No abstract

Summary Filamentous fungi are known as prolific untapped reservoirs of diverse secondary metabolites, where genes required for their synthesis are organized in clusters. The bioactive properties of these compounds are closely related to their functions in fungal biology, which are not well understood. In this study, we focused on the Podospora anserina gene cluster responsible for the biosynthesis of sterigmatocystin (ST). Deletion of the PaStcA gene encoding the polyketide synthase and overexpression (OE) of the PaAflR gene encoding the ST‐specific transcription factor in P. anserina were performed. We showed that growth of PaStcAΔ was inhibited in the presence of methylglyoxal, while OE‐PaAflR showed a little inhibition, indicating that ST production may enhance oxidative stress tolerance in P. anserina. We also showed that the OE‐PaAflR strain displayed an overpigmented thallus mediated by the melanin pathway. Overexpression of PaAflR also led to sterility. Interspecific confrontation assays showed that ST‐overexpressed strains produced a high level of peroxides and possessed a higher competitiveness against other fungi. Comparative metabolite profiling demonstrated that PaStcAΔ strain was unable to produce ST, while OE‐PaAflR displayed a ST overproduction. This study contributes to a better understanding of ST in P. anserina, especially with regard to its involvement in fungal physiology.

General. NMR spectra were recorded with a Bruker DPX 300, AV 400, or DMX 600 spectrometer in the solvents indicated; chemical shifts (δ) are given in ppm relative to TMS, coupling constants (J) in Hertz. The solvent signals were used as references and the chemical shifts converted to the TMS scale (CDCl 3 : δ C = 77.0 ppm; residual CHCl 3 in CDCl 3 : δ H = 7.24 ppm; CD 2 Cl 2 : δ C = 53.8 ppm; residual CH 2 Cl 2 in CD 2 Cl 2 : δ H = 5.32 ppm). Where indicated, the signal assignments are unambiguous; the numbering scheme is arbitrary and is shown in the inserts. The assignments are based upon 1D and 2D spectra recorded using the following pulse sequences from the Bruker standard pulse program library: DEPT; COSY (cosygs and Negishi carboalumination: Preparation of vinyl iodide 25.A solution of AlMe 3 (2.0 M in heptane, 21.2 mL, 42.4 mmol) was added to a suspension of Cp 2 ZrCl 2 (4.64 g, 15.9 mmol) in 1,2-dichloroethane (70 mL). After stirring for 0.5 h, a solution of alkyne 24 (3.56 g, 10.57 mmol) in 1,2-dichloroethane (15 mL) was added dropwise. The resulting yellow solution was stirred for 24 h at ambient temperature before the mixture was cooled to −20°C and a solution of iodine (16.10 g, 63.5 mmol) in THF (60 mL) was slowly added. After stirring for 20 min at −20°C and 30 min at 0°C, the reaction was carefully quenched with water (10 mL). A sat. aq. Na 2 SO 3 solution was then added and the layers were separated. The aqueous phase was extracted twice with CH 2 Cl 2 (40 mL each), the combined organic extracts were washed with brine, dried over MgSO 4 and evaporated. .7, 135.7, 134.0, 129.7, 127.8, 75.4, 61.8, 39.9, 37.7, 27.0, 20.5, 19.6, 19.3 was added dropwise to a solution of epoxide 7 (7.6 g, 30.84 mmol) in toluene (25 mL) at −78°C over the course of 1 h. The temperature was then raised to −40°C and stirring continued for 4 h before the reaction was carefully quenched at −60°C with a solution of tBuOH in THF (1:1, 25 mL). The resulting mixture was poured into an icecold solution of Rochelle's salt (85g in 250 mL water) and vigorously stirred for 2 h until a clear separation of the phases was reached. The aqueous layer was extracted with tert-butyl methyl ether, the combined organic phases were dried over Na 2 SO 4 and evaporated, and the residue was purified by flash chromatography (hexanes:EtOAc, 1:1) to give product 8 as a

General. NMR spectra were recorded with a Bruker DPX 300, AV 400, or DMX 600 spectrometer in the solvents indicated; chemical shifts (δ) are given in ppm relative to TMS, coupling constants (J) in Hertz. The solvent signals were used as references and the chemical shifts converted to the TMS scale (CDCl 3 : δ C = 77.0 ppm; residual CHCl 3 in CDCl 3 : δ H = 7.24 ppm; CD 2 Cl 2 : δ C = 53.8 ppm; residual CH 2 Cl 2 in CD 2 Cl 2 : δ H = 5.32 ppm). Where indicated, the signal assignments are unambiguous; the numbering scheme is arbitrary and is shown in the inserts. The assignments are based upon 1D and 2D spectra recorded using the following pulse sequences from the Bruker standard pulse program library: DEPT; COSY (cosygs and Negishi carboalumination: Preparation of vinyl iodide 25.A solution of AlMe 3 (2.0 M in heptane, 21.2 mL, 42.4 mmol) was added to a suspension of Cp 2 ZrCl 2 (4.64 g, 15.9 mmol) in 1,2-dichloroethane (70 mL). After stirring for 0.5 h, a solution of alkyne 24 (3.56 g, 10.57 mmol) in 1,2-dichloroethane (15 mL) was added dropwise. The resulting yellow solution was stirred for 24 h at ambient temperature before the mixture was cooled to −20°C and a solution of iodine (16.10 g, 63.5 mmol) in THF (60 mL) was slowly added. After stirring for 20 min at −20°C and 30 min at 0°C, the reaction was carefully quenched with water (10 mL). A sat. aq. Na 2 SO 3 solution was then added and the layers were separated. The aqueous phase was extracted twice with CH 2 Cl 2 (40 mL each), the combined organic extracts were washed with brine, dried over MgSO 4 and evaporated. .7, 135.7, 134.0, 129.7, 127.8, 75.4, 61.8, 39.9, 37.7, 27.0, 20.5, 19.6, 19.3 was added dropwise to a solution of epoxide 7 (7.6 g, 30.84 mmol) in toluene (25 mL) at −78°C over the course of 1 h. The temperature was then raised to −40°C and stirring continued for 4 h before the reaction was carefully quenched at −60°C with a solution of tBuOH in THF (1:1, 25 mL). The resulting mixture was poured into an icecold solution of Rochelle's salt (85g in 250 mL water) and vigorously stirred for 2 h until a clear separation of the phases was reached. The aqueous layer was extracted with tert-butyl methyl ether, the combined organic phases were dried over Na 2 SO 4 and evaporated, and the residue was purified by flash chromatography (hexanes:EtOAc, 1:1) to give product 8 as a