Abstract 9α-hydroxy-androst-4-ene-3,17-dione (9-OHAD), as an important precursor for a large number of steroidal drugs, can be produced by the mycobacterial degradation of phytosterols’ feedstock. Phytosterols are wastes separated from the production of vegetable oil. Three catabolic pathways were involved in the microbial conversion of phytosterols to form 9-OHAD in Mycobacterium neoaurum MS136, namely sterol uptake, sterol side-chain degradation, and steroid nucleus cleavage and degradation. To enhance the production of 9-OHAD, we systematically studied the expression pattern of the genes during the conversion of phytosterols to 9-OHAD. The genes with significant up-regulation (e.g., hsd, cyp125, fadD19, hsd4A and ksh) were overexpressed to examine their effects on the 9-OHAD production. Thus, the recombinant Mycobacterium neoaurum MS136-F was constructed with the overexpression of these four key genes (e.g., hsd, hsd4A, kshA1 and kshB) and the knockout of the gene kstd that led to the degradation of 9-OHAD. Upon adding 13.0 g/L of commercial phytosterols feedstock (including 6.2 g/L β-sitosterol, 3.0 g/L campesterol, 3.3 g/L stigmasterol and 0.5 g/L others), 6.8 g/L 9-OHAD was obtained by this recombinant strain MS136-F after 108 h’ fermentation, which was 1.45-fold of that of the parental M. neoaurum MS136 (4.7 g/L, the molar conversion rate is 77.2%). In the phytosterols feedstock, β-sitosterol and campesterol were converted to 9-OHAD with a much high molar conversion ratio of 98.5%. However, stigmasterol (3.3 g/L) was hardly used by mycobacteria, only 0.3 g/L of which was converted 9-OHAD. This study has a direct implication for the industrial production of 9-OHAD.

中文翻译：

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通过分枝杆菌代谢途径工程提高植物甾醇中 9α-羟基-雄甾醇-4-烯-3,17-二酮 (9-OHAD) 的产生

摘要 9α-羟基-雄甾醇-4-烯-3,17-二酮（9-OHAD）是大量甾体药物的重要前体，可通过分枝杆菌降解植物甾醇的原料产生。植物甾醇是从植物油生产中分离出来的废物。三种分解代谢途径参与植物甾醇在新金分枝杆菌 MS136 中形成 9-OHAD 的微生物转化，即甾醇摄取、甾醇侧链降解和甾醇核裂解和降解。为了提高 9-OHAD 的产生，我们系统地研究了植物甾醇向 9-OHAD 转化过程中基因的表达模式。过表达具有显着上调的基因（例如，hsd、cyp125、fadD19、hsd4A 和 ksh）以检查它们对 9-OHAD 产生的影响。因此，重组新金分枝杆菌 MS136-F 是通过这四个关键基因（例如 hsd、hsd4A、kshA1 和 kshB）的过表达和导致 9-OHAD 降解的基因 kstd 的敲除构建的。加入13.0 g/L商业植物甾醇原料（包括6.2 g/L β-谷甾醇、3.0 g/L菜油甾醇、3.3 g/L豆甾醇和0.5 g/L其他），由此得到6.8 g/L 9-OHAD重组菌株MS136-F经过108 h'发酵，是亲本M. neoaurum MS136（4.7 g/L，摩尔转化率为77.2%）的1.45倍。在植物甾醇原料中，β-谷甾醇和菜油甾醇以 98.5% 的高摩尔转化率转化为 9-OHAD。然而，豆甾醇 (3.3 g/L) 几乎不被分枝杆菌利用，只有 0.3 g/L 转化为 9-OHAD。