SEARCH

SEARCH BY CITATION

Summary

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References
  9. Supporting Information

Fungi are a rich source of bioactive secondary metabolites, and mushroom-forming fungi (Agaricomycetes) are especially known for the synthesis of numerous bioactive and often cytotoxic sesquiterpenoid secondary metabolites. Compared with the large number of sesquiterpene synthases identified in plants, less than a handful of unique sesquiterpene synthases have been described from fungi. Here we describe the functional characterization of six sesquiterpene synthases (Cop1 to Cop6) and two terpene-oxidizing cytochrome P450 monooxygenases (Cox1 and Cox2) from Coprinus cinereus. The genes were cloned and, except for cop5, functionally expressed in Escherichia coli and/or Saccharomyces cerevisiae. Cop1 and Cop2 each synthesize germacrene A as the major product. Cop3 was identified as an α-muurolene synthase, an enzyme that has not been described previously, while Cop4 synthesizes δ-cadinene as its major product. Cop6 was originally annotated as a trichodiene synthase homologue but instead was found to catalyse the highly specific synthesis of α-cuprenene. Coexpression of cop6 and the two monooxygenase genes next to it yields oxygenated α-cuprenene derivatives, including cuparophenol, suggesting that these genes encode the enzymes for the biosynthesis of antimicrobial quinone sesquiterpenoids (known as lagopodins) that were previously isolated from C. cinereus and other Coprinus species.


Introduction

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References
  9. Supporting Information

Fungi produce many well-known bioactive secondary metabolites including terpenoids, which represent the largest class of natural products (Davis and Croteau, 2000; Keller et al., 2005; Misiek and Hoffmeister, 2007; Spiteller, 2008). The diversity of terpene structures is in part the result of a biosynthetic process where prenyl diphosphate chains with 10, 15 or 20 carbons undergo numerous cyclization and rearrangement reactions catalysed by terpene synthases (also known as cyclases) that generate and then guide the procession of a reactive carbocation through a prenyl chain. Terpene synthases differ in their prenyl diphosphate chain length specificity [mono-(C10), sesqui-(C15) and diterpene-(C20) synthases], cyclization mechanism and whether they synthesize a narrow or broad range of cyclization products. Subsequent modifications of the terpene scaffolds further increase the structural diversity of this group of compounds (Sacchettini and Poulter, 1997; Davis and Croteau, 2000; Christianson, 2006; 2008).

Many terpene synthases have been described from plant species (Tholl, 2006), but relatively few microbial enzymes have been functionally characterized (Kawaide et al., 1997; Lesburg et al., 1997; Caruthers et al., 2000; Dairi et al., 2001; Rynkiewicz et al., 2001; Hamano et al., 2002; Toyomasu et al., 2004; 2007; Kawaide, 2006; Shishova et al., 2007; Pinedo et al., 2008). The availability of genome sequences recently led to the discovery of several sesquiterpene synthases from actinomycetes and cyanobacteria (Cane and Watt, 2003; Gust et al., 2003; Cane et al., 2006; Agger et al., 2008; Giglio et al., 2008; Komatsu et al., 2008; Zhao et al., 2008). However, so far only three genes encoding fungal sesquiterpene synthases have been cloned and functionally characterized. These enzymes are trichodiene synthase, aristolochene synthase and, very recently, presilphiperfolan-8β-ol synthase, which generate the sesquiterpene scaffolds of the trichothecene mycotoxins produced by Fusarium species, the PR-toxin made by Penicillium roqueforti and the botyrane phytotoxins synthesized by Botrytis cinerea respectively (Hohn and Beremand, 1989; Hohn and Plattner, 1989; Cane et al., 1995; Hohn et al., 1995; Cane and Kang, 2000; Caruthers et al., 2000; Rynkiewicz et al., 2001; Pinedo et al., 2008). Like their bacterial homologues (Tetzlaff et al., 2006; Agger et al., 2008; Zhao et al., 2008), fungal terpene synthases are frequently part of a biosynthetic cluster, which includes additional terpene-modifying genes such as cytochrome P450 monooxygenases. Trichothecene biosynthetic cluster, for example, have been studied in great detail (Tokai et al., 2007), and more recently botrydial biosynthesis has been characterized (Siewers et al., 2005; Collado et al., 2007; Pinedo et al., 2008).

No terpene biosynthetic enzymes have so far been described from mushroom-forming higher fungi of the class Agaricomycetes {Homobasidiomycetes [sensu (Hibbett et al., 2007)]}, despite the fact that this class of fungi is known to produce numerous bioactive and often cytotoxic sesquiterpenoid secondary metabolites (reviewed in Abraham, 2001). In part, this may be due to a general lack of genetic tools and genomic sequence information for this group of fungi. Unlike the many genome sequences released for filamentous fungi, only one Agaricales genome sequence, that of the model species Coprinus cinereus (also known as Coprinopsis cinerea), was known until very recently (Broad Institute). The genome sequence of another Agaricales species, Laccaria bicolor (Joint Genome Institute) (Martin et al., 2008), was released in early 2008; and a genomic survey of Moniliophthora perniciosa (LGE-UNICAMP, Brazil) (Mondego et al., 2008) was published in late 2008. Genome sequences of the following non-mushroom-forming Basidiomycota have been completed and published: Cryptococcus neoformans (Loftus et al., 2005), Phanerochaete chrysosporium (Martinez et al., 2004), Ustilago maydis (Kamper et al., 2006) and Postia placenta (Martinez et al., 2009).

In this study we describe the identification and cloning of six sesquiterpene synthase and two associated cytochrome P450 monooxygenase genes from C. cinereus. Expression in Escherichia coli and Saccharomyces cerevisiae allowed functional characterization of the corresponding enzymes. Major sesquiterpene hydrocarbons produced by these enzymes correspond to those detected in small quantities in cultures of C. cinereus. One of the characterized terpene synthases, a homologue of trichodiene synthase, produces α-cuprenene and appears to catalyse the first step in the biosynthesis of the antimicrobial lagopodins. Lagopodins are quinone sesquiterpenoids that were identified in C. cinereus cultures in a manuscript dating two decades ago (Bu'Lock and Darbyshire, 1976).

Results

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References
  9. Supporting Information

Sesquiterpene synthase homologues in fungi

A blast search of fungal genome sequences at NCBI, JGI and Broad Institute with experimentally characterized microbial sesquiterpene synthases resulted in the identification of a large number of sesquiterpene synthase homologues, of which only a handful have so far been functionally characterized (Fig. 1). Most fungi have several sesquiterpene synthase homologues. Fungi belonging to the genera Aspergillus, Gibberella, Phanerochaete, Postia, Coprinus and Laccaria have four or more putative sesquiterpene synthases, while sesquiterpene synthase genes appear to have undergone extensive gene duplication in P. chrysosporium, P. placenta and L. bicolor.

image

Figure 1. Unrooted Neighbor-Joining tree of fungal sesquiterpene synthase homologues. Protein sequences of known bacterial sesquiterpene sequences were used for homology searches in NCBI's non-redundant protein sequence database and in fungal genome sequences obtained from the Broad Institute and JGI. Sequences that did not contain the first conserved metal binding domain (DDXXD motif) of terpene synthases or appeared to be incorrectly annotated were not included in the alignment. Only a few representatives of fungal genera with several sequenced genomes (e.g. Aspergillus) were included in the search. Branches are labelled with their respective bootstrap values, gene accession numbers and strain names. Functionally characterized sesquiterpene synthases (including the Cop enzymes in this study) are also indicated. Question marks indicate that the function of Cop5 could not be determined in this study.

Download figure to PowerPoint

Phylogenetic analysis reveals that several terpene synthase homologues, including proteins from Phanerochaete, Laccaria and one protein from Coprinus, cluster together with the experimentally characterized trichodiene synthases (Hohn and Desjardins, 1992), suggesting that they may catalyse the same or a related cyclization reaction (Fig. 1). Other putative terpene synthases cluster together with the aristolochene synthases from Penicillium and Aspergillus (Proctor and Hohn, 1993; Cane and Kang, 2000), the recently characterized presilphiperfolan-8β-ol synthase (Pinedo et al., 2008) and fusicoccadiene synthase (Toyomasu et al., 2007). The majority of identified putative terpene synthase sequences cluster into clades without experimentally characterized representatives. These include several terpene synthase homologues from Coprinus, Laccaria and Phanerochate located at the top of the phylogenetic tree shown in Fig. 1.

To gain insight into the functions of the multiple terpene synthases present in many fungi, the six sesquiterpene synthase homologues (named Cop1 to Cop6) identified in C. cinereus were selected for cloning and functional characterization (see Table S1 for accession numbers and genome locus tags of the six cop genes). Three of the six putative terpene synthases (Cop1 to Cop3) cluster in one clade of the phylogenetic tree, while Cop4 and Cop5 are located in separate clades and Cop6 is part of a large clade that also includes the known trichodiene synthases (Fig. 1). The cop6 gene is the only putative sesquiterpene synthase gene identified in C. cinereus that appears to be part of a biosynthetic gene cluster. The cop6 ORF is flanked on either side by two ORFs encoding putative cytochrome P450 monooxygenases (Cox1 and Cox2).

Analysis of sesquiterpenes produced by C. cinereus

Coprinus cinereus is not generally considered as a terpenoid producer, although one study describes the isolation and structural identification of quinone sesquiterpenoids from this fungus (Bu'Lock and Darbyshire, 1976). Therefore, to determine whether any of the putative terpene synthase homologues identified in the C. cinereus genome sequence is functionally expressed under laboratory growth conditions, C. cinereus was cultured for several weeks in the dark and terpene accumulation was analysed periodically in the culture broth and headspace by solid phase microextraction (SPME) and gas chromatography-mass spectrometry (GC/MS). Very low levels of volatile terpenoid compounds were detected in the culture headspace after 1 month of growth, suggesting that C. cinereus expresses terpene synthases, albeit at very low levels under standard laboratory growth conditions (Fig. 2). Four major peaks with mass fragmentation pattern typical for sesquiterpenes hydrocarbons and with parent ions at 204 m/z were identified. Comparison of their mass spectra and retention indices (RI) with those from published reference spectra and authentic compounds identified them as pentalenene 1, α-muurolene 2, α-cuprenene 3 and δ-cadinene 4 (numbers in bold text placed after compound names correspond to structures, if known; shown in Fig. 3). In addition, several minor sesquiterpenes were present that yielded spectra that did not match any reference spectra.

image

Figure 2. GC/MS analysis of C. cinereus culture. The culture was grown for 1 month in the dark at 28°C with shaking at 125 r.p.m. Several different sesquiterpenes were detected including pentalenene 1, α-muurolene 2, α-cuprenene 3 and δ-cadinene 4.

Download figure to PowerPoint

image

Figure 3. Structures and names of sesquiterpenes described in this study.

Download figure to PowerPoint

Cloning and characterization of sesquiterpene synthases Cop1 to Cop6

To determine the products of the putative terpene synthases Cop1 to Cop6 from C. cinerea, their predicted coding sequences were amplified from cDNA using gene-specific primers for the gene models predicted by the Broad Institute (Table S1). PCR products were cloned into the E. coli expression vector pUCmodRBS and sequenced. PCR amplification products of the expected sizes and sequences were cloned for cop1, cop3 and cop4, while amplification products of cop2 and cop5 contained predicted introns despite several attempts at obtaining spliced products. Predicted introns in cop2 and cop5 (using the Broad Institute gene models) were therefore subsequently removed by overlap extension PCR.

The resulting plasmids containing the cop1 to cop6 sequences were transformed into E. coli JM109 for gene expression and subsequent analysis of terpene compounds produced by the recombinant cultures. For this, 50 ml of cultures were grown to mid-log phase before analysis of the culture headspace by SPME and GC/MS for the presence of terpene products. Terpene compounds were identified by comparing RI and mass spectra with published reference spectra and authentic standard compounds.

The culture headspaces of E. coli cells expressing either the terpene synthase Cop1 or Cop2 contained one major product along with several minor compounds. The major volatile compound in both cultures had a mass fragmentation pattern typical for a sesquiterpene with a parent ion at 204 m/z and characteristic daughter ions at 189, 147, 107, 93 and 81 m/z (Fig. 4A and B; see Fig. S2 for mass spectra). The RI and fragmentation pattern identified this compound as β-elemene 6, which is the heat-induced Cope rearrangement (thermal isomerization of a 1,5-diene) product of germacrene A 6a (see Fig. 3 for structures) (de Kraker et al., 1998; Faraldos et al., 2007). Cis-β-elemene 5, the Cope rearrangement product of the 4Z-isomer of germacrene A, (+)-helminthogermacrene A 5a, was also detected as a minor peak eluting just before β-elemene 6. Minor compounds produced by both Cop1 and Cop2 included α-muurolene 2 and δ-cadinene 4. Germacrene D 7 was detected only in the culture headspace of E. coli cultures expressing Cop1 but not in Cop 2 cultures.

image

Figure 4. GC/MS analysis of volatile organic compounds produced by E. coli transformants expressing sesquiterpene synthases from C. cinereus. E. coli cells expressing Cop1 (A) or Cop2 (B) produced as a major compound germacrene A 6a, which undergoes Cope rearrangement to β-elemene 6 under the high temperatures of the injection port. E. coli cultures expressing Cop3 (C) accumulated significant quantities of α-muurolene 2, while δ-cadinene 4 was the major product of E. coli cells transformed with Cop4 (D). Peaks are labelled with numbers that correspond to their identified structures shown Fig. 3. Mass spectra for individual peaks are shown in Fig. S2. Peak assignments were confirmed by comparing RI with that of authentic compounds.

Download figure to PowerPoint

Escherichia coli strains expressing either Cop3 or Cop4 produced several volatile sesquiterpene compounds (Fig. 4C and D). α-Muurolene 2 (major ions m/z 204, 161, 105) was the major terpenoid compound detected in Cop3 cultures, accounting for about 30% of the total sesquiterpenes detected. In addition, significant amounts of β-elemene 6, γ-muurolene 9, germacrene D 7 and δ-cadinene 4 were detected in the headspace of Cop3 cultures (Fig. 4C). E. coli strains expressing Cop4 accumulated δ-cadinene (major ions m/z 204, 161, 134, 119, 105) as the major terpenoid, accounting for about 40% of the total sesquiterpenes detected (Fig. 4D). However, unlike E. coli cultures that expressed Cop1, Cop2 or Cop3, cultures expressing Cop4 did not produce detectable levels of germacrene A 6a (β-elemene 6). Instead, Cop4 cultures produced several sesquiterpene compounds (β-cubebene 10, sativene 11, β-copaene 12, cubebol 13) not synthesized by any of the other three enzymes.

No terpene compounds were detected in E. coli cultures expressing the cop5 gene that was generated from an intron-containing PCR amplification product by overlap extension PCR based on the gene model annotated by the Broad Institute. Realizing that the cop5 transcript may be spliced differently than the annotated gene model in the genome sequence, two alternative gene models for cop5 were predicted using the Augustus gene prediction programme (Stanke et al., 2004; 2006; 2008) (Fig. S1). Splice site predictions between the three gene models differ in intron 2 and at the 3′ end of the transcript. The two alternative gene models were synthesized by overlap extension PCR from the original, intron-containing cop5 clone. In addition, a chimeric cop5 gene construct was generated that combines predictions of both alternative gene models (Fig. S1). However, none of the three designed cDNA constructs led to the production of sesquiterpenoid compounds in E. coli cultures transformed with the genes cloned into pUCmodRBS. In a further attempt to detect Cop5 enzyme activity, gene constructs were subcloned into a pET expression vector to achieve higher expression levels. However, while protein expression was detected by SDS-PAGE analysis (not shown), none of the expressed recombinant proteins were functional either in vivo in E. coli or under in vitro reaction conditions using E. coli cell lysates.

Escherichia coli cultures expressing trichodiene synthase homologue Cop6 accumulated one major sesquiterpene, accounting for 99% of total products detected (Fig. S3). The mass fragmentation pattern and RI identified this compound as α-cuprenene. α-Cuprenene has previously been isolated and structurally characterized from Hypericum perforatum (St John's wort) (Weyerstahl et al., 1995), and an extract of this plant was used to obtain an authentic standard for α-cuprenene. The retention time of the Cop6 product was identical to the authentic α-cuprenene and therefore identifies Cop6 as an α-cuprenene synthase (Fig. S3).

Characterization of P450 monooxygenases Cox1 and Cox2 located adjacent to Cop6

The cop6 gene is flanked by two putative cytochrome P450 monooxygenase genes (cox1 and cox2,‘cox’ stands for α-cuprenene oxidase). Biosynthetic genes are frequently clustered in fungi so that it was reasonable to assume that the P450 enzymes encoded by these two genes modify α-cuprenene to produce oxygenized sesquiterpenes. To characterize the activities of the two enzymes, S. cerevisiae was chosen for coexpression of cop6 and cox genes. S. cerevisiae expresses a NADPH cytochrome P450 reductase needed for functional expression of fungal microsomal P450 monooxygenases that is not present in E. coli (Tokai et al., 2007). The cuprenene synthase gene, cop6, was subcloned from pUCmodRBS into the galactose inducible yeast expression vector pESC-trp (Table S1). The cox1 and cox2 were first amplified from cDNA using gene-specific primers and directly cloned into the yeast plasmids pESC-leu (pESC-Cox1) and pESC-ura (pESC-Cox2) respectively (Table S1). Expression vector pESC-Cop6 was then transformed into S. cerevisiae alone, along with plasmid pESC-Cox1 or pESC-Cox2 and with both P450 expression vectors. Recombinant yeast cultures were grown up and induced with galactose. After 48 h post induction, culture media were analysed for the accumulation of α-cuprenene oxidation products using SPME followed by GC/MS analysis (Fig. 5).

image

Figure 5. GC/MS analysis of recombinant S. cerevisiae cultures expressing Cop6 and P450s Cox1 and Cox2. Culture media of S. cerevisiae expressing sesquiterpene synthase Cop6 alone (A) or together with Cox1 (B) or Cox2 (C) or both (D) were analysed for the accumulation of sesquiterpene compounds. Recombinant yeast cultures transformed with Cop6 made almost exclusively α-cuprenene 3. α-Cuparene 14 is detected as a minor compound in all P450 coexpressing yeast cultures. Cultures coexpressing Cox2 make α-cuparophenol 18, while mass spectra of other new peaks (numbers correspond to mass spectra in Fig. S2) detected in P450 and Cop6 coexpressing cultures yielded no matches in perused spectral libraries (indicated by questions marks). Parent ions for peaks are shown.

Download figure to PowerPoint

Saccharomyces cerevisiae cultures expressing only Cop6 produced as expected α-cuprenene 3 (Fig. 5A). However, when Cop6 and Cox1 were coexpressed, three new compound peaks (compounds 14, 15 and 16) in addition to α-cuprenene 3 were detected in the culture medium (Fig. 5B; Fig. S2 for mass spectra). Compound 14 had a parent ion of m/z 202 and, based on RI and mass fragmentation pattern, was identified as α-cuparene, an aromatized derivative of α-cuprenene (m/z 204) (see Fig. 3 for structures). The parent ion of m/z 220 of the most-abundant compound 15 suggests the addition of one oxygen atom to α-cuprenene, while the parent ion of m/z 218 of peak 14 indicates a similar oxidation reaction for α-cuparene. Comparison of RI and mass fragments of compounds 14 and 15 with reference data in the MassFinder and National Institute of Standards and Technology (NIST) databases yielded no match for structural assignment.

Coexpression of Cop6 with Cox2 resulted in the appearance of two new sesquiterpene peaks 17 and 18 in addition to α-cuprenene 3 and α-cuparene 14 in the culture medium (Fig. 5C; Fig. S2 for mass spectra). The RI and mass fragmentation pattern of the more-abundant compound 18 (m/z 218) matched those of the aromatic sesquiterpene alcohol cuparophenol 18 (Fig. 3). The mass of compound 17 (m/z 234) indicates an α-cuprenene derivative containing two oxygen atoms, but its mass fragmentation pattern and RI do not give any matches to compounds in reference databases.

When Cox1 and Cox2 together were coexpressed with Cop6, one new peak 19 occurred with a m/z of 232, suggesting again synthesis of an α-cuprenene derivative with two additional oxygen atoms but with slightly longer retention time compared with compound 17 (m/z 234), which is produced by S. cerevisiae coexpressing Cop6 and only Cox2 (Fig. 5D; Fig. S2 for mass spectra). Peaks corresponding to the sesquiterpene hydrocarbons α-cuprenene 3 and α-cuparene 14, the Cox2 oxidation product cuparophenol 18 and the two unknown Cox1 products 15 and 16 were also detected in yeast cultures expressing all three genes of the cop6 gene cluster.

Discussion

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References
  9. Supporting Information

Analysis of sequence data shows that sesquiterpene synthase homologues are widespread among fungi and that this class of enzymes appears to have undergone extensive gene duplication in some fungal species (Fig. 1). However, despite the preponderance of sesquiterpene synthases homologues in fungi, relatively little is known about their activities and biological functions. The few enzymes that have been characterized catalyse the first committed step in the biosynthesis of mycotoxins. Aristolochene synthase, characterized from Penicillium and Aspergillus (Caruthers et al., 2000; Shishova et al., 2007), synthesizes the hydrocarbon precursor of a number of eremophilane mycotoxins (Moule et al., 1977; Capasso et al., 1984; Proctor and Hohn, 1993). Another well-characterized fungal sesquiterpene synthase, trichodiene synthase (Rynkiewicz et al., 2001), makes the hydrocarbon scaffold of the trichothecene mycotoxins produced by, for example, Trichothecium, Stachybotrys and Fusarium; infection of grain crops with Fusarium species, for example, represents a major human and animal health concern (Brown et al., 2004; Desjardins and Proctor, 2007). Presilphiperfolan-8β-ol synthase from the gray mold fungus B. cinerea has recently been functionally characterized (Pinedo et al., 2008) and shown to produce the hydrocarbon scaffold that is modified to the phytotoxic bicyclic sesquiterpene botrydial and its derivatives (Collado et al., 2007).

Some Coprinus species are known to produce bioactive sesquiterpene compounds (Gonzalez del Val et al., 2003; Reina et al., 2004), but sesquiterpene synthases have not been characterized from these species. C. cinereus has long been used as model for fungal development, but it is generally not described as a sesquiterpene producer except for a study published in the 1970s (see below). In the current study, analysis of liquid culture and culture headspace revealed that this species produces sesquiterpenes at very low levels. Major peaks in the headspace analysis corresponded to pentalenene 1, α-muurolene 2, α-cuprenene 3 and δ-cadinene 4, suggesting the presence of sesquiterpene synthases with corresponding specificities. These sesquiterpenes have also been identified in fruiting bodies of wood-rotting basidiomycetes (Rosecke et al., 2000). Enzymes specific for α-cuprenene and α-muurolene have not yet been identified, and pentalenene synthase is only known from bacteria (Lesburg et al., 1997).

Among the six sesquiterpene synthase homologues identified in C. cinereus, Cop1, Cop2 and Cop3 are most closely related to one another (about 45% amino acid sequence identity, Fig. 1), suggesting that they may cyclize all-trans (E,E)-FPP via the same carbocation intermediate. Indeed, formation of the major products of all three enzymes can be proposed to proceed through a germacren-11-yl cation generated via 1,10 ring closure from a primary transoid-farnesyl cation (Fig. 6). Germacrene A 6a, the major product of Cop1 and Cop2, is then derived from this cation by deprotonation. This sesquiterpenoid is the product of several known plant sesquiterpene synthases, and many plant natural products are derived from germacrene A by subsequent oxidation steps (de Kraker et al., 1998; 1999; 2001; Bouwmeester et al., 2002; Prosser et al., 2002; 2004; Bertea et al., 2006). Surprisingly, neither germacrene A 6a nor its heat-induced Cope rearrangement product β-elemene 6 was detected in C. cinereus cultures, suggesting that transcription levels of their genes are very low under the cultivation conditions chosen. In fact, a correctly spliced Cop2 transcript could not be amplified from cDNA preparations.

image

Figure 6. Proposed reaction mechanisms for the formation of major products of C. cinereus sesquiterpene synthases Cop1–4 and Cop6. Shown cyclization pathways are based on previous investigations on cyclization reactions catalysed by various sesquiterpene synthases reported in the literature (Steele et al., 1998; Tholl et al., 2005; Vedula et al., 2008).

Download figure to PowerPoint

Cop3 also accumulates germacrene A 6a, but this enzyme is proposed to catalyse a hydride shift and ring closure of the germacren-11-yl cation to produce, after deprotonation, α-muurolene 2 as its major product. A dedicated muurolene synthase has not been previously characterized, to the best of our knowledge. Several plant species are known to produce this sesquiterpene, including e.g. Cedrela odorata (Brunke et al., 1986) and Cucumis melo (Portnoy et al., 2008). A sesquiterpene synthase was recently cloned from the latter species and shown to produce α-muurolene 2 as a minor compound when expressed in E. coli (Portnoy et al., 2008).

Cop4 and Cop6, which share only about 10–25% sequence identity with Cop1, Cop2 and Cop3, are proposed to catalyse a different reaction mechanism that requires first the isomerization of all-trans (E,E)-FPP at the 2,3 double bond to generate a cisoid-farnesyl cation. Cop4 then catalyses a 1,10 ring closure of this farnesyl cation isomer to form the secondary cis-germacrene-dienyl cation, while Cop6 catalyses a 1,6 ring closure to generate the secondary bisabolyl cation. Both enzymes subsequently generate tertiary cations (Fig. 6). Cop4 generates a tertiary cadinyl cation which upon deprotonation yields its major cyclization product δ-cadinene 4. The cadinyl cation reacts further to yield other cyclization products (β-cubebene 10, cubebol 13, β-copaene 12, sativene 11), observed for Cop4 (Fig. 6). The detection of δ-cadinene 4 and germacrene D 7 (derived from cis-germacrenyl-dienyl cation by deprotonation) in GC/MS traces of Cop1 and Cop2 suggests that these enzymes must also catalyse to some extent the isomerization of all-trans (E, E)-FPP to generate a cadinyl cation. Its major cyclization product identifies Cop4 as a δ-cadinene synthase. Sesquiterpene synthases with this activity have been cloned and characterized from cotton (Gossypium) where they catalyse the first committed step in the biosynthesis of the phytoalexin gossypol (Chen et al., 1995; 1996; Benedict et al., 2001; Yoshikuni et al., 2006).

Cop6 generates the tertiary cuprenyl cation which yields α-cuprenene after deprotonation (Fig. 6). Unlike all the other Coprinus sesquiterpene synthases characterized in this study, Cop6 is a highly specific enzyme that catalyses the cyclization of all-trans (E,E)-FPP into only one product: α-cuprenene (accounting for greater than 99% of all terpenoid products detected in the culture headspace). An α-cuprenene synthase has, to the best of our knowledge, not been described previously; although α-cuprenene is made as a very minor product by the Arabidopsis sesquiterpene synthase At5g44630 (Tholl et al., 2005). Cop6 was originally annotated as a trichodiene synthase and does share a similar reaction mechanism with trichodiene synthase. Both enzymes generate a cuprenyl cation, which in the case of Cop6 is protonated, while trichodiene synthase catalyses an additional methyl shift before releasing the final deprotonated cyclization product. Site-directed mutagenesis of trichodiene synthase supported by structural information has resulted in a variant that makes α-cuprenene as a minor cyclization product (Cane et al., 1996; Rynkiewicz et al., 2002).

The cop6 gene is the only C. cinerea sesquiterpene synthase gene that appears to be part of a biosynthetic gene cluster consisting of cop6 and two predicted genes, cox1 and cox2, encoding predicted P450 monooxygenases. Coexpression of Cop6 with the two P450s yielded six new oxidized α-cuprenene derivatives. Two of the new compounds could be identified as α-cuparene 14 and cuparophenol 18, while the unidentified compounds contained one (peaks 15, 16) or two (peaks 17, 19) oxygen atoms. Oxidized cuparene-type sesquiterpenoids have been described from several fungi and include the antimicrobial enokipodins from the edible mushroom Flammulina velutipes (Ishikawa et al., 2000; 2001), the helicobasidin pigments of the violet root rot fungus Helicobasidium mompa (Natori et al., 1964; 1967; Srikrishna and Ravikumar, 2005; 2006) and the antibiotic lagopodins from several Coprinus species (Bottom and Siehr, 1975; Bu'Lock and Darbyshire, 1976; Srikrishna et al., 2006; 2007). Lagopodin A and B have been isolated from C. cinereus shake flask cultures and structurally characterized by NMR (Bu'Lock and Darbyshire, 1976). Lagopodin A 20 can be derived from α-cuprenene by first oxidation of its cylohexadiene ring to yield an aromatic ring as in α-cuparene, followed by two additional oxidation reactions at positions 1 and 4 (see sesquiterpenoid numbering shown in Fig. 6) to yield a quinone ring. The pentane ring of lagopodin A 20 at position 9 is also oxidized to the corresponding ketone. Lagopodin A is likely the primary biosynthetic product of C. cinereus. Other lagopodins (e.g. lagopodin B containing additional hydroxy groups) that were isolated from C. cinereus cultures are suspected to be artifacts of the isolation procedure (Bu'Lock and Darbyshire, 1976).

The oxidation pattern of lagopodin A 20 allows us to make assumptions as to the function of the two P450s, Cox1 and Cox2. The identification of α-cuparene 14 and α-cuparophenol 18, and the detection of another peak with a parent ion of m/z 234 in yeast cultures coexpressing Cop6 and Cox2, suggests that Cox2 oxidizes the cyclohexadiene ring of α-cuprenene at positions 1 and 4. The resulting oxidation will first give α-cuparene 14 (m/z 202, ring-oxidation followed by H2O elimination upon ring-aromatization), followed by α-cuparophenol 18 (m/z 218, one oxygen atom added) and compound 17 (m/z 234, two oxygen atoms added). Cox1 will then likely catalyse the oxidation at position 9 of the pentane ring of α-cuprenene to give the corresponding hydroxy (m/z 220, compound 15) or ketone (m/z 218, compound 16) derivatives. Coexpression of Cop6 with Cox1 and Cox2 results in one new peak 19 with a parent ion of m/z 232. Considering the proposed activities of the two P450s, peak 19 likely corresponds to an oxidized α-cuparophenol derivative containing a keto-group at the pentane ring. Additional optimization of expression and cultivation conditions will be necessary to fully explore the catalytic activities of the two P450s in the heterologous expression host S. cerevisiae. Larger-scale synthesis of the more-oxidized lagopodin A 20 will be required for structural confirmation of oxidized α-cuprenene derivatives by NMR.

Pentalenene is the most-abundant volatile sesquiterpene produced by C. cinereus, but the sesquiterpene synthase responsible for its synthesis has not yet been identified. The cop5 ORF is a good candidate to encode this sesquiterpene synthase; but despite several attempts, we have been unable to obtain a cDNA that leads to the expression of a functional protein. Nevertheless, this study shows that basidiomycetes represent a rich source for the identification of new terpene synthases and associated biosynthetic genes which may lead to the discovery and subsequent recombinant production of new bioactive compounds, especially in light of the many bioactive sesquiterpenes already isolated from these fungi (Abraham, 2001). Fungal genome sequences with their numerous and often duplicated putative terpene synthase genes give us a glance into a potentially diverse terpenoid metabolism present in this class of organism. Multiple terpene synthase homologues can be identified in well-studied commercial fungi such as some Aspergillus species. It can be expected that these uncharacterized terpene synthases participate in the synthesis of yet unidentified bioactive terpenes. Experimental evidence supports a role of fungal secondary metabolites as antifeedants that provide protection against insect, amoebae, nematode and other invertebrate feeding (Fox and Howlett, 2008). Consequently, many of these secondary metabolites show potent bioactivities against higher eukaryotic organisms, underlining the potential of fungal natural products, including terpene derived compounds, for the discovery of new drugs.

Experimental procedures

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References
  9. Supporting Information

Strains and growth conditions

Coprinus cinereus 9/55 was obtained from University of Minnesota mycological culture collection in St Paul, Minnesota. C. cinereus was maintained on potato dextrose agar and grown for 3 weeks in the dark prior to inoculating liquid cultures. To start liquid cultures, a small agar plug containing mycelium was cut from a plate and ground into small particles using a sterile blender. The slurry was then added to fungal medium containing 1 g l−1 of potassium phosphate (monobasic), 3 g l−1 of sodium nitrate, 0.5 g l−1 of potassium chloride, 0.5 g l−1 of magnesium sulphate (heptahydrate), 5 g l−1 of corn steep powder and 40 g l−1 of glucose and grown in Fernbach flasks containing 1 l of medium at 28°C and 150 r.p.m. in the dark for 3 weeks.

All cloning and investigations of sesquiterpene biosynthesis were carried out in E. coli strain JM109 or S. cerevisiae strain YPH 500 (ura3–52 lys2–801amber ade2–101ochre trp1-Δ63 his3-Δ200 leu2-Δ1). E. coli cultures were grown in Luria–Bertani (LB) medium supplemented with ampicillin (100 μg ml−1) at 30°C and 250 r.p.m. S. cerevisiae was grown in synthetic dextrose minimal medium (SD) containing 6.7 g l−1 of yeast nitrogen base without amino acids, 20 g l−1 of dextrose, 1.3 g l−1 of amino acid dropout powder. Protein expression was induced in synthetic galactose minimal medium (SG), which has the same composition as SD medium except that dextrose is replaced with galactose. The specific dropout medium used corresponded to the auxotrophic strain requirements for the specific plasmids transformed into the yeast (Table S1).

Homology searches and phylogenetic tree construction

Homology searches were performed using the NCBI blast software based on protein sequences of experimentally characterized microbial sesquiterpene synthases from bacteria (Kawaide et al., 1997; Lesburg et al., 1997; Caruthers et al., 2000; Dairi et al., 2001; Rynkiewicz et al., 2001; Hamano et al., 2002; Cane and Watt, 2003; Gust et al., 2003; Toyomasu et al., 2004; 2007; Cane et al., 2006; Kawaide, 2006; Shishova et al., 2007; Agger et al., 2008; Giglio et al., 2008; Komatsu et al., 2008; Pinedo et al., 2008; Zhao et al., 2008) and fungi (Hohn and Beremand, 1989; Hohn and Plattner, 1989; Cane et al., 1995; Hohn et al., 1995; Cane and Kang, 2000; Caruthers et al., 2000; Rynkiewicz et al., 2001; Pinedo et al., 2008). Putative fungal terpene synthase sequences were identified in NCBI's non-redundant protein sequence database and in fungal genome sequences obtained from the Broad Institute and Joint Genome Institute. Sequence alignments were computed using ClustalW (Thompson et al., 2002) and the Mega 4.1 software interface (Tamura et al., 2007). For phylogenetic tree construction, alignments were manually inspected to eliminate sequences that either did not contain the first conserved metal binding domain (DDXXDD motif) of terpene synthases or seemed to be incorrectly annotated (e.g. sequences appeared to be too short or long). Phylogenetic analysis was conducted in Mega 4.1 (Tamura et al., 2007) using the Neighbor-Joining method (Saitou and Nei, 1987) with a bootstrap test of phylogeny (2000 replicates) (Felsenstein, 1992).

Cloning of sesquiterpene synthase and cytochrome P450 monooxygenase genes

For the cloning of the six sesquiterpene synthase genes (cop1 to cop6) and two cytochrome P450 monooxygenase genes (cox1 and cox2), approximately 1 g of freeze dried mycelium of C. cinereus was ground with a mortar and pestle, and mRNA was extracted using the Qiagen RNAeasy Plant Mini Kit (Valencia, CA, USA) followed by DNase I digestion with the DNA-free Kit (Ambion, Foster City, CA, USA). mRNA was annealed to oligo dT (Invitrogen, Carlsbad, CA, USA) followed by RT-PCR using Transcriptor Reverse Transcriptase (Roche, Basel, Switzerland). The genes were then amplified from cDNA by PCR with Vent polymerase (New England Biolabs, Ipswich, MA, USA) using gene-specific primers with added restriction sites. The PCR products were digested with the appropriate restriction enzymes, gel purified and ligated into one or more of the vectors pUCmodRBS (Agger et al., 2008), pHIS8 (Jez et al., 2000) (E. coli expression vector) or pESC (Stratagene, La Jolla, CA, USA) (yeast expression vector) for overexpression in either S. cerevisiae or E. coli (Table S1). Cloned genes were verified by DNA sequencing. Predicted introns in amplification products obtained for cop2 and cop5 were removed by overlap extension PCR (Pogulis et al., 1996).

Analysis of sesquiterpenes produced by recombinant E. coli

To investigate sesquiterpene production, single colonies of E. coli JM109 transformants harbouring the putative sesquiterpene synthases genes on pUCmodRBS were grown for 12 h in 4 ml of LB medium supplemented with ampicillin at 30°C. A 50 ml culture was then inoculated with 1 ml from the seed culture and allowed to shake for 20 h at 30°C. The culture headspace was then sampled for 10 min by SPME using a 100 μm polydimethlysiloxane fibre (Supelco Bellefonte, PA, USA). The fibre was inserted through a tin foil seal into the flask headspace (gas phase) and, after absorption, inserted into the injection port of a GC/MS for thermal desorption.

Analysis of sesquiterpenes produced by recombinant S. cerevisiae coexpressing Cop6 and cytochrome P450 genes

The sesquiterpene synthase gene cop6 from C. cinereus was subcloned from pUCmodRBS into pESC-trp under the control of the gal1–10 promoter. The two putative cytochrome P450 genes cox1 and cox2 flanking the cop6 ORF were cloned into pESC-leu and pEC-ura respectively (Table S1). Plasmid pESC-Cop6 was transformed into S. cerevisiae strain YPH500 alone or cotransformed with pESC-Cox1 or pESC-Cox2 or with both constructs by electroporation and plated onto selective SD minimal medium plates. Recombinant yeast strains were allowed to grow for 48 h at 30°C on agar plates at which time they were scraped off and resuspended to an OD600 of 1 in 50 ml of SG broth containing galactose for induction of protein expression. Yeast cultures were then grown at 30°C and 250 r.p.m. for 48 h before analysis of culture medium and headspace for the presence of sesquiterpene hydrocarbons and oxidized sesquiterpenes. Culture headspace was analysed by SPME as described above for recombinant E. coli cultures. Cells were removed by centrifugation prior to the analysis of terpene products in culture medium. For this, a SPME fibre was submerged into slowly stirring medium for 10 min before thermal desorption in the GC/MS injection port.

Gas chromatography-mass spectrometry analysis

Gas chromatography-mass spectrometry analysis was carried out on a Varion 3800 GC coupled to an ion-trap mass spectrometer (Saturn, Palo Alto, CA, USA). Separation was carried out on a HP-1ms capillary column (30 m × 0.25 mm inner diameter × 1.5 μm) with an injection port temperature of 250°C and helium as a carrier gas. Mass spectra were recorded in electron impact ionization mode. Volatiles were absorbed from the headspace over 10 min, and the fibre was desorbed for 5 min in the injection port. The temperature programme started at 60°C and ramped up 8°C min−1 to a final oven temperature of 250°C. Mass spectra were scanned in the range of 5–300 atomic mass units at 1 s intervals.

Structural identification of sesquiterpene compounds

Sesquiterpene compounds were identified by comparison of their mass spectra with those of published reference terpene spectra in MassFinder's (software version 3) terpene library (Konig et al., 1999) and in the NIST MS database. In addition, RI of sesquiterpene peaks (derived by calibration of GC runs with n-alkane standards) were compared with RI values of terpenoid compounds in MassFinder's terpene library. Essential oils with known terpene compositions served as authentic standards to further confirm the identity of sesquiterpenoid compounds produced by recombinant cultures. The following essential oils with relevant sesquiterpenoid compositions were purchased from Liberty Natural Products: Cedrela woods oil [α-muurolene (1% w/w), δ-cadinene (11.7% w/w), α-copaene (15.6% w/w)], Cubeb oil [germacrene D (1% w/w), γ-muurolene (4.2% w/w), β-cubebene (4.4% w/w), cubebol (15.2% w/w)], Amyris wood oil [β-elemene (germacrene A) (0.1% w/w)]. St John's wort oil (alcohol extract of young flowering tops of H. perforatum) was purchased from Natural Answers Inc. to provide an authentic standard for α-cuprenene (Weyerstahl et al., 1995).

Acknowledgements

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References
  9. Supporting Information

This research was supported by the Academic Health Center of the University of Minnesota (Grant 2005–12) and the National Institute of Health (Grant GM080299).

References

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References
  9. Supporting Information
  • Abraham, W.R. (2001) Bioactive sesquiterpenes produced by fungi: are they useful for humans as well? Curr Med Chem 8: 583606.
  • Agger, S.A., Lopez-Gallego, F., Hoye, T.R., and Schmidt-Dannert, C. (2008) Identification of sesquiterpene synthases from Nostoc punctiforme PCC 73102 and Nostoc sp. strain PCC 7120. J Bacteriol 190: 60846096.
  • Benedict, C.R., Lu, J.L., Pettigrew, D.W., Liu, J., Stipanovic, R.D., and Williams, H.J. (2001) The cyclization of farnesyl diphosphate and nerolidyl diphosphate by a purified recombinant delta-cadinene synthase. Plant Physiol 125: 17541765.
  • Bertea, C.M., Voster, A., Verstappen, F.W., Maffei, M., Beekwilder, J., and Bouwmeester, H.J. (2006) Isoprenoid biosynthesis in Artemisia annua: cloning and heterologous expression of a germacrene A synthase from a glandular trichome cDNA library. Arch Biochem Biophys 448: 312.
  • Bottom, C.B., and Siehr, D.J. (1975) Hydroxylagopodin B: sesquiterpenoid quinone from a mutant strain of Coprinus Macrorhizus Var Microsporus. Phytochem 14: 1433.
  • Bouwmeester, H.J., Kodde, J., Verstappen, F.W.A., Altug, I.G., De Kraker, J.W., and Wallaart, T.E. (2002) Isolation and characterization of two germacrene A synthase cDNA clones from chicory. Plant Physiol 129: 134144.
  • Brown, D.W., Dyer, R.B., McCormick, S.P., Kendra, D.F., and Plattner, R.D. (2004) Functional demarcation of the Fusarium core trichothecene gene cluster. Fungal Genet Biol 41: 454462.
  • Brunke, E.J., Hammerschmidt, E.J., and Koste, F.H. (1986) Essential oil of Cedrela odorata L. (Meliaceae) from Brazil: revised list of constituents. In Progress in Essential Oil Research. Brunke, E.J. (ed.). Berlin: Walter de Gruyter, pp. 117122.
  • Bu'Lock, J.D., and Darbyshire, J. (1976) Lagopodin metabolites and artifacts in cultures of Coprinus. Phytochemistry 15: 2004.
  • Cane, D.E., and Kang, I. (2000) Aristolochene synthase: purification, molecular cloning, high-level expression in Escherichia coli, and characterization of the Aspergillus terreus cyclase. Arch Biochem Biophys 376: 354364.
  • Cane, D.E., and Watt, R.M. (2003) Expression and mechanistic analysis of a germacradienol synthase from Streptomyces coelicolor implicated in geosmin biosynthesis. Proc Natl Acad Sci USA 100: 15471551.
  • Cane, D.E., Shim, J.H., Xue, Q., Fitzsimons, B.C., and Hohn, T.M. (1995) Trichodiene synthase. Identification of active site residues by site-directed mutagenesis. Biochemistry 34: 24802488.
  • Cane, D.E., Xue, Q., and Fitzsimons, B.C. (1996) Trichodiene synthase. Probing the role of the highly conserved aspartate-rich region by site-directed mutagenesis. Biochemistry 35: 1236912376.
  • Cane, D.E., He, X., Kobayashi, S., Omura, S., and Ikeda, H. (2006) Geosmin biosynthesis in Streptomyces avermitilis. Molecular cloning, expression, and mechanistic study of the germacradienol/geosmin synthase. J Antibiot (Tokyo) 59: 471479.
  • Capasso, R., Iacobellis, N.S., Bottalico, A., and Randazzo, G. (1984) Structure toxicity relationships of the eremophilane phomenone and PR-toxin. Phytochemistry 23: 27812784.
  • Caruthers, J.M., Kang, I., Rynkiewicz, M.J., Cane, D.E., and Christianson, D.W. (2000) Crystal structure determination of aristolochene synthase from the blue cheese mold, Penicillium roqueforti. J Biol Chem 275: 2553325539.
  • Chen, X.Y., Chen, Y., Heinstein, P., and Davisson, V.J. (1995) Cloning, expression, and characterization of (+)-delta-cadinene synthase: a catalyst for cotton phytoalexin biosynthesis. Arch Biochem Biophys 324: 255266.
  • Chen, X.Y., Wang, M., Chen, Y., Davisson, V.J., and Heinstein, P. (1996) Cloning and heterologous expression of a second (+)-delta-cadinene synthase from Gossypium arboreum. J Nat Prod 59: 944951.
  • Christianson, D.W. (2006) Structural biology and chemistry of the terpenoid cyclases. Chem Rev 106: 34123442.
  • Christianson, D.W. (2008) Unearthing the roots of the terpenome. Curr Opin Chem Biol 12: 141150.
  • Collado, I.G., Sanchez, A.J., and Hanson, J.R. (2007) Fungal terpene metabolites: biosynthetic relationships and the control of the phytopathogenic fungus Botrytis cinerea. Nat Prod Rep 24: 674686.
  • Dairi, T., Hamano, Y., Kuzuyama, T., Itoh, N., Furihata, K., and Seto, H. (2001) Eubacterial diterpene cyclase genes essential for production of the isoprenoid antibiotic terpentecin. J Bacteriol 183: 60856094.
  • Davis, E.M., and Croteau, R. (2000) Cyclization enzymes in the biosynthesis of monoterpenes, sesquiterpenes, and diterpenes. Topics Curr Chem 209: 5395.
  • Desjardins, A.E., and Proctor, R.H. (2007) Molecular biology of Fusarium mycotoxins. Int J Food Microbiol 119: 4750.
  • Faraldos, J.A., Wu, S., Chappell, J., and Coates, R.M. (2007) Conformational analysis of (+)-germacrene A by variable-temperature NMR and NOE spectroscopy. Tetrahedron 63: 77337742.
  • Felsenstein, J. (1992) Estimating effective population size from samples of sequences: a bootstrap Monte Carlo integration method. Genet Res 60: 209220.
  • Fox, E.M., and Howlett, B.J. (2008) Secondary metabolism: regulation and role in fungal biology. Curr Opin Microbiol 11: 481487.
  • Giglio, S., Jiang, J., Saint, C.P., Cane, D.E., and Monis, P.T. (2008) Isolation and characterization of the gene associated with geosmin production in cyanobacteria. Environ Sci Technol 42: 80278032.
  • Gonzalez del Val, A., Platas, G., Arenal, F., Orihuela, J.C., Garcia, M., Hernandez, P., et al. (2003) Novel illudins from Coprinopsis episcopalis (syn. Coprinus episcopalis), and the distribution of illudin-like compounds among filamentous fungi. Mycol Res 107: 12011209.
  • Gust, B., Challis, G.L., Fowler, K., Kieser, T., and Chater, K.F. (2003) PCR-targeted Streptomyces gene replacement identifies a protein domain needed for biosynthesis of the sesquiterpene soil odor geosmin. Proc Natl Acad Sci USA 100: 15411546.
  • Hamano, Y., Kuzuyama, T., Itoh, N., Furihata, K., Seto, H., and Dairi, T. (2002) Functional analysis of eubacterial diterpene cyclases responsible for biosynthesis of a diterpene antibiotic, terpentecin. J Biol Chem 277: 3709837104.
  • Hibbett, D.S., Binder, M., Bischoff, J.F., Blackwell, M., Cannon, P.F., Eriksson, O.E., et al. (2007) A higher-level phylogenetic classification of the fungi. Mycol Res 111: 509547.
  • Hohn, T.M., and Beremand, P.D. (1989) Isolation and nucleotide sequence of a sesquiterpene cyclase gene from the trichothecene-producing fungus Fusarium sporotrichioides. Gene 79: 131138.
  • Hohn, T.M., and Plattner, R.D. (1989) Purification and characterization of the sesquiterpene cyclase aristolochene synthase from Penicillium roqueforti. Arch Biochem Biophys 272: 137143.
  • Hohn, T.M., and Desjardins, A.E. (1992) Isolation and gene disruption of the Tox5 gene encoding trichodiene synthase in Gibberella pulicaris. Mol Plant Microbe Interact 5: 249256.
  • Hohn, T.M., Desjardins, A.E., and McCormick, S.P. (1995) The Tri4 gene of Fusarium sporotrichioides encodes a cytochrome P450 monooxygenase involved in trichothecene biosynthesis. Mol Gen Genet 248: 95102.
  • Ishikawa, N.K., Yamaji, K., Tahara, S., Fukushi, Y., and Takahashi, K. (2000) Highly oxidized cuparene-type sesquiterpenes from a mycelial culture of Flammulina velutipes. Phytochemistry 54: 777782.
  • Ishikawa, N.K., Fukushi, Y., Yamaji, K., Tahara, S., and Takahashi, K. (2001) Antimicrobial cuparene-type sesquiterpenes, enokipodins C and D, from a mycelial culture of Flammulina velutipes. J Nat Prod 64: 932934.
  • Jez, J.M., Ferrer, J.L., Bowman, M.E., Dixon, R.A., and Noel, J.P. (2000) Dissection of malonyl-coenzyme A decarboxylation from polyketide formation in the reaction mechanism of a plant polyketide synthase. Biochemistry 39: 890902.
  • Kamper, J., Kahmann, R., Bolker, M., Ma, L.J., Brefort, T., Saville, B.J., et al. (2006) Insights from the genome of the biotrophic fungal plant pathogen Ustilago maydis. Nature 444: 97101.
  • Kawaide, H. (2006) Biochemical and molecular analyses of gibberellin biosynthesis in fungi. Biosci Biotechnol Biochem 70: 583590.
  • Kawaide, H., Imai, R., Sassa, T., and Kamiya, Y. (1997) Ent-kaurene synthase from the fungus Phaeosphaeria sp. L487. cDNA isolation, characterization, and bacterial expression of a bifunctional diterpene cyclase in fungal gibberellin biosynthesis. J Biol Chem 272: 2170621712.
  • Keller, N.P., Turner, G., and Bennett, J.W. (2005) Fungal secondary metabolism – from biochemistry to genomics. Nat Rev Microbiol 3: 937947.
  • Komatsu, M., Tsuda, M., Omura, S., Oikawa, H., and Ikeda, H. (2008) Identification and functional analysis of genes controlling biosynthesis of 2-methylisoborneol. Proc Natl Acad Sci USA 105: 74227427.
  • Konig, W.A., Bulow, N., and Saritas, Y. (1999) Identification of sesquiterpene hydrocarbons by gas phase analytical methods. Flavour Frag J 14: 367378.
  • De Kraker, J.W., Franssen, M.C., De Groot, A., Konig, W.A., and Bouwmeester, H.J. (1998) (+)-Germacrene A biosynthesi: the committed step in the biosynthesis of bitter sesquiterpene lactones in chicory. Plant Physiol 117: 13811392.
  • De Kraker, J.W., Bouwmeester, H.J., Franssen, M.C.R., and De Groot, A. (1999) (+)-Germacrene A synthesis in chicory (Cichorium intybus L.); the first step in sesquiterpene lactone biosynthesis. Acta Bot Gall 146: 111115.
  • De Kraker, J.W., Franssen, M.C., Dalm, M.C., De Groot, A., and Bouwmeester, H.J. (2001) Biosynthesis of germacrene A carboxylic acid in chicory roots. Demonstration of a cytochrome P450 (+)-germacrene a hydroxylase and NADP+-dependent sesquiterpenoid dehydrogenase(s) involved in sesquiterpene lactone biosynthesis. Plant Physiol 125: 19301940.
  • Lesburg, C.A., Zhai, G.Z., Cane, D.E., and Christianson, D.W. (1997) Crystal structure of pentalenene synthase: mechanistic insights on terpenoid cyclization reactions in biology. Science 277: 18201824.
  • Loftus, B.J., Fung, E., Roncaglia, P., Rowley, D., Amedeo, P., Bruno, D., et al. (2005) The genome of the basidiomycetous yeast and human pathogen Cryptococcus neoformans. Science 307: 13211324.
  • Martin, F., Aerts, A., Ahren, D., Brun, A., Danchin, E.G., Duchaussoy, F., et al. (2008) The genome of Laccaria bicolor provides insights into mycorrhizal symbiosis. Nature 452: 8892.
  • Martinez, D., Larrondo, L.F., Putnam, N., Gelpke, M.D., Huang, K., Chapman, J., et al. (2004) Genome sequence of the lignocellulose degrading fungus Phanerochaete chrysosporium strain RP78. Nat Biotechnol 22: 695700.
  • Martinez, D., Challacombe, J., Morgenstern, I., Hibbett, D., Schmoll, M., Kubicek, C.P., et al. (2009) Genome, transcriptome, and secretome analysis of wood decay fungus Postia placenta supports unique mechanisms of lignocellulose conversion. Proc Natl Acad Sci USA 106: 19541959.
  • Misiek, M., and Hoffmeister, D. (2007) Fungal genetics, genomics, and secondary metabolites in pharmaceutical sciences. Planta Med 73: 103115.
  • Mondego, J.M., Carazzolle, M.F., Costa, G.G., Formighieri, E.F., Parizzi, L.P., Rincones, J., et al. (2008) A genome survey of Moniliophthora perniciosa gives new insights into Witches' Broom Disease of cacao. BMC Genomics 9: 548.
  • Moule, Y., Moreau, S., and Bousquet, J.F. (1977) Relationships between the chemical structure and the biological properties of some eremophilane compounds related to PR-toxin. Chem Biol Interact 17: 185192.
  • Natori, S., Inoue, Y., and Nishikawa, H. (1967) The structures of mompain and deoxyhelicobasidin and the biosynthesis of helicobasidin, quinonoid metabolites of Helicobasidium mompa Tanaka. Chem Pharm Bull 15: 380390.
  • Natori, S., Nishikawa, H., and Ogawa, H. (1964) Structure of helicobasidin, a novel benzoquinone from helicobasdium from Helicobasidium mompa Tanaka. Chem Pharm Bull 12: 236243.
  • Pinedo, C., Wang, C.M., Pradier, J.M., Dalmais, B., Choquer, M., Le Pecheur, P., et al. (2008) Sesquiterpene synthase from the botrydial biosynthetic gene cluster of the phytopathogen Botrytis cinerea. ACS Chem Biol 3: 791801.
  • Pogulis, R.J., Vallejo, A.N., and Pease, L.R. (1996) In vitro recombination and mutagenesis by overlap extension PCR. Methods Mol Biol 57: 167176.
  • Portnoy, V., Benyamini, Y., Bar, E., Harel-Beja, R., Gepstein, S., Giovannoni, J.J., et al. (2008) The molecular and biochemical basis for varietal variation in sesquiterpene content in melon (Cucumis melo L.) rinds. Plant Mol Biol 66: 647661.
  • Proctor, R.H., and Hohn, T.M. (1993) Aristolochene synthase. Isolation, characterization, and bacterial expression of a sesquiterpenoid biosynthetic gene (Ari1) from Penicillium roqueforti. J Biol Chem 268: 45434548.
  • Prosser, I., Phillips, A.L., Gittings, S., Lewis, M.J., Hooper, A.M., Pickett, J.A., and Beale, M.H. (2002) (+)-(10R) -Germacrene A synthase from goldenrod, Solidago canadensis; cDNA isolation, bacterial expression and functional analysis. Phytochemistry 60: 691702.
  • Prosser, I., Altug, I.G., Phillips, A.L., Konig, W.A., Bouwmeester, H.J., and Beale, M.H. (2004) Enantiospecific (+)- and (−)-germacrene D synthases, cloned from goldenrod, reveal a functionally active variant of the universal isoprenoid-biosynthesis aspartate-rich motif. Arch Biochem Biophys 432: 136144.
  • Reina, M., Orihuela, J.C., Gonzalez-Coloma, A., De Ines, C., De La Cruz, M., Gonzalez del Val, A., et al. (2004) Four illudane sesquiterpenes from Coprinopsis episcopalis. Phytochemistry 65: 381385.
  • Rosecke, J., Pietsch, M., and Konig, W.A. (2000) Volatile constituents of wood-rotting basidiomycetes. Phytochemistry 54: 747750.
  • Rynkiewicz, M.J., Cane, D.E., and Christianson, D.W. (2001) Structure of trichodiene synthase from Fusarium sporotrichioides provides mechanistic inferences on the terpene cyclization cascade. Proc Natl Acad Sci USA 98: 1354313548.
  • Rynkiewicz, M.J., Cane, D.E., and Christianson, D.W. (2002) X-ray crystal structures of D100E trichodiene synthase and its pyrophosphate complex reveal the basis for terpene product diversity. Biochemistry 41: 17321741.
  • Sacchettini, J.C., and Poulter, C.D. (1997) Creating isoprenoid diversity. Science 277: 17881789.
  • Saitou, N., and Nei, M. (1987) The Neighbor-Joining method: a new method for reconstructing phylogenetic trees. Mol Biol Evol 4: 406425.
  • Shishova, E.Y., Di Costanzo, L., Cane, D.E., and Christianson, D.W. (2007) X-ray crystal structure of aristolochene synthase from Aspergillus terreus and evolution of templates for the cyclization of farnesyl diphosphate. Biochemistry 46: 19411951.
  • Siewers, V., Viaud, M., Jimenez-Teja, D., Collado, I.G., Gronover, C.S., Pradier, J.M., et al. (2005) Functional analysis of the cytochrome P450 monooxygenase gene bcbot1 of Botrytis cinerea indicates that botrydial is a strain-specific virulence factor. Mol Plant Microbe Interact 18: 602612.
  • Spiteller, P. (2008) Chemical defence strategies of higher fungi. Chemistry 14: 91009110.
  • Srikrishna, A., and Ravikumar, P.C. (2005) Total synthesis of HM-1 and HM-2, aromatic sesquiterpenes isolated from the phytopathogenic fungus Helicobasidium mompa. Structure revision of HM-2. Tetrahedron Lett 46: 61056109.
  • Srikrishna, A., and Ravikumar, P.C. (2006) Structure revision of HM-3, an aromatic sesquiterpene isolated from the phytopathogenic fungus Helicobasidium mompa. First total syntheses of HM-3 and HM-4. Tetrahedron 62: 93939402.
  • Srikrishna, A., Lakshmi, B.V., and Ravikumar, P.C. (2006) The first total synthesis of (+/−)-lagopodin A. Tetrahedron Lett 47: 12771281.
  • Srikrishna, A., Babu, R.R., and Ravikumar, P.C. (2007) A regioselective total synthesis of the fungal sesquiterpene (+/−)-lagopodin A. Synlett 4: 655657.
  • Stanke, M., Steinkamp, R., Waack, S., and Morgenstern, B. (2004) AUGUSTUS: a web server for gene finding in eukaryotes. Nucleic Acids Res 32: W309W312.
  • Stanke, M., Keller, O., Gunduz, I., Hayes, A., Waack, S., and Morgenstern, B. (2006) AUGUSTUS: ab initio prediction of alternative transcripts. Nucleic Acids Res 34: W435W439.
  • Stanke, M., Diekhans, M., Baertsch, R., and Haussler, D. (2008) Using native and syntenically mapped cDNA alignments to improve de novo gene finding. Bioinformatics 24: 637644.
  • Steele, C.L., Crock, J., Bohlmann, J., and Croteau, R. (1998) Sesquiterpene synthases from grand fir (Abies grandis). Comparison of constitutive and wound-induced activities, and cDNA isolation, characterization, and bacterial expression of delta-selinene synthase and gamma-humulene synthase. J Biol Chem 273: 20782089.
  • Tamura, K., Dudley, J., Nei, M., and Kumar, S. (2007) MEGA4: Molecular Evolutionary Genetics Analysis (MEGA) software version 4.0. Mol Biol Evol 24: 15961599.
  • Tetzlaff, C.N., You, Z., Cane, D.E., Takamatsu, S., Omura, S., and Ikeda, H. (2006) A gene cluster for biosynthesis of the sesquiterpenoid antibiotic pentalenolactone in Streptomyces avermitilis. Biochemistry 45: 61796186.
  • Tholl, D. (2006) Terpene synthases and the regulation, diversity and biological roles of terpene metabolism. Curr Opin Plant Biol 9: 297304.
  • Tholl, D., Chen, F., Petri, J., Gershenzon, J., and Pichersky, E. (2005) Two sesquiterpene synthases are responsible for the complex mixture of sesquiterpenes emitted from Arabidopsis flowers. Plant J 42: 757771.
  • Thompson, J.D., Gibson, T.J., and Higgins, D.G. (2002) Multiple sequence alignment using ClustalW and ClustalX. Curr Protoc Bioinform Chapter 2: Unit 2 3.
  • Tokai, T., Koshino, H., Takahashi-Ando, N., Sato, M., Fujimura, M., and Kimura, M. (2007) Fusarium Tri4 encodes a key multifunctional cytochrome P450 monooxygenase for four consecutive oxygenation steps in trichothecene biosynthesis. Biochem Biophys Res Commun 353: 412417.
  • Toyomasu, T., Nakaminami, K., Toshima, H., Mie, T., Watanabe, K., Ito, H., et al. (2004) Cloning of a gene cluster responsible for the biosynthesis of diterpene aphidicolin, a specific inhibitor of DNA polymerase alpha. Biosci Biotechnol Biochem 68: 146152.
  • Toyomasu, T., Tsukahara, M., Kaneko, A., Niida, R., Mitsuhashi, W., Dairi, T., et al. (2007) Fusicoccins are biosynthesized by an unusual chimera diterpene synthase in fungi. Proc Natl Acad Sci USA 104: 30843088.
  • Vedula, L.S., Jiang, J., Zakharian, T., Cane, D.E., and Christianson, D.W. (2008) Structural and mechanistic analysis of trichodiene synthase using site-directed mutagenesis: probing the catalytic function of tyrosine-295 and the asparagine-225/serine-229/glutamate-233-Mg2+B motif. Arch Biochem Biophys 469: 184194.
  • Weyerstahl, P., Splittgerber, U., and Marshall, H. (1995) Constitutents of the leaf essential oil of Hypericum perforatum L. from India. Flavour Fragrance J 10: 365370.
  • Yoshikuni, Y., Martin, V.J., Ferrin, T.E., and Keasling, J.D. (2006) Engineering cotton (+)-delta-cadinene synthase to an altered function: germacrene D-4-ol synthase. Chem Biol 13: 9198.
  • Zhao, B., Lin, X., Lei, L., Lamb, D.C., Kelly, S.L., Waterman, M.R., and Cane, D.E. (2008) Biosynthesis of the sesquiterpene antibiotic albaflavenone in Streptomyces coelicolor A3 (2). J Biol Chem 283: 81838189.

Supporting Information

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References
  9. Supporting Information
FilenameFormatSizeDescription
MMI_6717_sm_Figures_S1-S3_and_Table_S1.pdf405KSupporting info item

Please note: Wiley Blackwell is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.