Deoxysugar Methylation during Biosynthesis of the Antitumor Polyketide Elloramycin by Streptomyces olivaceus

CHARACTERIZATION OF THREE METHYLTRANSFERASE GENES*

Eugenio P. PatalloDagger , Gloria BlancoDagger , Carsten Fischer§, Alfredo F. BrañaDagger , Jürgen Rohr§, Carmen MéndezDagger , and José A. SalasDagger ||

From the Dagger  Departamento de Biología Funcional e Instituto Universitario de Oncología del Principado de Asturias, Universidad de Oviedo, 33006 Oviedo, Spain and § Medical University of South Carolina, Department of Pharmaceutical Sciences, Charleston, South Carolina 29425-2303

Received for publication, February 8, 2001, and in revised form, February 23, 2001


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The anthracycline-like polyketide drug elloramycin is produced by Streptomyces olivaceus Tü2353. Elloramycin has antibacterial activity against Gram-positive bacteria and also exhibits antitumor activity. From a cosmid clone (cos16F4) containing part of the elloramycin biosynthesis gene cluster, three genes (elmMI, elmMII, and elmMIII) have been cloned. Sequence analysis and data base comparison showed that their deduced products resembled S-adenosylmethionine-dependent O-methyltransferases. The genes were individually expressed in Streptomyces albus and also coexpressed with genes involved in the biosynthesis of L-rhamnose, the 6-deoxysugar attached to the elloramycin aglycon. The resulting recombinant strains were used to biotransform three different elloramycin-type compounds: L-rhamnosyl-tetracenomycin C, L-olivosyl-tetracenomycin C, and L-oleandrosyl-tetracenomycin, which differ in their 2'-, 3'-, and 4'-substituents of the sugar moieties. When only the three methyltransferase-encoding genes elmMI, elmMII, and elmMIII were individually expressed in S. albus, the methylating activity of the three methyltransferases was also assayed in vitro using various externally added glycosylated substrates. From the combined results of all of these experiments, it is proposed that methyltransferases ElmMI, ElmMII, and ElmMIII are involved in the biosynthesis of the permethylated L-rhamnose moiety of elloramycin. ElmMI, ElmMII, and ElmMIII are responsible for the consecutive methylation of the hydroxy groups at the 2'-, 3'-, and 4'-position, respectively, after the sugar moiety has been attached to the aglycon.


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

A number of important bioactive compounds are produced by actinomycetes. Particularly, the streptomycetes are responsible for the biosynthesis of most of the bioactive compounds clinically useful or with application in veterinary and animal husbandry. Many of them are glycosylated compounds and, in this case, the biological activity is usually correlated with the presence of the sugars, being in some cases essential for activity. Most of these sugars belong to the large family of 6-deoxyhexoses (6DOHs),1 which form part of a great number of natural products, and at least 70 different 6DOHs have been identified in various metabolites (1-3). In recent years, a number of genes encoding deoxysugar biosynthetic enzymes from antibiotic-producing microorganisms have been characterized. The first and common step in most 6DOH biosynthesis is the activation of D-glucose 1-phosphate into TDP D-glucose in a reaction catalyzed by a glucose-1-phosphate:TTP thymidylyl transferase. Then a key dehydration step takes place, by the action of a TDP-D-glucose-4,6-dehydratase, generating TDP-D-4-keto-6-deoxyglucose, a key intermediate of the 6DOH biosynthesis. In this step, the 6-position of the sugar gets deoxygenated, and the typical 5-methyl group (=C-6) is generated that will remain in the structure of all 6DOHs. These two "initial" enzymes are quite well conserved in many pathways, thus facilitating the use of the corresponding genes as probes for the isolation of homologous genes from other pathways (4). Further steps in the biosynthesis will diversify the sugars into L- or D-6DOHs (upon the action of a 5- or a 3,5-epimerase), and other modifications can occur, affecting the substituents and/or the stereochemistry at carbon 2, 3, or 4. Typical are further deoxygenation steps, C- or O-methylations and transaminations, thus generating the large family of 6DOH.

Several genes have been characterized whose deduced products have been proposed to be sugar O-methyltransferases. Most of them participate in the modification of the sugar components of macrolide antibiotics. During the last two steps of the biosynthesis of tylosin, the intermediate demethylmacrocin is converted into macrocin and then to tylosin through two O-methylation steps catalyzed by the TylE and TylF O-methyltransferases, respectively (5, 6). These two reactions occur once all the sugars in tylosin have been attached to the aglycon. They modify 6-deoxy-D-allose, the sugar that has been transferred by a glycosyltransferase, into D-mycinose, the final sugar in tylosin. From the gene cluster of the structurally closely related macrolide antibiotic mycinamycin produced by Micromonospora griseorubida, another gene, mycF, was isolated and proposed to encode a macrocin O-methyltransferase (7). Another O-methyltransferase gene, oleY, was reported in Streptomyces antibioticus, the oleandomycin producer (8). Its product OleY is involved in the conversion of L-olivose into L-oleandrose (9). Finally, eryG encodes an O-methyltransferase that catalyzes the last step in the erythromycin biosynthesis in Saccharopolyspora erythraea, the 3-O-methylation of L-mycarose into L-cladinose, thus also acting on a completely glycosylated intermediate (10). With the exception of tylosin, only a single methylation event modifying a deoxysugar moiety occurs in all of the biosyntheses of these macrolides. However, there are some glycosylated bioactive compounds harboring three methoxy groups in a single 6DOH at carbons 2, 3, and 4. Such is the case of the anthracycline-like drug elloramycin, which is produced by Streptomyces olivaceus Tü2353 (11), along with various minor congeners (12). Elloramycin A (Fig. 1A) has antibacterial activity against Gram-positive bacteria and also exhibits antitumor activity (13). It is a glycosylated tetracyclic aromatic polyketide, and its aglycon elloramycinone is a structural isomer of tetracenomycin C, with the difference in the methylation pattern. Elloramycin has its C-12a-O methylated (OH in tetracenomycin C), while tetracenomycin C contains an 8-OCH3 group (OH in elloramycinone). However, the most striking difference between elloramycin and tetracenomycin C is that elloramycin contains a permethylated L-rhamnose residue attached at the C-8 hydroxy group. Most of the elloramycin congeners, such as elloramycins B to D, differ in the methylation pattern, mostly within the L-rhamnose moiety (Fig. 1A) (we will henceforth refer to elloramycin A simply as elloramycin).

In our studies on deoxysugar biosynthesis and glycosyltransfer in antibiotic biosyntheses, we were interested in identifying and characterizing methyltransferase genes responsible for the L-rhamnose permethylation. We report here the cloning, sequencing, and expression of the elmMI, elmMII, and elmMIII genes from the elloramycin producer S. olivaceus Tü2353 and experimental evidence for the preferred substrate each of the corresponding enzymes is acting on.

    MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Bacterial Strains, Culture Conditions, and Vectors

Streptomyces olivaceus Tü2353, elloramycin producer, was used as a source of chromosomal DNA. Streptomyces albus G (14) was used as transformation host. Growth was carried out on trypticase soya broth (TSB; Oxoid) or R5A medium (15). For sporulation, they were grown for 7 days at 30 °C on agar plates containing A medium (15). For protoplast formation, strains were cultivated on TSB medium containing 0.75% glycine. Generation of protoplasts and protoplast transformation were carried out using standard procedures (16). Escherichia coli XL1-Blue (17) was used as host for subcloning and was grown at 37 °C in TSB medium. Features of plasmid constructs and vectors used are shown in Table I. When plasmid-containing clones were grown, the medium was supplemented with the appropriate antibiotics: 2.5 or 25 µg/ml thiostrepton for liquid or solid cultures, respectively, and 100 µg/ml for ampicillin.

                              
View this table:
[in this window]
[in a new window]
 
Table I
Vectors and plasmid constructs used in this work

DNA Manipulation and Sequencing

Plasmid DNA preparations, restriction endonuclease digestions, alkaline phosphatase treatments, ligations, and other DNA manipulations were performed according to standard procedures for E. coli (18) and for Streptomyces (16). Sequencing was performed on double-stranded DNA templates in pUC18 by using the dideoxynucleotide chain termination method (19) and the Cy5 AutoCycle Sequencing Kit (Amersham Pharmacia Biotech). Both DNA strands were sequenced with primers supplied in the kits or with internal oligoprimers (17-mer) using an ALF-express automatic DNA sequencer (Amersham Pharmacia Biotech). Computer-aided data base searching and sequence analyses were carried out using the University of Wisconsin Genetics Computer Group programs package (20) and the BLAST program (21).

Expression of Elloramycin Methyltransferases

Genes encoding methyltransferases were amplified by PCR using the oligoprimers shown in Table II. Restriction sites for SpeI (at the forward primer) and XbaI (at the reverse primer) were introduced to facilitate subcloning. PCR conditions were as follows. 100 ng of template DNA was mixed with 30 pmol of each primer and 2 units of Vent DNA Polymerase (New England Biolabs) in a total reaction volume of 50 µl containing a 2 mM concentration of each dNTP and 10% Me2SO (Merck). The polymerization reaction was performed in a thermocycler (MinyCycler, MJ Research) under the following conditions: an initial denaturation of 3 min at 98 °C; 30 cycles of 30 s at 98 °C, 60 s at 67 °C, and 90 s at 72 °C; and after the 30 cycles an extra extension step of 5 min at 72 °C followed by cooling at 4 °C at the end of this program. The PCR products were digested with SpeI and XbaI and subcloned into the same sites of pBSK. For cloning of elmMI, the corresponding fragment was rescued as a SpeI-XbaI fragment from the pBSK construct and subcloned into the XbaI site of pIAGO in the correct orientation and immediately downstream of the erythromycin resistance promoter (ermE*p), generating pEPM1. For cloning of elmMII and elmMIII, the fragments were rescued as BamHI-XbaI fragments from the corresponding pBSK constructs and subcloned into the same sites of pIAGO, generating pEPM2 and pEPM3, respectively.

                              
View this table:
[in this window]
[in a new window]
 
Table II
Oligoprimers used for PCR amplification of elmMI, elmMII, and elmMIII
Underlined DNA sequence represents sites for SpeI (ACTAGT) and XbaI (TCTAGA) that were introduced at the oligoprimers to facilitate subcloning.

The three methyltransferase genes were also coexpressed with genes directing the biosynthesis of L-rhamnose coded by pRHAM (22). The three PCR fragments mentioned above were rescued from pBSKM1, pBSKM2, and pBSKM3 as SpeI-XbaI fragments and subcloned into the XbaI site of pRHAM, located immediately downstream of the rhamnose biosynthetic genes.

Preparation of Glycosylated Tetracenomycin Derivatives

Several glycosylated compounds were prepared to be used as substrates for biotransformation and for enzyme assays. These were L-rhamnosyl-tetracenomycin C (RHA-TCMC), L-olivosyl-tetracenomycin C (OLV-TCMC), L-oleandrosyl-tetracenomycin C (OLE-TCMC), and 2-methoxy-L-rhamnosyl-tetracenomycin C (MeRHA-TCMC). RHA-TCMC and MeRHA-TCMC were produced in a two-step process. In a first step, a common intermediate, 8-demethyl-tetracenomycin C (8DMTC), was produced by growing a strain of Streptomyces argillaceus ATCC 12956 (mithramycin producer) containing cos16F4 (23). This compound was then used as substrate for biotransformation experiments, using as hosts two different S. albus recombinant strains. (i) For the production of RHA-TCMC, a S. albus recombinant strain harboring pRHAM-GT (see Table I) able to direct the biosynthesis of L-rhamnose and harboring the elloramycin glycosyltransferase gene elmGT was used (23), and (ii) for the production of MeRHA-TCMC, a S. albus recombinant strain harboring pRH-M1 (see Table I and "Results") able to direct the biosynthesis of L-rhamnose and harboring the elloramycin glycosyltransferase gene elmGT was used (23). In both cases, spores of the respective clones were inoculated in TSB medium supplemented with 2.5 µg/ml thiostrepton and incubated at 30 °C and 250 rpm. After 24 h, the cultures were used to inoculate (at 2.5%, v/v) three 2-liter Erlenmeyer flasks, each containing 400 ml of R5A medium (14) with 2.5 µg/ml thiostrepton. After further incubation for 24 h in the same conditions, the cultures were supplemented with 100 µg/ml 8DMTC, and incubation continued for an additional 24-h period. HPLC analysis of culture supernatants showed the disappearance of most of the precursor and the formation of new tetracenomycin derivatives. The cultures were then centrifuged, filtered, extracted, and chromatographed as previously described (15). The mobile phase used for elution in isocratic conditions was a mixture of methanol and 0.1% trifluoroacetic acid in water (50:50) for RHA-TCMC or a mixture of acetonitrile and 0.1% trifluoroacetic acid in water (27:73) for MeRHA-TCMC. The peaks corresponding to the desired compounds were collected, diluted 4-fold with water, applied to a reverse phase extraction cartridge, washed with water to eliminate trifluoroacetic acid, and finally eluted with methanol and dried in vacuo.

OLV-TCMC and OLE-TCMC were obtained by growing the S. albus recombinant strain AL16-OLV that contains cos16F4 and pOLV and purified as described (22).

Characterization and Physicochemical Properties of the New Elloramycin Analogs RHA-TCMC and MeRHA-TCMC

All NMR spectra were recorded on a Varian Inova 400 NMR instrument operating at a magnetic field strength of 9.4 teslas. The positive fast atom bombardment (FAB) mass spectra and the high resolutions spectra were obtained using a VG70SQ double focusing magnetic sector MS instrument. Nitrobenzene amide was used as the matrix.

The structures of the previously unknown new elloramycin derivatives, RHA-TCMC and MeRHA-TCMC, were unambiguously identified using one- and two-dimensional NMR and FAB mass spectroscopy. All proton and carbon NMR signal assignments were initially based on comparison with other known elloramycin/tetracenomycin derivatives (11-13, 22) and further refined considering couplings of the H,H-COSY spectrum and the 1JC-H and 2-3JC-H couplings observed in the two-dimensional heteronuclear single quantum coherence and heteronuclear multiple bond correlation spectra, respectively.

RHA-TCMC could be identified from its 1H NMR spectrum showing seven hydroxy groups. The alpha -linkage of the L-rhamnose moiety follows from the small coupling constant observed for the anomeric proton 1'-H (J1'-H/2'-H = 1.5 Hz). The attachment of the sugar moiety at the expected 8-position was proven from the nuclear Overhauser effect correlation spectroscopy spectrum, in which a clear correlation between 1'-H of the sugar building block and 7-H of the aglycon moiety could be observed. The relative stereochemistry in the rigid six-membered L-rhamnose moiety could be unambiguously verified from the H,H coupling constants considering the Karplus rules. All pf the carbon and proton signals of the aglycon moiety match those of previously characterized elloramycin derivatives. The NMR signals and especially the high resolution positive FAB mass spectrum confirm the deduced molecular formula of C28H28O15.

8-Demethyl-8-alpha -- L-rhamnosyltetracenomycin C---C28H28O15 (604.51 g/mol) 1H NMR (400 MHz, acetone-d6) delta /ppm (J/Hz): 1.18 d, 3H (6.5), 6'-H3; 2.84 s, 3H, 10-CH3; 3.52 ddd (9, 9, 4.5), 4'-H; 3.61 dq (9, 6.5), 5'-H; 3.75 ddd (9, 6, 3.5), 3'-H; 3.82 s, 3H, 3-OCH3; 3.94 s, 3H, 9-COOCH3; 4.03 ddd (3.5, 3, 1.5), 2'-H; 4.04 d (6), exchangeable by D2O, 3'-OH; 4.16 d (4.5), exchangeable by D2O, 4'-OH; 4.35 d (3), exchangeable by D2O, 2'-OH; 4.92 d (8), exchangeable by D2O, 4-OH; 5.06 d (8), 4-H; 5.13 broad s, exchangeable by D2O, 4a-OH; 5.61 s, 2-H; 5.76 broad s, 12a-OH; 5.85 d (1.5), 1'-H; 7.80 s, 7-H; 8.03 s, 6-H; 14.03 s, exchangeable by D2O, 11-OH.

13C NMR (100.6 MHz, CD3OD): delta /ppm 18.3, C-6'; 21.1, 10-CH3; 52.9, 9-OCH3; 57.4, 3-OCH3; 70.7, C-4; 71.1, 71.4, 72.3, and 73.4, C-2', C-3', C-4', and C-5'; 83.8, C-12a; 99.3, C-1'; 100.2, C-2; 110.5, C-11a; 112.1; C-7; 121.4, C-6; 122.2, C-10a; 129.4, C-9; 130.9, C-6a; 138.8, C-10; 141.2, C-5a; 155.1, C-8; 168.2, 9-C=O; 175.7, C-3; 190.8, C-1; 194.1, C-5; 198.2, C-12; signals for C-11 and C-4a not observed.

Positive FAB MS (nitrobenzene amide): m/z 605 (12%, M-H+), high resolution calc.: 605.1506, found: 605.1516; 460 (100%, M-rhamnose).

8-Demethyl-8-(2-methoxy-alpha -L-rhamnosyl)-tetracenomycin C-- C29H30O15 (618.16 g/mol). 1H NMR (400 MHz, acetone-d6) delta /ppm (J/Hz): 1.17 d, 3H (6), 6'-H3; 2.84 s, 3H, 10-CH3; 3.45 dd (9.5, 9.5), 4'-H; 3.55 s, 3H, 2'-OCH3; 3.57 dq (9.5, 6), 5'-H; 3.64 dd (3.5, 1.5), 2'-H; 3.76 m, after D2O- exchange: dd (9.5, 3.5), 3'-H; 3.82 s, 3H, 3-OCH3; 3.95 s, 3H, 9-COOCH3; 3.91 broad s, exchangeable by D2O, 3'-OH; 4.17 broad s, exchangeable by D2O, 4'-OH; 4.93 d (7), exchangeable by D2O, 4-OH; 5.06 d (7), 4-H; 5.14 broad s, exchangeable by D2O, 4a-OH; 5.61 s, 2-H; 5.77 broad s, 12a-OH; 6.00 d (1.5), 1'-H; 7.81 s, 7-H; 8.01 s, 6-H; 14.20 s, exchangeable by D2O, 11-OH.

13C NMR (100.6 MHz, acetone-d6): delta /ppm 18.2, C-6'; 21.1, 10-CH3; 53.0, 9-OCH3; 57.4, 3-OCH3; 59.6, 2'-OCH3; 70.4, C-4; 71.0, C-5'; 71.8, C-3'; 73.3, C-4'; 80.8, C-2'; 83.2, C-12a; 96.1, C-1'; 99.9, C-2; 110.0, C-11a; 111.9; C-7; 121.2, C-6; 121.8, C-10a; 129.1, C-6a; 130.5, C-9; 138.4, C-10; 140.8, C-5a; 154.5, C-8; 168.1, 9-C=O; 175.3, C-3; 191.0, C-1; 193.9, C-5; 197.9, C-12; signals for C-11 and C-4a not observed.

Positive FAB MS (nitrobenzene amide): m/z 619 (100%, M - H+).

Biotransformation-- For small scale biotransformation experiments, spores of the appropriate S. albus recombinant strains were used to inoculate 5 ml of TSB liquid medium containing 2.5 µg/ml thiostrepton for 24 h at 30 °C and 250 rpm. Then 100 µl of this preinoculum were used to inoculate 5 ml of R5A liquid medium. After 24 h of incubation, OLV-TCMC, OLE-TCMC, RHA-TCMC, or MeRHA-TCMC was added at a 100 µg/ml final concentration, and the cultures were further incubated for 24-48 h. Samples (900 µl) were removed and mixed with 300 µl of ethyl acetate. After centrifugation to separate the aqueous and organic phases, the organic phase was removed and evaporated in vacuo, and the residue was resuspended in a small volume of methanol for further analysis.

TLC and HPLC Analysis-- For TLC analysis, 1-2-µl samples were applied onto precoated silica gel 60 F254 plates and developed by chromatography using dichloromethane/methanol (90:10, v/v) as a solvent. Detection was carried out by UV light absorption. HPLC analysis was performed in a reversed phase column (Symmetry C18, 4.6 × 250 mm; Waters) as previously described (24). Detection and spectral characterization of peaks were made with a photodiode array detector and Millennium software (Waters), extracting bidimensional chromatograms at 280 nm. In some cases, HPLC analysis was performed on supernatants obtained by centrifugation of culture samples without previous extraction.

Preparation of Cell-free Extracts-- For the in vitro enzyme assays of the methyltransferases, a preinoculum was prepared (see "Biotransformation") of the appropriate S. albus recombinant strains (containing pEPM1, pEPM2, and pEPM3). Then 1 ml of this preinoculum was used to inoculate 50 ml of TSB liquid medium, and, after 24 h of incubation, the mycelia was harvested by centrifugation and washed in distilled water and again in 50 mM Tris-HCl pH 8.0 buffer containing 1 mM dithiothreitol. The mycelia was finally suspended in 5 ml of the same buffer and disrupted by sonication (five pulses of 15 s each with intermittent cooling in an MSE ultrasonic disintegrator at 150 watts and 20 kHz). After the removal of unbroken cells and cellular debris by centrifugation, the soluble fraction was used for methylation assays.

Methylation Assays-- Methylation reactions consisted of the following components (300-µl final volume): 6 µl of S-adenosyl-L-[methyl-3H]methionine (0.1 mCi/ml; specific activity 68 Ci/mmol), 6 µl of 5 mg/ml stock solution of the different glycosylated tetracenomycin derivatives, and a variable volume of extract and buffer (to a total of 300 µl) depending on the protein concentration of the extracts. During incubation at 30 °C, samples (50 µl) were removed at different time intervals and extracted with 50 µl of ethyl acetate. After phase separation, the organic phase was recovered, the solvent was evaporated, and the residue was suspended in 100 µl of methanol. After the addition of scintillation mixture, the radioactivity in the samples was determined. Radioactivity in the organic phase corresponded to methylated tetracenomycin derivatives as verified by HPLC analysis (see "Results").

    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Cloning of the elmMI, elmMII, and elmMIII Genes-- From a cosmid library of chromosomal DNA from S. olivaceus Tü2353, elloramycin producer, several overlapping cosmid clones were isolated that contained part of the elloramycin biosynthetic gene cluster (25). One of these clones, cos16F4, was found to hybridize with probes containing different genes of the structurally related anthracycline tetracenomycin C (Fig. 1B), thus probably containing all or part of the genes necessary for the biosynthesis of the elloramycin aglycon. This was confirmed by expression of cos16F4 in Streptomyces lividans giving rise to the formation of the intermediate 8DMTC (25). Also, it contains the gene elmGT coding for a flexible glycosyltransferase and responsible for the attachment of L-rhamnose to the aglycon (23). Interestingly, when cos16F4 was expressed into a S. lividans recombinant strain harboring a plasmid (pOLV) containing several genes of the L-oleandrose biosynthetic gene cluster from the oleandomycin producer S. antibioticus (9), formation of three glycosylated tetracenomycin derivatives and also elloramycin was observed (22). The fact that elloramycin (containing a permethylated L-rhamnose) was synthesized by this recombinant strain strongly suggested that cos16F4 should contain the methyltransferase genes required for methylating the hydroxy groups at carbons 2, 3, and 4 of the L-rhamnose moiety. In order to isolate these genes, we used as probe a 1.1-kb EcoRI-XhoI fragment from S. antibioticus ATCC 11891 (oleandomycin producer) that contains most of the oleY gene (only the last 74 base pairs are absent), which codes for a methyltransferase involved in L-oleandrose biosynthesis in S. antibioticus (8). With this probe we initially restricted the hybridizing region to a 10-kb BamHI fragment located at the right side of cos16F4 and within this to three restriction fragments: 0.4-kb BamHI-PstI, 2.8-kb PstI, and 3-kb PstI (Fig. 1B). One of these fragments, 2.8-kb PstI, was previously shown to contain the elmGT glycosyltransferase gene (23).


View larger version (13K):
[in this window]
[in a new window]
 
Fig. 1.   A, structure of elloramycin. The arrows indicate the methyl groups that are introduced by the three methyltransferases described in this paper. B, schematic organization of cos16F4 and the region sequenced in this work that includes the methyltransferase-encoding genes. The black bars show DNA fragments that hybridized against the oleY gene probe. The gray bar indicates the region that has been shown to hybridize with the tcmAGNPKLM genes of the tetracenomycin C gene cluster (24). The elmGT gene (shown as a white arrow) was previously cloned and sequenced (22).

Sequencing and Deduced Functions of the elMI, elmMII, and elmMIII Gene Products-- DNA regions at both sides of elmGT gene were sequenced: 1,180 base pairs upstream and 2,030 base pairs downstream of elmGT. Analysis of the DNA sequence (it has been deposited in GenBankTM under the accession number AJ309821) for the presence of potential coding regions using the CODONPREFERENCE program (20) showed three open reading frames, one upstream and two downstream of elmGT, all transcribed in the same direction as elmGT. They were designated (Fig. 1B, left to right) as elmMI, elmMII, and elmMIII. All of them showed a high GC content characteristic of Streptomyces genes and the bias in the third codon position characteristic of the genes of this genus. Some features of the genes and their deduced products are shown in Table III. Two of the genes, elmMI and elmMII, showed similar sizes, but the third one, elmMIII, was smaller. Comparison of the deduced products of the three genes with other proteins in data bases using a BLAST search (21) shows similarities for the three gene products with several proteins that have been proposed to encode O-methyltransferases involved in the biosynthesis of different 6DOH (Table III). The three methyltransferases show conserved motifs (Fig. 2) that are involved in the binding of the S-adenosylmethionine cofactor and/or one of the products of the methylation reaction (S-adenosylhomocysteine) (26). The most conserved of these three motifs, motif I, a glycine-rich region that is usually separated from motif II by ~36-90 amino acid residues. Motif III is normally located between 12 and 38 amino acids from the end of motif II. In some methyltransferases, not all of these motifs are clearly identified. In ElmMI and ElmMII, the three motifs are clearly recognized (Fig. 2A), but only motifs I and III can be appreciated in ElmMIII (Fig. 2B). On the basis of the profiles deduced from these data base comparisons, it was assumed that the three proteins could code S-adenosylmethionine-dependent methyltransferases.

                              
View this table:
[in this window]
[in a new window]
 
Table III
Characteristics of elmMI, elmMII, and elmMIII genes and their deduced products and similarities with other related proteins


View larger version (100K):
[in this window]
[in a new window]
 
Fig. 2.   Alignment of the deduced amino acid sequences of the ElmMI, ElmMII, and ElmMIII proteins with different methyltransferases. TylE and TylF, O-methyltransferases from S. fradiae (5, 6); SnogY, O-methyltransferase from Streptomyces nogalater (35); OleY, O-methyltransferase from S. antibioticus (8); MtfB, O-methyltransferase from Mycobacterium tuberculosis (accession number ADD20362); MycF, O-methyltransferase from S. mycarofaciens (7); NovP, O-methyltransferase from S. sphaeroides (27). ElmMI, ElmMII, and ElmMIII are described in this paper.

In Vivo Assay of the Methyltransferase Activities-- In order to assign specific functions to the products of the three elm methyltransferase genes, we conducted a series of experiments for the in vivo assay of their activity through biotransformation experiments. First, the three genes were independently coexpressed with genes involved in the biosynthesis of L-rhamnose in S. albus. Plasmid pRHAM contains four genes (oleL, oleS, oleE, and oleU) from the L-oleandrose biosynthetic pathway of the oleandomycin producer S. antibioticus cloned under the transcriptional control of the promoter of the erythromycin resistance gene from S. erythraea (ermE*p). This plasmid has been shown to code the enzymatic activities necessary for the biosynthesis of dTDP-L-rhamnose (22). The elm methyltransferase genes (elmMI, elmMII, and elmMIII) were independently amplified by PCR and subcloned into pRHAM immediately downstream of the oleU gene, generating constructs pRH-M1, pRH-M2, and pRH-M3, respectively. These three constructs (and also pRHAM, which lacks any methyltransferase genes) were used to transform protoplasts of a recombinant strain of S. albus that harbors the elmGT glycosyltransferase integrated into the chromosome (23). Transformants were selected by resistance to thiostrepton (antibiotic marker in the plasmid constructs), and one transformant from each transformation (named RH, RHM1, RHM2, and RHM3) was selected for the biotransformation experiments. These four clones were then cultivated in the presence of the 8DMTC, and, after biotransformation, products were analyzed by HPLC. Since it was unknown whether the methylation of the three hydroxy groups at L-rhamnose occurs before or after transfer of the sugar to the aglycon, this experiment would allow both alternatives, because free TDP-L-rhamnose, the rhamnosyl glycosyltransferase, each methyltransferase, and the aglycon 8DMTC would coexist in the cytoplasm. A peak corresponding to RHA-TCMC (as compared with a standard) was observed in all of the experiments, indicating that the L-rhamnose was being synthesized and that the glycosyltransferase ElmGT was linking L-rhamnose to 8DMTC. In addition, some other peaks were found in the experiments in which the three methyltransferase-encoding genes were individually expressed (data not shown). It should be noticed that expression of elmMI gave higher biotransformation yields than elmMII and elmMIII. HPLC retention values of these peaks were higher than that of RHA-TCMC, suggesting that possibly they contained more hydrophobic compounds, probably caused by the methylation of one of the hydroxy groups of L-rhamnose. From these experiments, it can be concluded that the ElmMI methyltransferase was probably acting on its natural substrate (RHA-TCMC), while the other two methyltransferases were only weakly acting probably because they were not recognizing their natural substrates. However, it cannot be concluded whether these methylations occur before or after the attachment of L-rhamnose to the aglycon.

To further define the substrate of each methyltransferase and its site of action, elmMI, elmMII, and elmMIII genes were then individually expressed in the bifunctional expression vector pIAGO under the control of the ermEp (constructs pEPM1, pEPM2, and pEPM3) and introduced by protoplast transformation into S. albus. After selection of transformants by thiostrepton resistance, recombinant clones EPM1, EPM2, and EPM3, respectively, and a control strain (only harboring pIAGO) were used for biotransformation. Three different compounds (RHA-TCMC, OLV-TCMC, and OLE-TCMC; Fig. 3) were used as potential substrates. The 6DOH present in these three compounds (L-rhamnose, L-olivose, and L-oleandrose) differ in the hydroxy group at C-2, which is present in L-rhamnose and absent in L-olivose and L-oleandrose (both are 2,6-DOH). In addition, L-olivose and L-oleandrose differ in the equatorial substituent at C-3 that is OH in the former sugar and OCH3 in the latter one. These differences in the substituents at C-2 and C-3 make these compounds useful to differentiate the regiospecificity of the methylation reaction at these two carbons. Using as biotransformation host strain EPM1 (expressing elmMI), ~24% of the precursor RHA-TCMC was converted into a new more hydrophobic derivative, but no biotransformation was observed when OLV-TCMC was used as substrate (Table IV). The compound thus generated by the action of ElmMI was purified by HPLC, and its structure was elucidated by NMR and MS.


View larger version (36K):
[in this window]
[in a new window]
 
Fig. 3.   Chemical structures of tetracenomycin C derivatives.

                              
View this table:
[in this window]
[in a new window]
 
Table IV
Biotransformation of RHA-TCMC, OLV-TCMC, OLE-TCMC, and MeRHA-TCMC by different recombinant strains of S. albus
The values are the average of three determinations. MT, methyltransferase.

The positive FAB MS (m/z 619, MH+) confirmed the expected molecular formula C29H30O15, which indicates the presence of an additional methyl group when compared with RHA-TCMC. Indeed, an additional signal for a newly generated O-methyl group appears in the 1H and 13C NMR spectra at delta H 3.55 and delta C 59.6, respectively, and only six exchangeable OH groups appear in the 1H NMR spectrum. A long range 5JH, H coupling between 1'-H and 7-H observable in the relay H,H-COSY spectrum as well as the 3JC-H coupling between 1'-H (delta  6.00) and C-8 (delta  154.5) in the heteronuclear multiple bond correlation spectrum leave no doubt about the position of the sugar moiety at C-8. That the O-methylation occurred at the 2'-position can be deduced from the 3JC-H coupling between the protons of the OCH3 group and C-2', also observable in the heteronuclear multiple bond correlation spectrum. The assignment of C-2' follows from its direct 1JC-H coupling with 2'-H, which can easily be assigned from its H,H coupling pattern, considering the Karplus rules (with the e,a 2,3-coupling constant of 3.5 Hz and an e,e 1,2-coupling constant of 1.5 Hz). Thus, the structure of the new metabolite could be unambiguously assigned to MeRHA-TCMC. This clearly demonstrates that ElmMI methylates the hydroxy group at the C-2 position of L-rhamnose.

When strain EPM2 was the biotransformation host, ~25% RHA-TCMC was converted into a new methylated derivative with different retention time than MeRHA-TCMC. However, the bioconversion efficiency was much higher (61%) when OLV-TCMC was used in biotransformations. In this case, the new derivative obtained showed identical HPLC retention time and spectral characteristics as OLE-TCMC, a 3-O-methylated OLV-TCMC. In addition, and as further confirmation, no biotransformation was observed when EPM2 was grown in the presence of OLE-TCMC (which is already methylated at the hydroxy group at C-3). Consequently, the site of action of ElmMII must be the hydroxy group at C-3 of L-rhamnose.

When strain EPM3 (expressing elmMIII) was incubated in the presence of RHA-TCMC or OLV-TCMC, the efficiency of biotransformation was very low (4 and 3%, respectively). However, biotransformation of OLE-TCMC was highly efficient; actually, all of the substrate provided was converted into a new derivative. Since OLE-TCMC lacks the hydroxy group at C-2' and has an O-methyl group at C-3', the only free hydroxy group is that at C-4', thus indicating that the ElmMIII must act on the C-4 of the L-rhamnose. The result that OLE-TCMC can be converted efficiently in contrast to OLV-TCMC and RHA-TCMC also indicates that ElmMIII prefers 3'-OH to be methylated.

MeRHA-TCMC, the product of the action of the ElmMI methyltransferase, was also used as biotransformation substrate for strains EPM2 and EPM3 (Table IV). The ElmMII and ElmMIII methyltransferases converted ~31 and 29% of the MeRHA-TCMC supplied into double methylated derivatives.

All of the 6DOH derivatives tested so far were L-sugars. We also tested a possible action of the three methyltransferases on D-sugars. In this way, three glycosylated derivatives containing D-sugars were also tested. They were D-olivosyl-, D-mycarosyl-, and D-diolivosyl-tetracenomycin C. These compounds were produced by expressing cosmid 16F4 into the mithramycin producer S. argillaceus (27). When these compounds were individually fed into S. albus clones EPM1, EPM2, and EPM3, no new biotransformation products were found (data not shown), indicating that none of the methyltransferases act on D-sugars.

In Vitro Assay of the Methyltransferase Activities-- The activity of the three methyltransferases was also assayed in vitro using RHA-TCMC, OLV-TCMC, OLE-TCMC, and MeRHA-TCMC as substrates (Fig. 4). Cell-free extracts were prepared from S. albus recombinant strains EPM1, EPM2, and EPM3. Extracts of EPM1 and EPM2 were effective in methylating RHA-TCMC, while those of EPM3 were much less efficient. OLV-TCMC and OLE-TCMC were only methylated by extracts of EPM2 and EPM3, respectively. MeRHA-TCMC was methylated at a similar extent by extracts of EPM2 and EPM3. In all of the cases, methylation was dependent upon the presence of exogenously supplied S-adenosylmethionine, indicating that the three enzymes were S-adenosylmethionine-dependent methyltransferases. All of these results showed a good correlation with those described above for the in vivo biotransformation experiments.


View larger version (18K):
[in this window]
[in a new window]
 
Fig. 4.   Time course for the in vitro methylation of different glycosylated substrates by the ElmMI (open circles), ElmMII (closed circles) and ElmMIIII (closed triangles) methyl- transferases.

To confirm that methylation of each substrate conducts to the formation of the expected sugar methylated derivative, in vitro methylation assays were carried out in the presence of 3H-labeled S-adenosylmethionine, the reaction products were fractionated by HPLC, and radioactivity of the different peaks was counted (Fig. 5). In all cases, most of the radioactivity was associated with fractions corresponding to the HPLC retention time of the expected product.


View larger version (29K):
[in this window]
[in a new window]
 
Fig. 5.   Measurement of radioactivity in the different HPLC fractions after in vitro methylation of the various glycosylated substrates by the ElmMI, ElmMII, and ElmMIII methyltransferases. S and P, the mobility of each respective substrate and the corresponding expected product.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Many 6DOHs, being a structural part of glycosylated bioactive compounds, are methylated in some of the hydroxy groups. Several genes from antibiotic-producing organisms have been isolated and proposed to code for enzymes responsible for these methylation steps. These genes are mainly from macrolide producers and include tylE and tylF from the tylosin producer Streptomyces fradiae (5, 6), mycF from the mycinamycin producer M. griseorubida (7), oleY from the oleandomycin producer S. antibioticus (8), eryG from the erythromycin producer S. erythraea (10), and novP from the novobiocin producer S. sphaeroides (28). The involvement of some of these genes in O-methylation has been proved. However, experimental evidence on their action as methylating enzymes has been provided so far only for eryG (10) and mycF (7). Furthermore, most of the mentioned methyltransferases are the only methylating enzymes acting on the corresponding sugar that therefore only possess a single methoxy group. In contrast, the three methyltransferases we reported here produce a permethylated sugar by successively acting on the same sugar (i.e. L-rhamnose).

Permethylated L-rhamnose is not frequently found as a sugar moiety in glycosylated, bioactive natural compounds. As far as we know, this completely O-methylated sugar occurs only in the molecule studied in this work, elloramycin, produced by S. olivaceus (11), and in the macrolide insecticide spinosyn produced by Saccharomyces spinosa (29). From the elloramycin producer, we have identified three methyltransferase-encoding genes (elmMI, elmMII, and elmMIII) in a cosmid that contains part of the elloramycin gene cluster. Initial assignment of the respective gene products to the methyltransferase family came from data base comparisons with other proteins from antibiotic-producing microorganisms that have been proposed to act as methyltransferases. This assignment was further confirmed by assaying the methylating activity of these enzymes on their correspondent substrates in two independent ways: (i) by in vivo biotransformation of specific potential substrates of the enzymes and the analysis of the modified methylated biotransformation products and (ii) by in vitro assay of the methylating activities using cell-free extracts of recombinant clones individually expressing the methyltransferases. For the biotransformation approach, it was necessary to create appropriate biotransformation hosts. Using S. albus as a host, two different systems were used. In the first system, the organism was endowed with a triple capability. First, the organism had the ability to synthesize an activated sugar (dTDP-L-rhamnose) by transformation with plasmid pRHAM. Also, co-expressed were each of the respective individual methyltransferase-encoding genes and elmGT, the gene encoding the L-rhamnose glycosyltransferase. When these recombinant cell factories were incubated in the presence of the aglycon substrate of ElmGT, 8DMTC, all of them efficiently linked L-rhamnose to the aglycon; however, lesser amounts of products with methylated sugars could be observed, with the exception of MeRHA-TCMC, which was readily formed. Since this first system does not allow the principal distinction between (i) methylation before and (ii) methylation after the glycosyl transfer step, a second system was developed. Here, three different glycosylated TCMC derivatives, which were postulated as substrates for the different methyltransferases, were exogenously provided, taking advantage of the fact that these glycosylated tetracenomycin C derivatives were available from earlier experiments on the ElmGT (22, 26). The recombinant S. albus strains used for these biotransformation experiments only contained each individual methyltransferase gene to be investigated. Using these two systems, the role of the methyltransferases ElmI, ElmMII, and ElmMIII in the biosynthesis of elloramycin could be deduced as methylating the free hydroxy groups at 2'-, 3'-, and 4'-positions of L-rhamnose, respectively.

The site of action of ElmMI methyltransferase is the hydroxy group at C-2'. This assignment is based on two results. First, no methylation occurs on substrates lacking a hydroxy group at C-2' (OLV-TCMC and OLE-TCMC). Second, when using clone EPM1 (expressing ElmMI) for the biotransformation of RHA-TCMC, which possesses the three free hydroxy groups at carbons 2', 3', and 4', only a single HPLC biotransformation peak was found, whose product was purified and identified after structure elucidation as MeRHA-TCMC.

ElmMII was concluded to act on 3'-OH based on the comparison of the results obtained when EPM2 is incubated with either OLV-TCMC or OLE-TCMC. These two compounds only differ in that 3'-OH is methylated in OLE-TCMC but not methylated in OLV-TCMC. The absence of modification by biotransformation with OLE-TCMC and the highly efficient conversion of OLV-TCMC into OLE-TCMC indicates that ElmMII acts on the C-3' hydroxy group. Interestingly, ElmMII can also act on RHA-TCMC.

Finally, ElmMIII must act on C-4'. This could be concluded from comparing the biotransformation of EPM3 with OLV-TCMC and OLE-TCMC. Both compounds posses a free hydroxy group at C-4' but differ in position 3', which is methylated in OLE-TCMC and unmethylated in OLV-TCMC. The former was very efficiently biotransformed (all pf the precursor was converted into a new derivative), while the latter was nearly unaffected (only 3% conversion). Since OLE-TCMC has only one free hydroxy group (C-4'), ElmMIII should methylate this position. The results of these experiments also suggest that the ElmMIII activity is dependent on the presence of a methoxy group at C-2' or C-3', since both OLE-TCMC and MeRHA-TCMC were biotransformed in vitro. As a conclusion, it is proposed that ElmMI, ElmMII, and ElmMIII act on the free hydroxy groups at 2', 3', and 4' positions, respectively, of L-rhamnose during the late biosynthesis of elloramycin. The relaxed substrate specificity found for ElmMII and ElmMIII explains why elloramycin C (2'-OCH3, 3'-OH) and D (2'-OH, 3'-OCH3) can be found side-by-side as minor congeners of wild type strain S. olivaceus Tü2353 (12).

Two questions remain: (i) when and (ii) in which order do the three methyl transfer steps occur? If these three methylation events happen before the sugar has been attached to the aglycon, the three gene products should act during the deoxysugar biosynthesis, and the sugar co-substrate of ElmGT would be TDP-activated permethylated L-rhamnose. Alternatively, nonmethylated TDP-L-rhamnose could be the sugar co-substrate of the glycosyltransferase ElmGT, and the methylation of the different OH-groups would occur on glycosylated intermediates (i.e. as the last steps of the elloramycin biosynthesis). The experimental evidence shown in this paper strongly argues in favor of the latter hypothesis. When the different clones expressing the methyltransferase genes were used for the biotransformation of glycosylated derivatives, methylated derivatives were found and, in some cases, very efficient methylation rates. These results were quite improbable if the methyltransferases were to normally act on TDP-activated sugar derivatives, which are extremely different substrates compared with glycosylated tetracenomycins. Thus, these results support the view that the methylation events occur most likely after the sugar has been linked to the aglycon. In a few cases in which this has been established, sugar O-methylation occurs once the sugar has been attached to the aglycon. This is the case of the EryG (10), TylE and TylF (30, 31), and MycF (7). One exception was found for L-oleandrose during the avermectin biosynthesis; it contains a methoxy group at C-3' that it is introduced before the sugar is transferred (32).

The second question concerns the order of the methylation events. It can be clearly deduced that ElmMIII acts only when at least one methoxy group is already present (either at C-2' or C-3'), since it cannot methylate efficiently elloramycin derivatives lacking any OCH3 group in the sugar moiety (e.g. RHA-TCMC and OLV-TCMC). However, ElmMIII methylates MeRHA-TCMC and OLE-TCMC, both possessing methoxy groups although in different positions. The fact that ElmMIII is much more efficient methylating C-4' when the methoxy group at C-3' (introduced by ElmMII) is present indicates that it should act after ElmMII. Both ElmMI and ElmMII are able to methylate RHA-TCMC to a similar extent in the biotransformation experiments. However, the in vitro assays showed that RHA-TCMC is a much better substrate for ElmMI than for ElmMII. Thus, we favor ElmMI to act as the first methylating enzyme. Combining all of these conclusions, we propose the biosynthetic sequence shown in Fig. 6 for the methylation events of the elloramycin biosynthesis.


View larger version (19K):
[in this window]
[in a new window]
 
Fig. 6.   Proposed pathway for the L-rhamnose methylation steps during the biosynthesis of elloramycin by S. olivaceus.

All three enzymes described in this paper showed some relaxed substrate specificity, although to different degrees, when confronted with the glycosylated tetracenomycin C derivatives. Thus, they may have the potential to be used as tailoring enzymes for the decoration of sugar moieties and therefore the ability of generating a diversity of bioactive compounds. Various enzymes can be used in this sense to modify the architecture of bioactive compounds. Some of them affect the structure of the aglycons such as oxygenases and methyltransferases. So far, only a few O-methylating enzymes have been shown, which modify sugar moieties, including the already mentioned O-methyltransferases involved in macrolide biosyntheses. In addition, other sugar-modifying enzymes (acyltransferases) have been reported from macrolide producers (33-35). As far as we know, no methyltransferases or acyltransferases involved in modifying the glycosyl moieties of aromatic polyketides have been characterized before. The methyltransferases ElmMI, ElmMII, and ElmMIII from the elloramycin producer S. olivaceus represent the first examples of such enzymes and therefore add some degree of novelty to the pool of sugar-modifying enzymes. The broader biological activity of elloramycin B compared with all of the other natural elloramycins (12) shows also that the O-methylation pattern of deoxysugar moieties has a significant influence on the biological activity of a given compound.

    ACKNOWLEDGEMENTS

We thank David Rodriguez for helpful collaboration in the methylation assays and Drs. W. Cotham and M. Walla (Department of Biochemistry and Chemistry, University of South Carolina) for the FAB mass spectra.

    FOOTNOTES

* This work was supported by European Community Grant BIO4-CT96-0068 (to J. R. and J. A. S.), Spanish Ministry of Education and Science Grant BIO97-0771 through the "Plan Nacional en Biotecnologia" (to J. A. S.), and grants from the South Carolina Commission of Higher Education as well as the United States Department of Defense (to J. R.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AJ309821.

To whom correspondence should be addressed for chemical communications: Dept. of Pharmaceutical Sciences, Medical University of South Carolina, 171 Ashley Ave., Charleston, SC 29425-2303. Tel.: 843-953-6659; Fax: 843-953-6615; E-mail: rohrj@musc.edu.

|| To whom correspondence should be addressed for molecular biology communications: Dept. de Biología Funcional e Instituto Universitario de Oncología del Principado de Asturias, Universidad de Oviedo, 33006 Oviedo, Spain. Tel./Fax: 34-985-103652; E-mail: jasf@sauron. quimica.uniovi.es.

Published, JBC Papers in Press, February 28, 2001, DOI 10.1074/jbc.M101225200

    ABBREVIATIONS

The abbreviations used are: 6DOH, 6-deoxyhexose; PCR, polymerase chain reaction; RHA-TCMC, L-rhamnosyl-tetracenomycin C; MeRHA-TCMC, 2-methoxy-L-rhamnosyl-tetracenomycin C; OLV-TCMC, L-olivosyl-tetracenomycin C; OLE-TCMC, L-oleandrosyl-tetracenomycin C; FAB, fast atom bombardment; MS, mass spectrometry; kb, kilobase pair.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

1. Piepersberg, W. (1994) Crit. Rev. Biotechnol 14, 251-285[Medline] [Order article via Infotrieve]
2. Liu, H., and Thorson, S. J. (1994) Annu. Rev. Microbiol 48, 223-256[CrossRef][Medline] [Order article via Infotrieve]
3. Trefzer, A., Salas, J. A., and Bechthold, A. (1999) Nat. Prod. Rep 16, 283-299[CrossRef][Medline] [Order article via Infotrieve]
4. Decker, H., Gaisser, S., Pelzer, S., Schneider, P., Westrich, L., Wohlleben, W., and Bechthold, A. (1996) FEMS Microbiol. Lett. 141, 195-201[CrossRef][Medline] [Order article via Infotrieve]
5. Bate, N., and Cundliffe, E. (1999) J. Ind. Microbiol. Biotechnol. 23, 118-122[CrossRef][Medline] [Order article via Infotrieve]
6. Fouces, R., Mellado, E., Diez, B., and Barredo, J. L. (1999) Microbiology 145, 855-868[Abstract]
7. Inouye, M., Suzuki, H., Takada, Y., Muto, N., Horinouchi, S., and Beppu, T. (1994) Gene (Amst.) 141, 121-124[CrossRef][Medline] [Order article via Infotrieve]
8. Olano, C., Rodriguez, A. M., Michel, J. M., Méndez, C., Raynal, M. C., and Salas, J. A. (1998) Mol. Gen. Genet. 259, 299-308[CrossRef][Medline] [Order article via Infotrieve]
9. Aguirrezabalaga, I., Olano, C., Allende, N., Rodriguez, L., Braña, A. F., Méndez, C., and Salas, J. A. (2000) Antimicrob. Agents Chemother 44, 1266-1275[Abstract/Free Full Text]
10. Paulus, T. J., Tuan, J. S., Luebke, V. E., Maine, G. T., DeWitt, J. P., and Katz, L. (1990) J. Bacteriol 172, 2541-2546[Medline] [Order article via Infotrieve]
11. Drautz, H., Reuschenbach, P., Zähner, H., Rohr, J., and Zeeck, A. (1985) J. Antibiot. 38, 1291-1301[Medline] [Order article via Infotrieve]
12. Fiedler, H. P., Rohr, J., and Zeeck, A. (1986) J. Antibiot. 39, 856-859[Medline] [Order article via Infotrieve]
13. Rohr, J., and Zeeck, A. (1990) J. Antibiot. 43, 1169-1178[Medline] [Order article via Infotrieve]
14. Chater, K. F., and Wilde, L. C. (1980) J. Gen. Microbiol. 116, 323-334[Medline] [Order article via Infotrieve]
15. Fernández, E., Weißbach, U., Sánchez Reillo, C., Braña, A. F., Méndez, C., Rohr, J., and Salas, J. A. (1998) J. Bacteriol 180, 4929-4937[Abstract/Free Full Text]
16. Hopwood, D. A., Bibb, M. J., Chater, K. F., Kieser, T., Bruton, C. J., Kieser, H. M., Lydiate, D. J., Smith, C. P., Ward, J. M., and Schrempf, H. (1985) Genetic Manipulation of Streptomyces: A Laboratory Manual , the John Innes Foundation, Norwich, United Kingdom
17. Bullock, W. O., Fernández, J. M., and Short, J. N. (1987) BioTechniques 5, 376
18. Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual , 2nd Ed. , Cold Spring Harbor Laboratory, Cold Spring Harbor, NY
19. Sanger, F., Nicklen, S., and Coulson, A. R. (1977) Proc. Natl. Acad. Sci. U. S. A. 74, 5463-5467[Abstract]
20. Devereux, J., Haeberli, P., and Smithies, O. (1984) Nucleic Acids Res. 12, 387-395[Abstract]
21. Altschul, S. F., Gish, W., Miller, W., Myers, E. W., and Lipman, D. J. (1990) J. Mol. Biol. 215, 403-410[CrossRef][Medline] [Order article via Infotrieve]
22. Rodriguez, L., Oelkers, C., Aguirrezabalaga, I., Braña, A. F., Rohr, J., Méndez, C., and Salas, A. (2000) J. Mol. Microbiol. Biotechnol. 2, 271-276[Medline] [Order article via Infotrieve]
23. Blanco, G., Patallo, E. P., Braña, A. F., Trefzer, A., Bechthold, A., Rohr, J., Méndez, C., and Salas, J. A. (2001) Chem. Biol. 8, 253-263[CrossRef][Medline] [Order article via Infotrieve]
24. Fernández Lozano, M. J., Remsing, L. L.,., Quirós, L. M., Braña, A. F., Fernández, E., Sánchez, C., Méndez, C., Rohr, J., and Salas, J. A. (2000) J. Biol. Chem. 275, 3065-3074[Abstract/Free Full Text]
25. Decker, H., Rohr, J., Motamedi, H., Zähner, H., and Hutchinson, C. R. (1995) Gene (Amst.) 166, 121-126[CrossRef][Medline] [Order article via Infotrieve]
26. Kagan, R. M., and Clarke, S. (1994) Arch. Biochem. Biophys. 310, 417-427[CrossRef][Medline] [Order article via Infotrieve]
27. Wohlert, S. E., Blanco, G., Lombó, F., Fernández, E., Braña, A. F., Reich, S., Udvarnoki, G., Méndez, C., Decker, H., Salas, J. A., and Rohr, J. (1998) J. Am. Chem. Soc. 120, 10596-10601[CrossRef]
28. Steffensky, M., Muhlenweg, A., Wang, Z. X., Li, S. M., and Heide, L. (2000) Antimicrob. Agents Chemother. 44, 1214-1222[Abstract/Free Full Text]
29. Kirst, H. A., Michel, K. H., Martin, J. W., Creemer, L. C., Chio, E. H., Yao, R. C., Nakatsukasa, W. N., Boeck, L. D., Occolowitz, J. L., Paschal, J. W., Deeter, J. B., Jones, N. D., and Thompson, G. D. (1991) Tetrahedron Lett. 32, 4839-4842[CrossRef]
30. Kreuzman, A. J., Turner, J. R., and Yeh, W-K. (1988) J. Biol. Chem. 263, 15626-15633[Abstract/Free Full Text]
31. Seno, E. T., and Baltz, R. H. (1981) Antimicrob. Agents Chemother. 20, 370-377[Medline] [Order article via Infotrieve]
32. Schulman, M. D., Acton, S. L., Valentino, D. L., and Arison, B. H. (1990) J. Biol. Chem. 265, 16965-16970[Abstract/Free Full Text]
33. Epp, J. K., Huber, M. L., Turner, J. R., Goodson, T., and Schöner, B. E. (1989) Gene (Amst.) 85, 293-301[Medline] [Order article via Infotrieve]
34. Arisawa, A., Kawamura, N., Takeda, K., Tsunekawa, H., Okamura, K., and Okamoto, R. (1994) Appl. Env. Microbiol. 60, 2657-2660[Abstract]
35. Arisawa, A., Kawamura, N., Narita, T., Kojima, I., Okamura, K., Tsunekawa, H., Yoshioka, T., and Okamoto, R. (1996) J. Antibiot. 49, 349-354[Medline] [Order article via Infotrieve]
36. Ylihonko, K., Tuikkanen, J., Jussila, S., Cong, L., and Mäntsälä, P. (1996) Mol. Gen. Genet. 251, 113-120[CrossRef][Medline] [Order article via Infotrieve]


Copyright © 2001 by The American Society for Biochemistry and Molecular Biology, Inc.