Kegg Pathway: Aminoglycosides - Streptomyces

J Microbiol Biotechnol. 2008 May; 18(5): 866-73
Kurumbang NP, Oh TJ, Liou K, Sohng JK

2-Deoxystreptamine is a core aglycon that is vital to backbone formation in various Aminoglycosides. This core structure can be modified to develop hybrid types of aminoglycoside antibiotics. We obtained three genes responsible for 2-deoxystreptamine production, neo7, neo6, and neo5, which encode 2-deoxy-scyllo-inosose synthase, L-glutamine: 2-deoxy-scyllo-inosose aminotransferase, and dehydrogenase, respectively, from the neomycin gene cluster. These genes were cloned into pIBR25, a Streptomyces expression vector, resulting in pNDOS. The recombinant pNDOS was transformed into a non-aminoglycoside-producing host, Streptomyces venezuelae YJ003, for heterologous expression. Based on comparisons of the retention time on LC-ESI/MS and ESIMS data with those of the 2-deoxystreptamine standard, a compound produced by S. venezuelae YJ003/pNDOS was found to be 2-deoxystreptamine.

Pol J Microbiol. 2008; 57(1): 81-4
Saadoun I, Gharaibeh R

In this study, we revealed rapid detection of streptomycin-producing Streptomyces spp. by extraction of total soil DNA from 14 soil samples using a modified lysis method followed by PCR amplification ofa genus-specific sequence in the Streptomyces' 16S rDNA gene. DNA band of the expected size (438 bp) was seen with all the samples. Additionally, specific amplification of the streptomycin-coding gene (strb1) directly from soil revealed the presence of a single DNA band of 940 bp. These results indicate that PCR-amplification of Streptomyces specific genes could be used for direct detection of streptomycin-producing Streptomyces species from soil.

Proc Natl Acad Sci U S A. 2008 Jun 17; 105(24): 8399-404
Park JW, Hong JS, Parajuli N, Jung WS, Park SR, Lim SK, Sohng JK, Yoon YJ

Since the first use of streptomycin as an effective antibiotic drug in the treatment of tuberculosis, aminoglycoside antibiotics have been widely used against a variety of bacterial infections for over six decades. However, the pathways for aminoglycoside biosynthesis still remain unclear, mainly because of difficulty in genetic manipulation of actinomycetes producing this class of antibiotics. Gentamicin belongs to the group of 4,6-disubstituted Aminoglycosides containing a characteristic core aminocyclitol moiety, 2-deoxystreptamine (2-DOS), and the recent discovery of its biosynthetic gene cluster in Micromonospora echinospora has enabled us to decipher its biosynthetic pathway. To determine the minimal set of genes and their functions for the generation of gentamicin A(2), the first pseudotrisaccharide intermediate in the biosynthetic pathway for the gentamicin complex, various sets of candidate genes from M. echinospora and other related aminoglycoside-producing strains were introduced into a nonaminoglycoside producing strain of Streptomyces venezuelae. Heterologous expression of different combinations of putative 2-DOS biosynthetic genes revealed that a subset, gtmB-gtmA-gacH, is responsible for the biosynthesis of this core aminocyclitol moiety of gentamicin. Expression of gtmG together with gtmB-gtmA-gacH led to production of 2'-N-acetylparomamine, demonstrating that GtmG acts as a glycosyltransferase that adds N-acetyl-d-glucosamine (GLcNA) to 2-DOS. Expression of gtmM in a 2'-N-acetylparomamine-producing recombinant S. venezuelae strain generated paromamine. Expression of gtmE in an engineered paromamine-producing strain of S. venezuelae successfully generated gentamicin A(2), indicating that GtmE is another glycosyltransferase that attaches d-xylose to paromamine. These results represent in vivo evidence elucidating the complete biosynthetic pathway of the pseudotrisaccharide aminoglycoside.

J Antibiot (Tokyo). 2008 Feb; 61(2): 63-9
Yu Y, Hou X, Ni X, Xia H

Streptomyces tenebrarius H6 mainly produces three kinds of antibiotics: apramycin, carbamoyltobramycin and some carbamoylkanamycin B. In our present study, a dehydrogenase gene tacB in the tobramycin biosynthetic gene cluster was disrupted by in-frame deletion. The result of TLC bio-autograph analysis demonstrated the disruption mutant strain produced apramycin and a new antibiotic. The new antibiotic was identified as 3'-deoxy-carbamoylkanamycin C by MS and NMR analysis after isolation and purification. The disruption mutant was restored to produce carbamoyltobramycin in a complementation experiment by the intact tacB gene. Our studies suggested that the tacB gene encodes a 6'-dehydrogenase, which reduces the 6'-hydroxyl group of paromamine to a keto group, thus facilitating the transfer of an aminogroup to form neamine. This study is the first report on the generation of a tobramycin derivative by gene engineering, and will contribute to clarify the complete biosynthetic pathway of tobramycin.

J Microbiol Biotechnol. 2008 Mar; 18(3): 397-403
Ling HB, Wang GJ, Li JE, Tan HR

Nikkomycins are a group of peptidyl nucleoside antibiotics with potent fungicidal, insecticidal, and acaricidal activities. sanN was cloned from the partial genomic library of Streptomyces ansochromogenes 7100. Gene disruption and complementation analysis demonstrated that sanN is essential for nikkomycin biosynthesis in S. ansochromogenes. Primer extension assay indicated that sanN is transcribed from two promoters (sanN-P1 and sanN-P2), and sanN-P2 plays a more important role in nikkomycin biosynthesis. Purified recombinant SanN acts as a dehydrogenase to convert benzoate-CoA to benzaldehyde in a random-order mechanism in vitro, with respective Kcat/Km values of 3.8 mM-1s-1 and 12.0 mM-1s-1 toward benzoate-CoA and NADH, suggesting that SanN catalyzes the formation of picolinaldehyde during biosynthesis of nikkomycin X and Z components in the wild-type stain. These data would facilitate us to understand the biosynthetic pathway of nikkomycins and to consider the combinatorial synthesis of novel antibiotic derivatives.

J Bacteriol. 2008 Jun; 190(11): 4050-60
Ohnishi Y, Ishikawa J, Hara H, Suzuki H, Ikenoya M, Ikeda H, Yamashita A, Hattori M, Horinouchi S

We determined the complete genome sequence of Streptomyces griseus IFO 13350, a soil bacterium producing an antituberculosis agent, streptomycin, which is the first aminoglycoside antibiotic, discovered more than 60 years ago. The linear chromosome consists of 8,545,929 base pairs (bp), with an average G+C content of 72.2%, predicting 7,138 open reading frames, six rRNA operons (16S-23S-5S), and 66 tRNA genes. It contains extremely long terminal inverted repeats (TIRs) of 132,910 bp each. The telomere's nucleotide sequence and secondary structure, consisting of several palindromes with a loop sequence of 5'-GGA-3', are different from those of typical telomeres conserved among other Streptomyces species. In accordance with the difference, the chromosome has pseudogenes for a conserved terminal protein (Tpg) and a telomere-associated protein (Tap), and a novel pair of Tpg and Tap proteins is instead encoded by the TIRs. Comparisons with the genomes of two related species, Streptomyces coelicolor A3(2) and Streptomyces avermitilis, clarified not only the characteristics of the S. griseus genome but also the existence of 24 Streptomyces-specific proteins. The S. griseus genome contains 34 gene clusters or genes for the biosynthesis of known or unknown secondary metabolites. Transcriptome analysis using a DNA microarray showed that at least four of these clusters, in addition to the streptomycin biosynthesis gene cluster, were activated directly or indirectly by AdpA, which is a central transcriptional activator for secondary metabolism and morphogenesis in the A-factor (a gamma-butyrolactone signaling molecule) regulatory cascade in S. griseus.

J Appl Microbiol. 2008 Jul; 105(1): 300-8
Thapa LP, Oh TJ, Liou K, Sohng JK

AIMS: To obtain spectinomycin and spectinamine by heterologous expression into the biosynthetic deoxysugar (desosamine) gene-deleted host Streptomyces venezuelae YJ003. METHODS AND RESULTS: The 17-kb spectinomycin biosynthetic gene cluster from Streptomyces spectabilis ATCC 27741 was heterologously expressed into Streptomyces venezuelae YJ003. Furthermore, the speA, speB and spcS2 encoded in the spectinomycin biosynthetic gene cluster of cosmid pSPC8 were also heterologously characterized to be responsible for the production of spectinamine. CONCLUSIONS: The results of this study indicated that pSPC8 contains all the genes necessary for the biosynthesis of spectinomycin. We also concluded that SpeA, SpeB and SpcS2 are sufficient for the biosynthesis of spectinamine. We also verified that SpeB and SpcS2 show dual character in the biosynthetic pathway of spectinomycin in Streptomyces spectabilis. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the report regarding the expression of a biosynthetic gene cluster that gives rise to the production of aminoglycoside antibiotics in Streptomyces venezuelae YJ003. Therefore, this work may serve as a foundation for further research on spectinomycin biosynthesis and other Aminoglycosides.

Microbiology. 2008 Mar; 154(Pt 3): 905-14
Hirano S, Tanaka K, Ohnishi Y, Horinouchi S

AtrA, a transcriptional activator for actII-ORF4, encoding the pathway-specific transcriptional activator of the actinorhodin biosynthetic gene cluster in Streptomyces coelicolor A3(2), has been shown to bind the region upstream from the promoter of strR, encoding the pathway-specific transcriptional activator of the streptomycin biosynthetic gene cluster in Streptomyces griseus [Uguru et al. (2005) Mol Microbiol 58, 131-150]. The atrA orthologue (atrA-g) in S. griseus was constitutively transcribed throughout growth from a promoter located about 250 nt upstream of the translational start codon, as determined by S1 nuclease mapping. DNase I footprinting showed that histidine-tagged AtrA-g bound an inverted repeat located upstream of strR at positions -117 to -142 relative to the transcriptional start point of strR as +1. This AtrA-g-binding site was between two AdpA-binding sites at approximately nucleotide positions -270 and -50. AdpA is a central transcriptional activator in the A-factor regulatory cascade and essential for the transcription of strR. AtrA-g and AdpA simultaneously bound the respective binding sites. In contrast to AdpA, AtrA-g was non-essential for strR transcription; an atrA-g-disrupted strain produced streptomycin on routine agar media to the same extent as the wild-type strain. However, the atrA-g-disrupted strain tended to produce a smaller amount of streptomycin than the wild-type strain under some conditions, for example, on Bennett agar containing 1 % maltose and on a minimal medium. Therefore, AtrA-g had a conditionally positive effect on streptomycin production, as a tuner, probably by enhancing the AdpA-dependent transcriptional activation of strR in a still unknown manner.

Chem Biol. 2008 Feb; 15(2): 157-66
Kallio P, Liu Z, Mäntsälä P, Niemi J, Metsä-Ketelä M

Tailoring steps in aromatic polyketide antibiotic biosynthesis are an important source of structural diversity and, consequently, an intriguing focal point for enzymological studies. PgaE and PgaM from Streptomyces sp. PGA64 are representatives of flavoenzymes catalyzing early post-PKS reactions in angucycline biosynthesis. This in vitro study illustrates that the chemoenzymatic conversion of UWM6 into the metabolite, gaudimycin C, requires multiple closely coupled reactions to prevent intermediate degradation. The NMR structure of gaudimycin C confirms that the reaction cascade involves C12- and C12b-hydroxylation, C2,3-dehydration, and stereospecific ketoreduction at C6. Enzymatic 18O incorporation studies verify that the oxygens at C12 and C12b derive from O2 and H2O, respectively. The results indicate that PgaM deviates mechanistically from flavoprotein monooxygenases, and suggest an alternative catalytic mechanism involving a quinone methide intermediate.

J Antibiot (Tokyo). 2007 Dec; 60(12): 739-44
Ji Z, Wang M, Zhang J, Wei S, Wu W

Four streptothricin-group antibiotics (1~4) were isolated from the fermentation broth of Streptomyces qinlingensis sp. nov. Along with the known antibiotics streptothricins F (1) and D (3), two new members of this class (2, 4) were identified as 12-carbamoyl derivatives of 1 and 3, respectively, mainly by analysis of the IR, HR-MS and NMR spectral data. The antibacterial activities of 1~4 against Escherichia coli (MICs 3.1, 25.0, 3.1 and 12.5 microg/ml), Bacillus subtilis (MICs 6.3, 25.0, 3.1 and 50 microg/ml), Staphylococcus aureus (MICs 12.5, >100.0, 6.3, >100.0 microg/ml), Bacillus cereus (MICs 25.0, 50.0, 25.0 and 50.0 microg/ml) and Pseudomonas aeruginosa (MICs 50.0, >100.0, 50.0, >100.0 microg/ml) were assayed by micro-broth dilution. The results based on MIC data indicated that 2 and 4 exhibited significantly less potent antibacterial activities when compared to that of 1 and 3.

Tsitol Genet. 2007 Sep-Oct; 41(5): 3-8
Klymyshin DO, Hromyko OM, Fedorenko VO

Successful transfer of the studied plasmids into S. nogalater IMET43360 cells using bacterial conjugation in the system E. coli--Streptomyces is an appropriate method for constructing this strain. Using DNA-DNA hybridization the character of integration of pVWB and pRT801 plasmids has been studied. The influence of these plasmids on nogalamycin biosynthesis has been investigated as well. The obtained results enable detailed study of nogalamycin gene functioning in S. nogalater IMET43360. The use of conjugation for substitution, destruction of genes, and heterological expression allows to get new "hybrid" antibiotics produced by this strain.

Chembiochem. 2008 Mar 3; 9(4): 624-33
Olano C, Abdelfattah MS, Gullón S, Braña AF, Rohr J, Méndez C, Salas JA

Expression of the steffimycin gene cluster in Steptomyces albus in combination with plasmids directing the biosynthesis of different neutral and branched-chain deoxyhexoses led to the identification of twelve new glycosylated derivatives of steffimycin with different degrees of decoration in the tetracyclic core. These experiments demonstrate the flexibility of L-rhamnosyltransferase StfG for recognition of a variety of D- and L-deoxyhexoses, harboring different degrees of deoxygenation as 2-deoxyhexoses, 2,6-deoxyhexoses, and 2,3,6-deoxyhexoses, and their attachment to 8-demethoxy-10-deoxysteffimycinone. In addition, the flexibility of 3'-O-methyltransferase OleY, from Streptomyces, for the methylation of deoxyhexoses attached to the steffimycin aglycone is shown by expression of oleY in Streptomyces steffisburgensis, leading to the isolation of 3'-O-methylsteffimycin. Analysis of the biological activities of these compounds against three human tumor cell lines-breast adenocarcinoma, non-small cell lung cancer, and colon adenocarcinoma-revealed two of them, 3'-O-methylsteffimycin and D-digitoxosyl-8-demethoxy-10-deoxysteffimycinone, to possess improved antitumor activities, showing GI50 values below 1.0 microM, while steffimycin's GI50 values fluctuate between 2.61 to 6.79 microM depending upon the cell line used. The antitumor activity data provide some insights into the structure-activity relationships of the new steffimycin derivatives, in relation to the configuration of hydroxy groups at positions C-3' and C-4' of the sugar moiety and positions C-8 and C-10 of the tetracyclic core.

J Am Chem Soc. 2008 Feb 13; 130(6): 1822-3
Futamura Y, Sawa R, Umezawa Y, Igarashi M, Nakamura H, Hasegawa K, Yamasaki M, Tashiro E, Takahashi Y, Akamatsu Y, Imoto M

J Nat Prod. 2008 Feb; 71(2): 199-207
Baig I, Perez M, Braña AF, Gomathinayagam R, Damodaran C, Salas JA, Méndez C, Rohr J

Plasmid pLNBIV was used to overexpress the biosynthetic pathway of nucleoside-diphosphate (NDP)-activated l-digitoxose in the mithramycin producer Streptomyces argillaceus. This led to a "flooding" of the biosynthetic pathway of the antitumor drug mithramycin (MTM) with NDP-activated deoxysugars, which do not normally occur in the pathway, and consequently to the production of the four new mithramycin derivatives 1- 4 with altered saccharide patterns. Their structures reflect that NDP sugars produced by pLNBIV, namely, l-digitoxose and its biosynthetic intermediates, influenced the glycosyl transfer to positions B, D, and E, while positions A and C remained unaffected. All four new structures have unique, previously not found sugar decoration patterns, which arise from either overcoming the substrate specificity or inhibition of certain glycosyltransferases (GTs) of the MTM pathway with the foreign NDP sugars expressed by pLNBIV. An apoptosis TUNEL (=terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling) assay revealed that compounds 1 (demycarosyl-3D-beta- d-digitoxosyl-MTM) and 3 (deoliosyl-3C-beta- d-mycarosyl-MTM) show improved activity (64.8 +/- 2% and 50.3 +/- 2.5% induction of apoptosis, respectively) against the estrogen receptor (ER)-positive human breast cancer cell line MCF-7 compared with the parent drug MTM (37.8 +/- 2.5% induction of apoptosis). In addition, compounds 1 and 4 (3A-deolivosyl-MTM) show significant effects on the ER-negative human breast cancer cell line MDA-231 (63.6 +/- 2% and 12.6 +/- 2.5% induction of apoptosis, respectively), which is not inhibited by the parent drug MTM itself (2.6 +/- 1.5% induction of apoptosis), but for which chemotherapeutic agents are urgently needed.

Proc Natl Acad Sci U S A. 2008 Jan 15; 105(2): 494-9
Van Lanen SG, Lin S, Shen B

C-1027 is an enediyne antitumor antibiotic composed of four distinct moieties: an enediyne core, a deoxy aminosugar, a beta-amino acid, and a benzoxazolinate moiety. We now show that the benzoxazolinate moiety is derived from chorismate by the sequential action of two enzymes-SgcD, a 2-amino-2-deoxyisochorismate (ADIC) synthase and SgcG, an iron-sulfur, FMN-dependent ADIC dehydrogenase-to generate 3-enolpyruvoylanthranilate (OPA), a new intermediate in chorismate metabolism. The functional elucidation and catalytic properties of each enzyme are described, including spectroscopic characterization of the products and the development of a fluorescence-based assay for kinetic analysis. SgcD joins isochorismate (IC) synthase and 4-amino-4-deoxychorismate (ADC) synthase as anthranilate synthase component I (ASI) homologues that are devoid of pyruvate lyase activity inherent in ASI; yet, in contrast to IC and ADC synthase, SgcD has retained the ability to aminate chorismate identically to that observed for ASI. The net conversion of chorismate to OPA by the tandem action of SgcD and SgcG unambiguously establishes a new branching point in chorismate metabolism.

J Am Chem Soc. 2008 Jan 30; 130(4): 1126-7
Kurosawa K, Ghiviriga I, Sambandan TG, Lessard PA, Barbara JE, Rha C, Sinskey AJ

J Microbiol Biotechnol. 2007 Nov; 17(11): 1856-61
Lee EM, Park HR, Hwang JH, Park DJ, Chang KS, Kim CJ

Physiological cell conditions such as glucose deprivation and hypoxia play roles in the development of drug resistance in solid tumors. These tumor-specific conditions cause decreased expression of DNA topoisomerase IIalpha, rendering cells resistant to topo II target drugs such as etoposide. Thus, targeting tumor-specific conditions such as a low glucose environment may be a novel strategy in the development of anticancer drugs. On this basis, we established a novel screening program for anticancer agents with preferential cytotoxic activity in cancer cells under glucose-deprived conditions. We recently isolated an active compound, AA-98, from Streptomyces sp. AA030098 that can prevent stress-induced etoposide resistance in vitro. Furthermore, LC-MS and various NMR spectroscopic methods identified AA-98 as mithramycin, which belongs to the aureolic acid group of antitumor compounds. We found that mithramycin prevents the etoposide resistance that is induced by glucose deprivation. The etoposide-chemosensitive action of mithramycin was just dependent on strict low glucose conditions, and resulted in the selective cell death of etoposide-resistant HT-29 human colon cancer cells.

Arch Microbiol. 2008 Apr; 189(4): 419-26
Xu D, Kwon HJ, Suh JW

In the present study, a mechanism for S-adenosylmethionine (SAM) to promote secondary metabolism was characterized in terms of bldH sl) expression in Streptomyces lividans. A previous study demonstrated that SAM, on application at 2 microM, induces the transcription of the strR promoter (strRp), which originates from Streptomyces griseus, in S. lividans. An inactivation study verified that bldH sl is essential to strRp transcription in S. lividans and it was demonstrated that the effects of SAM on the induction of strRp activity, on the transcription of redZ and actII-orf4, and on antibiotic production were compromised when the unique leucine TTA-codon of bldH sl was changed to TTG. Western blot analysis revealed that SAM supplementation enhances the expression of bldH sl when the TTA-codon was intact but not when the TTG replacement was provided. This study validates the involvement of BldH sl in the potentiating effect of SAM on the antibiotic production and substantiates that SAM controls the expression of bldH sl through the TTA-codon control in translating bldH mRNA. It is argued here that the intracellular SAM-level modulates the maturation of bldA, which encodes the UUA-codon tRNA and controls secondary metabolism in S. lividans.

Chembiochem. 2008 Jan 4; 9(1): 150-6
Sun Y, Hong H, Gillies F, Spencer JB, Leadlay PF

The biosynthetic pathway to the unusual tetronate ring of certain polyketide natural products, including the antibiotics abyssomicin and tetronomycin (TMN) and the antitumour compound chlorothricin (CHL), is presently unknown. The gene clusters governing chlorothricin and tetronomycin biosynthesis both contain a gene encoding an atypical member of the FkbH family of enzymes, which has previously been shown to synthesise glyceryl-S-acyl carrier protein (ACP) as the first step in production of unusual extender units for modular polyketide biosynthesis. We show here that purified recombinant FkbH-like protein, Tmn16, from the TMN gene cluster catalyses the efficient transfer of a glyceryl moiety from D-1,3-bisphosphoglycerate (1,3-BPG) to either of the dedicated ACPs, Tmn7a and ChlD2, to form glyceryl-S-ACP, which directly implicates this compound as an intermediate in tetronate biosynthesis as well. Neither Tmn16 nor Tmn7a produced glyceryl-S-ACP when incubated, respectively, with analogous ACP and FkbH-like proteins from a known extender-unit pathway; this indicates a highly selective channelling of glycolytic metabolites into tetronate biosynthesis.

Biochemistry. 2007 Dec 18; 46(50): 14672-81
Beltran-Alvarez P, Cox RJ, Crosby J, Simpson TJ

The actinorhodin (act) minimal polyketide synthase (PKS) from Streptomyces coelicolor consists of three proteins: an acyl carrier protein (ACP) and two beta-ketoacyl ACP synthase components known as KSalpha and KSbeta. The act minimal PKS catalyzes at least 18 separate reactions which can be divided into loading, initiation, extension, and cyclization and release phases. Two quantitative kinetic assays were developed and used to measure individual rate and Michaelis constants for loading, initiation and extension steps. In the minimal PKS, the reaction between malonyl CoA and ACP to form malonyl ACP (loading) is the rate-limiting step (kcat = 0.49 min-1, KM = 207 microM). This reaction increases 5-fold in rate in the presence of KSalphaKSbeta (kcat = 2.3 min-1, KM = 215 microM). In the presence of S. coelicolor malonyl CoA:ACP transacylase (MCAT), the rate of loading increases and the kinetic parameters of malonyl-ACP as a substrate of KSalphaKSbeta can be measured (kcat = 20.6 min-1, KM = 2.4 microM). Under these conditions, it appears that decarboxylation of malonyl-ACP to form acetyl-ACP (initiation) is the rate-limiting step. When an excess of acetyl ACP is supplied, either chain extension, cyclization, or release steps become rate limiting (k approximately 60 min-1). No ACP-bound intermediates could be observed, suggesting that partially or fully extended chains do not accumulate because chain extension is rate limiting under these conditions and that cyclization and release are fast. apo-ACP acts as a mixed inhibitor of malonyl ACP binding to KSalpha/KSbeta (Kic = 50 microM, Kiu = 137 microM), but apo-ACP does not appear to inhibit MCAT.