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.