戈登氏菌3-甾酮-Δ1-脱氢酶基因定点突变及异源表达

Heterologous expression and site-directed mutagenesis of 3-ketosteroid-Δ1-dehydrogenase gene from Gordonia neofelifaecis

目的:将来源于戈登氏菌(Gordonia neofelifaecis)的3-甾酮-Δ1-脱氢酶[3-ketosteroid-Delta(1)-dehydrogenase,KstD]基因通过定点突变并在大肠杆菌中进行表达,获得具有活性的脱氢酶。方法:克隆戈登氏菌KstD基因与表达载体连接构建野生型重组载体pCold I-KstD;以野生型重组载体为模板,通过反向PCR定点突变构建突变体重组载体pCold I-F307A,把重组载体转入大肠杆菌Escherichia coli BL21(DE3)成功构建了表达脱氢酶KSDD的重组子菌株。结果:30℃下诱导表达后获得的重组酶的酶活为85217.08 U·mg-1,比野生型提高了2.27倍,酶动力学参数测定结果显示重组酶对雄烯二酮的催化效率比野生型提高了2.32倍。结论:通过定点突变改造野生型3-甾酮-Δ1-脱氢酶基因,将酶活性催化中心的苯丙氨酸突变为丙氨酸,其对甾体A环C1,2脱氢反应的活性提高。本研究验证了KstD基因的活性位点,为构建高效转化雄烯二酮的基因工程菌奠定了基础。

Objective:To obtain active dehydrogenase by site-directed mutagenesis of 3-ketosteroid-Delta(1)-dehydrogenase(KstD)which comes from Gordonia neofelifaecis and expressing it in Escherichia coli(E.coli).Methods:A wild type recombinant vector was constructed by linking KstD from Gordonia neofelifaecis with the pCold I-KstDG vector.The mutant body weight group vector pColdI-F307A was constructed by reverse PCR using wild type recombinant vector as template.The recombinant plasmid was transferred into E.coli BL21(DE3).The recombinant strain expressing KSDD was successfully constructed.Results:The recombinant strain E.coli pCold I-F307A was successfully constructed.It was induced to express the dehydrogenase by isopropyl-β-d-thiopyranogalactoside(IPTG)and the activity of the mutant was 85217.08 U·mg-1 in E.coli pCold I-F307A,2.27-fold as that of pCold I-KstDG at 30℃.The catalytic efficiency of F307A to the substrate was 2.32 times higher than that of pCold I-KstDG.Conclusion:The results showed that the phenylalanine was mutated to alanine in the catalytic center of enzyme activity by site-directed mutagenesis of KstD gene,and the activity for dehydrogenation of steroids A-cyclic C1,2 was increased.This research verified the active site of KstD gene,which laid a foundation for the construction of a highly efficient gene engineering strains which can efficiently transform androstenedione.