Isolation of compounds in the cultures of ORF23 null mutant and ORF27 null mutant.

The ORF23 null mutant and ORF27 null mutant were incubated in 100 ml culture with 0.1% (wt/vol) cholic acid, and a total volume of 500 ml of the culture of each mutant was extracted with 550 ml of ethyl acetate under neutral conditions. The ethyl acetate layer was treated with Na₂SO₄ and concentrated in vacuo, and then it was dissolved in a small amount of methanol. The methanol solution was subjected to HPLC (Waters 600), and the fractions containing each of these compounds were purified. The water layer then was extracted twice with 550 ml of ethyl acetate under acidic conditions (pH 2 with HCl), and the compounds in the ethyl acetate layer extracted under acidic conditions were isolated in the same manner as the ethyl acetate layer extracted under neutral conditions. Fractions containing new compounds were isolated with HPLC (Waters 600) for purification, dried, and dissolved in a suitable solvent (DMSO-D₆) for NMR and EI-MS analysis.