生物学杂志

生物学杂志 ›› 2019, Vol. 36 ›› Issue (5): 46-.doi: 10.3969/j.issn.2095-1736.2019.05.046

• 研究报告 • 上一篇    下一篇

维吉尼亚链霉菌P450-105D1在大肠杆菌中自诱导表达和雄烯二酮的羟化活性

  

  1. 1. 合肥师范学院 生命科学学院, 合肥 231601; 2. 华中科技大学 生命科学与技术学院, 武汉 430074; 3. 合肥停弦渡科技有限公司, 合肥 231601
  • 出版日期:2019-10-18 发布日期:2019-10-11
  • 通讯作者: 陈亚军，硕士，助理实验师，主要从事药物代谢动力学研究，E-mail:18225870205@163.com
  • 作者简介:张雁，博士，讲师，主要从事微生物次级代谢研究，E-mail:zhangyanwind@163.com
  • 基金资助:
    安徽省高校自然科学基金重点项目(KJ2018A0494)； 安徽省青年人才支持项目(gxyq2018056)；国家自然科学青年基金(31800049)

Self-induced expression in Escherichia coli for P450-105D1 of Streptomyces Virginia and identification of its hydroxylation activity for androstenedione

  1. 1. School of Life Sciences, Hefei Normal University, Hefei 231601; 
    2. College of Life Science and Technology, Huazhong University of Science and Technology, Wuhan 430074; 
    3. Hefei Tingxiandu Biotechnology Co.,Ltd., Hefei 231601, China
  • Online:2019-10-18 Published:2019-10-11

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摘要： 雄烯二酮是甾体药物生产的重要中间体，其C9位羟基化产物9-羟基雄烯二酮也是多种重要甾体药物的合成前体，具有重要的临床药用价值。微生物转化生产9-羟基雄烯二酮具有重要的经济价值。将维吉尼亚链霉菌IBL14中svu005与pET28a质粒融合，导入大肠杆菌BL21（DE3）。使用优化的自诱导培养基诱导表达其产物细胞色素P450-105D1（CYP105D1）蛋白，以雄烯二酮等多种甾体作为底物进行微生物羟化。结果显示，构建的重组质粒pET28a-svu005在大肠杆菌中成功表达，使用ZYM培养基，自诱导表达的活性蛋白产量（0.0916 nmol/L）显著高于传统的IPTG诱导方式的产量（0.0367 nmol/L）。表达产物CYP105D1可以羟化雄烯二酮，经HPLC检测，产物为9-羟基雄烯二酮。发现了微生物转化合成9-羟基雄烯二酮的新途径，具有很好的工业应用前景。

关键词: 9-羟基雄烯二酮, 微生物转化, 细胞色素P450-105D1, 自诱导表达

Abstract: Androstenedione is an important intermediate in the production of steroid medicine. Its C9 hydroxylation product, 9-hydroxyandrostenedione, is also a precursor of many important steroid medicines and has important clinical medicinal value. Therefore, the production of 9-hydroxyandrostenedione by microbial transformation has important economic value. In this study, the svu005 gene of Streptomyces Virginia IBL14 was fused with pET28a plasmid and introduced into Escherichia coli BL21(DE3). The expression of CYP105D1 protein was induced by optimized self-induction medium, and various substrates, including androstenedione, were used for microbial transformation. The results showed that the constructed recombinant plasmid pET28a-svu005 was successfully expressed in E. coli. The production of self-induced active protein was 0.0916 nmol/L in ZYM medium, which was significantly higher than that of traditional IPTG induction method (0.0367 nmol/L). The expressed product CYP105D1 protein has the function of biotransforming androstenedione to 9-hydroxyandrostenedione. This study has opened up a new way for the synthesis of 9-hydroxyandrostenedione by microbial transformation and has a good prospect of industrial application.

Key words: font-size:medium, ">9-hydroxyandrostenedione； microbial transformation； CYP105D1； self-induction

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