Production of testosterone from phytosterol using a single-step microbial transformation by a mutant of Mycobacterium sp.

A testosterone (TS)-producing mutant, ST2, was derived from a phytosterol-assimilating and androst-4-ene-3,17-dione (AD)-producing bacterium, Mycobacterium sp. B-3805S, using nitrosoguanidine (NTG) mutagenesis. Production of TS from phytosterol using a single-step microbial transformation process by ST2 was investigated in a 5-l surface-aeration microprocessor-controlled fermentor loaded with a synthetic medium supplemented with 0.1% phytosterol, 2% glucose and 1% peptone at 30 degrees C. An increase in dissolved oxygen at the initial stage of fermentation favored the side-chain degradation of phytosterol to AD. Later in the fermentation, a decrease in the dissolved oxygen to zero resulted in a decrease in pH to 6.0 as well as the reduction of AD to TS. Under optimal fermentation conditions, the maximum conversion ratio of phytosterol to TS was 31% after 120 h cultivation. It was concluded that the control of dissolved oxygen in the fermentation culture is the most important parameter for production of TS from phytosterol via AD. TS was isolated from the fermentation culture by addition of Amberlite XAD-7 resin and was further purified by flash chromatography on a silica gel column. After crystallization, TS was obtained as needle crystals with the correct melting point.