KR100564163B1 - The Enzyme Concerning in Biosynthesis of Ribostamycin and the Genes Thereof - Google Patents

Abstract

The present invention relates to an enzyme involved in ribostamycin (Rbm) biosynthesis and a gene thereof, and more specifically, a base sequence of a gene cluster involved in ribostamycin biosynthesis, a DOI synthase that is a ribostamycin biosynthetic enzyme [ 2-deoxy- scyllo- inosose (DOI) synthase], AAC (3) (aminoglycoside-N-3-acetyltransferase) and genes encoding the enzymes.

Gene clusters involved in ribostamycin biosynthesis, DOI synthase and AAC (3) according to the invention are useful for the preparation of ribostamycin and similar novel aminoglycoside based antibiotics.

Ribostamycin, Streptomyces, DOI Synthase, AAC (3)

Description

Enzyme Concerning in Biosynthesis of Ribostamycin and the Genes Thereof}             

Figure 1 shows the structure of aminoglycoside-aminocyclitol (AmAc), A represents butyrosine B (Bn B), B represents Rbm.

Figure 2 shows a gene map of the butyrosine and Rbm biosynthetic gene cluster. Proteins encoding similar genes are indicated by the same arrows, and B, E and H on the genetic map represent Bam HI, Eco RI and Hin dIII, respectively.

Figure 3 shows the expected route of Rbm synthesis in S. ribosidificus . 1 is D-glucose, 2 is glucose-6-phosphate, 3 is DOI, 4 is 2-deoxy-syl-inosamine, 5 is 2-deoxy-3-amino-syl-inosose, 6 is 2- Deoxystreptamine, 7 represents 2-amino-2-deoxy-D-glucose, 8 represents neamine and 9 represents ribostamycin.

Figure 4 shows the SDS-PAGE results of RbmA expressed for 3 days in S. lividans TK24. Lane 1 represents the total protein expressed in the control S. lividans TK24 / pIBR25, lane 2 represents the total protein of S. lividans TK24 / pBS2, M represents the protein marker (Novagen, USA). RbmA is shown as a box.

Figure 5 shows the results of HPLC analysis of DOI synthase activity of the crude enzyme solution purified from S. lividans transformant. Arrows indicate oxime derivative peaks of DOI.

Figure 6 shows the SDS-PAGE results of RbmI. Lane 1 shows bacterial lysate, lanes 2 and 3 are purified via Ni ²⁺ affinity chromatography, and lane 4 shows protein markers (Novagen, USA). Arrows indicate RbmI.

Figure 7 is a photograph analyzed by TLC whether the acetylation of AmAcs reacted with RbmI. Lanes 1, 2, 3, 4, 5 and 6 are control samples without coenzyme A added to kanamycin, ribostamycin, apramycin, neomycin, gentamicin and spectinomycin, respectively, A sample reacted by adding coenzyme A.

8 is an analysis of the antimicrobial activity of acetylated AmAcs. Apm, Km, Rbm, Nem and Gm represent apramycin, kanamycin, ribostamycin, neomycin and gentamicin, respectively, and Rxn, Ref and Std represent reaction samples, control samples and standard samples, respectively.

The present invention relates to an enzyme involved in ribostamycin (Rbm) biosynthesis and a gene thereof, and more specifically, a base sequence of a gene cluster involved in ribostamycin biosynthesis, a DOI synthase that is a ribostamycin biosynthetic enzyme [ 2-deoxy- scyllo -inosose (DOI) synthase], AAC (3) (aminoglycoside-N-3-acetyltransferase) and genes encoding the enzyme.

Aminoglycoside-aminocyclitol (AmAc) antibiotics, including DOS, have a broad antimicrobial spectrum and are one of the most important medical antibiotics. In terms of chemical structure, AmAc can be divided into two main forms. One form is 4,5-disubstituted DOS (deoxystreptamine), such as ribostamycin (Rbm), butyrosine (Bn), and neomycin (Nm), and the other is 4, such as kanamycin (Km) and gentamicin (Gm). , 6-disubstituted DOS.

Genetic biochemical studies of these antibiotics began with the isolation of Bn biosynthetic genes from Bacillus circulans (Ota, Y. et al ., J. antibiot., 53: 1158, 2000), followed by Actinomycetes Gm, Km and tobramycin (Tm) gene clusters from actinomycetes were found (Kharel, MK et al. , FEMS Microbiol. Lett ., 230: 185, 2004).

Although actinomycetes is the main producer of 4,5-disubstituted AmAc, reports on the biosynthetic genes of the antibiotics in actinomycetes have been reported by AAC (3) (aminoglycoside-) of M. chalcea and S. fradiae . N-3-acetyltransferase), Km kinase from S. fradiae and Rbm phosphotransferase from S. ribosidificus (Salauze, D. et al ., Gene , 101: 143, 1991, Bibb, MJ et al ., Mo. Gen. Genet ., 199: 26, 1985, Hoshiko, S. et al ., Gene , 68: 285, 1988).

Rbm consists of three subunits: DOS, neosamine C and ribose. This is a structure in which AHBA [2R-4-amino-2-hydroxy butric acid] is conjugated to C1-NH ₂ of Bn B (FIG. 1). This conjugation is also used for medical use in the form of Km B in the form of ABK (arbekacin) and Km A in the form of amikacin.

It is reported that Rbm is an intermediate in Bn biosynthesis (Baud, HJ et al ., Antibiot., 30: 720, 1977). 2-deoxy- scyllo- inosose (DOI). Synthase (BtrC) catalyzes the production of DOI from G-6-P (glucose-6-phosphate) (Kudo, F. et al ., J. Antibiot ., 52: 559, 1999). DOI is L- glutamine biosynthesis of DOS in B. circulans:. DOI by amino transferase dehydratase (GLA) is trans-amino Nation 2-deoxy- scyllo - is converted to inosamine (Tamegai, H. et al, J. Antibiot. , 55: 707, 2002). Using the DOI synthase activity used for the chem-enzymatic synthesis of catechol, the nature of BtrC and the role of the active site (Lys-141) have been revealed (Nango, E. et al ., J. Org Chem ., 69: 593, 2004). However, there have been no reports on the sequencing of Rbm biosynthetic gene clusters of S. ribosidificus and the chemical characterization of Rbm biosynthetic enzymes.

Accordingly, the present inventors cloned the gene fragments expected to be biosynthetic gene clusters of Rbm from the chromosomal DNA of S. ribosidificus to determine the sequencing, and to express the biosynthetic genes of Rbm in a large amount, and then biochemical analysis of the purified protein Through the cloned gene fragment confirmed that the biosynthetic gene cluster of Rbm was completed the present invention.

It is an object of the present invention to provide a nucleotide sequence of a gene cluster involved in ribostomycin biosynthesis.

Another object of the present invention is to provide a DOI synthase that is a ribotomycin biosynthetic enzyme and a gene encoding the same.

Still another object of the present invention is to provide an aminoglycoside-N-acetyltransferase [AAC (3)], which is a ribostamycin biosynthetic enzyme, and a gene encoding the same.

In order to achieve the above objects, the amino acid sequence of the invention SEQ ID NO: to 1 encoding the amino acid sequence of racP, racN encoding racO, amino acid sequence of SEQ ID NO: 3 encoding the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 4 encoding the amino acid sequence of encoding the amino acid sequence of encoding the amino acid sequence of encoding the amino acid sequence of encoding rbmI, SEQ ID NO: 5 rbmM, SEQ ID NO: 6 racL, SEQ ID NO: 7 rbmH, SEQ ID NO: 8 racK, SEQ ID NO: to 9 encoding the amino acid sequence of encoding the racJ, amino acid sequence of SEQ ID NO: 10 racI, racH encoding the amino acid sequence of SEQ ID NO: 11, rbmG encoding an amino acid sequence of SEQ ID NO: 12, SEQ ID NO: 13 encoding the amino acid sequence of rbmF amino acid sequence of SEQ ID NO: 14 that encodes the amino acid sequence of the rbmE, SEQ ID NO: rbmD to 15 encoding the amino acid sequence of, SEQ ID NO: 16 to Encoding the amino acid sequence of encoding the amino acid sequence of encoding rbmA, SEQ ID NO: 17 rbmB, SEQ ID NO: racA, SEQ ID NO: 20 of the 18 encoding the amino acid sequence of rbmC, encoding the amino acid sequence of SEQ ID NO: 19 racB, SEQ ID NO: racC to 21, encoding the amino acid sequence of, SEQ ID NO: 22 of the amino acid rph encoding the sequence of SEQ ID NO racD to 23, encoding the amino acid sequence of SEQ ID NO: encoding a racE, the amino acid sequence of SEQ ID NO: 25 to 24 encoding the amino acid sequence of RacF, and Ribostamycin (Rbm) biosynthetic gene cluster comprising racG encoding the amino acid sequence of SEQ ID NO: 26 is provided.

In the present invention, the gene cluster may be characterized as derived from Streptomyces ribosidificus .

The present invention also provides a DOI synthase, which is a ribotomycin biosynthetic enzyme having the amino acid sequence of SEQ ID NO: 4, and a gene ( rbmA ) encoding the DOI synthase.

The present invention also provides a method for producing a DOI synthase, comprising culturing the recombinant vector containing the gene, the bacteria transformed with the recombinant vector and the transformed bacteria.

The present invention also relates to an aminoglycoside-N-acetyltransferase [AAC (3)], which is a ribostamycin biosynthetic enzyme having the amino acid sequence of SEQ ID NO: 4, and a gene ( rbmI ) encoding the AAC (3). to provide.

The present invention also provides an aminoglycoside-N-acetyltransferase [AAC (3)] comprising culturing a recombinant vector containing the gene, a bacterium transformed with the recombinant vector, and the transformed bacterium. It provides a method of manufacturing.

Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are only for illustrating the present invention, it will be apparent to those skilled in the art that the scope of the present invention is not to be construed as being limited by these examples.

S. ribosidificus (ATCC21294) and S. lividans TK24 (US 20030142745 A1) used in the present invention were cultured using ISP2 (Difco, USA) and R2YE (US 5,843,735) medium at 28 ° C. In the present invention, E. coli XL1 blue (Stratagene, USA) was used as a subcloning host, and E. coli BL21 (DE3) (Stratagene, USA) was used as a host for gene expression. POJ446 (US 20040053274A1) was used to construct the chromosome library, and pET-32a (+) (Novagene, USA) was used for gene expression in E. coli . PIBR25 (Sthapit, B. et al., FEBS Lett. , 566: 201, 2004) was used for gene expression in S. lividans TK24. In order to maintain the recombinant vector in S. lividans TK24, thiostrepton (50 µg / ml) was added to the culture medium.

Example 1 DNA Library Construction and Cosmid Library Screening

To construct the DNA library, chromosomal DNA of S. ribosidificus was partially digested for several hours at 0.5-5 minute intervals using Mbo I, followed by agarose gel electrophoresis. A portion containing a 35-45 kb fragment was taken, treated with alkaline phosphatase and ligated with pOJ446 treated with Bam HI and Hpa I. In vitro packaging of the ligated samples was done using GigapackIII XL (Stratagene, USA).

Cosmid libraries of S. ribosidificus were screened using a partial sequence of DOI synthase GLA obtained from the chromosomal DNA of S. ribosidificus as a probe. The DOI synthase probe was labeled with 32P-dCTP (Perkin-Elmer Life Science, USA) using a random primer labeling kit (Stratagene, USA), purified by gel filtration and used as a probe.

Hybridization was performed in 10 ml of 2 × SSC solution at 65 ° C. for 6 hours. Sequencing was performed in an automated sequencer using dideoxy chain termination method, and the determined sequences were arranged using DNA star program package (DNASTAR, Inc., USA). Possible ORFs (open reading frames) were identified using FramePlot (http://www.nih.go.jp/~jun/cgi-bin/frameplot.pl) and BLAST (http: //www.ncbi.nlm) homology was searched using .nih.gov / BLAST /).

As a result of colony hybridization of the cosmid library of S. ribosidificus and the partial sequence of the DOI synthase, four independent clones were obtained. PCR was performed using primers of SEQ ID NOs: 27 to 30 designed from base sequences of DOI synthase and GLA. As a result, PCR products of 345bp and 270bp size were generated. Among them, fragments larger than 32 kb were named pBRM4 and sequenced.

SEQ ID NO: 27 (DOIS 1): 5'-ACTCSGTSCTSTCSCTSAAGCAGGCS-3 '

SEQ ID NO: 28 (DOIS 2): 5'-CGTGSCCSACSGTGTGSCCGTACT-3 '

SEQ ID NO: 29 (AT 1): 5'-TSGGSGCSGGSGACGAGGTSATC-3 '

SEQ ID NO: 30 (AT 2): 5'-TGSGCCTGSGCGCAGTCCTCGAT-3 '

Example 2. Sequencing of Ribostatamycin Biosynthetic Gene Clusters

A cluster with 26 ORFs in the 31.892kb region of pBRM4 was identified (FIG. 2). The cluster could contain Rbm biosynthetic genes (rbmA, rbmB, rbmC, rbmD , rbmG and rbmH), resistance genes (rbmI and rph) and transport genes (rbmE and rbmF).

All biosynthetic genes were transcribed in the same direction, while resistance genes were located on the side of the biosynthetic gene and transcribed in the opposite direction. All ORFs were using the typical actinomyces codon usage (average 73% G + C content).

(1) Rbm biosynthetic gene

expected amino acid of the protein encoded by the gene sequences of Tbm rbmA TbmA producing strain of S. tenebrarius (accession no. CAE22471) , Gm -producing strain of the M. echinospora GtmA (accession no. BAC41210 ) , and Bn-producing strain B. The homology of circulans with BtrC (accession no. 41210) was 62%, 55% and 37%, respectively. All these proteins are enzymes that catalyze the production of DOI using G-6-P in the presence of NAD ⁺ and Co ²⁺ (FIG. 3).

Other homologues include DHQ (3-dehydroquuinate) syntheses of various organisms. The ¹⁰² LGGGVTGNITGLM ¹¹⁴ sequence of RbmA is consistent with GXXGXXXG, the NAD ⁺ attachment motif found in the DHQ synthase. The results show that RbmA is involved in the generation of DOI.

RbmB encodes a 44.9 kDa, pi 5.45 polypeptide having similarities to GLA of several AmAc producing strains. TbmB (accession no. CAE22472) of S. tenebrarius, GtmB (accession no. CAE06512) of M. echinospora , and BtrS (accession no. 41204) of B. circulans , respectively, showed 66%, 60% and 40% homology. It was. BtrS catalyzes transamimination of DOI, producing 2-deoxy-scyllo-inosamine in the presence of L-glutamine and PLP in the butirosin biosynthetic pathway. The homology indicates that RbmB is involved in transamimination of the RbmA reaction phase upon DOS biosynthesis of S. ribosidificus .

RbmC encodes a polypeptide of 35.5 kDa, pI5.22, which shows 62% and 42% homology with TacD of S. tenebrarius and GacH of M. echinospora , respectively. These proteins are presumed to be involved in the oxidation of glucosamine subunits in the biosynthesis of the neosamine C or analog subunits of Tbm.

RbmG showed homology with the TacD of the Rbm cluster. As the neosamine C subunit does not change at Rbm, dehydrogenation of glucosamine by dihydrogenase or subsequent transamimination with RbmH is expected.

However, no RbmC and RbmG homologues have yet been found in the Bn gene cluster sequence. Like BtrM in Bn clusters, glycosylation of DOS due to RbmD activity is expected. All of these biosynthetic genes are in good agreement with the structure of Rbm.

(2) resistance genes

In the Rbm biosynthetic gene cluster, two resistance genes ( rbmI and rph ) are located on the side of the Rbm biosynthetic gene. The polypeptide encoded by rbmI had homology with AAC (3) of various AmAc producing bacteria. AACC8 (accession no. P29809) in S. fradiae, AACC7 of S. rimosus (accession no. P30180) , showed a Streptomyces griseus of kan (accession no. BAA78619) and a 70% similarity of 58% and 55%, respectively.

Hoshiko et al . Found that the protein encoded by rph phosphorylates Rbm in S. ribosidificus and confers resistance to the host (Hoshiko, S. et al ., Gene , 68: 285, 1988). The homology between the proteins encoded by rbmF and rbmE and several antibiotic transport proteins revealed that these two proteins play a role in transporting Rbm from cells. Some other hypothetical proteins of the Rbm cluster are shown in Table 1.

Example 3. rbmA, btrC  And rbmI Expression Plasmid Construction

In order to identify rbmA encoding DOI synthase, a plasmid was produced to produce DOI synthase (18 kDa) fused to have threonine and histidine at the amino group end. To amplify rbmA from pBRM4 , primers of SEQ ID NOs: 31 and 32 were used.

SEQ ID NO: 31 (RB-DOI-1): 5'-GAGGATCCGGAGGGAACATGCAG-3 '

SEQ ID NO: 32 (RB-DOI-2): 5'-TGCAAGCTTACCGGCTACGGCAG-3 ')

The amplified fragment was digested with Bam HI and Hin dIII and cloned into pET-32a (+) (Novagen, USA) and pIBR25 to construct pBS1 and pBS2, respectively. Recombinant pDOIO-2 for expression of btrC (1.1 kb) was constructed by known methods (Kharel, MK et al ., FEMS Microbiol. Lett ., 230: 185, 2004).

In order to confirm the rbmI, cut to SEQ ID NO: 33 and 34 amplify the rbmI using primers, and Bam HI and Hin dIII was cloned in the pET-32a (+) was constructed by pBS3.

SEQ ID NO: 33 (Rac-1): 5'-TCGGATCCATGGACGAGAGGGAACT-3 '

SEQ ID NO: 34 (Rac-2): 5'-GCAAGCTTCCTCAGGAGCCGTCG-3 '

PCR was carried out for 25 cycles in one cycle of 0.5 minutes at 95 ℃, 1 minute at 55 ℃, 1 minute at 72 ℃. All PCR products were cloned into the vector and sequenced to confirm that there were no mutant sequences.

The obtained pBS1 and pBS3 were introduced into E. coli BL21 (DE3) by heat shock method, and pBS2 was introduced into S. lividans TK24 by protoplasm method.

Example 4. Gene Expression

To express rbmA , E. coli BL21 (DE3) / pBS1 was first shaken for 8 hours using 10 ml LB medium with ampicillin at 37 ° C. The culture solution was transferred to 100 ml of LB medium and cultured at 37 ° C. When the absorbance of the culture solution reached 0.6 to OD ₆₀₀ , IPTG was added to induce the expression of rbmA to reach a final concentration of 0.4 mM, followed by 25 ° C. Incubated for an additional 20 hours. The culture solution was centrifuged at 6000 g for 10 minutes, washed with Tris-HCl buffer (50 mM Tris-HCl, pH7.5, 0.2 mM Co ²⁺ ), and stored at -20 ° C for 6 hours.

In addition, S. lividans TK24 / pBS2 was incubated in R2YE medium with 25 ml of thiostrepton (50 µg / ml) for 3 days, and then 5 ml of the culture solution was added to 250 ml of R2YE medium for 5 days under the same conditions. Incubated. 50 ml of the culture solution was collected at various times (24 hours, 38 hours, 96 hours, and 120 hours), washed with Tris-HCl buffer (50 mM Tris-HCl, pH7.5, 0.2 mM Co ²⁺ ), and then- It was stored at 20 ° C. for 6 hours.

On the other hand, to, E. coli BL21 (DE3) / pBS3 to express the rbmI, to the E. coli BL21 (DE3) / in the same manner as pBS1, additional incubation for 12 hours at 20 ℃ After inducing expression with IPTG Incubated.

Example 5. Purification and Analysis of Enzymes

The cells prepared in Example 4 were dissolved, suspended in Tris-HCl buffer, crushed by ultrasonication, and the supernatant was removed by centrifuging the crushed bacteria at 12,000 g for 20 minutes. RbmI was purified by Ni ²⁺ affinity chromatography (Invistrogen, USA), and then the concentration of the purified protein was determined by the Bradford method (Bradford, MM, Anal. Biochem ., 72: 248, 1976).

RbmA coenzyme solution was obtained from S. lividans TK24 / pBS2 similarly to the above, and used for enzymatic analysis after dialysis. Thereafter, the concentration of CoCl ₂ was maintained at 0.1 mM in all operations of RbmA. Standard DOI was prepared by a modification of the method of Kudo et al . Using BtrC coenzyme (Kudo, F. et al ., J. Antibiot ., 52: 559, 1999).

Example 6. RbmA Function of

The reaction was performed under various conditions on the crude enzyme solution derived from E. coli purified by the method of Example 5, but no DOI formation was observed. This inactivation of RbmA expressed in E. coli is presumably due to poor folding. On the other hand, RbmA expressed in S. ividans TK24 showed the maximum expression in the culture on the 3rd day, the concentration of the target protein was confirmed by SDS-PAGE decreases with the date (Fig. 4).

The reaction product (DOI) was treated with NBHA [O- (4-nitrobenzyl) hydroxylamine hydrochloride] solution containing pyridine (250 µg / ml), and reacted at 72 ° C. for 2 hours to prepare an oxime derivative. The oxime derivatives were separated using a mixture of methanol and chloroform (1: 5) using silica gel column chromatography.

RbmA analysis was performed in the same manner as the BtrC coenzyme analysis method. The reaction product formed a derivative by NBHA and was separated at 280 nm by HPLC (Shimazu, Japan). HPLC analysis was eluted with C-18 column (MIGHTYSIL-RP-18, Japan) at an elution rate of 30 ° C., 1 mL / min with water acidified with acetonitrile and 0.1% trifluoroacetic acid.

DOI synthase activity was confirmed in HPLC purified liquid of the crude enzyme solution obtained from the transformants cultured for 3 days. However, activity could not be observed in the coenzyme solution of S. lividans TK24 / pIBR25 as a control (FIG. 5). The results indicate that rbmA encodes a DOI synthase and is involved in the biosynthesis of the DOS subunit of Rbm in S. ribosidificus .

Example 7 Encoding Aminoglycoside 3-N-acetyl Transferase rbmI

As shown in the homology assay shown in Example 2, rbmI encodes a protein homologous to ACC (3), which protein acetylates Rbm, thereby making S. ribosidificus resistant to Rbm. It is estimated.

, the RbmI in rbmI the overexpressed E. coli and purified in Example 4 to confirm that the function of rbmI. The molecular weight of the purified protein was confirmed to be 49 kDa on SDS-PAGE (FIG. 6), which is consistent with the prediction by sequencing.

RbmI analysis was performed in 100 μl of 50 mM phosphate buffer (pH 7.5) containing 1 mM Coenzyme A (Acetyl-CoA), 0.5 mM AmAc antibiotic, 31.2 μg RbmI. Acetylation was confirmed by thin layer chromatography (TLC). In addition, the antimicrobial susceptibility of AmAcs and acetylated derivatives against Bacillus subtilis was compared in soft agar. RbmI activity was measured using Nn, Km, Rbm, Gm, Apramycin (Apm) and spectinomycin (Spn).

As a result of measuring the antibiotics reacted with RbmI by TLC, acetylation was confirmed in all antibiotics except Gm and Spn (FIG. 7). In addition, all of the acetylated derivatives showed no antimicrobial activity against B. subtilis (FIG. 8).

On the other hand, a result of measuring the in vivo activity in E.coli RbmI (XL1-Blue), E.coli ( XL1-Blue) / pBS3 was a high concentration of Rbm (800㎍ / ㎖), Km (300㎍ / ㎖) , Resistance at Nem (150 μg / ml) and Apm (200 μg / ml). In contrast, E. coli (XL1-Blue) with control pET32a (+) showed sensitivity even at the same antibiotics at concentrations lower than that (50 μg / ml). As expected in the in vitro experiments, E. coli (XL1-Blue) / pBS3 showed sensitivity at low concentrations (30 μg / ml) of Gm and Spn. The results indicate that rbmI encodes a protein that makes S. ribosidificus resistant to Rbm.

Having described the specific part of the present invention in detail, it is obvious to those skilled in the art that such a specific description is only a preferred embodiment, thereby not limiting the scope of the present invention. something to do. Therefore, the substantial scope of the present invention will be defined by the appended claims and their equivalents.

The present invention has the effect of providing a nucleotide sequence of the gene cluster involved in ribostomycin biosynthesis. The present invention also has an effect of providing a DOI synthase that is a ribostamycin biosynthetic enzyme and a gene encoding the same, and an AAC (3) that is a ribostamycin biosynthetic enzyme and a gene encoding the same.

Gene clusters involved in ribostamycin biosynthesis, DOI synthase and AAC (3) according to the present invention can be usefully used for the preparation of ribostamycin and similar novel aminoglycoside based antibiotics.

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Claims (12)

delete delete DOI synthase, which is a ribostamycin biosynthetic enzyme having the amino acid sequence of SEQ ID NO: 16. Gene encoding the DOI synth of claim 3 ( rbmA ). Recombinant vector containing the gene of claim 4. Bacteria transformed with the recombinant vector of claim 5. A method for producing a DOI synthase, comprising culturing the transformed bacterial bacterium of claim 6. Aminoglycoside-N-acetyltransferase [AAC (3)], which is a ribostamycin biosynthetic enzyme having the amino acid sequence of SEQ ID NO: 4. Gene ( rbmI ) encoding the AAC (3) of claim 8 . A recombinant vector containing the gene of claim 9. Bacteria transformed with the recombinant vector of claim 10. A method for producing an aminoglycoside-N-acetyltransferase [AAC (3)], comprising culturing the transformed bacterial bacterium of claim 11.

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