Abstract:

The original strain Mycobacterium MN2 which is capable of selectively cleaving the side chain of phytosterol to
generate androst-4-ene-3,17-dione (AD) and androsta-1,4-diene-3,17-dione (ADD) was induced by UV irradiation and dithyl
sulfate (DES). The mutant strains were selected and grown with higher concentration substrate by gradient plate method
and their fermentation performance was evaluated. Then the biotransformation medium of the selected mutant strains was
optimized by orthogonal array design. One mutant strain MN4 was obtained. When the concentration of substrate phytosterol
was 20 g/L and shaking flask fermentation was proceeded for 144 hours, the conversion rate of androst-4-ene-3,17-dione
(AD) from phytosterol by MN4 was 40.9%, which was 15.1% higher than that of the original strain. In addition, the mutant
strain showed high genetic stability after five continuous passages. The optimum medium composition for AD production
was found to be 1 g/L glucose, 25 g/L corn steep liquor, 0.6 g/L Na2HPO4, 6.2 g/L NaNO3, and 160 g/L soybean oil. Under
the optimal conditions, the conversion rate of androst-4-ene-3,17-dione was 50.6%. A method for screening substrateresistant
strain with high yield of androst-4-ene-3,17-dione has been established, and the mutant strain MN4 with high
biotransformation ability and excellent genetic stability had been obtained after UV and DES mutation.

Key words: Mycobacterium neoaurum, substrate-resistant strain, mutation breeding, androst-4-ene-3,17-dione, biotransformation