An 8.8-kb fragment of A. fumigatus DNA containing the genes dmaW, easF, easE, and easC was successfully amplified by PCR and integrated into the genome of two A. nidulans transformants, yielding strains WFEC33 and WFEC35. The presence of each of the four A. fumigatus genes in the WFEC strains was verified by PCR primed from internal gene primer-annealing sites (Figure 2). The recipient strain A. nidulans A767 did not contain any of the four genes. The absence of the ergot alkaloid gene cluster in A. nidulans was further supported by the lack of sequences with an E value less than 10 in a BLASTn search between the A. fumigatus dmaW, easF, easE, and easC cluster sequence with the genome of A. nidulans FGSC A4.

Both WFEC strains and A. nidulans A767 were analyzed by HPLC with fluorescence detection to investigate the presence of ergot alkaloids. Transformed strains accumulated an analyte that co-eluted with chanoclavine-I at approximately 33 min, whereas A. nidulans A767 did not (Figure 3). Extracts of WFEC35 and A. nidulans A767 also were analyzed by electrospray ionization (ESI) LC-MS in positive mode. Again WFEC35 contained a peak that co-eluted with chanoclavine-I standard. The analyte had an m/z of 257.1 consistent with the [M + H]⁺ of chanoclavine-I (Figure 4). HPLC with fluorescence detection of mutant strain WFEC35 also revealed the presence of the early pathway intermediate N-Me-DMAT which eluted at approximately 44 min (Figure 3). The identity of this analyte as N-Me-DMAT was supported by an ion of m/z 287.1 in ESI LC-MS analyses (corresponding to [N-Me-DMAT + H]⁺) and by co-elution with authentic standard (Figure 4).

Conidiating cultures produced modest quantities of chanoclavine-I. Aspergillus nidulans strain WFEC35 produced approximately 0.3 fg chanoclavine-I per conidium, or about 3 ng chanoclavine-I per mm² of culture surface area and comparable quantities of N-Me-DMAT. Strain WFEC33 produced lesser quantities of chanoclavine-I and did not accumulate measurable quantities of N-Me-DMAT.

Two additional mutant strains of A. nidulans were generated by transformation with subsets of three of the four ergot alkaloid cluster genes: strains WFC (containing dmaW, easF, and easC) and WFE (containing dmaW, easF, and easE). The presence of each gene in the genomes of the different mutants was verified by PCR (Figure 2). Chanoclavine-I was not detectable by HPLC (Figure 5) or ESI LC-MS (data not shown) in either strain. Expression of dmaW and easF from the inserted construct in both strains was validated by the accumulation of the early pathway intermediate, N-Me-DMAT, which eluted from the column at approximately 44 min (Figure 5). In addition, WFC cultures accumulated a novel molecule with an elution time of 41 min and that had an m/z value of 259.0 in ESI LC-MS analyses. The unidentified metabolite displayed a higher fluorescence at excitation and emission wavelengths of 310 nm and 410 nm, respectively, than at 272 nm excitation and 372 nm emission settings (Figure 5). Ergot alkaloids with a double bond between carbons 9 and 10 of the ergot alkaloid ring system fluoresce stronger at the 310 nm (excitation) and 410 nm (emission) setting compared to their fluorescence at the 272 nm/372 nm setting; conversely, ergot alkaloids with a 8,9 double bond or those lacking a double bond at either of those positions fluoresce stronger at the 272 nm/372 nm setting [15]. The novel alkaloid was not produced in sufficient quantities to allow the acquisition of additional structural data at this time.

Aspergillus fumigatus isolate Af293, as a source of genomic DNA, was grown in malt extract broth (15 g malt extract/L) overnight at 37 °C. The uridine auxotrophic A. nidulans isolate FGSC A767 (Fungal Genetic Stock Center, Kansas City, MO, USA) was grown at 37 °C on SYE medium (20 g/L sucrose, 10 g/L yeast extract, 10 g/L MgSO₄, 2 mL/L trace element solution [17], 2.2 g/L uracil, 2.2 g/L uridine, with or without 15 g/L agar). For preparation of protoplasts, A. nidulans was grown in liquid SYE medium overnight. Transformants were regenerated on selective SYE agar medium which did not contain uracil or uridine and was supplemented with 304 g/L sucrose (to serve as osmoticum as well as carbon source). To test alkaloid accumulation, cultures were grown on SYE agar medium at 37 °C for one week.

DNA was extracted from A. fumigatus according to the protocol described by Richards et al. [18]. An 8774-bp fragment containing dmaW, easF, easC, and easE was amplified by PCR primed with oligonucleotides primer 1 (5'-TACCTATACCTAATCGAAGCCGCACGCAGTGCACC-3') and primer 2 (5'-GCCGCCCATTCACCAAGATTTTTGCACAAATCTGCG-3'). A 25 μL reaction contained 1× LongAmp Taq reaction buffer, 200 μM of each deoxynucleotide triphosphate, 1 μM of each primer, and 2.5 units LongAmp Taq DNA polymerase (New England BioLabs, Ipswich, MA, USA). The reaction began with an initial denaturing step of 3 min at 94 °C, followed by 35 cycles of 30 s at 94 °C and 10 min at 65 °C, with a final extension of 5 min at 65 °C.

A 6123-bp fragment containing dmaW, easF, and easE was amplified by PCR primed with oligonucleotides primer 3 (5'-GAGAGCTACTTGACATATTGTGTCGGCAGGTGCGCA-3') and primer 2 (5'-GCCGCCCATTCACCAAGATTTTTGCACAAATCTGCG-3'). The PCR conditions were the same as stated above except the elongation time was 7 min.

In order to generate transformant WFC (containing dmaW, easF, and easC), two separate DNA fragments were amplified for co-transformation. The first fragment containing dmaW and easC was primed with primer 1 (5'-TACCTATACCTAATCGAAGCCGCACGCAGTGCACC-3') and primer 5 (5'-CTACTCCATTGTCCTGTGAGTTG-3') which primed amplification of a 4846-bp fragment. The PCR conditions were the same as stated above except the elongation time was 5 min. The second fragment (2943 bp) contained only easF and was amplified by PCR primed with oligonucleotides easFF (5'-TCCATTCTTCGCTCGTTCAACCAGCAGG-3') and easFR (5'-CAGGACCTGTACCTAAAGCCTGGTAACC-3') in a 25 μL reaction containing 1× GoTaq Flexi buffer, 200 μM each deoxynucleotide triphosphate, 1.5 mM MgCl₂, 1 μM of each primer, and 2.5 units of Taq DNA polymerase (Promega, Madison, WI, USA). The reaction began with an initial denaturing step of 3 min at 94 °C, followed by 35 cycles of 30 s at 94 °C, 30 s at 55 °C, and 3 min at 72 °C, with a final extension of 5 min at 72 °C.

PCR products were purified with QIAquick gel extraction kits (Qiagen, Valencia, CA, USA) prior to their inclusion in fungal transformations. The selectable marker pPyrG was obtained from the Fungal Genetics Stock Center and was digested with XhoI (New England BioLabs, Ipswich, MA, USA) and purified with QIAquick gel extraction kit prior to transformation.

Protoplasts of A. nidulans FGSC A767 were prepared by incubating overnight cultures with 50 mg driselase (Sigma-Aldrich, St. Louis, MO, USA), which was filter sterilized with a 0.22 μm filter, and 75 mg lysing enzyme (Sigma-Aldrich, St. Louis, MO, USA) in 15 mL of 0.7 M sodium chloride. Protoplasts were purified and co-transformed with PCR product and selectable marker (pPyrG) as described by Coyle et al. [19]. Protoplasts were plated on SYE agar medium containing no uracil or uridine. Transformation plates were incubated at 37 °C, and transformants were collected approximately three days after transformation.

Each transformant was screened by PCR for the presence or absence of dmaW, easF, easC, and easE. An internal region of dmaW was primed with dmaWF (5'-TTGATCTGGAGTGGTTCCGC-3') and dmaWR (5'-CGTTCATGCCGAAGGTTGTG-3') yielding a 651-bp fragment. Primers for amplifying an internal region of easE were easEF (5'-CCAGATACATTGCCATCGCATG-3') and easER (5'-TGTTCCAACTGCTTGGCCAGAT-3'), which primed amplification of a 897-bp fragment. The presence or absence of easC was tested by PCR with primers easCF (5'-GAATTCGAGGTATTGATCTCC-3') and easCR (5'-AGCCAGGCAAAGATCCATAGTT-3') which flank a 1036-bp fragment. An internal region of easF was primed with easFF (5'-AAGTTGTCGAAGGTCTCACGAA-3') and easFR (5'-GTGATTAGAGATGCTTCTGTC-3'), yielding an 820-bp fragment. Each of these PCRs were 25 μL reactions containing 1× GoTaq Flexi buffer, 200 μM deoxynucleotide triphosphate, 1.5 mM MgCl₂, 1 μM of each primer, and 2.5 units of Taq DNA polymerase (Promega, Madison, WI, USA). PCR conditions began with an initial denaturing step of 3 min at 94 °C, followed by 35 cycles of 30 s at 94 °C followed by 30 s at 55 °C, and 1 min and 30 s at 72 °C, followed by a final extension of 5 min at 72 °C.

Transformants positive for the presence of the target genes were analyzed by HPLC for the accumulation of chanoclavine-I. Ergot alkaloids were extracted by incubating a spore-rich sample of the colony, containing approximately 1 cm² of culture surface area, in 400 μL of methanol for 30 min and clarifying the extract by centrifugation. Extracts were injected into a C₁₈ column (Prodigy 5-μm ODS3 (150 mm by 4.6 mm); Phenomenex, Torrance, CA, USA) and subjected to a multilinear binary gradient from 5% (v/v) acetonitrile plus 95% (v/v) aqueous 50 mM ammonium acetate to 75% acetonitrile plus 25% aqueous 50 mM aqueous ammonium acetate at a flow rate 1 mL/min, as previously described [15]. Ergot alkaloids were detected using two fluorescence settings. The first setting had an excitation wavelength of 272 nm and emission wavelength of 372 nm; the second setting was 310 nm (excitation) and 410 nm (emission). Chanoclavine-I (generously provided by Brian Tapper, AgResearch, New Zealand) and N-Me-DMAT (gift from Sarah O’Connor, University of East Anglia) were run as standards. Peaks of interest were isolated from the HPLC by collecting the runoff from the discard tube for 30 s after the peak was visualized on the detector.

Ergot alkaloid extracts were prepared by washing the surface of a conidiating colony with 2 mL of methanol, rotating the extract at 30 rpm for 30 min, and then clarifying the extract by centrifugation. Clarified extracts were then concentrated to a volume of 50 μL in a speed-vac apparatus, and 10 μL of concentrated extract were injected into the LC-MS. To isolate individual analytes, concentrated extracts were analyzed by fluorescence HPLC, peaks were collected manually, and concentrated to a volume of 50 μL in a speed-vac apparatus prior to LC-MS analysis. Concentrated samples were analyzed on a Finnigan LCQDecaXP plus mass spectrometer equipped with a Surveyor HPLC system. Analytes were separated on a C₁₈ column (Phenomenex 4-μm polar-RP (150 mm by 2 mm)), maintained at 30 °C, by combining mobile phases A (5% acetonitrile, 0.1% formic acid) and B (75% acetonitrile, 0.1% formic acid). Initial conditions were 86% A + 14% B, which ramped linearly to 73% A + 27% B at 25 min, 25% A + 75% B at 40 min, and 0% A + 100% B at 42 min. The flow rate was 200 μL/min. Analytes were ionized by electrospray ionization in positive mode and were detected by scanning for ions with a m/z between 200–400.