Cloning the 3-ketosteroid 9-α-hydroxylase gene from Arthrobacter simplex

• Main Authors: Che-Ming Chang, 張哲銘
• Other Authors: Menghsiao Meng
• Format: Others
• Language: zh-TW
• Online Access: http://ndltd.ncl.edu.tw/handle/u6qhgh

碩士 === 國立中興大學 === 生物科技學研究所 === 98 === Steroids, which are focused by many pharmaceutical companies, possess considerable bioactivities and huge economic value. In early period, chemical synthesis of steroid compounds was the major process, however the synthetic efficiency was low and the waste disposal caused a serious environmental problem. Therefore, the steroid synthesis coupled the chemical and biological approaches. The 3-ketosteroid 9-α-hydroxylase (ksh) is the key enzyme in the steroid degradation and has been discovered in various actinobacterial genera, e.g., Arthrobacter, Nocardia, Mycobacterium, and Rhodococcus. The steroid industry utilize the 3-ketosteroid- Δ1,2-dehydrogenase or ksh enzymes mutant Mycobacterium to accumulate the steroid pharmaceutical precursors, 9α-hydroxyandrost-4-ene-3,17-dione or 1,4-androstadiene- 3,17-dione, to synthesize high value steroid. A. simplex is a potential strain in industrial fermentation on the basis of higher tolerance to cholesterol. Besides, more detailed research of A. simplex has not yet been published. Because of these reasons, we choose A. simplex as the material in this study. At first, we blasted some ksh enzymes and related oxygenase sequences published and search the conserve regions to design degenerated primer. After PCR amplification, the fragment were sequenced and cloned. In order to obtain the entire sequence, we performed inverse PCR and DNA walking, the result of which showed that ksh enzymes in A. simplex consisted of two components, kshA (terminal oxygenase) and kshB (reductase). Moreover, there are two kshA isoforms, kshA(S1A2) and kshA(S3A2). We next used global alignment to align these two isoforms in workbench website. It showed 70.5％ identity in DNA and 58.7％ in amino acid level between these two isoforms. We shall assay the enzyme activity of these two isoforms to identify their role and the activity in steroid degradation. Finally, we shall use restriction enzyme to generate truncated 3-ketosteroid 9-α-hydroxylase, and perform genetic recombination to generate mutant A. simplex. In the future, we will assay whether the 3-ketosteroid 9-α-hydroxylase knockout strain has higher accumulation of 4-androstene-3,17-dione and 1,4-androstadiene-3,17-dione.