Title |
Enhancing Expression of 3-Ketosteroid-9α-Hydroxylase Oxygenase, an Enzyme with Broad Substrate Range and High Hydroxylation Ability, in Mycobacterium sp. LY-1
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Published in |
Applied Biochemistry and Biotechnology, September 2018
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DOI | 10.1007/s12010-018-2876-2 |
Pubmed ID | |
Authors |
Hui Li, Xiangdong Wang, Longfei Zhou, Yang Ma, Wanjuan Yuan, Xiaomei Zhang, Jinsong Shi, Zhenghong Xu |
Abstract |
3-Ketosteroid-9α-hydroxylase (KSH) consists of two protein systems, KshA and KshB, and is a key enzyme in microbial degradation pathway of natural sterols. 9α-Hydroxy-4-androstene-3,17-dione (9α-OH-AD) is a valuable steroid pharmaceutical intermediate. The expression of a 3-ketosteroid-9α-hydroxylase oxygenase (KshA1) with a broad substrate range and high hydroxylation ability was enhanced in Mycobacterium sp. LY-1 to improve the yield of 9α-OH-AD. Through whole-genome sequence mining and homologous comparison, the putative genes (kshA1 and kshB) in wild strain LY-1 were firstly identified. Then they were heterogeneously co-expressed in Escherichia coli BL21. The transformation results of recombinant BL21-KshA1/B demonstrated KshA1/B had high hydroxylation ability to AD. Moreover, substrate preference analysis suggested that KshA1LY-1 had a broad substrate range. After enhancing expression of kshA1 and kshB in the strain LY-1, the maximum productivity of 9α-OH-AD in recombinant LY-1-KshA1/B reached 0.064 g/L/h in a 5-L stirred fermenter. |
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