Two-Step Bioprocess for Reducing Nucleus Degradation in Phytosterol Bioconversion by Mycobacterium neoaurum NwIB-R10 hsd4A

In order to obtain high 4-androstene-3,17-dione (AD) yield, nucleus degradation needs to be avoided during phytosterol bioconversion process with Mycobacterium neoaurum NwIB-R10hsd4A. 3-Ketosteroid-Δ1-dehydrogenase (KstD) catalyzes 1,2-desaturation of steroids and is a key enzyme involved in steroid nucleus oxidation. Heterogeneous expression and characterization of two KstDs (KstD2, KstD3) from M. neoaurum NwIB-R10hsd4A showed that their activities were inhibited by shifting temperature from 30 to 37 °C. However, the total activities of KstD2 and KstD3 were replenished when M. neoaurum NwIB-R10hsd4A was cultured at 37 °C because the transcription levels of kstD2 and kstD3 were upregulated 1.61- and 1.43-fold respectively compared with the cultivation at 30 °C. As the optimal temperature for cell growth was 30 °C, we developed a two-step bioprocess, cell culture at 30 °C and bioconversion with resting cells at 37 °C avoiding higher transcriptional level of kstD2 and kstD3. This process repressed the activities of KstDs, resulted in the decrease of 1,2-desaturation products, and reduced the nucleus degradation (17.6%). AD production increased to 24.7 g l−1 at higher substrate concentration (50 g l−1). These results indicated that the two-step bioprocess was potential in phytosterol biotransformation industrially.