Uracil → Orotidine 5′-monophosphate

Participants

Reaction Steps

After UV irradiation of the suspension of Penicillium multicolor IAM7153 conidia, the spores were coated on a 5-fluoroorotic acid (5-FOA) medium (Boeke et al., Mol. Gen. Genet. 197: 345-346, 1984) and incubated at 27° C. for 6 to 8 days. The 5-FOA-resistant strain grown was picked up respectively onto a minimal medium (0.3% NaNO3: 0.05% KCl, 0.05% MgSO4.7H2O, 0.001% FeSO4.7H2O, 0.1% KH2PO4, 2% glucose, 1.5% agar, pH 6.0) and a minimal medium containing 5 mM uracil respectively, and the media were incubated at 27° C. for 3 to 5 days. The uracil-demanding strain growing only on the minimal medium containing 5 mM uracil was inoculated into a YPM medium containing 5 mM uridine (1% yeast extract, 2% peptone, 2% maltose, pH 6.0) and incubated at 27° C. and 140 rpm for 3 days by shaking culture. 1 g of the hypha and 1 g of sea sand were placed in a mortar cooled on ice and were pulverized sufficiently therein, and 10 ml of 50 mM Tris-HCl (pH 8.0) was added to the mortar, to give a suspension of the hypha. The suspension was centrifuged at 36,000×g for 10 minutes, for separation of the supernatant. The orotic acid phosphoribosyltransferase (OPRTase) activity of the supernatant was determined according to the method of Belser et al., (Meth. Enzymol. 51: 155-167, 1978), and the active strain was regarded as an orotidine-5′-phosphate decarboxylase-deficient strain (IAM7153ΔpyrG) (Horiuchi et al., Curr. Genet. 27: 472-478, 1995).

Provenance & Evidence