3-甾酮-9α-羟基化酶基因在分枝杆菌中的异源表达与9α-羟基雄烯二酮的制备

Accumulation of 9α-hydroxy-4-androstene-3,17-dione by co-expressing kshA and kshB encoding component of 3-ketosteroid-9α-hydroxylase in Mycobacterium sp. NRRL B-3805

摘要:

9α-羟基雄烯二酮 (9-OH-AD) 是制备甾体类药物的重要中间产物。3-甾酮-9α-羟基化酶 (KSH) 能够转化雄烯二酮 (AD) 产生9α-羟基雄烯二酮 (9-OH-AD)，该酶由KshA和KshB两个亚基构成。为获得高效积累9-OH-AD的重组菌株，本研究选择耻垢分枝杆菌Mycobacterium smegmatis mc2155和戈登氏菌Gordonia neofelifaecis NRRL B-59395，对其在胆固醇为唯一碳源条件下表达明显上调的kshA和kshB候选基因进行克隆，插入到分枝杆菌表达载体pNIT中，构建共表达质粒，并将它们导入分枝杆菌Mycobacterium sp. NRRL B-3805中，获得重组菌株。利用重组菌株分别对植物甾醇、胆固醇和谷甾醇进行生物转化，分离纯化转化产物，采用光谱学方法鉴定其化学结构，确定该转化产物为9α-羟基雄烯二酮，说明分枝杆菌Mycobacterium sp. NRRL B-3805由积累雄烯二酮变为积累9α-羟基雄烯二酮 (9-OH-AD)，进而证明导入的候选基因kshA和kshB确实为有功能的基因。生物转化实验表明，与胆固醇、谷甾醇相比，植物甾醇作为底物更易于转化；而用来源于耻垢分枝杆菌的kshA、kshB构建的重组菌转化率更高，可达90%，具有较高的应用价值。本研究通过对KSH编码基因的异源表达，成功地进行了分枝杆菌生物转化特性的改造，为探索各种甾体药物中间体的工业生产奠定了基础。

Abstract:

9α-hydroxy-4-androstene-3,17-dione (9-OH-AD) is an important intermediate in the steroidal drugs production. 3-ketosteroid-9α-hydroxylase (KSH), a two protein system of KshA and KshB, is a key-enzyme in the microbial steroid ring B-opening pathway. KSH catalyzes the transformation of 4-androstene-3,17-dione (AD) into 9-OH-AD specifically. In the present study, the putative KshA and KshB genes were cloned from Mycobacterium smegmatis mc2155 and Gordonia neofelifaecis NRRL B-59395 respectively, and were inserted into the expression vector pNIT, the co-expression plasmids of kshA-kshB were obtained and electroporated into Mycobacterium sp. NRRL B-3805 cells. The recombinants were used to transform steroids, the main product was characterized as 9α-hydroxy-4-androstene-3,17-dione (9-OH-AD), showing that kshA and kshB were expressed successfully. Different from the original strain Mycobacterium sp. NRRL B-3805 that accumulates 4-androstene-3,17-dione, the recombinants accumulates 9α-hydroxy-4-androstene-3,17-dione as the main product. This results indicates that the putative genes kshA, kshB encode active KshA and KshB, respectively. The process of biotransformation was investigated and the results show that phytosterol is the most suitable substrate for biotransformation, kshA and kshB from M. smegmatis mc2155 seemed to exhibit high activity, because the resultant recombinant of them catalyzed the biotransformation of phytosterol to 9-OH-AD in a percent conversion of 90%, which was much higher than that of G. neofelifaecis NRRL B-59395. This study on the manipulation of the ksh genes in Mycobacterium sp. NRRL B-3805 provides a new pathway for producing steroid medicines.