Xu Li-Qin, Liu Yong-Jun, Yao Kang, Liu Hao-Hao, Tao Xin-Yi, Wang Feng-Qing, Wei Dong-Zhi
State Key Laboratory of Bioreactor Engineering, Newworld Institute of Biotechnology, East China University of Science and Technology, Shanghai 200237, China.
Sci Rep. 2016 Feb 22;6:21928. doi: 10.1038/srep21928.
The catabolism of sterols in mycobacteria is highly important due to its close relevance in the pathogenesis of pathogenic strains and the biotechnological applications of nonpathogenic strains for steroid synthesis. However, some key metabolic steps remain unknown. In this study, the hsd4A gene from Mycobacterium neoaurum ATCC 25795 was investigated. The encoded protein, Hsd4A, was characterized as a dual-function enzyme, with both 17β-hydroxysteroid dehydrogenase and β-hydroxyacyl-CoA dehydrogenase activities in vitro. Using a kshAs-null strain of M. neoaurum ATCC 25795 (NwIB-XII) as a model, Hsd4A was further confirmed to exert dual-function in sterol catabolism in vivo. The deletion of hsd4A in NwIB-XII resulted in the production of 23,24-bisnorcholenic steroids (HBCs), indicating that hsd4A plays a key role in sterol side-chain degradation. Therefore, two competing pathways, the AD and HBC pathways, were proposed for the side-chain degradation. The proposed HBC pathway has great value in illustrating the production mechanism of HBCs in sterol catabolism and in developing HBCs producing strains for industrial application via metabolic engineering. Through the combined modification of hsd4A and other genes, three HBCs producing strains were constructed that resulted in promising productivities of 0.127, 0.109 and 0.074 g/l/h, respectively.
分枝杆菌中甾醇的分解代谢非常重要，因为它与致病菌株的发病机制密切相关，以及非致病菌株在甾体合成中的生物技术应用。然而，一些关键的代谢步骤仍然未知。在本研究中，对新金色分枝杆菌ATCC 25795的hsd4A基因进行了研究。编码的蛋白质Hsd4A被鉴定为一种双功能酶，在体外具有17β-羟基类固醇脱氢酶和β-羟基酰基辅酶A脱氢酶活性。以新金色分枝杆菌ATCC 25795的kshAs缺失菌株（NwIB-XII）为模型，进一步证实Hsd4A在体内甾醇分解代谢中发挥双功能。NwIB-XII中hsd4A的缺失导致23,24-双降胆甾烯类固醇（HBCs）的产生，表明hsd4A在甾醇侧链降解中起关键作用。因此，提出了两种竞争途径，即AD途径和HBC途径用于侧链降解。所提出的HBC途径在阐明甾醇分解代谢中HBCs的产生机制以及通过代谢工程开发用于工业应用的HBCs生产菌株方面具有重要价值。通过对hsd4A和其他基因的联合修饰，构建了三株HBCs生产菌株，其生产率分别为0.127、0.109和0.074 g/l/h，前景良好。
