分枝杆菌甾酮C1,2位脱氢酶基因敲除的研究

利用分枝杆菌对植物甾醇进行边链降解可产生4-AD（4-烯-雄甾-3,17-二酮）和ADD（1,4-二烯-雄甾-3,17-二酮），ADD由4-AD在C1,2位脱氢酶(ksdD)作用下脱氢产生，这两种物质在化学结构上高度相似，难以分离。本文首先扩增出部分ksdD基因，大小为631bp，并以此为基础构建打靶载体pUC19-MK。将pUC19-MK电转分枝杆菌感受态，通过同源重组敲除分枝杆菌染色体上正常的ksdD基因，使C1,2位脱氢酶失活，以达到4-AD大量积累的目的。结果通过初筛筛选出5株转化子，进行甾体转化实验，发酵144h时，1号转化子的4-AD生成率达到17.52％，比出发菌株提高了192％，而此时ADD的生成率仅为6.12％，比出发菌株降低了89.9％。

Studies on Gene Knocking Out of 3-Ketosteriod-1-Dehydrogenase in Mycobacterium neoaurum

The selective side-chain cleavage of phytosterol to 4-androstene-3,17-dione(4-AD)and 1,4-androstadiene-3,17-dione(ADD)by Mycobacterium sp.was described.Because of the similarity in chemical structure between 4-AD and ADD,it is difficult to separate them from the fermentation broth.So far,it has been verified that the ADD can be produced by dehydrogenation of 4-AD.In this reaction,3-Ketosteriod-1-Dehydrogenase(ksdD)plays an important role.The gene knocking out method was used to solve the problem.Partial sequence of ksdD was obtained by PCR which was 631bp in length.Then,a targeting vector pUC19-MK was constructed,which was electroporate into the original strain Mycobacterium neoauru.The method of homologous recombination was used to knock out ksdD gene located in the chromosome of Mycobacterium neoauru.In this way,ksdD would lose its enzyme activity.In the result,5 transformants were screened.The experiments of steroid transformation by the transformants were carried out.The productivity of 4-AD reached 17.52% after 144h,which is 192% higher than the original strain.Meanwhile,the productivity of ADD reached 6.12%,which is 89.9% lower than the original strain.