Mutation breeding of high 4-androstene-3,17-dione-producing Mycobacterium neoaurum ZADF-4 by atmospheric and room temperature plasma treatment

Abstract

Steroid medication is used extensively in clinical applications and comprises a large and vital part of the pharmaceutical industry. However, the difficulty of separating 4-androstene-3,17-dione (AD) from 1,4-androstadiene-3,17-dione (ADD) restricts the application of the microbial transformation of phytosterols in the industry. A novel atmospheric and room temperature plasma (ARTP) treatment, which employs helium as the working gas, was used to generate Mycobacterium neoaurum mutants producing large amounts of AD. After treatment of cultures with ARTP, four mutants were selected using a novel screening method with a color assay. Among the mutants, M. neoaurum ZADF-4 was considered the best candidate for industrial application. When the fermentation medium contained 15 g/L phytosterols and was cultivated on a rotary shaker at 160 r/min at 30°C for 7 d, (6.28±0.11) g/L of AD and (0.82±0.05) g/L of ADD were produced by the ZADF-4 mutant, compared with (4.83±0.13) g/L of AD and (2.34±0.06) g/L of ADD by the original strain, M. neoaurum ZAD. Compared with ZAD, the molar yield of AD increased from 48.3% to 60.3% in the ZADF-4 mutant. This result indicates that ZADF-4 may have potential for industrial production of AD.