The Kanamycin Biosynthetic Gene Cluster from Streptomyces Kanamyceticus Microbiological Resources and Technology Laboratories, M
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VOL. 57 NO. 5, MAY 2004 THE JOURNAL OF ANTIBIOTICS pp. 351-354 The Kanamycin Biosynthetic Gene Cluster collection which is highly developed for kanamycin from Streptomyces kanamyceticus production, was cultivated in YEME liquid medium16) at 28℃ for 24 hours, after which time, genomic DNA was prepared using an ISOPLANT DNA extraction kit (Wako KOJI YANAI* and TAKESHI MURAKAMI Pure Chemicals, Japan). Genomic DNA was then partially digested with MboI, dephosphorylated with calf intestinal Microbiological Resources and Technology Laboratories, alkaline phosphatase, and subsequently ligated to SuperCos Meiji Seika Kaisha, Ltd., 788 Kayama, Odawara-shi, Kanagawa 250-0852, Japan 1 vector (Stratagene, USA) that had been digested with XbaI, dephosphorylated with bacterial alkaline phosphatase (Received for publication March 5, 2004) and then digested with BamHI. Following packaging of the ligated DNA into the head of λ phage using a Gigapack III in vitro packaging kit (Stratagene, USA), a cosmid library Kanamycin is an aminoglycoside antibiotic produced by was propagated using Escherichia coli XLI-Blue MRA. Streptomyces kanamyceticus1). Although it possesses broad The kmr gene encoding 16S rRNA methyltransferase was range antibacterial activity, bacteria resistant to this amplified by PCR using two primers (forward; 5'- antibiotic readily appear, and therefore its clinical use has ATGTCGCAGTCCGCGTCC-3', reverse; 5'- been restricted to tuberculosis. Resistant bacteria inactivate TCAGCCCTTCGTGACCACG-3'), and employed as a kanamycin by modifying its specific amino- or hydroxy- probe for the screening of the cosmid library using an ECL group using acetyltransferase, phosphotransferase and direct DNA/RNA detection system (Amersham nucleotidyltransferase2). Investigations concerning bacterial Biosciences, USA) according to the manufacturer's resistance mechanisms led to the development of semi- instructions. From approximately 1,000 colonies that were synthetic kanamycin derivatives such as dibekacin3), screened, one positive clone was obtained. As determined amikacin4) and arbekacin5). These derivatives show good from restriction endonuclease and partial nucleotide activities against kanamycin-resistant bacteria and, except sequence analyses of a cosmid pKM9 prepared from the arbekacin, have been used as chemotherapeutic agents positive clone, this cosmid was found to contain only part against resistant bacteria of clinical importance. Arbekacin of the target cluster genes. Then, we carried out has been used as an effective anti-MRSA (methicillin- chromosome walking in order to isolate an additional DNA resistant Staphylococcus aureus) orphan drug in Japan fragment. since 1990. Southern blot analysis of chromosomal DNA revealed Combinatorial biosynthesis, which has mainly dealt with that a left-side DNA fragment of pKM9 (Fig. 1) hybridized investigations concerning polyketide synthases and non- to an approximate 10-kbp SphI fragment. The genomic ribosomal peptide synthetases, is an attractive and potential DNA was completely digested with SphI, and the DNA technology for the production of modified or novel fragments around 10kbp were purified from a low melting- antibiotics6). The identification of biosynthetic gene clusters point agarose gel following electrophoresis. A genomic for other type secondary metabolites can offer additional DNA library containing these DNA fragments was possibilities for combinatorial biosynthesis. In the case of constructed in pUC119 and screened using the DNA aminoglycosides, biosynthetic gene clusters have been fragment described above as a probe. Restriction enzyme isolated and characterized for streptomycin7), fortimicin analysis of 5 positive clones showed that they contained the A8), butirosin9), tobramycin10) and gentamicin (only same SphI fragment. One of these was named pKM95 and sequence data available; AY524043). Concerning used for further analysis. kanamycin, however, molecular biological studies have The nucleotide sequence of the insert of pKM95, been restricted to its resistance genes11-15) and little is including the overlapping region to the NdeI site of pKM9, known about kanamycin biosynthetic genes. In this report consisting of 25,467bp, was determined and has been we describe the isolation of the kanamycin biosynthetic deposited in DDBJ/EMBL/GenBank nucleotide sequence gene cluster from S. kanamyceticus. databases with accession number AB164642. Frame Streptomyces kanamyceticus 21-18, a strain from our analysis of the nucleotide sequence performed using * Corresponding author: [email protected] 352 THE JOURNAL OF ANTIBIOTICS MAY 2004 Fig. 1. Restriction enzyme map of the isolated DNA fragments and schematic representation of the ORFs identified on the kanamycin biosynthetic gene cluster. 1 kbp Table 1. Summary of genes identified on the kanamycin biosynthetic gene cluster. a: ID; % identity, SM; % similarity. VOL. 57 NO. 5 THE JOURNAL OF ANTIBIOTICS 353 Fig. 2. Southern blot analyses of genomic DNAs of the wild type and high-producing strains using the insert DNA of pKM95 (A) and the 4.95-kbp SphI fragment (B) as probes. A: lane1; pKM95 digested with SphI, lane2; genomic DNA of the wild type strain digested with SphI, lane3; genomic DNA of the high-producing strain digested with SphI. B: lanel; pKM9 digested with BamHI, lane2; pKM9 digested with BamHI and NdeI, lane3; pKM9 digested with SphI, lane4; genomic DNA of the wild type strain digested with BamHI, lane5; genomic DNA of the high-producing strain digested with BamHI, lane6; genomic DNA of the wild type strain digested with BamHI and NdeI, lane7; genomic DNA of the high-producing strain digested with BamHI and NdeI, lane8; genomic DNA of the wild type strain digested with SphI, lane9; genomic DNA of the high-producing strain digested with SphI. FramePlot17)revealed that there are 24 open reading frames ECL direct DNA/RNA detection system. When the entire (ORFs) as shown in Fig. 1. The deduced gene products of insert DNA of pKM95 was used as a probe (Fig. 2A), a the individual ORFs were analyzed using the BLAST single 10.3-kbp band corresponding to the probe was program18) (ver. 2.2.8) available through the National detected in both SphI-digested genomic DNA samples. The Center for Biotechnology Information, and the results are intensity of the band is likely stronger with the high- summarized in Table 1. orf19 corresponded to the kmr gene producing strain DNA than with the wild type strain DNA. used as a probe. orf1 corresponded to the aminoglycoside When the 4.95-kbp SphI fragment containing orf20-24 was 6'-N-acetyltransferase gene (kac)11,12) which is one of the used as a probe (Fig. 2B), two bands at positions 9.6- and kanamycin resistance genes previously isolated from S. 10.8-kbp were detected in the BamHI digested genomic kanamyceticus. The product of orf11 showed significant DNA sample of the high-producing strain, where the 10.8- homology to 2-deoxy-scyllo-inosose synthase, a key kbp band corresponded to a BamHI fragment derived from enzyme involved in the biosynthesis of 2- pKM9. On the other hand, only a single band at position deoxystreptamine19). In addition to these genes, several 9.6-kbp was detected in the sample derived from wild type genes likely involved in kanamycin biosynthesis, such as strain, while identical size bands were detected in both aminotransferase and glycosyltransferase genes, were also samples digested with SphI or double-digested with BamHI found in the gene cluster. These facts indicated that the and NdeI. These results indicated that the high-producing isolated gene cluster constitutes at least part of the strain had another copy of the biosynthetic gene cluster, the kanamycin biosynthetic gene cluster, although further right-side end of which was different from the original analyses, such as gene disruption and heterologous cluster present in the wild type strain. Although cosmid expression, would be required for the final confirmation. clone pKM9 had the insert derived from the high-producing The genomic DNAs of the wild type strain (NBRC strain specific gene cluster given that a 10.8-kbp band 13414) and a high-producing strain 12-1, which is a parent corresponding to the high-producing strain specific gene of the high-producing strain 21-18 described above, were cluster was detected in the BamHI digested sample of compared by Southern blot analysis using the DNA pKM9, the nucleotide sequence determined in this study fragments contained in the isolated cluster as probes and an was based on the common region between the original and 354 THE JOURNAL OF ANTIBIOTICS MAY 2004 high-producing strain specific gene clusters, from SphI to biosynthetic gene cluster from Bacillus circulans. J. NdeI. The increase in copy number of the biosynthetic gene Antibiotics 53: 1158-1167, 2000 10) KHAREL, M. K.; D. B. BASNET, H. C. LEE, K. LIOU, J. cluster in the high-producing strain, although it remains S. WOO, B. G. KIM & J. K. SOHNG: Isolation and unknown whether the entire cluster is present in an extra characterization of the tobramycin biosynthetic gene copy, might account for the improved production of cluster from Streptomyces tenebrarius. FEMS Microbiol. kanamycin. Lett. 230: 185-190, 2004 11) MURAKAMI, T.; C. NOJIRI,H. TOYAMA, E. HAYASHI, K. KATUMATA, H. ANZAI, Y. MATSUHASHI, Y. YAMADA & K. NAGAOKA: Cloning of antibiotic-resistance genes in Acknowledgment Streptomyces. J. Antibiotics 36: 1305-1311, 1983 12) MATSUHASHI, Y.; T. MURAKAMI, C. NOJIRI, H. TOYAMA, We are greatly indebted to Dr. S. HOSHIKO