J. Antibiot. 59(8): 471–479, 2006 THE JOURNAL OF ORIGINAL ARTICLE [_ ANTIBIOTICSJ Geosmin Biosynthesis in Streptomyces avermitilis. Molecular Cloning, Expression, and Mechanistic Study of the Germacradienol/Geosmin Synthase David E. Cane, Xiaofei He, Seiji Kobayashi, Satoshi O¯ mura, Haruo Ikeda This paper is submitted in honor of the late Professor Kenneth L. Rinehart, a pioneer in the study of natural products and their biosynthesis. Received: May 30, 2006 / Accepted: August 3, 2006 © Japan Antibiotics Research Association Abstract Geosmin (1) is responsible for the Keywords sesquiterpene, biosynthesis, geosmin, characteristic odor of moist soil. The Gram-positive soil Streptomyces avermitilis, Streptomyces coelicolor bacterium Streptomyces avermitilis produces geosmin (1) as well as its precursor germacradienol (3). The S. avermitilis gene SAV2163 (geoA) is extremely similar to Introduction the S. coelicolor A3(2) SCO6073 gene that encodes a germacradienol/geosmin synthase. S. avermitilis mutants Geosmin (1) is a well-known, odoriferous metabolite with a deleted geoA were unable to produce either produced by a wide variety of microorganisms, including germacradienol (3) or geosmin (1). Biosynthesis of both Streptomyces, cyanobacteria, myxobacteria, and various compounds was restored by introducing an intact geoA fungi, as well as by higher plants such as liverworts and gene into the mutants. Incubation of recombinant GeoA, sugar beets [1ϳ7]. Geosmin, with an especially low encoded by the SAV2163 gene of S. avermitilis, with detection threshold of 10ϳ100 parts per trillion, is farnesyl diphosphate (2) in the presence of Mg2ϩ gave a responsible for the characteristic odor of freshly turned mixture of (4S,7R)-germacra-1(10)E,5E-diene-11-ol (3) earth and is associated with unpleasant off-flavors in water, (66%), (7S)-germacrene D (4) (24%), geosmin (1) (8%), wine, and fish [8, 9]. and a hydrocarbon, tentatively assigned the structure of Streptomyces avermitilis (S. avermectinius is a junior octalin 5 (2%). Incubation of this germacradienol/geosmin homotypic synonym of S. avermitilis), an industrially 2 synthase with [1,1- H2]FPP (2a) gave geosmin-d1 (1a), important Gram-positive bacterium used for the production as predicted. When recombinant GeoA from either of the potent anthelminthic macrolide avermectin, also S. avermitilis or S. coelicolor A3(2) was incubated with produces geosmin. The 9.03-Mb linear chromosome of S. nerolidyl diphosphate (8), only the acyclic elimination avermitilis harbors genes encoding at least six apparent products b-farnesene (10), (Z)-a-farnesene (11), and (E)- terpene synthases that are individually implicated in a-farnesene (12) were formed, thereby ruling out nerolidyl carotene, hopanoid, and sesquiterpene biosynthesis [10, diphosphate as an intermediate in the conversion of 11]. Among the sesquiterpene synthases, the 2178-bp geoA farnesyl diphosphate to geosmin, germacradienol, and gene (SAV2163) encodes a putative protein of 725 amino germacrene D. acids (aa) with significant similarity to the S. coelicolor D. E. Cane (Corresponding author), X. He: Department of Minato-ku, Tokyo 108-8642, Japan Chemistry, Brown University, Box H, Providence, Rhode Island S. Kobayashi, H. Ikeda: Kitasato Institute for Life Sciences, 02912-9108 USA, E-mail: [email protected] Kitasato University, 1-15-1, Kitasato, Sagamihara, Kanagawa S. O¯ mura: The Kitasato Institute, 9-1, Shirokane 5-chome, 228-8555, Japan 472 - - ~- GS~ Y)nY~~:OPP M·Cf)·W 3 4 5 1 2, FPP Scheme 1 Conversion of farnesyl diphosphate (FPP, 2) to geosmin (1). Scheme 2 Mechanism of the cyclization of FPP (2) to geosmin (1), germacradienol (3), germacrene D (4), and octalin 5. A3(2) SCO6073 gene product [12]. The latter protein has biochemical characterization of the homologous S. been shown to catalyze the Mg2ϩ-dependent conversion of avermitilis protein GeoA, and the confirmation that the farnesyl diphosphate (FPP, 2) to a mixture of (4S,7R)- recombinant enzyme is indeed a germacradienol/geosmin germacra-1(10)-diene-11-ol (3), germacrene D (4), a C12 synthase. The function of the geoA gene product is also hydrocarbon tentatively identified as octalin 5, and geosmin supported by the demonstration that a geoA deletion mutant (1) (Scheme 1) [13ϳ15]. does not produce either geosmin or germacradienol. The production of both germacradienol (3) and Finally, we describe experiments that rule out the tertiary germacrene D (4) by the S. coelicolor A3(2) allylic isomer of FPP, nerolidyl diphosphate, as an germacradienol/geosmin synthase has been shown to intermediate in the enzymatic reaction. involve the partitioning of a common reaction intermediate, proposed to correspond to carbocation 6, in which loss of the original H-1si proton of FPP leads to formation of 3 Experimental while a competing 1,3-hydride shift of H-1si results in the generation of 4 (Scheme 2) [14]. The H-1re proton of FPP General Procedures is retained at C-6 of both germacradienol and germacrene General analytical, molecular biological and biochemical D, as well as at (most likely) C-6 of geosmin. The relative methods were as previously described [13, 15, 16]. proportion of geosmin to germacradienol in the product Recombinant germacradienol/germacrene D synthase, mixtures can be enhanced by increasing either the amount encoded by the SCO6073 gene of S. coelicolor A3(2) of enzyme or the incubation time, suggesting that was expressed from E. coli BL21(DE3)pLysS/pRW31, germacradienol is transiently released from the cyclase and resolubilized from inclusion bodies, purified to then rebound before conversion to geosmin [15]. homogeneity, and assayed as previously described [13ϳ15]. 2 Although the N- and C-terminal halves of the S. Farnesyl diphosphate, [1,1- H2]farnesyl diphosphate and coelicolor A3(2) germacradienol/geosmin synthase share nerolidyl diphosphate were each prepared as previously a high level of sequence similarity, only the N-terminal described [17, 18]. domain catalyzes the conversion of FPP to germacradienol (2), while the C-terminal domain has no assigned GC-MS Analysis of Volatile Organic Products of biochemical function [13]. Molecular genetic experiments S. avermitilis with S. coelicolor A3(2) deletion mutants have established The spores of wild-type and mutants (see below) of S. that the N-terminal domain alone can support geosmin avermitilis were used to inoculated a 100-ml flask formation [14]. We now report the expression and containing 10 ml of vegetative medium (glucose (0.5 g), soy 473 flour (1.5 g), and yeast extract (0.5 g) per 100 ml, pH 7.2) (thiostreptone resistance), geoA-deletion mutants, and the culture was allowed to grow while shaking at 30°C generated by double homologous recombination upstream for 2 days. Geosmin production was observed in several and downstream of geoA, were obtained by selection for media, but a synthetic medium was most suitable for the thiostreptone-sensitivity and streptomycin/spectinomycin- GC-MS analysis because many peaks were detected in the resistance. extract of the complex medium. A 0.1 ml of portion of the culture was used to inoculate a 100-ml flask containing Transfer of an Extra-copy of geoA Gene into 10 ml of synthetic medium consisting of glucose (60 g), S. avermitilis geoA-deletion Mutants (NH4)2SO4 (2 g), MgSO4·7H2O (0.1 g), K2HPO4 (0.5 g), Cosmid CL_228_H07 was digested with KpnI and the NaCl (2 g), FeSO4·7H2O (0.05 g), ZnSO4·7H2O (0.05 g), 4,594-bp segment (nt 2,634,026 to 2,638,620) carrying MnSO4·4H2O (0.05 g), CaCO3 (5 g), and yeast extract (2 g) geoA was purified by agarose gel electrophoresis and per liter, pH 7.0. After incubation while shaking at 28°C for ligated with the KpnI-cut fC31-based integrating vector 4 days, the culture was filtered. The supernatant was carrying aphII as selectable marker. The resulting plasmid extracted with 1 ml of n-hexane or pentane and the organic was used to transform E. coli recA dcm/pUB307::Tn7 and layer was dried over Na2SO4 and filtered through a 1-cm then transferred into the above-described S. avermitilis Љ column of Na2SO4 in a Pasteur pipette. A 1 to 5-ml portion DgeoA::aad3 by conjugation. The S. avermitilis of the extract was analyzed by GC-MS (Shimadzu GC- DgeoA::aad3Љ/geoA exoconjugants, were obtained by 17A, 70 eV, EI, positive ion mode; 30 mϫ0.25 mm neutral selection for neomycin resistance. bond-5 capillary column (5% phenylmethylsilicon), using a temperature program of 50ϳ280°C, temperature gradient Germacradienol/Geosmin Synthase (GeoA, SAV2163p) of 20°C/minute). Geosmin (1) and germacradienol (3) were The geoA gene (SAV2163) was amplified by PCR from identified by comparison with the spectra of the template DNA from S. avermitilis cosmid CL_228_H03 corresponding reference compounds in the NIST/EPA/NIH using the forward (5Ј-GGTAGGGAATTCCCATGAC- MS Library (2002 version). GCAGCCGTTCC-3Ј) and reverse (5Ј-CTGTCCCTCGAG- TCAGCGCGCCACC-3Ј) primers to introduce EcoRI and Molecular Cloning and Expression of Recombinant XhoI restriction sites (bold) flanking the normal start and S. avermitilis Construction of geoA-deletion Mutant stop codons, respectively. The resulting amplicon and the Two segments were amplified by PCR from S. avermitilis pET21d(ϩ) vector were digested separately with EcoRI cosmid CL_228_H03 (http://avermitilis.ls.kitasato- and XhoI before ligation with T4 DNA ligase (15 : 1 u.ac.jp/). A segment upstream of geoA (nt 2,639,510 to insert:vector, 4°C, 13 hours) and transformation of 2,637,781) was amplified using the forward (5Ј- electrocompetent E. coli XL-1 Blue cells. The resultant CTCGAGAAGCTTTGTCGTTGCCCGCGACCGTCAG- pXH17 plasmid was purified using a QIAprep Miniprep Kit CA-3Ј) and reverse (5Ј-CTCGAGTCTAGACATG- and the sequence of the insert was confirmed by direct GCCGGGCCCTACCCAGGGCC-3Ј) primers to introduce sequencing of both strands. (This construct appends the HindIII and XbaI restriction sites (bold), respectively. peptide MASMTGGQQMGRIRIP upstream of the native The forward (5Ј-CTCGAGTCTAGATGAGGGGGGGGA- methionine start codon.) The purified pXH17 plasmid was CAGGCGGCTG-3Ј) and reverse (5Ј-CTCGAGAAG- then used to transform E.