nidulans argB as a selectable marker. Transformants were streak purified and verified for correct integration into see more the IS1 site (Hansen et al., 2011) by two complementing diagnostic PCRs. Strains were inoculated as three point stabs on solid media and incubated for 7 days at 37 °C in the dark. Metabolite extraction was performed according to the micro extraction procedure (Smedsgaard, 1997). Extracts were analyzed by two methods:
(1) Ultra-high performance liquid chromatography-diode array detection (UHPLC-DAD) analyses using a Dionex RSLC Ultimate 3000 (Dionex, Sunnyvale, CA) equipped with a diode-array detector. Separation of 1 μL extract was obtained on a Kinetex C18 column (150 × 2.1 mm, 2.6 μm; Phenomenex, Torrence, CA) at 60 °C using a linear water–acetonitrile gradient starting from AZD5363 price 15% CH3CN to 100% (50 ppm trifluoroacetic acid) over 7 min at a flow rate of 0.8 mL min−1. (2) Exact mass, HPLC-DAD-HRMS, was performed on a 5 cm × 3 μm, Luna C18(2) column (Phenomenex) using a water–acetonitrile gradient from 15% CH3CN to 100% over 20 min (20 mM formic acid). LC-DAD-MS analysis was performed on a LCT oaTOF mass spectrometer (Micromass, Manchester, UK) as in Nielsen & Smedsgaard (2003) and Nielsen et al. (2009). 3,5-Dimethylorsellinic acid and dehydroaustinol
were purified from large-scale ethyl acetate extracts prepared from 100 MM agar plates after 4 days’ cultivation in darkness at 37 °C. The compounds were purified using a 10 × 250 mm Phenomenex pentafluorophenyl column (5 μm particles) with a water–acetonitrile gradient from 15% to 100% CH3CN in 20 min using a flow of 5 mL min−1. Arugosin A was isolated from an ethyl acetate extract of the reference strain grown on 200 CYAs agar plates using a Waters 19 × 300 mm C18 Delta Pak column (15 μm particles), gradient from 80% to 90% CH3CN in 10 min, and a flow of 30 mL min−1. The NMR spectra were acquired on a Varian Unity Inova 500 MHz spectrometer using standard Amino acid pulse sequences. Additional details about the compound identification can be found in the supporting information.
The principle of using different media and/or incubation conditions for fungal secondary metabolite production has often been promoted (Oxford et al., 1935; Davis et al., 1966; Pitt et al., 1983; Bode et al., 2002; Scherlach & Hertweck, 2006). Based on our previous experiences (Frisvad, 1981; Frisvad & Filtenborg, 1983; Filtenborg et al., 1990; Frisvad et al., 2007), eight different media, CYA, CYAs, CY20, MM, RT, RTO, YE and YES, were initially selected for the analysis (Fig. 1a). HPLC analyses revealed a large number of different secondary metabolites produced by the A. nidulans reference strain on CYA, CYAs, CY20, RT, RTO and YES (Fig. 1b) and these metabolites served as a source for further investigation. To investigate whether any of the compounds observed in Fig. 1 could be genetically linked to a PKS gene, we decided to take a global approach and individually deleted all 32 (putative and known) PKS genes in the A.