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Proc Natl Acad Sci U S A. May 27, 2008; 105(21): 7422–7427.
Published online May 20, 2008. doi:  10.1073/pnas.0802312105
PMCID: PMC2387273
Biochemistry

Identification and functional analysis of genes controlling biosynthesis of 2-methylisoborneol

Abstract

To identify the genes for biosynthesis of the off-flavor terpenoid alcohol, 2-methylisoborneol (2-MIB), the key genes encoding monoterpene cyclase were located in bacterial genome databases by using a combination of hidden Markov models, protein–family search, and the sequence alignment of their gene products. Predicted terpene cyclases were classified into three groups: sesquiterpene, diterpene, and other terpene cyclases. Genes of the terpene cyclase group that form an operon with a gene encoding S-adenosyl-l-methionine (SAM)-dependent methyltransferase were found in genome data of seven microorganisms belonging to actinomycetes, Streptomyces ambofaciens ISP5053, Streptomyces coelicolor A3(2), Streptomyces griseus IFO13350, Streptomyces lasaliensis NRRL3382R, Streptomyces scabies 87.22, Saccharopolyspora erythraea NRRL2338, and Micromonospora olivasterospora KY11048. Among six microorganisms tested, S. ambofaciens, S. coelicolor A3(2), S. griseus, and S. lasaliensis produced 2-MIB but M. olivasterospora produced 2-methylenebornane (2-MB) instead. The regions containing monoterpene cyclase and methyltransferase genes were amplified by PCR from S. ambofaciens, S. lasaliensis, and Saccharopolyspora erythraea, respectively, and their genes were heterologously expressed in Streptomyces avermitilis, which was naturally deficient of 2-MIB biosynthesis by insertion and deletion. All exoconjugants of S. avermitilis produced 2-MIB. Full-length recombinant proteins, monoterpene cyclase and methyltransferase of S. lasaliensis were expressed at high level in Escherichia coli. The recombinant methyltransferase catalyzed methylation at the C2 position of geranyl diphosphate (GPP) in the presence of SAM. 2-MIB was generated by incubation with GPP, SAM, recombinant methyltransferase, and terpene cyclase. We concluded that the biosynthetic pathway involves the methylation of GPP by GPP methyltransferase and its subsequent cyclization by monoterpene cyclase to 2-MIB.

Keywords: genome mining, methyltransferase, monoterpene cyclase

Terpenoid metabolites, monoterpenes, sesquiterpenes, and diterpenes, have been isolated from terrestrial and marine plants or from fungi, with only a relatively minor fraction from prokaryotes. Their compounds are used as antibiotics, hormones, flavor or odor constituents, and pigments. Some of them possess other physiologically or commercially important properties (1, 2). Three terpenoid compounds (Fig. 1), 2-methylisoborneol (2-MIB), geosmin, and albaflavenone are known as odorous and volatile microbial metabolites. The former two terpenoid alcohols are the most frequently found secondary metabolites of actinomycetes (35), filamentous cyanobacteria (68), myxobacteria (9), and fungi (10, 11), and account for many odor problems encountered with freshwater or with fish (7, 1214). Geosmin is also known to contribute to the characteristic earthy red beet flavor (15). 2-MIB is related to the musty-earthy notes in Brie and Camembert cheese flavor (16). An α,β-unsaturated sesquiterpene ketone, albaflavenone, was isolated from highly odorous Streptomyces culture and is an unusual odorous metabolite with antibacterial activity (17). The structures of both 2-MIB and geosmin originally isolated from actinomycetes were determined by Gerber (18, 19) and Medsker et al. (20). Geosmin is a degraded sesquiterpenoid alcohol, and biochemical and molecular genetic studies of its biosynthesis have been carried out in actinomycete strains Streptomyces coelicolor A3(2) (21) and Streptomyces avermitilis (22). Another sesquiterpene cyclase generating epi-isozizaene that could be an intermediate of albaflavenone biosynthesis, epi-isozizaene synthase (SCO5222p) of S. coelicolor A3(2), has been characterized (23).

Fig. 1.
Structures of microbial volatile terpenoid metabolites, 2-MIB (Left; 1,2,7,7-tetramethyl-exo-bicycloheptan-2-ol), geosmin (Center; 4,8a-dimethyl-octahydro-naphthalen-4a-ol) and albaflavenone (Right; 2,6,7,7-tetramethyltricyclo [6.2.1.01,5]undec-5-en-4-one). ...

Plant monoterpene, sesquiterpene, and diterpene synthases have strongly conserved amino acid sequences, as well as similar intron organization and exon sizes, suggesting a common evolutionary origin (24). This feature has allowed homology-based physical and bioinformatic searching methods to be very successful in identifying new terpene synthase genes from not only plant but also fungi (25). Conversely, the amino acid sequence of microbial sesquiterpene cyclase (pentalenene synthase, germacradienol/geosmin synthase, and epi-isozizaene synthase) has shown no significant similarity to any other known sesquiterpene synthase. Indeed, microbial terpene synthases in general show no overall sequence similarity either to one another (except for orthologs synthesizing the same terpene product) or to any other protein. This divergence in primary sequence has thwarted attempts to prospect for additional microbial terpene synthases by techniques such as Southern hybridization or PCR amplification using consensus nucleotide sequences (26). Microbial sesquiterpene cyclases have been investigated, but information of microbial diterpene and monoterpene cyclases is limited and not only biochemical but also genetic approaches of microbial monoterpene cyclases have not been elucidated to date.

Labeling experiments conducted by Bentley and Meganathan (27) supported earlier assumptions that 2-MIB is a methylated monoterpene alcohol, the additional methyl-group being derived from S-adenosyl-l-methionine (SAM). Recent feeding experiments suggested that the methylation of geranyl diphosphate (GPP) would generate the substrate for the cyclization to form 2-MIB (9). However, no further direct evidence on the biosynthesis of 2-MIB in actinomycetes, cyanobacteria, myxobacteria, and fungi is available. More than 590 kinds of microbial genome analyses have been completed. Although existing databases contain above microorganisms, many of their protein-coding genes are still annotated as hypothetical proteins. Consequently, we assumed that genes connected with the biosynthesis of 2-MIB would be buried in the genome data. Because the amino acid sequences of microbial terpene cyclase bear little significant similarity, it was unlikely that screening biosynthetic genes for 2-MIB by sequence similarities would prove fruitful. However, if polypeptides have similar catalytic activities, they would have conserved sequence signature. We therefore adopted a searching method based on hidden Markov models Pfam search (28, 29) for the primary selection of desired terpene cyclases. Profile hidden Markov models are more suitable for sensitive database searching using statistical descriptions of a sequence family's consensus. The terpene cyclases predicted were classified by phylogenetic analysis. Final candidates were harvested as the gene formed an operon with SAM-dependent methyltransferase gene, because 2-MIB is a methylated monoterpenoid alcohol and methyl residue at the C2 position of 2-MIB is derived from the methyl residue of methionine in feeding experiments (9, 27).

We have identified two genes encoding 2-MIB biosynthesis from existing microbial genome databases, and they have been characterized. Here, we elucidate the biosynthetic mechanism from isopentenyl diphosphate (IPP) to 2-MIB in actinomycetes.

Results and Discussion

Terpene Cyclases in Bacteria.

The production of monoterpenoid metabolites, 2-MIB and so on, from microbial origin was first reported many years ago, but the biosynthesis of the microbial monoterpenoid metabolites is still not understood. Microbial sesquiterpene cyclases, pentalenene synthase, germacradienol/geosmin synthase and epi-isozizaene synthase, and diterpene cyclase were identified from actinomycete strains and have been characterized. The amino acid sequences of these microbial terpene cyclases have shown no significant similarities to that of plant origin. However, a significant conserved motif containing an acid-rich domain, associated with binding of catalytically essential magnesium ions, was found in these microbial terpene cyclases.

To elucidate the biosynthesis of the microbial monoterpenoid alcohol, 2-MIB, all microbial terpene cyclases were harvested from the genome-sequenced bacterial database of National Center for Biotechnology Information, Streptomyces scabies and Streptomyces griseus genome data, Micromonospora olivasterospora draft sequence data, and limited sequence data from a linear plasmid pKSL of Streptomyces lasaliensis [putative terpene cyclase and methyltransferase genes were found near the genes for modular polyketide synthases (30)] by searching on the basis of the profile hidden Markov models using a model of PF03936 (terpene synthase family, metal binding domain). The E-value used was <10−5 for setting the parameter of hidden Markov models search because proteins selected using >10−5 E-value were also selected by other Pfam models with lower E-values. Of 1,922,990 proteins, 41 proteins were selected with E-value ranges from 2.2 × 10−6 to 1.8 × 10−82. These proteins were classified into three major groups on the basis of phylogenetic analysis (Fig. 2). The sesquiterpene cyclases, pentalenene, germacradienol/geosmin and epi-isozizaene synthases, were classified in group II. Group III contained diterpene cyclase in terpentecin biosynthesis of Kitasatospora griseola (31) and undefined terpene cyclase of Salinispora arenicola. The proteins classified into group I were uncharacterized, and the sequence similarities of these proteins against known sesquiterpene and diterpene cyclases were low level. Sequence alignment with sesquiterpene and diterpene cyclases revealed that the proteins of group I also had two significant conserved motifs containing a metal-binding domain (3237).

Fig. 2.
Phylogenetic analysis of terpene cyclases from bacterial databases. Abbreviations: alr4685 (322 aa; NP_488725), Nostoc sp. PCC 7120; Ava_1982 (322 aa; ...

As shown in Fig. 3, two conserved motifs of the proteins in group I were located near the C terminus, in comparison with those of sesquiterpene and diterpene cyclases. The distance between the first and second conserved motifs of the proteins in group I (112 aa) was longer than those of sesquiterpene and diterpene cyclases (104–106 aa). The first motif in sesquiterpene cyclases was acid-rich domain with a high proportion of aromatic amino acids, –FFxxDDxxD– (pentalenene and epi-isozizaene synthases) or –FxFDDHFLE– (germacradienol/geosmin synthase). Although the first motif in diterpene cyclase (–LIVNDDRWD–) and the proteins of group I (–xVDDxxx[DE]–) also possessed an acid-rich domain, the content of aromatic amino acids was lower than that in sesquiterpne cyclases. The second motif in all proteins were conserved, –xxNxxxSxxxE–, in which the triad of residues in bold has also been implicated in the binding of magnesium ion (33, 34, 36). Because the SAV (deduced amino acid sequence of truncated terpene cyclase; 1,245,680 to 1,246,412 nt of the S. avermitilis genome) was defined as a pseudogene of S. avermitilis, the annotation was not completed. The deduced polypeptide was very similar to SAML0357 and SCO7700, but the second motif sequence was lacked in this deduced polypeptide. According to phylogenic analysis, we assumed that the proteins in group I are monoterpene cyclases and that 2-MIB synthase has to be found in this group.

Fig. 3.
Alignment of amino acid sequences of bacterial terpene cyclases with predicted cyclases. Shadow boxes indicate metal (Mg2+)-binding motif of terpene cyclase. The strain name of each protein is described in Fig. 2. SAV indicates deduced amino acid sequence ...

Because feeding experiments with labeled precursors suggested that 2-MIB was methylated monoterpenoid alcohol, the biosynthesis should involve at least two steps, methylation of GPP and cyclization of methylated GPP. In general, the genes involving secondary metabolite biosynthesis in bacteria are found as a cluster in a specific location of the genome and some of these genes form a single transcriptional unit. Consequently, it suggests that the genes encoding monoterpene cyclase for 2-MIB biosynthesis form an operon with a specific GPP methyltransferase. The genes flanking the monoterpene cyclase gene of group I were annotated and functions determined by several bioinformatics analyses. Each monoterpene cyclase gene from Streptomyces ambofaciens, Streptomyces coelicolor A3(2), S. griseus, S. lasaliensis, S. scabies (SCAB5041), Saccharopolyspora erythraea, and M. olivasterospora formed an operon with a gene encoding SAM-dependent methyltransferase (Fig. 4). Furthermore, genes encoding cyclic nucleotide-binding protein were also located upstream of these seven monoterpene cyclase genes. On the other hand, methyltransferase gene(s) was/were not found in or around monoterpene cyclase genes predicted in Pseudomonas fluorescens and S. scabies (SCAB82161). These two monoterpene cyclases would be involved in other monoterpenoid metabolite biosynthesis. Interestingly, a similar methyltransferase gene, SAV983, was found in S. avermitilis genome data (http://avermitilis.ls.kitasato-u.ac.jp/). Furthermore, truncated cyclic nucleotide-binding protein and monoterpene cyclase genes were found upstream of SAV983. The truncation will be induced by a deletion mutation, and deduced polypeptide of truncated gene product lacked the second conserved metal-binding motif (Fig. 3 Upper). Once S. avermitilis would be capable of producing 2-MIB, but the deletion of the region encoding the second conserved metal-binding motif in monoterpene cyclase gene prevented production of 2-MIB in this organism.

Fig. 4.
Organization of genes encoding predicted monoterpene cyclases and flanking genes. All predicted monoterpene cyclase genes are located in the chromosomes, but the gene of S. lasaliensis resided in a giant linear plasmid pKSL. The grayed, filled, and oblique-lined ...

Production of Volatile Terpenoid Metabolites in Actinomycetes Carrying Predicted Monoterpene Cyclase/Methyltransferase Genes.

The results of bioinformatics indicate that seven actinomycetes possess the ability to produce the monoterpenoid metabolite, 2-MIB. Because a plant pathogen S. scabies 87.22 was not stored in our culture stock, six actinomycete strains were examined. All six strains produced terpenoid metabolites, including sesquiterpenoid alcohol, geosmin, in varying quantity (Fig. 5). Five strains, S. ambofaciens, S. coelicolor A3(2), S. griseus, S. lasaliensis, and Sa. erythraea, produced a monoterpenoid metabolite that had identical retention time and mass spectrum ([M+], m/z 168) to those of the authentic 2-MIB. Interestingly, M. olivasterospora did not produce 2-MIB, but homomonoterpene hydrocarbon ([M+], m/z 150) was accumulated as a major component. The hydrocarbon was identical to 2-methylenebornane (2-MB), a dehydration product of 2-MIB in comparison with retention time and mass spectrum of the synthetic sample. Then inspection of mass spectra obtained from the extracts of all Streptomyces strains and Sa. erythraea allowed us to detect as a minor component. Thus, the monoterpene cyclase of M. olivasterospora is slightly different from those of other actinomycete strains.

Fig. 5.
Volatile monoterpenoid metabolites, 2-MIB (A) and 2-MB (B), and sesquiterpenoid metabolite, geosmin (C) produced by actinomycete strains carrying predicted monoterpene cyclase and methyltransferase genes. All microorganisms were grown on SFM (left half ...

The maximum production of 2-MIB and the corresponding 2-MB in each microorganism depended on the growth period (Fig. 5 A and B). Significant increase of monoterpene production in microorganisms except for M. olivasterospora was accomplished by growth on soy flour-mannitol (SFM) medium. All Streptomyces and M. olivasterospora reached the maximal production at 5–9 days of growth, but Sa. erythraea required a longer growth period for maximum production (Fig. 5A). This microorganism did not develop morphological differentiation until 7 days of growth, in which the mycelium grew as substrate mycelium, and sporulation started after this growth period. The morphological differentiation of other actinomycetes except for M. olivasterospora was completed within 5 days of growth. Furthermore, abundant sporulation was observed by the growth on the SFM medium rather than on inorganic salts-starch-yeast extract (M4YE) medium. All known bacterial producers of 2-MIB and geosmin (i.e., actinomycetes, cyanobacteria, and myxobacteria) exhibit complex morphological differentiation, including the formation of multicellular complexes. The aerial mycelium-negative mutants of Streptomyces antibioticus and Streptomyces sulfurous failed to produce both 2-MIB and geosmin (27). These observations assumed that complex morphological differentiation in these microorganisms compromised production of volatile terpenoid metabolites.

It has been reported that telomeric regions in S. ambofaciens are frequently deleted by induction of mutagenesis (38). Analysis revealed that monoterpene cyclase (SAML0357) and methyltransferase (SAML0358) genes were located near the left telomere. Once we had isolated mutants of S. ambofaciens (39), all mutants induced by UV irradiation were screened by PCR using primers for SAML0357 and SAML0358. One of the mutants, U1717R, was deficient of both genes, although the complex morphological differentiation was not affected. As expected, the mutant strain U1717R was unable to produce 2-MIB but produced another type of sesquiterpenoid metabolite, geosmin (Fig. 5). This feature was the same as that of S. avermitilis that was naturally deficient of a gene encoding monoterpene cyclase (Fig. 5). Absence of 2-MIB production in both S. ambofaciens mutant U1717R and S. avermitilis was presumed to be due to impact of monoterpene cyclases in group I (Figs. 2 and and3),3), which are involved in the biosynthesis of 2-MIB.

Heterologous Expression of Monoterpene Cyclase/Methyltransferase Genes.

Because S. avermitilis was deficient of a gene encoding monoterpene cyclase and its operon was naturally disrupted by an IS-insertion and deletions, so the microorganism is suitable for the heterologous expression of the foreign monoterpene cyclase genes. One of the large-deletion mutants, SUKA16, was used as a host strain for heterologous expression because the mutant lacks biosynthetic gene clusters for the main products of S. avermitilis and for terpenoid metabolites. Segments carrying predicted monoterpene cyclase and methyltransferase genes were amplified from two Streptomyces strains, S. ambofaciens and S. lasaliensis, and two non-streptomycete strains, Sa. erythraea and M. olivasterospora. The two Streptomyces produced large amounts of 2-MIB. The 2-MB-generating monoterpene cyclase of M. olivasterospora also proved interesting, as did the smallest size of monoterpene cyclase gene found in Sa. erythraea. These amplified segments were joined just downstream of the rpsJ promoter, which was constitutively expressed and had very strong transcriptional activity in S. avermitilis. Exoconjugants of S. avermitilis SUKA16 carrying monoterpene cyclase/methyltransferase genes of S. ambofaciens and S. lasaliensis, respectively, were grown in agar medium, and the extracts of whole culture were directly analyzed by GC-MS analyzer. Unfortunately, exoconjugant carrying the genes of M. olivasterospora did not exhibit monoterpenoid metabolites, but three exoconjugants carrying the genes of S. ambofaciens, S. lasaliensis, and Sa. erythraea, respectively, gave a single major product, 2-MIB (2.8 μg per plate, 1.1 μg per plate, and 65 μg per plate, respectively; supporting information (SI) Fig. S1). The exoconjugant carrying the genes from Sa. erythraea did produce a very large amount of 2-MIB accompanied with small amounts of three corresponding hydrocarbons, 2-MB, 2-methyl-2-bornene (2-M2B), and 1-methylcamphene (1-MC), were also produced (Fig. S2). When only the monoterpene cyclase gene of Sa. erythraea was introduced into S. avermitilis, resultant exoconjugants produced none of the monoterpenoid metabolites, indicating that the methyltransferase is essential for the biosynthesis of 2-MIB.

The results of heterologous expression of monoterpene cyclase/methyltransferase genes strongly suggest that S. avermitilis has the ability to supply GPP for the biosynthesis of monoterpenoid metabolites. Moreover, two genes encoding monoterpene cyclase and methyltransferase are necessary for the biosynthesis of 2-MIB.

Enzymatic Reaction of Recombinant Proteins.

Because exoconjugants produced 2-MIB, enzymatic reactions were examined by using recombinant proteins prepared in Escherichia coli. Two genes of S. lasaliensis were amplified and joined with pET22b(+) or pET28a(+) vectors. After four combinations of His6 residue at the N- or C-terminal ends were examined, soluble proteins were obtained from His6-tagged monoterpene cyclase at the C terminus (52.4 kDa) and methyltransferase at the N terminus (35.0 kDa), as judged by SDS/PAGE, respectively (Fig. S3). To test the predicted 2-MIB synthesis activities of the S. lasaliensis gene products, both soluble recombinant cyclase and methyltransferase were incubated with GPP and SAM in the presence of MgCl2. The organic-soluble product was analyzed by GC-MS analyzer. The enzymatic reaction gave a single major product with mass, [M+] m/z 168, consistent with formation of a monoterpenoid alcohol, and the product was identical to 2-MIB [by comparison with the synthetic sample of 2-MIB (Fig. S4)]. When SAM or the recombinant methyltransferase was removed from the reaction mixture, 2-MIB was not produced. Furthermore, incubation with recombinant methyltransferase, GPP, and SAM followed by alkaline phosphatase treatment gave a single major product, which was identified as 2-methylgeraniol by comparison of its retention time and mass spectrum ([M+], m/z 168) to those of the authentic 2-methygeraniol synthesized (Fig. S5). This observation strongly indicated that the methyltransferase catalyzes conversion of GPP to 2-methyl GPP. These results were also supported with those of heterologous expression of cloned genes. The enzymatic reaction suggests that a substrate for monoterpene cyclase predicted is a methylated GPP.

Three types of terpene cyclases, monoterpene, sesquiterpene, and diterpene, of microbial origin have been identified. The comparative analysis of monoterpene cyclases presented here, along with other microbial terpene cyclases, promises to illustrate the reaction mechanism of cyclization in detail. Experimental results have unambiguously elucidated the biosynthetic mechanism of the monoterpenoid odorous compound, 2-MIB, as follows: (i) GPP generated from isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP) by GPP synthase was converted to methylated GPP by a specific methyltransferase (GPP methyltransferase)l; (ii) 2-methyl GPP was cyclized to generate 2-MIB by a monoterpene cyclase (2-MIB synthase) in the presence of Mg2+ (Fig. 6). This finding is the first reported observation of the biochemical features of microbial monoterpene cyclase and of the methylation of GPP by a specific methyltransferase.

Fig. 6.
Biosynthetic mechanism from GPP to 2-MIB (or 2-MB).

Unfortunately, monoterpene cyclase genes encoding 2-MIB synthase were not found in genome-sequenced cyanobateria (Anabaena, Gloeobacter, Nostoc, Prochlorococcus, Synechococcus, Synecocyctis, Thermosynechococcus, and Trichodesmium) and myxobacteria (Myxococcus). Consequently, the productivity of monoterpenoid metabolites from these bacteria was not examined. In the near future, genome analysis of 2-MIB-producing cyanobacteria (Oscillatoria curviceps, Oscillatoria tenuis, and Lyngbya sp.) (7, 8) and myxobacteria (Nannocystis exedens) (9) will be completed, and their monoterpene cyclases will be characterized. Recently, we have found two genes from an actinomycete strain, Kitasatospora setae (produced 2-MIB), that formed an operon and bore significant similarities to 2-MIB synthase and GPP methyltransferase genes, respectively. Our work has confirmed that many genes encoding for 2-MIB synthase and GPP methyltransferase can be identified and characterized from a range of actinomycetes.

Materials and Methods

Bioinformatics.

Genome-sequenced bacterial genome data (total 1,906,368 proteins from 595 strains) was obtained from the National Center for Biotechnology Information (ftp://ftp.ncbi.nih.gov/genomes/Bacteria/) and the Pfam database version 22.0 (July 2007, 9,318 families) from the Howard Hughes Medical Institute (ftp://selab.janelia.org/pub/Pfam/). Streptomyces scabies 87.22 sequence data (8,984 protein-coding sequences annotated) was produced by the Streptomyces scabies Sequencing Group at the Sanger Institute U.K. and was obtained from ftp://ftp.sanger.ac.uk/pub/pathogens/ssc/. Other unpublished actinomycete genome data, S. griseus IFO13350 (40), Micromonospora olivasterospora KY11048 (draft sequence data), and some sequence data of a giant linear plasmid pKSL of S. lasaliensis NRRL3382R were also used. The extraction of Pfam model of PF03936 (terpene synthase family, metal-binding domain) and searching proteins from database used binary programs made by the compilation of a source package of hidden Markov models for biological sequence analysis, hmmer-2.3.2 (ftp://selab.janelia.org/pub/software/hmmer/2.3.2/). Alignment of sequences was analyzed by using MAFFT (41) version 6.24 obtained from Kyushu University (http://align.bmr.kyushu-u.ac.jp/mafft/software/source.html). Phylogenetic analysis of aligned sequences was done by the bootstrap method (bootstrap number, 1,000; seed number, 111) of CLUSTALW (42) version 1.83 (ftp://ftp.ebi.ac.uk/pub/software/unix/clustalw/). Drawing of the bootstrap tree was done by njplot (http://pbil.univ-lyon1.fr/software/njplot.html).

Bacterial Strains and Plasmid Vectors.

S. avermitilis K139 (isogenic to MA-4680), S. ambofaciens ISP5053, S. ambofaciens U1717R (39), S. coelicolor A3(2), S. griseus IFO13350, S. lasaliensis NRRL3382R, and Saccharopolyspora erythraea NRRL2338 were obtained from the culture collection of the Kitasato Institute. M. olivasterospora KY11048 was provided by Kyowa Hakko Co. Ltd Japan. S. avermitilis large deletion mutant SUKA16 (Δ79,460-1,595,563 nt Δptl::ermE ΔgeoA::aadA Δolm Δ8,892,894-8,917,256 nt) was used as a host strain for the heterologous expression of the operon carrying genes encoding monoterpene cyclase/methyltransferase predicted. E. coli DH5α, E. coli F dcm Δ(srl-recA)306::Tn10 carrying pUB307-aph::Tn7, and E. coli BL21(DE3) were used for general DNA manipulation, for E. coli/Streptomyces conjugation, and for the preparation of recombinant monoterpene cyclase and methyltransferase, respectively. Integrating vector pKU469 was used for the heterologous expression of actinomycete monoterpene cyclase/methyltransferase genes in S. avermitilis SUKA16. E. coli expression vectors pET22b(+) and pET28a(+) were used for preparation of recombinant monoterpene cyclase and methyltransferase, respectively. Culturing of E. coli used Luria–Bertani broth (containing 10.0 g/liter tryptone, 5.0 g/liter yeast extract, and 5.0 g/liter NaCl; pH 7.4). Agar was supplied at 15.0 g/liter for solid media (LA).

Cultivation and Extraction of Volatile Terpenoid Metabolites from Actinomycete Strains.

For the production of volatile terpenoid metabolites, spores were spread on agar media: M4YE (containing 10.0 g/liter soluble starch, 1.0 g/liter K2HPO4, 1.0 g/liter MgSO4·7H2O, 1.0 g/liter NaCl, 2.0 g/liter (NH4) 2SO4, 2.0 g/liter CaCO3, 0.001 g/liter FeSO4·7H2O, 0.001 g/liter MnSO4·4H2O, 0.001 g/liter ZnSO4·7H2O, 2.0 g/liter yeast extract, and 15.0 g/liter agar; pH 7.0); SFM (containing 20.0 g/liter defatted soy flour, 20.0 g/liter mannitol, and 15.0 g/liter agar; not adjusted pH); and YMG (containing 4.0 g/liter yeast extract, 10.0 g/liter malts extract, 10.0 g/liter glucose, and 20.0 g/liter agar; pH 7.4 for exoconjugants). SFM or YMS (43) medium was used for the sporulation of Streptomyces strains. The actinomycete strains spread on the agar plates (diameter 90 mm) were grown at 30°C. After cultivation, 5 ml of methanol was directly added to each plate, and the plates were allowed to stand at room temperature for 30 min. The methanol extract was collected into glass tubes. Volatile metabolites were extracted with 0.5 ml of n-hexane, and the upper n-hexane layer was carefully collected. The n-hexane extract was dehydrated over anhydrous Na2SO4, and a portion of extract was directly evaluated in a capillary GC-MS analyzer.

GC-MS Analysis of Volatile Metabolites.

A 1- to 5-μl portion of the extract was analyzed by capillary GC-MS [Shimadzu GC-17A, 70 eV, EI, positive ion mode; 30 m × 0.25-mm neutral bond-5 capillary column (5% phenylmethylsilicon), using a temperature program of 50°C–280°C, temperature gradient of 20°C/minute or 60°C for 3 min, 60°C–160°C, temperature gradient of 10°C/min, then 160°C–240°C, temperature gradient 40°C/min]. 2-MIB, 2-MB, geosmin, and germacradienol were identified by comparison with the spectra of the corresponding reference compounds in the database. Furthermore, chemically synthesized 2-MIB, 2-MB, 2-M2B, and 1-MC (SI Materials and Methods), and authentic sample of geosmin purified from S. avermitilis were also compared with each extract of actinomycete strain.

Supplementary Material

Supporting Information:

Acknowledgments.

We thank Kyowa Hakko Co. Ltd. Japan for kindly distribution of Micromonospora olivasterospora KY11048. We also thank Andy Crump for a critical reading of the manuscript. This work was supported by a Grant-in-Aid for Scientific Research on Priority Areas “Applied Genomics” from the Ministry of Education, Culture, Sports, Science and Technology, Japan (MEXT) (H.I.) and by Grants-in-Aid for Scientific Research from the Japan Society for the Promotion of Science (JSPS) (A) 17208010 (H.O.) and 20310122 (H.I.).

Footnotes

The authors declare no conflict of interest.

This article contains supporting information online at www.pnas.org/cgi/content/full/0802312105/DCSupplemental.

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