Tobramycin is an important broad spectrum ami-noglycoside antibiotic widely used against severe Gram-negative bacterial infections. It is produced by base-catalyzed hydrolysis of carbamoyltobramy-cin (CTB) generated by S. tenebrarius. We herein report the construction of a genetically engineered S. tenebrarius for direct fermentative production of tobramycin by disruption of aprK and tobZ. A unique putative NDP-octodiose synthase gene aprK was disrupted to optimize the production of CTB, resulting in the blocking of apramycin biosynthesis and the obvious increase in CTB production of aprK disruption mutant S. tenebrarius ST316. Additional mutation on the carbamoyltransferase gene tobZ in S. tenebrarius ST316 generated a strain ST318 that produces tobramycin as a single metabolite. ST318 could be used for industrial fermentative produc-tion of tobramycin.

Key words: carbamoyltobramycin; genetic engi-neering; Streptomyces tenebrarius; tobramycin

Introduction

　Streptomyces tenebrarius is an important industrial microorganism, producing a series of aminoglycoside antibi-otics that mainly consist of apramycin, carbamoyltobramy-cin (CTB) and carbamoylkanamycin B (CKB). Tobramycin and kanamycin B are produced by S. tenebrarius as minor constituents (Park et al., 2010), CTB and CKB are biosyn-thesized by the O-carbamoylation reaction of tobramycin and kanamycin B, respectively (Parthier et al., 2012). Tobramycin is a broad spectrum aminoglycoside antibiotic used against various types of bacterial infections, particu-larly Gram-negative infections. It is the preferred bacteri-

cidal agent for the prevention and treatment of Pseudomonas aeruginosa pulmonary infections in patients with cystic fibrosis. Currently, tobramycin shows the potency of suppressing premature translation termination, which is the molecular basis of many genetic diseases (Altamura et al., 2013).　Commercial-scale production of tobramycin is carried out with base-catalyzed hydrolysis of CTB. Thus, when S. tenebrarius is used for tobramycin production, apramycin and other components are all undesired byproducts. The biosynthesis of byproducts, especially the biosynthesis of apramycin, not only reduced the yield of carbamoyltobra-mycin, but also caused trouble to the subsequent purification process (Borodina et al., 2005). Furthermore, in the tobramy-cin manufacturing process, neutralization of the base with acid might hydrolyze tobramycin to nebramine and kanosamine, while kanamycin B might be hydrolyzed to neamine and kanosamine (Manyanga et al., 2013). Kanamy-cin B, nebramine, neamine and kanosamine are four known impurities, which are often responsible for side effects. Due to the similar properties of these compounds, it is very difficult to purify tobramycin effectively, which leads to a decrease in the product yield and an increase in the produc-tion costs. Consequently, it is imperative to develop more effective approaches to replace the traditional method of chemical modification for tobramycin.　Biosynthetic studies of aminoglycoside antibiotics have progressed remarkably during the past decade (Kudo et al., 2009a; Park et al., 2013). In particular, the advances in biosynthetic enzymes in neomycin and butirosin biosynthet-ic pathways have greatly facilitated the biochemistry and molecular genetics studies of aminoglycosides in S. tenebrarius (Kudo et al., 2009b). The tobramycin biosyn-thetic gene cluster (tob-cluster) responsible for the biosyn-thesis of CTB and CKB has been cloned (Kharel et al., 2004. GenBank accession number AJ579650; Piepersberg. GenBank accession number AJ810851). In tob-cluster,

Full Paper

Concentrated biosynthesis of tobramycin by genetically engineered Streptomyces tenebrarius

(Received March 20, 2014; September 20, 2014)

Jianping Xiao, Hui Li, Shuping Wen, and Wenrong Hong*College of Biological Science and Technology, Fuzhou University, Fuzhou, Fujian 350116, China

＊ Corresponding author: Dr. Wengrong Hong, College of Biological Science and Technology, Fuzhou University, Xue Yuan Road 2, University Town, Fuzhou, Fujian 350116, China.E-mail: hongwr56@163.com

None of the authors of this manuscript has any financial or personal relationship with other people or organizations that could inappropriately influence their work.

J. Gen. Appl. Microbiol., 60, 256‒261 (2014)doi 10.2323/jgam.60.256©2014 Applied Microbiology, Molecular and Cellular Biosciences Research Foundation

Concentrated biosynthesis of tobramycin by Streptomyces tenebrarius 257

tobS1 coding for L-glutamine: 2-deoxy-scyllo-inosose aminotransferase (Kharel et al., 2005), tobQ coding for 6′-dehydrogenase (Yu et al., 2008), tobM2 coding for 6-glucosyltransferase (Park et al., 2011) and tobZ (also termed tacA) coding for O-carbamoyltransferase (Parthier et al., 2012) have been characterized. The apramycin biosyn-thetic gene cluster (apr-cluster) was also isolated from S. tenebrarius (Piepersberg. GenBank accession number AJ629123). In this gene cluster, aprD3 and aprD4, which are responsible for C3′ deoxygenation of pseudodisaccha-rides, have been functionally characterized through gene disruption in vivo (Ni et al., 2011) and recombinant protein expression in vitro (Park et al., 2011). These advances greatly improved our understanding of the antibiotic biosyn-thesis in S. tenebrarius and eventually allowed us to manipu-late the biosynthetic pathway for direct fermentative produc-tion of tobramycin.　In 2012, we reported the mutant strain T106 (ΔaprH-M), in which total antibiotic production capacity was seriously decreased, while CTB was primarily produced without synthesizing apramycin (Hong et al., 2012). However, it is

of great significance to find the key genes of apramycin biosynthesis and construct a tobramycin-overproducing strain. A unique putative NDP-octodiose synthase gene, AprK, is located in the apr-cluster, which plays a critical role in the formation of the octodiose moiety in apramycin. The inactivation of aprK may block the biosynthesis of apramy-cin and direct the metabolite flux toward CTB instead of apramycin. Moreover, in the biosynthesis of CTB, the process for converting tobramycin to CTB is catalyzed by carbamoyltransferase encoded by tobZ in the tob-cluster (Fig. 1). In this study, we described the construction of a single-compound tobramycin producing a mutant strain of S. tenebrarius by inactivation of aprK and tobZ. Therefore, we could directly produce tobramycin by the application of this mutant strain.

Materials and Methods

　Bacterial strains and plasmids.　 The strains and plasmids used in this work are listed in Table 1. E. coli DH5α was used as a host for plasmid construction. E. coli ET12567/pUZ8002 was used for transferring DNA from E. coli to Streptomyces by conjugation. E. coli-Streptomyces shuttle vector pKC1139 was used to construct a recombinant plasmid for gene disruption (Flett et al., 1997). The plasmid pAGe was donated by Dr. Shao, L. (Shanghai Institute of Pharmaceutical Industry, Shanghai). S. tenebrarius Tt-49 (Tt-49) was stored in our laboratory and used as wild-type strain. The mutant strains S. tenebrarius ST316 and S. tenebrarius ST318 were constructed in this study.　Media and culture conditions.　 E. coli was used as a cloning host, grown in Luria-Bertani (LB) liquid or solid medium (Sambrook and Russell, 2001). Mannitol soya flour (MS) medium was used for the conjugation, and yeast extract-malt extract (YEME) medium was used for Strepto-myces mycelium formation (Kieser et al., 2000). Tt-49 and the mutant strain were grown on agar slant medium, at 37°C for sporulation. The seed medium contained glucose 0.5%, corn flour 2%, cornstarch 2%, soya bean meal 1.5%, KCl 0.1%, CaCl2 0.025%, KH2PO4 0.05%, and MgSO4 0.5%, pH 7.4. The fermentation medium consisted of glucose 1.5%, cornstarch 2.0%, soya bean meal 3.5%, bean oil 1.0%, (NH4)2SO4 0.7%, CaCO3 0.7%, ZnSO4 0.01%, and MgSO4

Fig. 1.　 Proposed biosynthetic pathway of tobramycin and apramy-cin.

Table 1.　Strains and plasmids used in this study.

Strains or plasmids Relevant characteristics Reference or source

E. coli DH5α F-, φ80d/lacZΔM15, Δ(lacZYA-argF)U169, deoR, recA1, endA1, hsdR17(rk- mk+), phoA, upE44, λ-, thi-1, gyrA96, relA1

Invitrogen

E. coli ET12567 Methylation defective, strain used in E. coli-Streptomyces intergeneric conjugation Senthamaraikannan et al. (2003)Bacillus subtilis 168 Indicator strain for antibiotics bioassay Stored in our lab.S. tenebrarius Tt-49 Wild-type strain, mainly producing apramycin, CTB and CKB Yushuang et al. (2008)S. tenebrarius ST316 aprK gene deletion mutant of S. tenebrarius Tt49 This studyS. tenebrarius ST318 aprK and tobZ gene deletion mutant of S. tenebrarius Tt49 This studypAGe pUC19 derivative containing ermE, AmpR, and EryR Wenrong et al. (2012)pKC1139 lacZα, oripUC19, oriTRK2, oripsG5, AmR Fiona et al. (1997)pBK3 pKC1139 derivative containing aprK upstream fragment, AmR This studypBK4 pBK3 derivative containing aprK downstream fragment, AmR This studypBK5 pBK4 derivative containing ermE, AmR, and EryR This studypBZ3 pKC1139 derivative containing tobZ upstream fragment, AmR This studypBZ4 pBZ3 derivative containing tobZ downstream fragment, AmR This studypBZ5 pBZ4 derivative containing ermE. AmR, EryR This study

　AmpR, ampicillin resistant; AmR, apramycin resistant; EryR, erythromycin resistant.

XIAO et al.258

1.1%, pH 7.0‒7.2. For fermentation, an agar piece of about 1 cm2 was inoculated into a 250 ml flask containing 50 ml of the seed medium and incubated at 37°C, 300 rpm for 16 h. A 250 ml flask containing 50 ml of the fresh fermentation medium was then inoculated with 5 ml of the seed culture, and incubation was continued at 37°C and 300 rpm for 120 h (Hong et al., 2012). Ampcillin (Am, 100 μg ml-1), apramy-cin (Apr, 50 μg ml-1), kanamycin (Km, 25 μg ml-1), and chloramphenicol (Cm, 25μg ml-1) were added to LB medium; erythromycin (Ery, 50 μg ml-1) and nalidixic acid (Na, 25 μg ml-1) were added to MS medium when necessary.　DNA manipulation.　 DNA isolation and manipulation in E. coli were carried out according to standard methods (Sambrook and Russell, 2001); the preparation of chromo-somal DNA from S.tenebrarius and the E.coli-Streptomyces intergeneric conjugation were described previously (Kieser et al., 2000). Polymerase and restriction endonucleases were purchased from TaKaRa Biotechnology (Dalian, China) and used according to the product manufacturer. PCR amplifica-tions were carried out on an authorized thermal cycler (S1000, Bio-Rad, Hercules, CA). All primers used in this work are listed in Table 2. Primers synthesis and DNA sequencing were performed at Sangon Biotech (Shanghai, China).　Construction of aprK recombinant plasmid.　 The plasmid pBK5 used for the disruption of aprK was constructed as follows: Firstly, a 1,991-bp DNA fragment (upstream homologous exchange arm BK1) that contains the upstream and the first 108 bp sequence of aprK was amplified from genomic DNA of Tt-49 by PCR using the primers P1 and P2. The PCR product was digested with EcoRI and XbaI and cloned into the XbaI and EcoRI restriction sites of pKC1139 to generate pBK3. Subsequently, primers P3 and P4 were used to amplify a 2,057-bp fragment (downstream homolo-gous exchange arm BK2) containing the last 63-bp sequence of aprK and the downstream sequence, BK2, was digested with XbaI and HindIII. The obtained XbaI and HindIII fragment was ligated into pBK3 cut with the same enzymes

to generate pBK4. Finally, the ermE gene was rescued from pAGe as a 1,746-bp fragment and inserted into the unique EcoRI site of pBK4 to generate recombinant plasmid pBK5.　Construction of tobZ recombinant plasmid.　 The recombi-nant plasmid pBZ5 for dispuption of tobZ was constructed as follows: Primers P11 and P12 were used to amplify a 2,030-bp tobZ upstream fragment from genomic DNA of Tt-49; the PCR product was digested with HindIII/XbaI and was ligated into the pKC1139 cut with the same enzymes to generate pBZ3. A 2,022-bp tobZ downstream fragment amplified with the primers P13 and P14, and was digested with XbaI and EcoRI; the resulting XbaI and EcoRI fragment was cloned into the same sites of pBZ3 to generate pBZ4. Finally, the ermE gene was rescued from pAGe as a 1,746-bp EcoRI fragment and inserted into the unique EcoRI site of pBZ4 to generate recombinant plasmid pBZ5.　Construction of aprK disruption strain.　The recombi-nant plasmid pBK5 was transferred into E. coli ET12567 (pUZ8002) to obtain unmethylated DNA, and then introduced into Tt-49 by conjugation with Ery as the resistance selection marker. After incubation on MS medium at 37°C for 4 days, the grown colonies that conferred Ery resistance were harvested. Because pBK5 bears a tempera-ture-sensitive replication origin which is unable to replicate at temperatures above 34°C, the grown colonies at 37°C were identified as conjugants in which the recombinant plasmid had integrated into the Tt-49 chromosome through single-crossover recombination. The single-crossover mutants were cultured in plates without Ery for three genera-tions. Clones were printed onto plates with and without erythromycin for screening; then the erythromycin-sensitive (EryS) strains were selected. These strains with EryS exhibit-ed the same phenotypes. They were wild-type revertant strains or aprK gene disruptants.　To obtain the desired double-crossover mutant, PCR analysis of the genomic DNA from EryS strains was performed using primers P5 and P6. PCR amplification with primers P5/P6 in the aprK disruptant chromosome would

Table 2.　Primers used in this study.

Primer Sequencea Restriction enzyme

P1 5′-CCGGAATTCCAACCAGGTCCCCGTCTACC-3′ EcoRIP2 5′-CTAGTCTAGAAGGAGGTCGAGGCGGTCAAC-3′ XbaIP3 5′-CTAGTCTAGACTTCGTGGGAGCTGACACTGG-3′ XbaIP4 5′-CCCAAGCTTACCGAGTTCCTCAGGACGCT-3′ HindIIIP5 5′-CCTGGTCGACGAGT CGAAG-3′P6 5′-GTCTACCAACCGATGTCGCG-3′P7 5′-AACGAGATCGACGCGGACGC-3′P8 5′-TTGACCTTG ACGCCGTCCGC-3′P9 5′-ACAGCTCCTGCTTGACCACC-3′P10 5′-ACTCGGTCCTGTCGCTGAAG-3′P11 5′-CCCAAGCTTCTGCGTTGTCCACTGTGGA-3′ HindIIIP12 5′-CTAGTCTAGATGTCTCTACCTCCGATGGGGAAG-3′ XbaIP13 5′-CTAGTCTAGATAGGCCACCACGCGTCAA-3′ XbaIP14 5′-CCGGAATTCAGGTGCCGACGAGGTTCT-3′ EcoRIP15 5′-CCGACATCCTCACCAAGGCA-3′P16 5′-TTGTAGGCG GCGAAGTCCCT-3′P17 5′-GTCTCCAAGGGACTGGCCAA-3′P18 5′-TTGTAGGCGG CGAAGTCCCT-3′P19 5′-ACCGACATCCTCACCAAGGC-3′P20 5′-ACCGACATCCTCACCAAGGC-3′

　aRestriction enzyme site is indicated by single underline.

Concentrated biosynthesis of tobramycin by Streptomyces tenebrarius 259

generate a 1,020-bp fragment which was replaced with a 1,420-bp fragment in wild-type revertant strains. Primers P7 and P8 and Primers P9 and P10 were designed for further identification of the desired double-crossover mutant; a 2,454-bp and a 2,532-bp fragment would be generated by PCR amplification with P7/P8 and P9/P10 respectively.　Construction of tobZ disruption strain.　 After the aprK gene disruption mutant strain was acquired, the carbamoyl-transferase gene tobZ was also disrupted using the same method. The PCR analysis of the genomic DNA from EryS

strains was performed using primers P15 and P16. PCR amplification with primers P15/P16 in the tobZ disruptant chromosome would generate a 794-bp fragment which was replaced with a 2,507-bp fragment in the aprK disruptant mutant strain. Primers P17 and P18 and Primers P19 and P20 were designed for further identification of the desired double-crossover mutant; a 2,545-bp and a 2,606-bp fragment would be generated by PCR amplification with P17/P18 and P19/P20 respectively.　Extraction and analysis of fermentation products.　 The pH of the fermentation broth was adjusted to 1.5 with concentrated sulfuric acid for 30 min and then readjusted to 6.5 using NaOH. The broth was centrifuged at 12,000 rpm for 15 min. The supernatant was processed by strongly acidic resin 732-NH4

+ to adsorb aminoglycosides. After 6 h of adsorption, the attached aminoglycosides were eluted with 5% NH4OH, followed by evaporation to remove NH4OH. The pH was adjusted to 7.0 with sulfuric acid.　The supernatant of the fermentation broth was used directly for bioassay with Bacillus subtilis 168 by agar diffusion. The purified products were analyzed by HPLC with a C18 column (Shimadzu, Osaka, Japan). The mobile phase consisted of sodium heptanesulfonate-sodium sulfate solution and methanol (v⁄v, 85 : 15), with an elution speed of 1.0 ml min-1 and a 30°C column temperature. The precise composition of elution fractions was futher determined by electrospray ionization mass spectrometry (ESI/MS) analysis. The Q-TOF-MS scan range was set at m/z 100‒800 in positive ion mode. The drying gas (N2) flow rate was set at 8.0 L/min, drying gas temperature at 350, nebulizer at 35 psig, capillary voltage at 3,500 V, and fragmentor at 135 V. Data analysis was performed using Agilent Masshunter (B.04.00).

Results

Enhancing the production of CTB by gene inactivation of aprK　In order to obtain an engineering strain that mainly produces CTB, the recombinant plasmid pBK5 containing the upstream and downstream sequences of aprK and ermE was constructed and successfully introduced into S. tenerar-ius Tt-49 by conjugation to inactivate aprK. The EryR conjugants were obtained and cultivated for 5 generations of sporulation in the absence of Ery. A desired double-crossover mutant of aprK disruption was screened by PCR analysis with primers P5/P6, and was further identified with primers P7/P8 and P9/P10 (Fig. 2B). PCR amplification products were subjected to sequencing analysis; the result showed that the internal 396 bp fragment of aprK was deleted. The desired double-crossover mutant was denoted by S. tenebrar-

ius ST316 (Fig. 2A).　Both the mutant strain ST316 and wild-type strain Tt-49 were cultivated under identical conditions for 96 h for antibi-otic fermentation. The fermentation broth was subsequently purified, and analysis for antibiotic components was performed.　The HPLC analysis of Tt-49 and ST316 metabolites is shown in Fig. 3. As shown in Fig. 3A, peak 1 represents apramycin, peak 2 represents tobramycin, and peak 3 represents CTB, while in Fig. 3B, a peak of apramycin was not observed, indicating that the mutant strain ST316 mainly produced CTB. It also showed that the proportion of CTB accounting for the total product had increased significantly from 36.9% in Tt-49 to 75.4% in ST316, and the proportion of tobramycin had increased from 15.2% to 24.6%. MS analysis revealed that apramycin (m/z 540.292) could not be detected in the fermentation products of ST316, while CTB (m/z 511.276) and apramycin (m/z 540.292) could be detect-ed in Tt-49 (Fig. 3D and E). This observation was consistent with the results of the HPLC analysis. Furthermore, the result of antimicrobial activity assay showed that the total antibiotics titer of the wild-type strain was 5,000 µg ml-1; a lower titer of 3,750 µg ml-1 was obtained in ST316, while 1,900 µg ml-1 was obtained in the previous mutant strain T106.

Concentrated biosynthesis tobramycin by gene inactivation of tobZ　After the construction of ST316 by inactivation of aprK, the carbamoyltransferase gene tobZ was further disrupted from ST316 by the same strategy. The desired double-crossover mutant was selected by PCR analysis with primers P15/P16 and identified with primers P17/P18 and P19/P20 (Fig. 2C). The tobZ disruption mutant was denoted by S. tenebrarius ST318 (Fig. 2A).　ST318 was fermented and the resulting products were

Fig. 2.　Targeted gene inactivation in Tt-49.　(A) Genotype of Tt-49 and its recombinational strains. (B) PCR analysis of the genomic DNA from Tt-49, using primers P5/P6 (lane 1); PCR analysis of the genomic DNA from ST316, using primers P5/P6 (lane 2), P7/P8 (lane 3), and P9/P10 (lane 4). (C) PCR analysis of the genomic DNA from ST316, using primers P15/P16 (lane 5); PCR analysis of the genomic DNA from ST318, using primers P15/P16 (lane 6), P17/P18 (lane 7), and P19/P20 (lane 8).

XIAO et al.260

analyzed. The results of HPLC and MS analyses (Fig. 3C and F) showed that CTB was undetectable in ST318, and the proportion of tobramycin was greatly increased. This confirmed that ST318 only produced tobramycin.

Investigation of the stability of the mutant strain ST316 and ST318　The mutant strains ST316 and ST318 showed no notice-able difference in growth phenotype from the wild-type strain; the colony shape, color of spores and the mycelia growth were all identical to those of the wild-type strain. The mutant strains ST316 and ST318 were further ferment-ed for five times to investigate the changes in products and productivity; the wild-type strain was used as a control under identical conditions. The result of bioassay showed that the mutant strains were also stable in production titer, and the average total antibiotics titer of ST318 was close to 4,000 µg ml-1.

Discussion

　With the development of Streptomyces genetics, genetic engineering has become a viable approach for the optimiza-tion of antibiotic biosynthesis in S. tenebrarius. A kanamy-cin B-overproducing strain was constructed by gene inacti-vation of aprD3-D4 and tacA (Ni et al., 2011), in which the biosynthesis of apramycin and CTB were both blocked. An apramycin-overproducing strain was also constructed by inactivation of tobS1-C, whose gene products were predict-ed to be a 2-deoxy-scyllo-inosose aminotransferase and a 2-deoxy-scyllo-inosose synthase (Lin and Hong, 2012). In

this study, we successfully obtained a stable single-compound tobramycin-producing strain of ST318 by inacti-vation of aprK and tobZ. As far as we know, this is the first reported case of using genetic engineering technology to acquire a single-compound tobramycin-producing strain.　By comparing the biosynthetic pathways of apramycin and CTB, we can see that they primarily share the same biosynthetic pathway for 2-DOS, but ramify into different pathways; one is for apramycin biosynthesis, while the other is for CTB biosynthesis. The NDP-octodiose synthase gene aprK is an essential gene in the apramycin tributary biosyn-thesis pathway, as elucidated by gene inactivation in this study. The apramycin biosynthesis was blocked and the proportion of CTB was increased substantially when aprK was disrupted. When the aprK gene was disrupted, the common intermediate 2-DOS was unable to be incorporated into the biosynthesis of apramycin, and the secondary metabolic intermediate turned to the CTB biosynthetic pathway.　In contrast to the previous mutant strain T106 (ΔaprH-M), we found an increase of 97.4% in the total antibiotic production capacity of ST316. This might be due to two factors. The mutant strain T106, obtained through gene replacement, had introduced the exogenous gene tct, which might affect the biosynthesis of CTB. On the other hand, apart from aprK, multiple genes were also disrupted simulta-neously in the mutant strain T106, some of which might play a role in the biosynthesis of CTB.　To obtain a singlecompound tobramycin-producing strain, the carbamoyltransferase gene, tobZ, was disrupted since it catalyzes an ancient enzymatic reaction for the biosynthesis

Fig. 3.　 HPLC analysis and MS analysis of metabolites from Tt-49, ST316 and ST318.　(A) Liquid chromatography of extract from wild-type strain Tt-49. (B) Liquid chromatography of extract from aprK disruption mutant strain ST316; (C) Liquid chromatography of extract from tobZ disruption mutant strain ST318. (D) Mass spectra of extract from wild-type strain Tt-49. (E) Mass spectra of extract from aprK disruption mutant strain ST316; (F) Mass spectra of extract from tobZ disruption mutant strain ST318.

Concentrated biosynthesis of tobramycin by Streptomyces tenebrarius 261

of CTB. When the mutant strain ST318 is applied in industri-al production, the elimination of apramycin will simplify the subsequent purification. Furthermore, the base-catalyzed hydrolysis of CTB will be omitted, which not only greatly simplifies the tobramycin manufacturing process, but also prevents the generation of byproducts such as neamine, kanosamine and nebramine.

Acknowledgments

　This study was supported by the National Natural Science Founda-tion of China (No. 3107009) and China National Key Hi-Tech Innova-tion Project for the R&D of Novel Drugs (No. 2012ZX09201101-008).

References

Altamura, N., Castaldo, R., Finotti, A., Breveglieri, G., Salvatori, F. et al. (2013) Tobramycin is a suppressor of premature termination codons. J. Cyst. Fibros., 12, 806‒ 811.

Borodina, I., Scholler, C., Eliasson, A., and Nielsen, J (2005) Metabol-ic network analysis of Streptomyces tenebrarius, a Streptomyces species with an active Entner-Doudoroff pathway. Appl. Environ. Microb., 71, 2294‒ 2302.

Flett, F., Mersinias, V., and Smith, C. P. (1997) High efficiency inter-generic conjugal transfer of plasmid DNA from Escherichia coli to methyl DNA-restricting Streptomycetes. FEMS Microbiol. Lett., 155, 223‒ 229.

Hong, W. and Yan, S. (2012) Engineering Streptomyces tenebrarius to synthesize single component of carbamoyltobramycin. Lett. Appl. Microbiol., 55, 33‒ 39.

Kharel, M. K., Basnet, D. B., Lee, H. C., Liou, K., Woo, J. S. et al. (2004) Isolation and characterization of the tobramycin biosyn-thetic gene cluster from Streptomyces tenebrarius. FEMS Micro-biol. Lett., 230, 185‒ 190.

Kharel, M. K., Subba, B., Lee, H. C., Liou, K., and Sohng, J. K. (2005) Characterization of L-glutamine: 2-deoxy-scyllo-inosose amino-transferase (tbmB) from Streptomyces tenebrarius. Bioorg. Med. Chem. Lett., 15, 89‒ 92.

Kieser, T., Bibb, M. J., Buttner, M. J., Chater, K. F., and Hopwood, D. A. (2000) Practical streptomyces genetics, The John Innes Foundation, Norwich.

Kudo, F. and Eguchi, T. (2009a) Biosynthetic genes for aminoglyco-side antibiotics. J. Antibiot., 62, 471‒ 481.

Kudo, F. and Eguchi, T. (2009b) Biosynthetic enzymes for the amino-glycosides butirosin and neomycin. Method. Enzymol., 459, 493‒ 519.

Lin, Y. and Hong, W. (2012) Construction of Streptomyces tenebrarius mutant with knock-out of tobS1-C genes. J. Chin. Pharm. Univ., 43, 92‒ 96.

Lin, Y., Hong, W., Lin, Y., and Liang, Z. (2008) Study on the breeding and fermentation characteristics of tobramycin by Streptomyces tenebrarius. Chin. J. Antibiot., 33, 442‒ 445.

Manyanga, V., Elkady, E., Hoogmartens, J., and Adams, E. (2013) Improved reversed phase liquid chromatographic method with pulsed electrochemical detection for tobramycin in bulk and pharmaceutical formulation. J. Pharmaceut. Anal., 3, 161‒ 167.

Ni, X., Li, D., Yang, L., Huang, T., Li, H. et al. (2011) Construction of kanamycin B overproducing strain by genetic engineering of Streptomyces tenebrarius. Appl. Microbiol. Biot., 89, 723‒ 731.

Parthier, C., Gorlich, S., Jaenecke, F., Breithaupt, C., Brauer, U. et al. (2012) The O-carbamoyltransferase TobZ catalyzes an ancient enzymatic reaction. Angew. Chem. Int. Edit., 51, 4046‒ 4052.

Paranthaman, S. and Dharmalingam, K. (2003) Intergeneric conjuga-tion in Streptomyces peucetius and Streptomyces sp. strain C5: Chromosomal integration and expression of recombinant plasmids carrying the chiC gene. Appl. Environ. Microb., 69, 84‒ 91.

Park, J. W., Park, S. R., Han, A. R., Ban, Y. H., Yoo, Y. J. et al. (2010) The nebramycin aminoglycoside profiles of Streptomyces tenebrarius and their characterization using an integrated liquid chromatography-electrospray ionization-tandem mass spectro-metric analysis. Anal. Chim. Acta, 661, 76‒ 84.

Park, J. W., Park, S. R., Nepal, K. K., Han, A. R., Ban, Y. H. et al. (2011) Discovery of parallel pathways of kanamycin biosynthesis allows antibiotic manipulation. Nat. Chem. Biol., 7, 843‒ 852.

Park, S. R., Park, J. W., Ban, Y. H., Sohng, J. K., and Yoon, Y. J. (2013) 2-Deoxystreptamine-containing aminoglycoside antibiotics: Recent advances in the characterization and manipulation of their biosynthetic pathways. Nat. Prod. Rep., 30, 11‒ 20.

Sambrook, J. F. and Russell, D.W. (2001) Molecular Cloning: A Laboratory Manual, 3rd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.

Yu, Y., Hou, X., Ni, X., and Xia, H. (2008) Biosynthesis of 3′-deoxy-carbamoylkanamycin C in a Streptomyces tenebrarius mutant strain by tacB gene disruption. J. Antibiot., 61, 63‒ 69.