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Hydroxylation of 1-Deoxypentalenic Acid Mediated by CYP105D7 (SAV 7469) of Streptomyces Avermitilis

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Hydroxylation of 1-Deoxypentalenic Acid Mediated by CYP105D7 (SAV 7469) of Streptomyces Avermitilis The Journal of Antibiotics (2011) 64, 65–71 & 2011 Japan Antibiotics Research Association All rights reserved 0021-8820/11 $32.00 www.nature.com/ja ORIGINAL ARTICLE Pentalenic acid is a shunt metabolite in the biosynthesis of the pentalenolactone family of metabolites: hydroxylation of 1-deoxypentalenic acid mediated by CYP105D7 (SAV_7469) of Streptomyces avermitilis Satoshi Takamatsu1,4, Lian-Hua Xu2,4, Shinya Fushinobu2, Hirofumi Shoun2, Mamoru Komatsu1, David E Cane3 and Haruo Ikeda1 Pentalenic acid (1) has been isolated from many Streptomyces sp. as a co-metabolite of the sesquiterpenoid antibiotic pentalenolactone and related natural products. We have previously reported the identification of a 13.4-kb gene cluster in the genome of Streptomyces avermitilis implicated in the biosynthesis of the pentalenolactone family of metabolites consisting of 13 open reading frames. Detailed molecular genetic and biochemical studies have revealed that at least seven genes are involved in the biosynthesis of the newly discovered metabolites, neopentalenoketolactone, but no gene specifically dedicated to the formation of pentalenic acid (1) was evident in the same gene cluster. The wild-type strain of S. avermitilis, as well as its derivatives, mainly produce pentalenic acid (1), together with neopentalenoketolactone (9). Disruption of the sav7469 gene encoding a cytochrome P450 (CYP105D7), members of which class are associated with the hydroxylation of many structurally different compounds, abolished the production of pentalenic acid (1). The sav7469-deletion mutant derived from SUKA11 carrying pKU462Hptl-clusterDptlH accumulated 1-deoxypentalenic acid (5), but not pentalenic acid (1). Reintroduction of an extra-copy of the sav7469 gene to SUKA11 Dsav7469 carrying pKU462Hptl-clusterDptlH restored the production of pentalenic acid (1). Recombinant CYP105D7 prepared from Escherichia coli catalyzed the oxidative conversion of 1-deoxypentalenic acid (5) to pentalenic acid (1) in the presence of the electron-transport partners, ferredoxin (Fdx) and Fdx reductase, both in vivo and in vitro. These results unambiguously demonstrate that CYP105D7 is responsible for the conversion of 1-deoxypentalenic acid (5) to pentalenic acid (1), a shunt product in the biosynthesis of the pentalenolactone family of metabolites. The Journal of Antibiotics (2011) 64, 65–71; doi:10.1038/ja.2010.135; published online 17 November 2010 Keywords: biosynthesis; cytochrome P450; pentalenolactone; sesquiterpene; Streptomyces avermitilis INTRODUCTION muscle cell proliferation.7 The first step in pentalenolactone biosynth- Pentalenolactone is a sesquiterpenoid antibiotic first discovered in esis is the cyclization of farnesyl diphosphate, the universal precursor 1957 in the culture extracts of Streptomyces roseogriseus,1 and subse- of all sesquiterpenes, to pentalenene (2), which is the parent hydro- quently isolated from numerous Streptomyces microorganisms.2–4 carbon of the pentalenolactone family of metabolites. From the results Pentalenolactone is active against both Gram-positive and Gram- of feeding experiments for pentalenolactone biosynthesis, a variety of negative strains of bacteria, as well as pathogenic and saprophytic plausible intermediates in the conversion of pentalenene to pentale- fungi.5 Pentalenolactone is also a potent and specific antiviral agent, nolactone have been isolated, including 1-deoxypentalenic acid (5;as inhibiting the replication of DNA viruses, such as the causal agent of glucuronate ester),4 as well as pentalenic acid (1),8,9 a demonstrated herpes simplex HSV-1 and HSV-2,6 and can inhibit vascular smooth shunt metabolite of the main pentalenolactone biosynthetic pathway. 1Laboratory of Microbial Engineering, Kitasato Institute for Life Sciences, Kitasato University, Minami-ku, Sagamihara, Kanagawa, Japan; 2Department of Biotechnology, Graduate School of Agriculture and Life Sciences, The University of Tokyo, Bunkyo-ku, Tokyo, Japan and 3Department of Chemistry, Brown University, Providence, RI, USA 4These authors contributed equally to this work. Correspondence: Professor H Ikeda, Laboratory of Microbial Engineering, Kitasato Institute for Life Sciences, Kitasato University, 1-15-1 Kitasato, Minami-ku, Sagamihara, Kanagawa 252-0373, Japan. E-mail: [email protected] Dedicated to the late Dr C Richard Hutchinson for his exceptional contributions to natural product biosynthesis, engineering, and drug discovery and in honor of his friendship. Received 15 August 2010; revised 16 October 2010; accepted 17 October 2010; published online 17 November 2010 Hydroxylation of 1-deoxypentalenic acid S Takamatsu et al 66 Figure 1 AseI-physical map of S. avermitilis and distribution of biosynthetic genes encoding terpenoid compounds (sav76, gene for avermitilol biosynthesis; crt, genes for isoneriatene biosynthesis; hop, genes for squalene and hopanoid biosynthesis; sav2163, gene for germacradienol and geosmin biosynthesis; sav3032,geneforepi-isozizaene biosynthesis) (a); and gene cluster for neopentalenoketolactone biosynthesis (sav2989, gene encoding MarR-family transcriptional regulator; gap1, gene for pentalenolactone-insensitive glyceraldehyde-3-phosphate dehydrogenase; ptlH, gene for 1-deoxypentalenic acid 11-b hydroxylase; ptlG, gene for transmembrane efflux protein; ptlF, 1-deoxy-11b-hydroxypentalenic acid dehydrogenase; ptlE, gene for Baeyer-Villiger monooxygenase; ptlD, gene for dioxygenase; ptlC, gene for hypothetical protein; ptlB, gene for farnesyl diphosphate synthase; ptlA, gene for pentalenene synthase; ptlI, gene for pentalenene C13 hydroxylase CYP183A1; sav3000, gene for AraC-family transcriptional regulator; sav3001, gene for lyase; sav3002, gene for hypothetical protein) (b). Two genes, sav7469 and sav7470, encode CYP105D7 and FdxH, respectively. The fact that tritium label from (1R)-[1-3H] pentalenene (2) was lost We have thus, elucidated all but the final step in the biosynthetic upon formation of pentalenic acid (1), but was retained in the derived pathway for the pentalenolactone variant, neopentalenoketolactone pentalenolactone has definitively ruled out 1 as an intermediate in the (9), whereas the genetic and biochemical basis for the accumulation of formation of pentalenolactone.9 A plausible biosynthetic pathway for pentalenic acid (1)inS. avermitilis remained to be clarified. The shunt pentalenolactone biosynthesis has been proposed on the basis of the product pentalenic acid (1) is thought to be formed by C-1 hydro- structures of these isolated pentalenolactone metabolites, but until xylation of 1-deoxypentalenic acid (5), an intermediate in the bio- recently, however, there had been no direct experimental evidence for synthesis of the pentalenolactone and neopentalenolactone family of biosynthetic sequence leading from pentalenene (2) to the pentaleno- metabolites, presumably under the control of a suitable hydroxylase or lactone family of metabolites. cytochrome P450 (CYP). Significantly, however, no candidate gene Streptomyces avermitilis is a Gram-positive soil bacterium respon- encoding such a hydroxylase is evident in the 13.4-kb ptl cluster. In sible for the production of the anthelminthic macrocyclic lactone fact, several S. avermitilis CYP genes are located within the biosyn- avermectins that are widely used in human and veterinary medicine. thetic clusters for avermectin, oligomycin, filipin, albaflavenone and Sequencing of the complete 9.03-Mb linear genome of S. avermitilis neopentalenolactone biosynthesis. Other CYP genes are distributed revealed at least 37 presumed biosynthetic gene clusters related independently throughout the linear chromosome of S. avermitilis.20 to secondary metabolism, with at least six of them encoding It is, therefore, of considerable interest to identify which S. avermitilis putative terpenoid biosynthetic pathways as shown in Figure 1a.10–13 CYP gene is responsible for the conversion of 1-deoxypentalenic acid Among the latter, a 13.4-kb gene cluster centered at 3.75 Mb in the (5)topentalenicacid(1). We now provide evidence that CYP105D7 S. avermitilis genome and containing 13 unidirectionally transcribed encoded by sav7469 is responsible for hydroxylation at the C-1 open reading frames (sav2990—sav3002) has been implicated in the position of 1-deoxypentalenic acid (5), resulting in accumulation of biosynthesis of pentalenolactone-like metabolites (Figure 1b). pentalenic acid (1) as a shunt metabolite of pentalenolactone or Although pentalenolactone itself has not been detected in the organic neopentalenolactone biosynthesis. extract of S. avermitilis, we have isolated the common pentalenolac- tone shunt metabolite pentalenic acid (1) from these cultures.14 MATERIALS AND METHODS Deletion of the 13.4-kb operon from S. avermitilis abolished the Bacterial strains production of both neopentalenoketolactone (9) and pentalenic acid The large-deletion mutants of S. avermitilis, SUKA5, SUKA7 (SUKA5 (1), whereas transfer of the entire gene cluster to Streptomyces. lividans Dsav7469), SUKA11 (SUKA5 Dptl-clusterHermE) and SUKA13 (SUKA11 1326, a strain that normally does not produce pentalenolactones, Dsav7469),21 were used for production of neopentalenolactone and related resulted in generation of pentalenic acid (1). In parallel with these compounds. Culture conditions for the sporulation and regeneration of molecular genetic investigations, we have been systematically expres- protoplasts after PEG-mediated protoplast transformation have been pre- 22,23 sing in E. coli the individual genes
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