受SnpR激活的snpA启动子调控的柔红霉素C-14羟化酶基因的克隆、表达和应用研究

Study on cloning and expression of daunorubicin C-14 hydroxylase gene under the control of SnpR-activated snpA promoter

尝试将柔红霉素经微生物转化法合成多柔比星,从Streptomyces sp.C5中通过PCR的方法扩增了含核糖体结合位点、大小为1.3kb的编码C-14柔红霉素羟化酶的doxA基因及受SnpR调控的snpA启动子。最终构建的重组质粒pYG908,能表达一大小为46.6ku的蛋白条带,CO结合差光谱分析表明所表达的酶在450nm有吸收峰。重组质粒转化Streptomyces lividans TK24后,工程菌能转化柔红霉素生成和多柔比星保留时间一样的产物。

Doxorubicin is produced through chemical semisynthesis initiated from daunorubicin. In this paper, the process of microbial conversion was studied for the production of doxorubicin. A DNA fragment containing C-14 hydroxylase encoding gene doxA, approximate 1.3kb including a ribosome binding site GGAGG was amplified from daunorubicin-producing strain Streptomyces sp. C5. At the same time, the SnpR-activated snpA promoter sequence was also amplified from Streptomyces sp. C5. The recombinant plasmid, pYG908, was constructed for the expression of doxA under the control of snpA promoter, while snpA was regulated by SnpR protein. SDS-PAGE indicated that the engineered strain could apparently express a 46. 6ku band according to the deduced molecular weight of daunorubicin C-14 hydroxylase. DoxA activity was confirmed by CO-binding differential absorbance at 450nm. pYG908 was introduced into Streptomyces lividans TK24 and the resultant engineering strains could convert daunorobicin into a product which has a similar retention time with doxorubicin by HPLC.