Selection and characterization of new microorganisms for the manufacture of 9-OH-AD from sterols

Using a special selection procedure, several mutants of Mycobacterium vaccae were isolated which were capable of converting sterols to 9 alpha-hydroxyandrost-4-ene-3,17-dion (9-OH-AD). Two mutants, Mycobacterium vaccae ZIMET 11052 and 11053, respectively, were further investigated. Strains of the species Mycobacterium fortuitum are mainly used for commercially obtaining 9-OH-AD from sterols. In contrast to the species Mycobacterium fortuitum the species Mycobacterium vaccae has not been reported to contain pathogenic strains. This seems an advantage for industrial application. Mutants with the ability of converting sterols to 9 alpha-hydroxysteroids have a defect in the steroid-1-dehydrogenase activity which is, however, only a partial one. The remaining activity may cause an undesirable degradation of the steroid nucleus. The steroid-1-dehydrogenase activity was tested using an assay developed by ATRAT (1986). We confirmed two apparently distinct steroid-1-dehydrogenases in Mycobacterium fortuitum NRRL B-8119 as reported by WOVCHA et al. (1979). One of them has an activity on androst-4-ene-3,17-dion (AD). The activity is increased by induction with sitosterol. The other one is active on 9-OH-AD. But Mycobacterium vaccae does not possess steroid-1-dehydrogenase activity on 9-OH-AD, and the AD specific steroid-1-dehydrogenase is not effected by sitosterol. The consequence is a high level of protection against steroid nucleus degradation yielding an effective accumulation of 9-OH-AD in fermentations with Mycobacterium vaccae mutants.