中国生物工程杂志

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. In this research, the gene knocking out method was used to solve the problem. In this research, 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.