Mycobacterium neoaurum strains can transform phytosterols to 4-androstene-3,17-dione (4-AD), a key intermediate for the synthesis of advanced steroidal medicines. In this work, we presented the complete genome sequence of the M. neoaurum strain HGMS2, which transforms β-sitosterol to 4-AD. Through genome annotation, a phytosterol-degrading pathway in HGMS2 was predicted and further shown to form a 9,10-secosteroid intermediate by five groups of enzymes. These five groups of enzymes included three cholesterol oxidases (ChoM; group 1: ChoM1, ChoM2 and Hsd), two monooxygenases (Mon; group 2: Mon164 and Mon197), a set of enzymes for side-chain degradation (group 3), one 3-ketosteroid-1,2-dehydrogenase (KstD; group 4: KstD211) and three 3-ketosteroid-9a-hydroxylases (Ksh; group 5: KshA226, KshA395 and KshB122). A gene cluster encoding Mon164, KstD211, KshA226, KshB122 and fatty acid β-oxidoreductases constituted one integrated metabolic pathway, while genes encoding other key enzymes were sporadically distributed. All key enzymes except those from group 3 were prepared as recombinant proteins and their activities were evaluated, and the proteins exhibited distinct activities compared with enzymes identified from other bacterial species. Importantly, we found that the KstD211 and KshA395 enzymes in the HGMS2 strain retained weak activities and caused the occurrence of two major impurities, i.e., 1,4-androstene-3,17-dione (ADD) and 9-hydroxyl-4-androstene-3,17-dione (9OH-AD) during β-sitosterol fermentation. The concurrence of these two 4-AD analogs not only lowered 4-AD production yield but also hampered 4-AD purification. HGMS2 has the least number of genes encoding KstD and Ksh enzymes compared with current industrial strains. Therefore, HGMS2 could be a potent strain by which the 4-AD production yield could be enhanced by disabling the KstD211 and KshA395 enzymes. Our work also provides new insight into the engineering of the HGMS2 strain to produce ADD and 9OH-AD for industrial application.

中文翻译：

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具有残留植物甾醇降解途径的产雄烯二酮分枝杆菌菌株的全基因组和酶促分析

新分枝杆菌菌株可以将植物甾醇转化为4-雄甾烯-3,17-二酮（4-AD），后者是合成高级甾体药物的关键中间体。在这项工作中，我们介绍了新农杆菌HGMS2的完整基因组序列，该菌株将β-谷甾醇转化为4-AD。通过基因组注释，可以预测HGMS2中的植物甾醇降解途径，并进一步显示通过五组酶形成9,10-secosteroid中间体。这五种酶包括三种胆固醇氧化酶（ChoM；第1组：ChoM1，ChoM2和Hsd），两种单加氧酶（星期一；第2组：Mon164和Mon197），一套用于侧链降解的酶（第3组），一种。 3-酮类固醇1,2-脱氢酶（KstD;组4：KstD211）和三个3-酮类固醇9a-羟基酶（Ksh;组5：KshA226，KshA395和KshB122）。编码Mon164，KstD211，KshA226，KshB122和脂肪酸β-氧化还原酶构成了一条整合的代谢途径，而编码其他关键酶的基因则零星分布。除第3组中的那些酶外，所有关键酶均被制备为重组蛋白并对其活性进行了评估，与从其他细菌物种中鉴定出的酶相比，这些蛋白表现出独特的活性。重要的是，我们发现HGMS2菌株中的KstD211和KshA395酶保留了弱活性，并导致了两种主要杂质的发生，即1,4-雄烯3,17-二酮（ADD）和9-羟基-4-雄烯β-谷甾醇发酵过程中的-3,17-二酮（9OH-AD）。这两个4-AD类似物的同时存在，不仅降低了4-AD的产量，而且还阻碍了4-AD的纯化。与目前的工业菌株相比，HGMS2具有最少数量的编码KstD和Ksh酶的基因。因此，HGMS2可能是强效菌株，通过禁用KstD211和KshA395酶可以提高4-AD的产量。我们的工作还为HGMS2菌株的工程设计提供了新的见解，以生产ADD和9OH-AD用于工业应用。