文献类型：期刊文献

英文题名：Characterization of 3-ketosteroid O1-dehydrogenases (KstD4&5) of Mycobacterium neoaurum and their effects on phytosterol transformation

作者：Xu, Lvqing[1];Wang, Xiao[1];Ma, Siyuan[1];Zhang, Jian[1];Wang, Xuedong[1]

机构：[1]East China Univ Sci & Technol, 130 Meilong Rd, Shanghai, Peoples R China

年份：2026

卷号：173

外文期刊名：BIOORGANIC CHEMISTRY

收录：;WOS:【SCI-EXPANDED(收录号:WOS:001708476200001)】；

语种：英文

外文关键词：3-ketosteroid O1-dehydrogenase (KstD); 22-hydroxy-23,24-bisnorchol-4-ene-3-one (HBC); Biotransformation; CRISPR; M. neoaurum

摘要：Mycobacterium neoaurum can convert phytosterols (PS) into steroidal intermediates such as HBC (22-hydroxy-23,24-bisnorchol-4-ene-3-one). However, 3-ketosteroid O1-dehydrogenase (KstD) can catalyze the dehydrogenation at the C1 position of the steroid nucleus, which leads to the accumulation of byproducts such as 22-hy-droxy-23,24-bisnorchol-1,4-diene-3-one (1,4-HBC). This is further accompanied by 9-hydroxylation of the steroid framework, resulting in spontaneous degradation of the B ring. Using the known KstD1-3 sequences as probes, bioinformatic analysis identified two novel 3-ketosteroid O1-dehydrogenases (KstD4 and KstD5) in M. neoaurum WIII. Recombinant E. coli BL21 strains expressing kstD4 and kstD5 exhibited 1-position dehydrogenation activity toward HBC and androstenedione (AD). Purified enzyme assays revealed that KstD5 preferentially catalyzes the C-19 steroid intermediate such as AD, whereas KstD4 favors the incomplete side-chain degradation intermediate HBC. These findings suggest that KstD4 and KstD5 have different physiological function but both contribute to steroid nucleus degradation and byproduct accumulation during PS transformation. Inactivation of KstD4 and KstD5 in M. neoaurum reduced the 1,4-HBC byproduct concentration from 0.27 g/L to 0.12 g/L and increased the HBC yield from 32.85 g/L to 34.20 g/L. Moreover, the mutant strain showed improved performance under industrially relevant conditions, including in a system with reduced cyclodextrin ratio and simulated autolyzed cells.