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Expression and Mechanistic Analysis of a Germacradienol Synthase from Streptomyces Coelicolor Implicated in Geosmin Biosynthesis

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Expression and Mechanistic Analysis of a Germacradienol Synthase from Streptomyces Coelicolor Implicated in Geosmin Biosynthesis Expression and mechanistic analysis of a germacradienol synthase from Streptomyces coelicolor implicated in geosmin biosynthesis David E. Cane* and Rory M. Watt Department of Chemistry, Brown University, Providence, RI 02912 Communicated by Christopher T. Walsh, Harvard Medical School, Boston, MA, December 16, 2002 (received for review October 11, 2002) The PCR has been used to amplify a 2,181-bp ORF from Strepto- -SCO6073), encod ؍) myces coelicolor A3(2), designated SC9B1.20 ing a protein of 726 amino acids and showing significant sequence similarity at the deduced amino acid level in both the N-terminal and C-terminal halves to the known sesquiterpene synthase pen- talenene synthase. The full-length recombinant protein was ex- pressed at high levels in Escherichia coli and shown to catalyze the 2؉ Mg -dependent conversion of farnesyl diphosphate to the ses- Scheme 1. Cyclization of FPP 2 to pentalenene 3, catalyzed by pentalenene quiterpene alcohol (4S,7R)-germacra-1 (10)E,5E-diene-11-ol. The synthase (PS). ؊3 ؊1 enzymatic cyclization had a kcat of 6.2 ؎ 0.5 ؋ 10 s and a Km -for farnesyl diphosphate of 62 ؎ 8 nM. Expression of the N terminal (366 amino acids) domain of the SC9B1.20 protein also synthase. Indeed, microbial sesquiterpene synthases in general gave a fully functional cyclase which converted farnesyl diphos- show no overall sequence similarity either to one another (except phate to the identical sesquiterpene alcohol with a slightly lower for orthologs synthesizing the same sesquiterpene product) or to ؊3 ؊1 kcat of 3.2 ؎ 0.4 ؋ 10 s and a twofold greater km of 115 ؎ 14 any other protein. This divergence in primary sequence has nM. By contrast, the expressed C-terminal domain of SC9B1.20 had frustrated attempts to prospect for additional microbial syn- no farnesyl diphosphate cyclase activity. The formation of the thases by techniques such as Southern hybridization or consensus germacradienol seems to be the committed step in the formation PCR amplification (12). By contrast, the more than three dozen of geosmin, the characteristic odoriferous constituent of Strepto- known plant monoterpene, sesquiterpene, and diterpene syn- myces species. thases have strongly conserved amino acid sequences, as well as similar intron organization and exon sizes, suggesting a common secondary metabolism ͉ sesquiterpene synthase ͉ Streptomyces gene evolutionary origin (13). As a consequence, homology-based physical and bioinformatic screening methods have been very erpenoid metabolites, the largest known group of natural successful in identifying new plant terpene synthase genes. Tproducts, include hormones, antibiotics, anti-tumor agents, Interestingly, although plant and microbial sesquiterpene syn- flavor and odor constituents, and pigments, among a wealth of thases differ significantly in primary structure, crystal structures other physiologically or commercially important properties (1, of four different sesquiterpene cyclases of diverse biological 2). Of the Ͼ25,000 known terpenoids, the vast majority have origin have indicated a very high degree of conservation in the been isolated from terrestrial or marine plants or from fungi, and three-dimensional structure of all such synthases (14–18). only a relatively minor fraction from prokaryotes. No more than Streptomyces coelicolor A3(2) serves as the prototype of acti- a dozen terpenoid metabolites have been obtained from Strep- nomycetes, a group of Gram-positive, soil-dwelling organisms, tomyces species, even though these actinomycetes are a rich many of which exhibit a complex lifecycle involving mycelial source of natural products of all structural classes, including over growth and spore formation. The genetics of S. coelicolor is two-thirds of all naturally occurring antibiotics and a range of highly developed (19), and the genome sequence of the entire other medically useful metabolites, such as antitumor agents and 8.7-Mb linear chromosome has recently been completed (20, 21). immunosuppressants. In early studies of the odoriferous con- Among the predicted 7,825 proteins is a 2-kb ORF found by a stituents of actinomycetes, Gerber (3) reported the isolation of TBLASTN search to have significant similarity to the pentalenene several sesquiterpene alcohols, including epicubenol, as well as synthase gene. We now report the functional cloning of the methyl isoborneol and geosmin 1, the latter two being modified full-length 2-kb gene, as well its 5Ј-terminal and 3Ј-halves, and monoterpene and sesquiterpene alcohols, respectively (4). The the demonstration that it encodes a sesquiterpene synthase. A earthy odorant geosmin has itself been especially widely inves- companion paper in this issue of PNAS by Gust et al. demon- tigated because of its major contribution to the ‘‘off-flavor’’ of strates the role of this same gene in geosmin production in S. contaminated drinking water, wines, and other foodstuffs (5–8). coelicolor (22). Among Streptomyces-derived sesquiterpenes, the most thor- oughly studied have been the pentalenolactone class of antibi- Materials and Methods otics, which have been isolated from several species of Strepto- Materials. S. coelicolor A3(2) cosmid St9B1 (SC9B1, EMBL BIOCHEMISTRY myces (9). Pentalenene synthase (EC 4.6.1.5) catalyzes the accession no. AL049727; ref. 20) was a gift from David A. 2ϩ Mg -dependent cyclization of farnesyl diphosphate (FPP; Hopwood (John Innes Centre, Norwich, U.K.). S. coelicolor Scheme 1 2) to pentalenene (Scheme 1 3), the parent hydrocar- CH999, a derivative of S. coelicolor A3(2) in which the actino- bon of the pentalenolactone family of metabolites (10). The rhodin biosynthetic gene cluster has been deleted and the pentalenene synthase gene from Streptomyces sp. UC5319 en- codes a protein of MD 38 kDa, which has been cloned and expressed at high levels in Escherichia coli (11). Abbreviations: ESI-MS, electrospray ionization–MS; FPP, farnesyl diphosphate. The amino acid sequence of pentalenene synthase has shown *To whom correspondence should be addressed at: Department of Chemistry, Box H, no significant similarity to any other known sesquiterpene Brown University, Providence, RI 02912-9108. E-mail: [email protected]. www.pnas.org͞cgi͞doi͞10.1073͞pnas.0337625100 PNAS ͉ February 18, 2003 ͉ vol. 100 ͉ no. 4 ͉ 1547–1551 Downloaded by guest on September 28, 2021 biosynthesis of prodigiosin has been blocked, was a gift from immediately by using a Qiaquick PCR purification kit (Qiagen), Chaitan Khosla (Stanford University, Stanford, CA). Oligonu- eluted with 50–60 ␮l of 10 mM Tris⅐HCl, pH 8.5, then double- cleotide primers were purchased from Integrated DNA Tech- digested with NcoI and XhoIat37°C for 2–2.5 h, and repurified nologies (Coralville, IA). E. coli XL1-Blue-, BL21(DE3)pLysS-, with the Qiaquick purification kit. and BL21(DE3-RP)-competent cells, NcoI and XhoI restriction The digested PCR products were ligated into NcoI͞XhoI- endonucleases, Pfu turbo DNA polymerase, and agarose (low digested pET21d by using a 3:1 molar ratio of insert:vector and EEO) were purchased from Stratagene. T4 DNA ligase, 1-kbp T4 DNA ligase; the ligation mixture was used to transform and 100-bp DNA ladders, nucleotide triphosphates, isopropyl competent cells of E. coli XL1-Blue under standard conditions. ␤-D-thiogalactoside (IPTG), and prestained protein ladders The resulting transformants were grown overnight at 37°Con were purchased from GIBCO͞BRL. The pET21d(ϩ) expression LB-agar plates containing ampicillin (100 ␮g͞ml). Plasmid DNA vector was obtained from Novagen. [1-3H]FPP [20 Ci͞mmol (1 was isolated from 10 individual colonies after overnight growth Ci ϭ 37 GBq)] was purchased from Dupont͞NEN, diluted with at 37°C in LB-ampicillin (100 ␮g͞ml) medium and purified by synthetic FPP to a specific activity of 80 Ci͞mol, and preex- using Qiagen spin miniprep kits. Plasmids were screened by tracted with pentane to remove any contaminating alcohols. appropriate restriction digests and sequenced to confirm the Qiaprep Spin miniprep kits and Qiaquick PCR purification kits integrity of the inserted DNA sequences. Three plasmids were were obtained from Qiagen (Chatsworth, CA). Superdex 200 selected for expression: pRW31 (full-length SC9B1.20), pRW22 and Q-Sepharose fast-flow resins were purchased from Amer- (N-terminal domain), and pRW19 (C-terminal domain). sham Pharmacia Biotech (Uppsala, Sweden). Chemicals and other buffer components were purchased from Sigma and were Expression of Recombinant Terpene Cyclase. In a typical procedure, of the highest grade available. pRW31 was used to transform competent cells of E. coli BL21(DE3)pLysS. Transformants were screened for the pres- Methods. DNA sequencing of plasmid constructs was performed by ence of the correct inserts by restriction digestion of isolated the Howard Hughes Medical Institute Biopolymer͞Keck Founda- plasmid. For expression of SC9B1.20 protein, a 50-ml culture of ͞ tion Biotechnology Resource Laboratory (Yale University School E. coli BL21(DE3)pLysS pRW31 was grown at 37°CtoanOD600 of Medicine, New Haven, CT) on an ABI 377 DNA sequencing of 0.7. The culture was cooled to 28°C and induced with 0.4 mM system (Applied Biosystems) using fluorescently labeled IPTG, then incubated for 6 h at 28°C. The cells were harvested dideoxynucleotides (big-dye terminators.) N-terminal protein se- by centrifugation (7,500 ϫ g) and resuspended in 7.5 ml of cell quencing and electrospray-ionization MS (ESI-MS) were carried lysis buffer [50 mM Tris⅐HCl͞20% (vol͞vol) glycerol͞1mM out at the Keck Laboratory. PCR was performed with a PTC-150 EDTA͞pH 8.2]. 2-Mercaptoethanol (10 mM), benzamidine (0.2 minicycler (MJ Research, Cambridge, MA). Radioactivity mea- mM), PMSF (0.2 mM), and lysozyme (1.5 mg͞ml) were added, surements used glass vials containing 7 ml of Optifluor scintillation and the suspended cells were incubated at 32–35°C, with periodic mixture (Packard) using an LS5801 Liquid Scintillation Counter inversion, for 7–10 min until cell lysis was complete. DNase I ␮ ͞ (Beckman Coulter). Optical rotations were measured on a Perkin– (10–20 g ml), MgCl2 (10 mM), and Triton X-100 (0.1%) were Elmer 241 polarimeter.
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