文献类型：期刊文献

英文题名：Engineered 3-ketosteroid 9a-hydroxylases in Mycobacterium neoaurum: An efficient platform for production of steroid drugs

作者：Liu, Hao-Hao[1]; Xu, Li-Qin[1]; Yao, Kang[1]; Xiong, Liang-Bin[1]; Tao, Xin-Yi[1]; Liu, Min[1]; Wang, Feng-Qing[1]; Wei, Dong-Zhi[1]

机构：[1] State Key Laboratory of Bioreactor Engineering, Newworld Institute of Biotechnology, East China University of Science and Technology, Shanghai, China

年份：2018

卷号：84

期号：14

外文期刊名：Applied and Environmental Microbiology

收录：EI(收录号：20182905565558)

语种：英文

外文关键词：Metabolic engineering - Lipids - Alcohols - Metabolism

摘要：3-Ketosteroid 9α-hydroxylase (Ksh) consists of a terminal oxygenase (KshA) and a ferredoxin reductase and is indispensable in the cleavage of steroid nucleus in microorganisms. The activities of Kshs are crucial factors in determining the yield and distribution of products in the biotechnological transformation of sterols in industrial applications. In this study, two KshA homologues, KshA1N and KshA2N, were characterized and further engineered in a sterol-digesting strain, Mycobacterium neoaurum ATCC 25795, to construct androstenone-producing strains. kshA1N is a member of the gene cluster encoding sterol catabolism enzymes, and its transcription exhibited a 4.7-fold increase under cholesterol induction. Furthermore, null mutation of kshA1N led to the stable accumulation of androst- 4-ene-3,17-dione (AD) and androst-1,4-diene-3,17-dione (ADD). We determined kshA2N to be a redundant form of kshA1N. Through a combined modification of kshA1N, kshA2N, and other key genes involved in the metabolism of sterols, we constructed a high-yield ADD-producing strain that could produce 9.36 g liter-1 ADD from the transformation of 20 g liter-1 phytosterols in 168 h. Moreover, we improved a previously established 9α-hydroxy-AD-producing strain via the overexpression of a mutant KshA1N that had enhanced Ksh activity. Genetic engineering allowed the new strain to produce 11.7 g liter-1 9α-hydroxy-4-androstene- 3,17-dione (9-OHAD) from the transformation of 20.0 g liter-1 phytosterol in 120 h. ? 2018 American Society for Microbiology.