In vivo labeling method of cyclosporin A with [¹⁴C-methyl]-S-adenosyl-L-methionine can evaluate a potency of cyclosporin A-producing mutant

Abstract

Cyclosporin A synthetase activity ofTolypocladium inflatum can be estimated by measuring its N-methyltransferase activity. In vivo N-methyltransferase activity of cyclosporin A synthetase of cells was measured by in vivo [¹⁴C-methyl] labeling assay, which was designed for actively growing cells. After the cells were incubated with 0.025 μCi of [¹⁴C-methyl]-S-adenosyl-l-methionine, [¹⁴C-methyl] labelled cyclosporin A and its analogs inside the cells were extracted with ethylacetate and¹⁴C radioactivity of the ethylacetate extract of the cells was counted. When various mutant cells grown on agar plate medium after ultraviolet irradiation or N-methyl-N’-nitroso-guanidine treatment were applied to in vivo [¹⁴C-methyl] labeling assay, these mutants showed a broad range of in vivo N-methyltransferase activity. Poor correlation was found between in vivo N-methyltransferase activity of cyclosporin A synthetase of the mutant grown on agar plate and the actual amount of cyclosporin A production in shake-flask culture. However, when the cells grown on the shake-flask culture were applied in the in vivo [¹⁴C-methyl] labeling assay, a better correlation was resulted. In vivo N-methyltransferase activity reached the maximum value at about 150 h, and then declined quickly, but cyclosporin A was synthesized for 200 h during fermentation. Specific in vivo N-methyltransferase activity was not greatly influenced by culture age during fermentation. The major product of in vivo [¹⁴C-methyl] labeling assay was identified as cyclosporin A, and only trace amounts of other cyclosporin analogues were detected. Therefore, the results suggest that in vivo labeling method with [C¹⁴-methyl]-S-adenosyl-l-methionine can easily compare a potency of cyclosporin A-producing mutant during fermentation.