Identification of steroid C27 monooxygenase isoenzymes involved in sterol catabolism and stepwise pathway engineering of Mycobacterium neoaurum for improved androst-1,4-diene-3,17-dione production
Identification of steroid C27 monooxygenase isoenzymes involved in sterol catabolism and stepwise pathway engineering of Mycobacterium neoaurum for improved androst-1,4-diene-3,17-dione production
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类固醇 C27 单加氧酶同工酶的鉴定，涉及新金分枝杆菌的甾醇分解代谢和逐步途径工程，以改善雄激素 1,4-二烯-3,17-二酮生产
DOI：
10.1007/s10295-018-02135-5
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发表时间：
2019-05-01
影响因子：
3.4
通讯作者：
Yang, Shangtian
中科院分区：
文献类型：
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作者：
Shao, Minglong;Zhang, Xian;Yang, Shangtian
胆固醇氧化酶、类固醇C27单加氧酶和3-酮类固醇-(1)-脱氢酶是参与微生物甾醇分解代谢的关键酶。在此，首次从新金分枝杆菌中鉴定出三种类固醇 C27 单加氧酶同工酶，作为类固醇 C27-羟基化的关键酶。在这三种同工酶中，类固醇C27单加氧酶2在甾醇分解代谢中表现出最强的功能。为了提高雄激素-1,4-二烯-3,17-二酮的产生，共表达胆固醇氧化酶、类固醇C27单加氧酶2和3-酮类固醇-(1)-脱氢酶，以增强雄激素-1,4-二烯-3,17-二酮和3-酮类固醇9-羟化酶的代谢通量，后者催化M. neoaurum JC-12 中的雄激素 1,4-二烯-3,17-二酮分解代谢被破坏，从而阻断雄激素-1,4-二烯-3,17-二酮降解途径。最后，重组菌株JC-12(S2-choM-ksdd/kshA)产生了20.1g/L的androst-1,4-diene-3,17-dione，这是报道的以甾醇为底物的最高产量。因此，这项工作希望为通过代谢工程高效生产雄激素-1,4-二烯-3,17-二酮铺平道路。
Cholesterol oxidase, steroid C27 monooxygenase and 3-ketosteroid-(1)-dehydrogenase are key enzymes involved in microbial catabolism of sterols. Here, three isoenzymes of steroid C27 monooxygenase were firstly characterized from Mycobacterium neoaurum as the key enzyme in sterol C27-hydroxylation. Among these three isoenzymes, steroid C27 monooxygenase 2 exhibits the strongest function in sterol catabolism. To improve androst-1,4-diene-3,17-dione production, cholesterol oxidase, steroid C27 monooxygenase 2 and 3-ketosteroid-(1)-dehydrogenase were coexpressed to strengthen the metabolic flux to androst-1,4-diene-3,17-dione, and 3-ketosteroid 9-hydroxylase, which catalyzes the androst-1,4-diene-3,17-dione catabolism, was disrupted to block the androst-1,4-diene-3,17-dione degradation pathway in M. neoaurum JC-12. Finally, the recombinant strain JC-12(S2-choM-ksdd/kshA) produced 20.1g/L androst-1,4-diene-3,17-dione, which is the highest reported production with sterols as substrate. Therefore, this work is hopes to pave the way for efficient androst-1,4-diene-3,17-dione production through metabolic engineering.