Abstract
Genes homologous to 2-deoxystreptamine (DOS) biosynthetic genes were isolated from aminoglycoside producers, Micromonospora and Streptomyces spp., using PCR primers based on the core sequences of 2-deoxy-scyllo-inosose (DOI) synthase and l-glutamine: scyllo-inosose aminotransferase (GIA). Identities of 40–45% were observed for DOI synthases, and 65–75% were observed for GIAs. The gene cluster of tobramycin biosynthesis was isolated from the genomic library of Streptomyces tenebrarius using DOI synthase as a probe. Sequencing of 33.9 kb revealed 24 putative open reading frames including the tobramycin biosynthetic gene cluster (13.8 kb) and a transport protein. This cluster encodes proteins homologous to 2-deoxystreptamine biosynthetic enzymes, glycosyltransferase and other aminocyclitols biosynthetic enzymes. Sequence analysis revealed the evolution of DOI synthases from 3-dehydroquinate (DHQ) synthases in actinomycetes. DOI synthases and GIA are therefore useful for cloning biosynthetic genes of DOS-containing aminocyclitol antibiotics or for screening such metabolites producers.
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References
Ahilert J, Distler J, Mansouri K, Pipersberg W (1997) Identification of stsC, the gene encoding the L-glutamine-scyllo-inosose aminotransferase from streptomycin producing actinomycetes. Arch. Microbiol. 168: 102–113.
Cho J, Rando RR (1999) Specificities in the binding of aminoglycosides to HIV-RRE RNA. Biochemistry 38: 8548–8554.
Jeanmougin F, Thompson, JD, Gouy M, Higgins DG, Gibson TJ (1998) Multiple sequence alignment with clustal X. Trends Biochem. Sci. 23: 403–405.
Kakinuma, K, Ogawa Y, Sasaki T, Seto H, Otake N (1989) Mechanism and stereochemistry of biosynthesis of 2-deoxystreptamine and neosamine C. J. Antibiotics 42: 926–933.
Kiesser T, Bibb MJ, Buffner J, Charter KF, Hopwood DA (2000) Practical Streptomyces Genetics. Norwich, UK: John Innes Foundation, pp. 161–171.
Koch KF, Davis FA, Rhoades JA (1973) Nebramycin: separation of the complex and identification of factors 4, 5 and 5′. J. Antibiotics 12: 745–751.
Kudo F, Tamegai H, Fusiwara T, Tamagi U, Hirayama K, Kakinuma K (1999) Molecular cloning of the gene for the key carbocycleforming enzye in the biosynthesis of 2-deoxystreptaminecontaining aminocyclitol antibiotics and its comparison with dehydroquinate synthase. J. Antibiotics 52: 559–571.
Ota Y, Tamegai H, Kudo F, Kuriki H, Takeshita-Koike A, Eguchi T, Kakinuma K (2000) Butiurosin-biosynthetic gene cluster from Bacillus circulans. J. Antibiotics 53: 1158–1167.
Sambrook, J, Russel DW (2001) Molecular Cloning, 3rd edn., Cold Spring Harbor, New York: Cold Spring Harbor Laboratory Press.
Tamegai H, Nango E, Kuwahara M, Yamamoto H, Ota Y, Kuriki H, Eguchi T, Kakinuma K (2002) Identification of L-glutamine:2-deoxy-scyllo-inosose aminotransferase required for the biosynthesis of butorisin in Bacillus circulans. J. Antibiotics 55: 707–714.
Yamauchi N, Kakinuma K (1992) Confirmation of in vitro synthesis if 2-deoxy-scyllo-inosose, the earliest intermediate in the biosynthesis if 2-deoxystreptamine, using cell free preparations of Streptomyces fradiae. J. Antibiotics 45: 774–780.
Yamauchi N, Kakinuma K (1995) Enzymatic carbocycle formation in microbial secondary metabolism. The mechanism of 2-deoxy-scyllo-inosose synthase reaction as a crucial step in the 2-deoxystreptamine biosynthesis in Streptomyces fradiae. J. Org. Chem. 60: 5614–5619.
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Kharel, M.K., Subba, B., Lee, H.C. et al. An approach for cloning biosynthetic genes of 2-deoxystreptamine-containing aminocyclitol antibiotics: isolation of a biosynthetic gene cluster of tobramycin from Streptomyces tenebrarius . Biotechnology Letters 25, 2041–2047 (2003). https://doi.org/10.1023/B:BILE.0000007063.66429.90
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DOI: https://doi.org/10.1023/B:BILE.0000007063.66429.90