Alkaloid Cluster Gene ccsA of the Ergot Fungus Claviceps purpurea ...

APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Mar. 2010, p. 1822–1830 0099-2240/10/$12.00 doi:10.1128/AEM.00737-09 Copyright © 2010, American Society for Microbiology. All Rights Reserved.

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Alkaloid Cluster Gene ccsA of the Ergot Fungus Claviceps purpurea Encodes Chanoclavine I Synthase, a Flavin Adenine Dinucleotide-Containing Oxidoreductase Mediating the Transformation of N-Methyl-Dimethylallyltryptophan to Chanoclavine I䌤 ˇulc,2 and Paul Tudzynski1* Nicole Lorenz,1 Jana Olsˇovska´,2 Miroslav S Institut fu ¨r Botanik, Westfalische Wilhelms-Universita ¨t Mu ¨nster, Schlossgarten 3, D-48149 Mu ¨nster, Germany,1 and Institute of Microbiology, Academy of Sciences of the Czech Republic, v.v.i., Víden ˇska ´ 1083, 14220 Prague 4, Czech Republic2 Received 31 March 2009/Accepted 16 January 2010

Ergot alkaloids are indole-derived secondary metabolites synthesized by the phytopathogenic ascomycete Claviceps purpurea. In wild-type strains, they are exclusively produced in the sclerotium, a hibernation structure; for biotechnological applications, submerse production strains have been generated by mutagenesis. It was shown previously that the enzymes specific for alkaloid biosynthesis are encoded by a gene cluster of 68.5 kb. This ergot alkaloid cluster consists of 14 genes coregulated and expressed under alkaloid-producing conditions. Although the role of some of the cluster genes in alkaloid biosynthesis could be confirmed by a targeted knockout approach, further functional analyses are needed, especially concerning the early pathwayspecific steps up to the production of clavine alkaloids. Therefore, the gene ccsA, originally named easE and preliminarily annotated as coding for a flavin adenine dinucleotide-containing oxidoreductase, was deleted in the C. purpurea strain P1, which is able to synthesize ergot alkaloids in axenic culture. Five independent knockout mutants were analyzed with regard to alkaloid-producing capability. Thin-layer chromatography (TLC), ultrapressure liquid chromatography (UPLC), and mass spectrometry (MS) analyses revealed accumulation of N-methyl-dimethylallyltryptophan (Me-DMAT) and traces of dimethylallyltryptophan (DMAT), the first pathway-specific intermediate. Since other alkaloid intermediates could not be detected, we conclude that deletion of ccsA led to a block in alkaloid biosynthesis beyond Me-DMAT formation. Complementation with a ccsA/gfp fusion construct restored alkaloid biosynthesis. These data indicate that ccsA encodes the chanoclavine I synthase or a component thereof catalyzing the conversion of N-methyl-dimethylallyltryptophan to chanoclavine I.

homolog of dmaW (AY259840) possessing a similar function could also be isolated in C. purpurea, as was confirmed by a knockout approach (N. Lorenz and P. Tudzynski, unpublished data). Using genome walking combined with cDNA screening, a 68.5-kb genomic region surrounding dmaW could be sequenced and revealed 14 open reading frames (ORFs) (putative genes) encoding, among others, nonribosomal peptide synthetases (NRPSs), a putative catalase, a CYP450-1 monooxygenase, a putative methyltransferase, and several oxidoreductases (6, 13, 19) (Fig. 2). Some of these genes were functionally and biochemically analyzed by a gene replacement approach which revealed their function within the pathway (2, 5, 7). However, there is still a deficit in functional analyses, especially with respect to the early steps within this pathway. The conversion from N-methyl-dimethylallyltryptophan (Me-DMAT) to agroclavine via chanoclavine I and chanoclavine I aldehyde includes successive oxidation and reduction steps mediated by a specific class of enzymes, the oxidoreductases (15) (Fig. 1). These enzymes are involved in the biosynthesis of many fungal secondary metabolites. A prominent example is the family of the cytochrome P450 monooxygenases (named after the characteristic peak of 450 nm when complexed with carbon monoxide). Cytochrome P450 (CYP450) monooxygenases catalyze the transfer of one oxygen atom from molecular oxygen

The ergot fungus Claviceps purpurea is a phytopathogenic ascomycete which infects the ears of several grasses, replacing the ovary and producing a hibernation structure, the so-called sclerotium, in which the ergot alkaloids are formed. These substances show a high level of structural homology to some neurotransmitters like serotonin and dopamine and can therefore bind to the same receptors in the central nervous system (CNS), which is the basis for the application of ergot alkaloids in a variety of clinical conditions (15). The biochemistry of ergot alkaloid biosynthesis was first investigated by isolation of intermediates and postulation of a hypothetical pathway as well as enzymes needed for the successive biosynthetic steps of the production (Fig. 1). Most of the data were collected by pursuing the fate of radiolabeled precursors in feeding experiments (4). The first enzyme which could be assigned to alkaloid production was dimethylallyltryptophan synthetase (DMATS), which is the key enzyme of the pathway and is encoded by the gene dmaW (18). These analyses were performed with a Claviceps fusiformis strain, but a * Corresponding author. Mailing address: Institut fu ¨r Botanik, Westfalische Wilhelms-Universita¨t Mu ¨nster, Schlossgarten 3, D-48149 Mu ¨nster, Germany. Phone: 49-251-8324998. Fax: 49-251-8321601. E-mail: [email protected]. 䌤 Published ahead of print on 29 January 2010. 1822

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FIG. 1. Biosynthetic pathway of the ergot alkaloid biosynthesis of C. purpurea. Genes analyzed so far have been assigned to the corresponding enzyme at the corresponding position within the pathway. DMAPP, dimethylallyldiphosphate; DMAT, dimethylallyltryptophan; Me-DMAT, N-methyl-DMAT. (Adapted from reference 7 with permission of Wiley-VCH Verlag GmbH & Co. KGaA.)

to various substrates, mostly accomplished by the involvement of NAD(P)H as an electron donor. The eas cluster of C. purpurea also includes a gene encoding a CYP450 monooxygenase: cloA is involved in the oxidation of elymoclavine, leading to the formation of paspalic acid (7). No further monooxygenase-encoding genes seem to be present in the eas cluster, but several genes code for putative oxidoreductases (easA, easD, easE, easG, and easH). These oxidoreductases are most likely involved in the early steps

within the pathway, but none of them has been functionally analyzed so far (15). We initiated a functional analysis of the putative oxidoreductase-encoding gene ccsA (formerly easE) (Fig. 2). The coding region of ccsA (AJ011965; 1,503 bp) is composed of two exons interrupted by an intron of 52 bp, yielding a coding capacity of 483 amino acids (aa). The gene product shows highest similarity to putative oxidoreductases of other ergot alkaloid-producing fungi: EasE of C. fusiformis (e⫺160;

FIG. 2. Alkaloid biosynthesis gene cluster of C. purpurea. Highlighted in white is the gene of interest ccsA. (Adapted from reference 7 with permission of Wiley-VCH Verlag GmbH & Co. KGaA.)

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FIG. 3. Construction of a replacement vector for knockout of the putative oxidoreductase gene ccsA. Top: genomic situation; orientation of arrows indicates direction of transcription. Positions of primers utilized for amplification of the flanks are marked by black arrows. The 5⬘ flank of 1,149 bp was amplified by the primer pair ccsA_VF_v2 and ccsA_VF_h and cloned in front of the gpdA promoter of the phleomycin resistance cassette using the restriction sites KpnI and EcoRI. The 3⬘ flank of 1,030 bp was amplified by the primer pair ccsA_HF_v and ccsA_HF_h and cloned behind the gpdA terminator of the phleomycin resistance cassette using the restriction sites NotI and SacII. The complete vector RV-ccsA (8,935 bp) is based on vector pAN8.1_UM. phleo, phleomycin resistance gene; prom, gpdA promoter (Aspergillus nidulans); term, gpdA terminator (A. nidulans). Relative positions of introns are marked by light gray boxes.

ABV57823), EasE of Neotyphodium lolii (e⫺118; ABM91450) and CpoX1 of Aspergillus fumigatus (e⫺96; XM_751049). Analyses of the protein sequence using the program PROSITE revealed a flavin adenine dinucleotide (FAD)-binding domain (pfam01565) spanning the region from amino acids 14 to 161 and a berberine bridge enzyme domain (BBE domain; pfam08031) from amino acids 412 to 457. The role of CcsA in the alkaloid biosynthesis pathway was investigated by knockout of the corresponding gene, followed by functional and biochemical analyses of the deletion mutants. MATERIALS AND METHODS Strains and culture conditions. The ku70-deficient strain derived from Claviceps purpurea strain P1 (ATCC 20102 [8, 19), which produces mainly ergotamine with small amounts of ergocryptine, was described earlier (5), as were the standard media and culture conditions (19). For alkaloid production, the fungus was cultivated in T25N medium with low (0.5 g/liter KH2PO4) and high (2.0 g/liter KH2PO4) levels of phosphates. Molecular biology techniques. Standard cloning and DNA analysis techniques were performed according to the methods of Sambrook et al. (14). The Escherichia coli strains used for cloning by using plasmids pUC19 (Fermentas, St. Leon-Rot, Germany) and pCR2.1-TOPO (Invitrogen, Karlsruhe, Germany) and propagation of clones was TOP10F⬘ (Invitrogen, Karlsruhe, Germany). Extraction of genomic DNA, Southern and Northern blot analyses, and DNA sequencing were performed as described previously (11). For sequence comparisons and multiple sequence alignments, DNA STAR (Madison, Wisconsin) was used. For

further analyses, the programs BLAST (http://www.ncbi.nlm.nih.gov/blast/Blast .cgi) and PROSITE (http://www.expasy.ch/prosite/) were utilized. Design of a replacement vector and transformation of a ku70-deficient strain of C. purpurea. The replacement vector RV-ccsA is based on the vector pAN8.1_UM, including two multiple cloning sites in front of the gpdA promoter (Aspergillus nidulans) and downstream of the gpdA terminator (Aspergillus nidulans) of the phleomycin resistance cassette, an ampicillin resistance gene for selection in bacteria, and an origin of replication (Fig. 3). (For primers used for amplification of the flanks, see below.) Transformation of protoplasts of C. purpurea was performed as described previously (2). RNA isolation. Total RNA extraction was done as described earlier (17) from 7-day-old mycelia using the RNAgents total RNA isolation system (Promega, Mannheim, Germany). Concentrations of purified RNA were determined using a BioPhotometer (Eppendorf, Hamburg, Germany), and RNA integrity was examined by electrophoresis in 1% formaldehyde agarose gels. PCR—primers and conditions. For PCR analysis, BioTherm (Genecraft, Lu ¨dinghausen, Germany) polymerase was used according to the manufacturer’s instructions. For the construction of the replacement vector, the flanks were amplified by the primer pairs ccsA_VF_v2 (5⬘-CCA TTC AGG TAC CCG TCC AG-3⬘) and ccsA_VF_h (5⬘-AAG GAC AGA ATT CCT CCA CGC-3⬘) for the 5⬘ flank and ccsA_HF_v (5⬘-CGG AGC TAT CTA GAT AGC ATC-3⬘) and ccsA_HF_h (5⬘-TTG GAT GCC GCG GTG AGT GCA T-3⬘) for the 3⬘ flank. To check the transformants for homologous integration events, diagnostic PCR was performed using the primer pair d_ccsA_v (5⬘-GCA TCA AGA GCA CAA CAA CAC GAG-3⬘) and PAN3 (5⬘-GGT CAC CAG TCG CTG GCT TCC CG-3⬘) for the 5⬘ flank and the primer pair PAN2 (5⬘-CCG TAA CAC CCA ATA CGC CGG-3⬘) and d_ccsA_h (5⬘-TGC AGC ATA GAC CCC AGA CAG AC-3⬘) for

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FIG. 4. Gene replacement strategy for ccsA (left) and diagnostic PCR of ⌬ccsA (5) and ⌬ccsA (6) deletion mutants compared to the wild type (right). Primers used for verification of homologous integration of the 5⬘ flank were d_ccsA_v (1) and PAN3 (2), yielding a fragment of 1.8 kb. Integration of the 3⬘ flank was checked using the primer pair PAN2 (3) and d_ccsA_h (4), resulting in a fragment of 1.3 kb. Purification of the transformants was tested by the primer pair ccsA_easF_U (5) and ccsA_R (6), yielding a fragment of 1.1 kb, exclusively observed in the wild-type P1. SacI restriction sites used for Southern analyses (Fig. 5) and the sizes of the fragments achieved were also indicated. phleo, phleomycin resistance gene; prom, gpdA promoter (Aspergillus nidulans); term, gpdA terminator (A. nidulans); WT, wild-type band.

the 3⬘ flank. Purification of the mutants was checked by the primer pair ccsA_ easF_U (5⬘-ACC GTG GGT GCA GTA GGA GGC-3⬘) and ccsA_R (5⬘-GCT TCC GGC AAA TAC CTT CTG-3⬘). For complementation of the deletion mutant, the gene ccsA was amplified by the primer pair ccsA_GFP_v (5⬘-CAC CAA TTC TAG AAG CAC CG-3⬘) and ccsA_GFP_h (5⬘-GGT ACC CAT TCC GAT AAG ACT GGA CGC-3⬘) and cloned into the pUC19 vector in front of the start codon of the gfp (green fluorescent protein) gene from Aequorea jellyfish (16), leading to a fusion protein where GFP is located at the C terminus of CcsA. The vector already contained a 1-kb fragment of the nitrate reductase gene (niaD) of C. purpurea (see Fig. 9). The niaD gene could be used for targeted integration of the complementation construct because of the increased rate of homologous integration of the ku70deficient strain of C. purpurea. Additionally, a hygromycin resistance cassette (consisting of the oliC promoter, the hygromycin resistance gene, and the trpC terminator of Aspergillus nidulans) was cloned via the XbaI restriction site into the vector, enabling direct selection of the transformants. Successful integration was tested by diagnostic PCR using the primer GFP_h (5⬘-TCG AAT TCT TAC TTG TAC AGC TCG TCC-3⬘). Extraction and analyses of ergot alkaloids. For alkaloid extraction and determination, cultures were adjusted to pH 11 with concentrated aqueous ammonium hydroxide and extracted three times with chloroform, and after concentration, the resulting liquid was applied onto thin-layer chromatography (TLC) plates (silica gel 60; Merck, Darmstadt, Germany). Alkaloids were identified by comparison with the corresponding standards in chloroform-methanol-ammonium (80:20:0.2; vol/vol/vol). After separation, TLC plates were sprayed with Ehrlich’s reagent for alkaloid visualization. Standards used were 1 mg/ml ergotamine (Sigma, Taufkirchen, Germany), 1 mg/ml ergocryptine (Sigma, Taufkirchen, Germany), 1 mg/ml ergocristine (Sigma, Taufkirchen, Germany), 1 mg/ml agroclavine (Sandoz AG, Basel, Switzerland), and 1 mg/ml D-lysergic acid (Sandoz AG, Basel, Switzerland). High-pressure liquid chromatography (HPLC) separation was carried out on a LiChrospher 100 RP-18 (Merck/Hitachi, Darmstadt, Germany) column (250 ⫻ 4 mm inner diameter; 5-␮m particle size), tempered at 25°C, and operated at a flow rate of 1 ml/min. Compounds were isocratically eluted within 40 min using a binary mobile phase containing 10 mM ammonium carbonate and acetonitrile (50:50, vol/vol). Ergot alkaloids (EA) were detected with an L-7400 UV detector operating at 230 nm. EA standards were used as described above. UPLC method. An Acquity ultrapressure liquid chromatography (UPLC) system (Waters, Milford, Massachusetts), equipped with a 2996 polydiode array (PDA) detector operating at 225 nm and 310 nm, was used for EA analysis under UPLC conditions (12). Data were processed with Empower 2 software (Waters). Samples were analyzed on a Waters BEH C18 column (50 ⫻ 2.1 mm inner diameter; 1.7-␮m particle size) under the following conditions: column temperature, 35°C; data sample rate, 20 data points (pts)/s; filter constant, 0.5; injection

volume, 5 ␮l; analysis time, 12 min; flow rate, 0.4 ml/min. Mobile phases consisted of water (phase A) and acetonitrile (phase B), both containing 0.04% NH4OH. Gradient elution started at 5% acetonitrile (0 min), increasing linearly to 61% acetonitrile within 12 min. Each analysis was followed with a columnwashing step (95% acetonitrile, 1 min) and equilibration step (1 min). Mass spectrometry analysis. Mass spectra were measured on a matrix-assisted laser desorption–ionization reflectron time-of-flight (MALDI-TOF) mass spectrometer (BIFLEX; Bruker-Franzen, Bremen, Germany) equipped with a nitrogen laser (337 nm) and gridless delayed extraction ion source. The ion acceleration voltage was 19 kV, and the reflectron voltage was set to 20 kV. Spectra were

FIG. 5. Southern analysis of three pure knockout transformants of ⌬ccsA and the wild-type P1. Genomic DNA of the wild type and three deletion mutants of ccsA were digested with the restriction enzyme SacI, separated in an agarose gel, blotted to a nylon membrane, and hybridized with the 5⬘ flank of the replacement vector. In the wild type, a band of 4.8 kb could be observed, whereas in the deletion mutants a truncated fragment of 3.4 kb is detected, due to an additional SacI restriction site in the replacement fragment (see also Fig. 4).

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MALDI-TOF and postsource decay (PSD) spectra were collected in reflectron mode.

RESULTS

FIG. 6. Northern analysis of ⌬ccsA (5) and ⌬ccsA (6) mutants and wild-type P1. Total RNA of the wild-type P1 and of two deletion mutants of ccsA were isolated, separated in a formaldehyde agarose gel (1%), and transferred to a nylon membrane. Hybridization was carried out using fragments of the genes ccsA, easD, and easF and ribosomal DNA genes (rDNA) as a loading control.

calibrated externally using the monoisotopic [M⫹H]⫹ ion of matrix peaks 190.1 m/z and 379.1 m/z and peptide MRFA (Met-Arg-Phe-Ala) 524.3 m/z. Solutions (10 mg/ml) of ␣-cyano-4-hydroxy-cinnamic acid or 2,5-dihydrobenzoic acid in 50% acetonitrile with 0.3% acetic acid were used as MALDI matrices. Each collected UPLC fraction was dried on a vacuum concentrator, dissolved in water, and sonicated for 5 min prior to mass spectrometry analysis. A 1-␮l sample of matrix solution was mixed with 1 ␮l of the aqueous solution of the sample, and 1 ␮l of this premix was loaded on the target and allowed to dry at ambient temperature. The

Generation of knockout mutants of ccsA in a ku70-deficient strain of C. purpurea P1. For deletion of ccsA, a double-crossover approach was used, resulting in replacement of the ccsA locus by a phleomycin resistance cassette by homologous recombination (Fig. 4). The replacement construct was based on the phleomycin resistance cassette of vector pAN8.1_UM flanked by 1,149-bp and 1,030-bp 5⬘ and 3⬘ regions, respectively, of ccsA (Fig. 4). As the recipient strain, a ku70-deficient derivative of the producer strain P1 was used, characterized by an enhanced efficiency for homologous recombination (5). Successful integration was checked by diagnostic PCR using the primer pair d_ccsA_v and PAN3 for homologous integration of the 5⬘ flank (yielding a fragment of 1.8 kb) and primer pair PAN2 and d_ccsA_h for verification of the 3⬘ flank (yielding a band of 1.4 kb; Fig. 4). Of 22 transformants checked, 13 showed homologous integration. Interestingly, 6 out of these 13 also missed the wild-type band when the primer pair ccsA_easF_U and ccsA_R was used, indicating that these transformants were already homokaryotic and contained only transformed nuclei. Successful knockout was also confirmed by Southern analysis using SacI-digested DNA of wild-type strain P1 as well as five homokaryotic transformants (Fig. 5). The gene ccsA lacks any SacI restriction site, so in the wild type only one band of 4.8 kb could be observed. In contrast, the gene replacement con-

FIG. 7. UPLC analyses of the transformant ⌬ccsA (14). A peak at time point 4.32 min could be detected with a protonated molecule at m/z 287.2 in MALDI-TOF analysis. The compound possessed a UV spectrum typical for ergot alkaloids showing maxima at 224 nm and 280 nm. The same UV spectrum could be observed for the compound detected at time point 4.58 min with a MALDI-TOF signal at m/z 272.2. AU, arbitrary units.

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FIG. 8. MALDI-TOF analysis of the ⌬ccsA transformant (14). The main product accumulating in the deletion mutant provided a protonated molecule at m/z 287.2 in full MS (left). The PSD experiment of this signal (middle) using a DHB matrix revealed the structure of Me-DMAT (N-methyl-dimethylallyltryptophan) with proposed fragmentation (right).

struct includes two SacI restriction sites, resulting in the detection of a reduced band of 3.4 kb in the deletion mutants when hybridized with the 5⬘ flank and a band of 3.1 kb when the 3⬘ flank was used as a probe (Fig. 4). Northern analyses confirmed the successful knockout of the gene: no ccsA signal could be detected in the knockout mutants, whereas the genes easD and easF were expressed as in the wild type, confirming that expression of the neighboring cluster genes was not affected by the gene replacement approach (Fig. 6). The integrity of the neighboring cluster genes was also verified by sequencing of genomic PCR fragments (data not shown). Biochemical characterization of ⌬ccsA mutants. The alkaloid-producing capabilities of five deletion mutants were analyzed by TLC (thin-layer chromatography), HPLC (high-pressure liquid chromatography), and UPLC (ultrapressure liquid chromatography [12]) with PDA detection (polydiode array),

and MS (mass spectrometry), and the spectra were compared to the alkaloid spectrum of the wild type. The first preliminary HPLC analyses showed that all knockout mutants had lost the ability to produce clavine alkaloids, and it was concluded, therefore, that the pathway was blocked at a step prior to the steps catalyzing ergoline ring formation. Moreover, TLC analyses confirmed not only the lack of clavines but also the lack of more complex alkaloids such as ergopeptines (data not shown). To address the production of possible intermediates in more detail, UPLC analyses were performed. In these analyses, the accumulation of a substance (after 4.32 min) was reproducibly detected showing the typical UV pattern of ergot alkaloids (maxima at wavelengths of 224 nm and 280 nm, respectively) (Fig. 7). Structure determination of the compound was performed by MS analysis. The acquired MALDI-TOF spectra revealed that the isolated intermediate had a main signal at m/z

FIG. 9. Scheme of the complementation vector and diagnostic PCR of a complemented mutant (cCcsA) and the wild type (P1). The complementation vector is based on the vector pUC19 and includes the complementation construct, consisting of the genes ccsA (white) with endogenous promoter (light gray) and gfp (dark gray), 1.0 kb of the niaD gene (black) for targeted integration, and a hygromycin resistance gene (gray) as a selection marker. Restriction enzymes relevant for cloning of the vector are indicated (for details see also Materials and Methods). Primers for diagnostic PCR were used as described for Fig. 4. Additionally, the whole complementation construct of 3.1 kb was amplified with the primer pair ccsA_GFP_v and GFP_h, whereas the gene ccsA (with endogenous promoter) was amplified with ccsA_GFP_h as a reverse primer delivering a fragment of only 2.3 kb.

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FIG. 10. HPLC analysis of the wild type (P1), the deletion mutant (⌬ccsA), and the complemented mutant (cCcsA:gfp). Peaks: 1, ergotamine; 2, ergocryptine; 3, ergotaminine; 4, methyl-dimethylallyltryptophan.

287.2, and manual interpretation of its MS/MS fragmentation resulted in identification of N-methyl-dimethylallyltryptophan (Me-DMAT) as the main end product of the deletion mutants (Fig. 8). Dimethylallyltryptophan (DMAT) was also found in minor amounts in the UPLC fraction at the time point 4.58 min, with a MALDI-TOF signal at m/z 272.2. The identification of Me-DMAT as the main product in contrast to DMAT strengthens the assumption that the pathway is blocked at the biosynthetic step following DMAT methylation, which explains why Me-DMAT accumulates whereas DMAT is constantly

converted to Me-DMAT and thus present in only minor amounts. The next pathway-specific intermediate not detected in the deletion mutants is chanoclavine I, supporting the presumption that CcsA is essential for the conversion from MeDMAT to chanoclavine I. Therefore, the gene—originally called easE (15)—was named ccsA (for chanoclavine synthase). Complementation of the ⌬ccsA mutants. To confirm that the failure of the mutants to produce clavine and more-complex alkaloids was due to the targeted knockout, the transformants were complemented with a gene construct designed to express

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FIG. 11. Early steps of the ergot alkaloid pathway, from N-methyl-dimethylallyltryptophan (Me-DMAT) to chanoclavine I. For details, see text.

a CcsA::GFP fusion protein. This construct could allow not only testing for restoration of alkaloid production but also analysis of the cellular localization of alkaloid biosynthesis, which is described elsewhere as compartmentalized (15). The complementation vector cCcsA/GFP contained the full coding region of ccsA with its endogenous promoter (to avoid overexpression), the gfp gene fused in frame to the 3⬘ end of ccsA, and part of the niaD (nitrate reductase) locus for targeted integration in this nonessential gene (for details, see Materials and Methods) (Fig. 9). Integration of the complementation fragment in the niaD locus was confirmed by PCR (Fig. 9) as well as Southern analysis (data not shown), verifying also that—due to the integration outside the eas cluster—the gene replacement situation was still present. The expression pattern, as well as the alkaloid spectrum of the complemented mutants, was analyzed by Northern blotting and HPLC. The expression of the ccsA/gfp fusion construct was confirmed by the presence of a larger ccsA homologous transcript (data not shown), and functionality of the resulting fusion protein was shown by restoration of ergotamine production in the complemented mutant (Fig. 10). The GFP signal obtained, however, was rather diffuse, not supporting the assumption that the biosynthesis is located in specialized cellular compartments (data not shown). DISCUSSION A knockout of the eas cluster gene ccsA resulted in accumulation of Me-DMAT and traces of DMAT, as indicated by UPLC and MS analyses, strongly suggesting that CcsA is involved in the “chanoclavine I synthase” activity converting Me-DMAT to chanoclavine I (Fig. 1). Two successive oxidation steps are required for chanoclavine I formation from Me-DMAT (Fig. 11). The reaction most likely starts with the generation of the diene by desaturation of the C8-C9 bond and loss of a proton at C17 (4). This is followed by rotation around the C8-C9 bond and oxidation of the C7-C8, proposed to give an epoxide intermediate (4). Decarboxylation of this intermediate by a (spontaneous) SN2⬘ reaction could be coupled with bond formation between C5 and C10 and cleavage of the epoxide. In addition to binding the FAD cofactor, it is conceivable that the BBE domain is involved in the subsequent cyclization, either as a catalyst or by determining stereospecificity, as has been suggested for the BBE (3, 9). Following this, ring opening of the epoxide occurs by proton attack with shift of the double bond between C9 and C10 to the position between C8 and C9.

We showed here that CcsA is essential for this biosynthetic step, but it remains an open question whether the reaction is carried out by CcsA alone or whether CcsA belongs to a complex of enzymes catalyzing the two oxidation steps to form the diene and then the epoxide, as was postulated by Schardl et al. (15). Experiments involving the knockout of other putative oxidoreductase-encoding genes of the eas cluster, e.g., easA, easD, easG, or easH, could help in further elucidating this process. ACKNOWLEDGMENTS We thank S. Pazˇoutova´ and M. Flieger, Prague, Czech Republic, for discussions and U. Keller, Berlin, Germany, for critical reading of the manuscript. The DFG (special focus program Evolution of Metabolic Diversity) provided financial support. REFERENCES 1. Reference deleted. 2. Correia, T., N. Grammel, I. Ortel, U. Keller, and P. Tudzynski. 2003. Molecular cloning and analysis of the ergopeptine assembly system in the ergot fungus Claviceps purpurea. Chem. Biol. 10:1281–1292. 3. Facchini, P. J., C. Penzes, A. G. Johnson, and D. Bull. 1996. Molecular characterization of berberine bridge enzyme genes from opium poppy. Plant Physiol. 112:1669–1677. 4. Gro ¨ger, D., and H. G. Floss. 1998. Biochemistry of ergot alkaloids—achievements and challenges, p. 171. In G. A. Cordell (ed.), Alkaloids, vol. 50. Academic Press, New York, NY. 5. Haarmann, T., N. Lorenz, and P. Tudzynski. 2008. Use of a non-homologous end-joining deficient strain (⌬ku70) of the ergot fungus Claviceps purpurea for identification of a nonribosomal peptide synthetase gene involved in ergotamine biosynthesis. Fungal Gen. Genet. 45:35–44. 6. Haarmann, T., C. Machado, Y. Lu ¨bbe, T. Correia, C. L. Schardl, D. G. Panaccione, and P. Tudzynski. 2005. The ergot alkaloid gene cluster in Claviceps purpurea: extension of the cluster sequence and intra species evolution. Phytochemistry 66:1312–1320. 7. Haarmann, T., I. Ortel, P. Tudzynski, and U. Keller. 2006. Identification of the cytochrome P450 monooxygenase that bridges the clavine and ergoline alkaloid pathways. Chembiochem 7:645–652. 8. Keller, U., M. Han, and M. Sto ¨ffler-Meilicke. 1988. D-lysergic acid activation and cell-free synthesis of D-lysergyl peptides in enzyme fractions from the ergot fungus Claviceps purpurea. Biochemistry 27:6164–6170. 9. Kutchan, T. M., and H. Dittrich. 1995. Characterization and mechanism of the berberine bridge enzyme, a covalently flavinylated oxidase of benzophenanthridine alkaloid biosynthesis in plants. J. Biol. Chem. 270:24475– 24481. 10. Reference deleted. 11. Mey, G., K. Held, J. Scheffer, K. B. Tenberge, and P. Tudzynski. 2002. CPMK2, a SLT2-homologous mitogen-activated protein (MAP) kinase, is essential for pathogenesis of Claviceps purpurea on rye: evidence for a second conserved pathogenesis-related MAP kinase cascade in phytopathogenic fungi. Mol. Microbiol. 46:305–318. ˇ ulc, P. Nova 12. Olsˇovska ´, J., M. S ´k, S. Pazˇoutova ´, and M. Flieger. 2008. Liquid chromatography-tandem mass spectrometry characterization of ergocristam degradation products. J. Chromatogr. B Analyt. Technol. Biomed. Life Sci. 873:165–172. 13. Rigbers, O., and S. M. Li. 2008. Ergot alkaloid biosynthesis in Aspergillus fumigatus. Overproduction and biochemical characterization of a 4-dimethylallyltryptophan N-methyltransferase. J. Biol. Chem. 283:26859–26868. 14. Sambrook, J., E. F. Fritsch, and T. Maniatis. 1989. Molecular cloning: a

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