Lab of Biorefinery, Shanghai Advanced Research Institute, Chinese Academy of Sciences, No. 99 Haike Road, Pudong, 201210, Shanghai, China.
School of Life Science and Technology, ShanghaiTech University, Shanghai, 201210, China.
Microb Cell Fact. 2018 May 18;17(1):77. doi: 10.1186/s12934-018-0916-9.
3-Ketosteroid-Δ1-dehydrogenase (KstD) is a key enzyme in the metabolic pathway for chemical modifications of steroid hormones. Only a few KstDs have thus far been characterized biochemically and applied for the production of steroidal pharmaceutical intermediates. Three KstDs, KstD1, KstD2, and KstD3, were identified in Mycobacterium neoaurum DSM 1381, and they shared up to 99, 85 and 97% amino acid identity with previously reported KstDs, respectively. In this paper, KstDs from M. neoaurum DSM 1381 were investigated and exemplified their potential application for industrial steroid transformation.
The recombinant KstD2 from Bacillus subtilis exhibited higher enzymatic activity when 4-androstene-3,17-dione (AD) and 22-hydroxy-23, 24-bisnorchol-4-ene-3-one (4HP) were used as the substrates, and resulted in specific activities of 22.40 and 19.19 U mg, respectively. However, the specific activities of recombinant KstD2 from Escherichia coli, recombinant KstD1 from B. subtilis and E. coli, and recombinant KstD3, also fed with AD and 4HP, had significantly lower specific activities. We achieved up to 99% bioconversion rate of 1,4-androstadiene-3,17-dione (ADD) from 8 g L AD after 15 h of fermentation using E. coli transformant BL21-kstD2. And in vivo transcriptional analysis revealed that the expression of kstD1 in M. neoaurum DSM 1381 increased by 60.5-fold with phytosterols as the substrate, while the mRNA levels of kstD2 and kstD3 were bearly affected by the phytosterols. Therefore, we attempted to create a 4HP producing strain without kstD1, which could covert 20 g L phytosterols to 14.18 g L 4HP.
In vitro assay employing the recombinant enzymes revealed that KstD2 was the most promising candidate for biocatalysis in biotransformation of AD. However, in vivo analysis showed that the cellular regulation of kstD1 was much more active than those of the other kstDs in response to the presence of phytosterols. Based on the findings above, we successfully constructed E. coli transformant BL21-kstD2 for ADD production from AD and M. neoaurum DSM 1381 ΔkstD1 strain for 4HP production using phytosterols as the substrate.
3-酮类固醇-Δ1-脱氢酶（KstD）是类固醇激素化学修饰代谢途径中的关键酶。迄今为止，只有少数 KstD 已通过生化方法进行了表征，并应用于甾体药物中间体的生产。在新金色分枝杆菌 DSM 1381 中鉴定出三种 KstD，即 KstD1、KstD2 和 KstD3，它们分别与先前报道的 KstD 具有高达 99%、85%和 97%的氨基酸同一性。在本文中，研究了来自新金色分枝杆菌 DSM 1381 的 KstD，并举例说明了它们在工业甾体转化中的潜在应用。
枯草芽孢杆菌的重组 KstD2 对 4-雄烯-3,17-二酮（AD）和 22-羟基-23,24-双降胆甾-4-烯-3-酮（4HP）作为底物表现出更高的酶活性，其比活性分别为 22.40 和 19.19 U mg。然而，重组 KstD2 来自大肠杆菌、重组 KstD1 来自枯草芽孢杆菌和大肠杆菌以及重组 KstD3 的比活性明显较低，也以 AD 和 4HP 为底物。我们使用大肠杆菌转化子 BL21-kstD2 实现了高达 99%的 1,4-雄二烯-3,17-二酮（ADD）从 8 g L AD 的生物转化转化率，经过 15 小时的发酵。体内转录分析显示，当以植物甾醇为底物时，新金色分枝杆菌 DSM 1381 中的 kstD1 表达增加了 60.5 倍，而 kstD2 和 kstD3 的 mRNA 水平几乎不受植物甾醇的影响。因此，我们试图创建一种没有 kstD1 的 4HP 生产菌株，它可以将 20 g L 的植物甾醇转化为 14.18 g L 的 4HP。
体外酶测定表明 KstD2 是 AD 生物转化中生物催化最有前途的候选酶。然而，体内分析表明，在植物甾醇存在的情况下，kstD1 的细胞调节比其他 kstD 更为活跃。基于上述发现，我们成功构建了用于 AD 生产 ADD 的大肠杆菌转化子 BL21-kstD2 和用于植物甾醇作为底物生产 4HP 的新金色分枝杆菌 DSM 1381 ΔkstD1 菌株。
