR, NDP-hexose 3,4-dehydratase from Streptomyces cyanogenus (38% identity); respectively. A gel retardation assay showed that KasT, a putative pathway-specific regulator for this gene cluster, bound to the upstream region of kasN and to the intergenic region of kasQ-kasR, suggesting that the expression of these operons is under the control of the regulator protein.
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5. Molnár  I, Hill  DS, Zirkle  R, Hammer  PE, Gross  F, Buckel  TG, Jungmann  V, Pachlatko  JP, Ligon  JM,     ( 2005 )

Biocatalytic conversion of avermectin to 4"-oxo-avermectin: heterologous expression of the ema1 cytochrome P450 monooxygenase.

Applied and environmental microbiology 71 (11)
PMID : 16269733  :   DOI  :   10.1128/AEM.71.11.6977-6985.2005     PMC  :   PMC1287623    
Abstract >>
The cytochrome P450 monooxygenase Ema1 from Streptomyces tubercidicus R-922 and its homologs from closely related Streptomyces strains are able to catalyze the regioselective oxidation of avermectin into 4"-oxo-avermectin, a key intermediate in the manufacture of the agriculturally important insecticide emamectin benzoate (V. Jungmann, I. Moln?r, P. E. Hammer, D. S. Hill, R. Zirkle, T. G. Buckel, D. Buckel, J. M. Ligon, and J. P. Pachlatko, Appl. Environ. Microbiol. 71:6968-6976, 2005). The gene for Ema1 has been expressed in Streptomyces lividans, Streptomyces avermitilis, and solvent-tolerant Pseudomonas putida strains using different promoters and vectors to provide biocatalytically competent cells. Replacing the extremely rare TTA codon with the more frequent CTG codon to encode Leu4 in Ema1 increased the biocatalytic activities of S. lividans strains producing this enzyme. Ferredoxins and ferredoxin reductases were also cloned from Streptomyces coelicolor and biocatalytic Streptomyces strains and tested in ema1 coexpression systems to optimize the electron transport towards Ema1.
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6. Jungmann  V, Molnár  I, Hammer  PE, Hill  DS, Zirkle  R, Buckel  TG, Buckel  D, Ligon  JM, Pachlatko  JP,     ( 2005 )

Biocatalytic conversion of avermectin to 4"-oxo-avermectin: characterization of biocatalytically active bacterial strains and of cytochrome p450 monooxygenase enzymes and their genes.

Applied and environmental microbiology 71 (11)
PMID : 16269732  :   DOI  :   10.1128/AEM.71.11.6968-6976.2005     PMC  :   PMC1287622    
Abstract >>
4"-Oxo-avermectin is a key intermediate in the manufacture of the agriculturally important insecticide emamectin benzoate from the natural product avermectin. Seventeen biocatalytically active Streptomyces strains with the ability to oxidize avermectin to 4"-oxo-avermectin in a regioselective manner have been discovered in a screen of 3,334 microorganisms. The enzymes responsible for this oxidation reaction in these biocatalytically active strains were found to be cytochrome P450 monooxygenases (CYPs) and were termed Ema1 to Ema17. The genes for Ema1 to Ema17 have been cloned, sequenced, and compared to reveal a new subfamily of CYPs. Ema1 to Ema16 have been overexpressed in Escherichia coli and purified as His-tagged recombinant proteins, and their basic enzyme kinetic parameters have been determined.
KeywordMeSH Terms
7. Kim  H, Kim  SH, Ying  YH, Kim  HJ, Koh  YH, Kim  CJ, Lee  SH, Cha  CY, Kook  YH, Kim  BJ,     ( 2005 )

Mechanism of natural rifampin resistance of Streptomyces spp.

Systematic and applied microbiology 28 (5)
PMID : 16094866  :   DOI  :   10.1016/j.syapm.2005.02.009    
Abstract >>
In a previous phylogenetic study of the genus Streptomyces using the rpoB gene, N531, which stands for an aspargine residue in position 531 of RpoB instead of serine (S531), known to be associated with natural rifampin resistance in several organisms, was also observed in the RpoB of several Streptomyces species. To determine whether N531 is associated with the rifampin resistance of Streptomyces strains, we analyzed the rifampin minimum inhibitory concentrations (MICs) of 11 strains of the N531 RpoB type (putative rifampin resistant strains) and of 12 strains of the S531 RpoB type. (putative rifampin susceptible strains). In general, the N531 RpoB types showed higher MIC levels (16-128 microg/ml) than the S531 RpoB types (0-8 microg/ml). To determine the isolation frequencies of N531 RpoB types versus rifampin concentration, we applied screening methods involving different rifampin concentrations (0, 20 and 100 microg/ml) to Korean soils. Higher isolation frequencies of the N531 RpoB types were observed at the higher rifampin concentrations. In addition, during the course of this study we developed an allele specific PCR method to detect rifampin resistant Streptomyces strains. Our results strongly suggested that N531 might be involved in a major mechanism of natural rifampin resistance in strains of the genus Streptomyces.
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8. Kasuga  K, Sasaki  A, Matsuo  T, Yamamoto  C, Minato  Y, Kuwahara  N, Fujii  C, Kobayashi  M, Agematu  H, Tamura  T, Komatsu  M, Ishikawa  J, Ikeda  H, Kojima  I,     ( 2017 )

Heterologous production of kasugamycin, an aminoglycoside antibiotic from Streptomyces kasugaensis, in Streptomyces lividans and Rhodococcus erythropolis L-88 by constitutive expression of the biosynthetic gene cluster.

Applied microbiology and biotechnology 101 (10)
PMID : 28243709  :   DOI  :   10.1007/s00253-017-8189-5    
Abstract >>
Kasugamycin (KSM), an aminoglycoside antibiotic isolated from Streptomyces kasugaensis cultures, has been used against rice blast disease for more than 50 years. We cloned the KSM biosynthetic gene (KBG) cluster from S. kasugaensis MB273-C4 and constructed three KBG cassettes (i.e., cassettes I-III) to enable heterologous production of KSM in many actinomycetes by constitutive expression of KBGs. Cassette I comprised all putative transcriptional units in the cluster, but it was placed under the control of the P neo promoter from Tn5. It was not maintained stably in Streptomyces lividans and did not transform Rhodococcus erythropolis. Cassette II retained the original arrangement of KBGs, except that the promoter of kasT, the specific activator gene for KBG, was replaced with P rpsJ , the constitutive promoter of rpsJ from Streptomyces avermitilis. To enhance the intracellular concentration of myo-inositol, an expression cassette of ino1 encoding the inositol-1-phosphate synthase from S. avermitilis was inserted into cassette II to generate cassette III. These two cassettes showed stable maintenance in S. lividans and R. erythropolis to produce KSM. Particularly, the transformants of S. lividans induced KSM production up to the same levels as those produced by S. kasugaensis. Furthermore, cassette III induced more KSM accumulation than cassette II in R. erythropolis, suggesting an exogenous supply of myo-inositol by the ino1 expression in the host. Cassettes II and III appear to be useful for heterologous KSM production in actinomycetes. Rhodococcus exhibiting a spherical form in liquid cultivation is also a promising heterologous host for antibiotic fermentation.
KeywordMeSH Terms
Heterologous production
Rhodococcus erythropolis
Streptomyces lividans
Multigene Family
9. Zhu  C, Kang  Q, Bai  L, Cheng  L, Deng  Z,     ( 2016 )

Identification and engineering of regulation-related genes toward improved kasugamycin production.

Applied microbiology and biotechnology 100 (4)
PMID : 26521251  :   DOI  :   10.1007/s00253-015-7082-3    
Abstract >>
Kasugamycin, produced by Streptomyces kasugaensis and Streptomyces microaureus, is an important amino-glycoside family antibiotic and widely used for veterinary and agricultural applications. In the left flanking region of the previously reported kasugamycin gene cluster, four additional genes (two-component system kasW and kasX, MerR-family kasV, and isoprenylcysteine carboxyl methyltransferase kasS) were identified both in the low-yielding S. kasugaensis BCRC12349 and high-yielding S. microaureus XM301. Deletion of regulatory gene kasT abolished kasugamycin production, and its overexpression in BCRC12349 resulted in an increased titer by 186 %. Deletion of kasW, kasX, kasV, and kasS improved kasugamycin production by 12, 19, 194, and 22 %, respectively. qRT-PCR analysis demonstrated that the transcription of kas genes was significantly increased in all the four mutants. Similar gene inactivation was performed in the high-yielding strain S. microaureus XM301. As expected, the deletion of kasW/X resulted in a 58 % increase of the yield from 6 to 9.5 g/L. However, the deletion of kasV and over-expression of kasT had no obvious effect, and the disruption of kasS surprisingly decreased kasugamycin production. In addition, trans-complementation of the kasS mutant with a TTA codon-mutated kasS increased the kasugamycin yield by 20 %. A much higher transcription of kas genes was detected in the high-yielding XM301 than in the low-yielding BCRC12349, which may partially account for the discrepancy of gene inactivation effects between them. Our work not only generated engineered strains with improved kasugamycin yield, but also pointed out that different strategies on manipulating regulatory-related genes should be considered for low-yielding or high-yielding strains.
KeywordMeSH Terms
Genetic engineering
Gene Expression Regulation, Bacterial
Metabolic Engineering
10.     ( 1996 )

Mechanism of multiple aminoglycoside resistance of kasugamycin-producing Streptomyces kasugaensis MB273: involvement of two types of acetyltransferases in resistance to astromicin group antibiotics.

The Journal of antibiotics 49 (7)
PMID : 8784431  :   DOI  :   10.7164/antibiotics.49.682    
Abstract >>
The biochemical basis for the multiple resistance to aminoglycoside antibiotics (AGs) of kasugamycin-producing Streptomyces kasugaensis MB273 was studied. The strain was resistant to a wide range of deoxystreptamine (DOS)-containing AGs as well as astromicin (ASTM) group antibiotics. These AGs strongly inhibited in vitro polyU-directed polyphenylalanine-synthesis using ribosomes from the strain, while they were acetylated and inactivated by the MB273 cell free extract supplemented with acetyl-CoA. It seemed thus likely that the acetyltransferase activity played a critical role for the multiple AG resistance. The acetylation was selective to AGs with 2'-NH2, suggesting the involvement of aminoglycoside 2'-N-acetyltransferase, AAC (2'). Interestingly, the acetylation of istamycin B (ISM-B; an ASTM group AG) resulted in the formation of two different products (1-N-acetyl ISM-B and 2"-N-acetyl ISM-B) at a similar ratio. In this context, an AAC (2') gene cloned as an ISM-B resistance gene from the strain MB273 directed the conversion of ISM-B to only 1-N-acetyl ISM-B. It seemed likely that two types of AACs [AAC(2') and a novel one] were involved in the mechanism of resistance to ASTM group AGs.
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11.     ( 1996 )

Enzymatic 2'-N-acetylation of arbekacin and antibiotic activity of its product.

The Journal of antibiotics 49 (5)
PMID : 8682723  :   DOI  :   10.7164/antibiotics.49.458    
Abstract >>
Aminoglycoside antibiotics (AGs) with a free 2'-amino group were subjected to enzymatic N-acetylation using a cell free extract that contained an aminoglycoside 2'-N-acetyltransferase, AAC (2'), derived from a kasugamycin-producing strain of Streptomyces kasugaensis. TLC and antibiotic assay of the incubated reaction mixtures revealed that a modified compound retaining substantial antibiotic activity was formed from arbekacin (ABK), while modification of the other AGs resulted in the marked decrease in antibiotic activity. Structure determination following isolation from a large scale reaction mixture showed the modified ABK to be 2'-N-acetyl ABK. In addition, 2',6'-di-N-acetyl ABK was formed as a minor product. The same conversion also occurred with dibekacin (DKB) resulting in the formation of 2'-N-acetyl DKB and 2',6'-di-N-acetyl DKB. MIC determination showed antibacterial activity (1.56 approximately 3.13 micrograms/ml) of 2'-N-acetyl ABK against a variety of organisms. By contrast, 2'-N-acetyl DKB showed no substantial antibiotic activity. We believe 2'-N-acetyl ABK has the highest and broadest antibacterial activity, compared with known N-acetylated AGs.
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12. Hirasawa  K, Ichihara  M, Okanishi  M,     ( 1985 )

Nucleotide sequence of Mec+ gene region of Streptomyces kasugaensis.

The Journal of antibiotics 38 (12)
PMID : 4093339  :   DOI  :   10.7164/antibiotics.38.1795    
Abstract >>
KeywordMeSH Terms
Genes, Bacterial
13.     ( 1998 )

A 7.6kb DNA region from Streptomyces kasugaensis M338-M1 includes some genes responsible for kasugamycin biosynthesis.

The Journal of antibiotics 51 (3)
PMID : 9589071  :   DOI  :   10.7164/antibiotics.51.341    
Abstract >>
A 7.6kb PstI-KpnI DNA fragment including a sequence highly similar to kasugamycin acetyltransferase gene (kac) was isolated from Streptomyces kasugaensis M338-M1 and sequenced. Nine open reading frames (ORFs), designated as ORF A, B, C, D, E, F, G, H and I, were recognized in this region, although ORF A was incomplete. ORF G runs in the opposite direction to the others. The amino acid sequence deduced from ORF H showed 98% similarity to that of the kasugamycin acetyltransferase from S. kasugaensis MB273-C4, another kasugamycin (KSM) producer. Transformation of E. coli JM109 with ORF H made the strain highly resistant to KSM. The deduced amino acid sequences of the ORF A, C and D products were similar, respectively, to glucosyltransferase I from E. coli (26%), beta-alanine: pyruvate transaminase from Pseudomonas putida (32%) and dTDP-D-glucose 4,6-dehydratase (StrE) from Streptomyces griseus (37%). The strE-like ORF (ORF D) seems to be the gene responsible for formation of the 6-deoxy structure of the kasugamine moiety. ORF A and ORF C are also likely to have roles in KSM biosynthesis. Taken together, our analyses strongly suggest that this DNA region includes at least a part of the gene cluster of KSM biosynthesis.
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