5-azacytidine Increases Tanshinone Production in Endophytic Fungi of Salvia abrotanoides (Kar.) Sytsma

Endophytic fungi are often producing host plant metabolites. Tanshinones are secondary metabolites of the Salvia genus which are also produced by some endophytic fungi. Efficient secondary metabolite production in endophytic fungi drops significantly after sequential subcultures. 5-azacytidine (5-AC) is an analog of the naturally occurring pyrimidine nucleoside cytidine and a DNA methyltransferase inhibitor. In this relation, 5-AC is an effective tool to induce the expression of silenced secondary metabolite genes in fungi. We isolated 4 endophytic fungi from the roots of Salvia abrotanoides which produced tanshinone. Cryptotanshinone and tanshinone IIA were produced by Penicillium canescens, Penicillium nodositatum, and Penicillium pinophilum, while Paraphoma radicina only produced tanshinone IIA. The maximum amount of tanshinones was extracted from P. pinophilum culture with 130.826 mg cryptotanshinone /g of dry weight and 50.155 mg Tanshinone IIA/g of dry weight. These amounts were significantly more than tanshinones produced in plant roots (0.55 mg cryptotanshinone/g of dry weight, 1.3 mg Tanshinone IIA/g of dry weight). In the third subculture, tanshinone production decreased significantly. 5-azacytidine as an epigenetic modifier retrieved tanshinone production in the third subculture of P. pinophilum. Also, 5- azacytidine treatment made a big jump in Tanshinone IIA production in P. radicina (63.176 mg TIIA/g of dry weight) besides increasing Tanshinone IIA production in P. nodositatum cultures. This is the first report using 5- azacytidine to improve tanshinone production in endophytic fungi. Our results confirm that 5- azacytidine is an efficient, easy, and quick chemical to felicitate secondary metabolite production in endophytic fungi.