Using mutant pheS gene as the counter-selection marker to delete the kstD genes in Rhodococcus equi

• Main Authors: Tzu-wei Kuo, 郭子瑋
• Other Authors: Meng-Hsiao Meng
• Format: Others
• Language: zh-TW
• Published: 2017
• Online Access: http://ndltd.ncl.edu.tw/handle/32102293835051724216

碩士 === 國立中興大學 === 生物科技學研究所 === 105 === 4-Androstene-3,17-dione (AD), an important precursor in steroids industry, can be transformed into a variety of steroids via a series of chemical synthesis. These steroids have been widely used as anti-inflammatory drugs, immunosuppressant, and contraceptives etc. In nature, some microorganisms are able to degrade sterol; therefore, utilization of these microorganisms to accumulate AD is considerably potential. Rhodococcus equi, a gram-positive bacterium, use the 3-ketosteroid Δ1-dehydrogenase (kstD) to converse AD into 1,4-androstadiene-3,17-dione (ADD) in steroid metabolism. According to the genomic information of R. equi, there are probably five kstD paralogs, which include Req_06770, Req_10320, Req_13080, Req_15820, and Req_30200, in R. equi. Nonetheless, the role of these genes in the sterol pathway is still unclear. In this study, an unmarked gene knockout method applicable to R. equi was established to delete the putative kstD genes and the resulting effects on AD accumulation in the culture medium were assayed by thin layer chromatography (TLC). So far Req_10320, Req_13080, and Req_15820、Req_06770 have been deleted respectively. Unfortunately, none of the four kstD-deleted strains could accumulate AD. We presume either the major gene responsible for AD to ADD conversion has not been deleted or all the KSTD proteins are involved in AD catabolism so that single kstD deletion cannot result in AD accumulation. In the future, we will continue to delete Req_30200 and create mutant strains with multiple kstD gene deletions in order to obtain the ideal strain for AD production.