Abstract
Brasilicardin A (BCA), produced by Nocardia brasiliensis IFM 0406 (currently referred to as N. terpenica), has a unique structure consisting of a diterpene skeleton with L-rhamnose, N-acetylglucosamine, amino acid, and 3-hydroxybenzoate moieties, and exhibits potent biological activities. To understand the biosynthetic machinery of this unique compound, we have cloned the corresponding gene cluster. Firstly, we cloned a gene by PCR that encodes geranylgeranyl diphosphate synthase (GGPPS), which produces a direct precursor of diterpene compounds. We obtained four candidate genes and one of the genes was confirmed to encode a GGPPS. By sequence analysis of regions flanking the GGPPS gene, we identified eleven genes (bra1-11), all oriented in the same direction. We did not, however, detect any genes related to L-rhamnose and N-acetylglucosamine biosyntheses in the flanking regions. A gene disruption experiment did indeed show that this gene cluster was responsible for BCA biosynthesis.
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Connolly JD, Hill RA . Dictionary of Terpenoids. Chapman & Hall, New York, ( 1992)
Dewick PM . The biosynthesis of C5~C25 terpenoid compounds. Nat Prod Rep 19: 181–222 ( 2002)
Wise ML, Croteau R . Comprehensive Natural Products Chemistry No. 2: Isoprenoids, Including Carotenoids and Steroids. Elsevier Science, Oxford, pp. 97–153 ( 1999)
Cane DE . Comprehensive Natural Products Chemistry No. 2: Isoprenoids, Including Carotenoids and Steroids. Elsevier Science, Oxford, pp. 155–200 ( 1999)
MacMillan J, Beale MH . Comprehensive Natural Products Chemistry No. 2: Isoprenoids, Including Carotenoids and Steroids. Elsevier Science, Oxford, pp. 217–243 ( 1999)
Bohlmann J, Meyer-Gauen G, Croteau R . Plant terpenoid synthases: Molecular biology and phylogenetic analysis. Proc Natl Acad Sci USA 95: 4126–4133 ( 1998)
Trapps S, Croteau R . Defensive resin biosynthesis in conifers. Annu Rev Plant Physiol Plant Mol Biol 52: 689–724 ( 2001)
Keeling CI, Bohlmann J . Genes, enzymes and chemicals of terpenoid diversity in the constitutive and induced defense of conifers against insects and pathogens. New Phytol 170: 657–675 ( 2006)
Cane DE, Sohng JK, Lamberson CR, Rudnicki SM, Wu Z, Lloyd MD, Oliver JS, Hubbard BR . Pentalenene synthase: purification, molecular cloning, sequencing, and high-level expression in Escherichia coli of a terpenoid cyclase from Streptomyces UC5319. Biochemistry 33: 5846–5857 ( 1994)
Lin X, Hopson R, Cane DE . Genome mining in Streptomyces coelicolor: molecular cloning and characterization of a new sesquiterpene synthase. J Am Chem Soc 128: 6022–6023 ( 2006)
Jiang J, He X, Cane DE . Geosmin biosynthesis. Streptomyces coelicolor germacradienol/germacrene D synthase converts farnesyl diphosphate to geosmin. J Am Chem Soc 128: 8128–8129 ( 2006)
Dairi T, Hamano Y, Kuzuyama T, Itoh N, Furihata K, Seto H . Eubacterial diterpene cyclase genes essential for production of the isoprenoid antibiotic terpentecin. J Bacteriol 183: 6085–6094 ( 2001)
Hamano Y, Kuzuyama T, Itoh N, Furihata K, Seto H, Dairi T . Functional analysis of eubacterial diterpene cyclases responsible for biosynthesis of a diterpene antibiotic, terpentecin. J Biol Chem 277: 37098–37104 ( 2002)
Kawasaki T, Kuzuyama T, Kuwamori Y, Matsuura N, Itoh N, Furihata K, Seto H, Dairi T . Presence of copalyl diphosphate synthase gene in an actinomycete possessing the mevalonate pathway. J Antibiot 57: 739–747 ( 2004)
Kawasaki T, Hayashi Y, Kuzuyama T, Furihata K, Itoh N, Seto H, Dairi T . Biosynthesis of a natural polyketide-isoprenoid hybrid compound, furaquinocin A: identification and heterologous expression of the gene cluster. J Bacteriol 188: 1236–1244 ( 2006)
Dairi T . Studies on biosynthetic genes and enzymes of isoprenoids produced by actinomycetes. J Antibiot 58: 227–243 ( 2005)
Komaki H, Nemoto A, Tanaka Y, Takagi H, Yazawa K, Mikami Y, Shigemori H, Kobayashi J, Ando A, Nagata Y . Brasilicardin A, a new terpenoid antibiotic from pathogenic Nocardia brasiliensis: fermentation, isolation and biological activity. J Antibiot 52: 13–19 ( 1999)
Komaki H, Tanaka Y, Yazawa K, Takagi H, Ando A, Nagata Y, Mikami Y . Antitumor activity of brasilicardin A, a novel terpenoid antibiotic from Nocardia brasiliensis. J Antibiot 53: 75–77 ( 2000)
Shigemori H, Komaki H, Yazawa K, Mikami Y, Nemoto A, Tanaka Y, Sasaki T, In Y, Ishida T, Kobayashi J . Brasilicardin A. A novel tricyclic metabolite with potent immunosuppressive activity from actinomycete Nocardia brasiliensis. J Org Chem 163: 6900–6904 ( 1998)
Komatsu K, Tsuda M, Tanaka Y, Mikami Y, Kobayashi J . SAR studies of brasilicardin A for immunosuppressive and cytotoxic activities. Bioorg Med Chem 13: 1507–1513 ( 2005)
Maniatis T, Fritsch EF, Sambrook J . Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press, New York, ( 1982)
Hamano Y, Dairi T, Yamamoto M, Kawasaki T, Kaneda K, Kuzuyama T, Itoh N, Seto H . Cloning of a gene cluster encoding enzymes responsible for the mevalonate pathway from a terpenoid-antibiotic-producing Streptomyces strain. Biosci Biotechol Biochem 65: 1627–1635 ( 2001)
Kawasaki T, Kuzuyama T, Furihata K, Itoh N, Seto H, Dairi T . A relationship between the mevalonate pathway and isoprenoid production in actinomycetes. J Antibiot 56: 957–966 ( 2003)
Dairi T, Ohta T, Hashimoto E, Hasegawa M . Organization and nature of fortimicin A (astromicin) biosynthetic genes studied using a cosmid library of Micromonospora olivasterospora DNA. Mol Gen Genet 236: 39–48 ( 1992)
Kawasaki T, Hamano Y, Kuzuyama T, Itoh N, Seto H, Dairi T . Interconversion of the product specificity of type I eubacterial farnesyl diphosphate synthase and geranylgeranyl diphosphate synthase through one amino acid substitution. J Biochem 133: 83–91 ( 2003)
Hopwood DA, Bibb MJ, Chater KF, Kieser T, Bruton CJ, Kieser HM, Lydiate DJ, Smith CP, Ward JM, Schremp H . Gene Manipulation of Streptomyces. In: A Laboratory Manual. The John Innes Foundation, Norwich, ( 1985)
Schafer A, Tauch A, Jager W, Kalinowski J, Thierbach G, Puhler A . Small mobilizable multi-purpose cloning vectors derived from the Escherichia coli plasmids pK18 and pK19: selection of defined deletions in the chromosome of Corynebacterium glutamicum. Gene 145: 69–73 ( 1994)
Onaka H, Taniguchi S, Igarashi Y, Furumai T . Characterization of the biosynthetic gene cluster of rebeccamycin from Lechevalieria aerocolonigenes ATCC 39243. Biosci Biotechol Biochem 67: 127–138 ( 2003)
Bentley SD, Chater KF, Cerdeno-Tarraga AM, Challis GL, Thomson NR, James KD, Harris DE, Quail MA, Kieser H, Harper D, Bateman A, Brown S, Chandra G, Chen CW, Collins M, Cronin A, Fraser A, Goble A, Hidalgo J, Hornsby T, Howarth S, Huang CH, Kieser T, Larke L, Murphy L, Oliver K, O'Neil S, Rabbinowitsch E, Rajandream MA, Rutherford K, Rutter S, Seeger K, Saunders D, Sharp S, Squares R, Squares S, Taylor K, Warren T, Wietzorrek A, Woodward J, Barrell BG, Parkhill J, Hopwood DA . Complete genome sequence of the model actinomycete Streptomyces coelicolor A3(2). Nature 417: 141–147 ( 2002)
Omura S, Ikeda H, Ishikawa J, Hanamoto A, Takahashi C, Shinose M, Takahashi Y, Horikawa H, Nakazawa H, Osonoe T, Kikuchi H, Shiba T, Sakaki Y, Hattori M . Genome sequence of an industrial microorganism Streptomyces avermitilis: Deducing the ability of producing secondary metabolites. Proc Natl Acad Sci USA 98: 12215–12220 ( 2001)
Hayashi Y, Onaka H, Itoh N, Seto H, Dairi T . Cloning of the gene cluster responsible for biosynthesis of KS-505a (longestin), a unique tetraterpenoid. Biosci Biotechol Biochem 71: 3072–3081 ( 2007)
Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ . Basic local alignment search tool. J Mol Biol 215: 403–410 ( 1990)
Pearson WR, Lipman DJ . Improved tools for biological sequence comparison. Proc Natl Acad Sci USA 85: 2444–2448 ( 1988)
Durr C, Schnell HJ, Luzhetskyy A, Murillo R, Weber M, Welzel K, Vente A, Bechthold A . Biosynthesis of the terpene phenalinolactone in Streptomyces sp. Tü6071: Analysis of the gene cluster and generation of derivatives. Chem Biol 13: 365–377 ( 2006)
Shigemori H, Komaki H, Yazawa K, Mikami Y, Nemoto A, Tanaka Y, Kobayashi J . Biosynthesis of diterpene moiety of brasilicardin A via non-mevalonate pathway in Nocardia brasiliensis. Tetraherdon Lett 40: 4353–4354 ( 1999)
Trefzer A, Salas JA, Bechthold A . Genes and enzymes involved in deoxysugar biosynthesis in bacteria. Nat Prod Rep 16: 283–399 ( 1999)
Gullon S, Olano C, Abdelfattah MS, Brana AF, Rohr J, Mendez C, Salas JA . Isolation, characterization, and heterologous expression of the biosynthesis gene cluster for the antitumor anthracycline steffimycin. Appl Environ Microbiol 72: 4172–4183 ( 2006)
Onwueme KC, Ferreras JA, Buglino J, Lima CD, Quadri LEN . Mycobacterial polyketide-associated proteins are acyltransferases: Proof of principle with Mycobacterium tuberculosis PapA5. Proc. Natl. Acad. Sci. USA 101: 4608–4613 ( 2004)
Crecy-Lagard V, Blanc V, Gil P, Naudin L, Lorenzon S, Famechon A, Bamas-Jacques M, Crouzet J, Thibaut D . Pristinamycin I biosynthesis in Streptomyces pristinaespiralis: molecular characterization of the first two structural peptide synthetase genes. J Bacteriol 179: 705–713 ( 1997)
Ishiyama D, Vujaklija D, Davies J . Novel pathway of salicylate degradation by Streptomyces sp. Strain WA46. Appl Environ Microbiol 70: 1297–1306 ( 2004)
Silakowski B, Kunze B, Nordsiek G Blöcker H, Höfle G, Muller R . The myxochelin iron transport regulon of the myxobacterium Stigmatella aurantiaca Sg a15. Eur J Biochem 267: 6476–6485 ( 2000)
Nakano C, Okamura T, Sato T, Dairi T, Hoshino T . Mycobacterium tuberculosis H37Rv 3377c encodes the diterpene cyclase for producing halimane skeleton. Chem Commun Issue 8: 1016–1018 ( 2005)
Thompson JD, Higgins DG, Gibson TJ . CLUSTALW. Nucleic Acids Res 22: 4673–4680 ( 1994)
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Hayashi, Y., Matsuura, N., Toshima, H. et al. Cloning of the Gene Cluster Responsible for the Biosynthesis of Brasilicardin A, a Unique Diterpenoid. J Antibiot 61, 164–174 (2008). https://doi.org/10.1038/ja.2008.126
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DOI: https://doi.org/10.1038/ja.2008.126
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