Unmarked gene deletion mutagenesis of kstD, encoding 3-ketosteroid Δ1-dehydrogenase, in Rhodococcus erythropolis SQ1 using sacB as counter-selectable marker
Unmarked gene deletion mutagenesis of kstD, encoding 3-ketosteroid Δ1-dehydrogenase, in Rhodococcus erythropolis SQ1 using sacB as counter-selectable marker
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DOI：
10.1016/s0378-1097(01)00464-5
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发表时间：
2001-12-18
影响因子：
2.1
通讯作者：
Dijkhuizen, L
中科院分区：
文献类型：
--
作者：
van der Geize, R;Hessels, GI;Dijkhuizen, L
本文报道了第一个在红球菌属中构建无标记基因缺失突变体的方法。使用 sac B 反选择系统实现了红平红球菌 SQ1 中编码 3-酮类固醇 Delta (I)-脱氢酶 (KSTD1) 的 kstD 基因的无标记删除。使用来自大肠杆菌 S17-1 的诱变质粒与红平红细胞菌株 SQ1 的接合动员来避免其随机基因组整合。 kstD基因缺失突变体，命名为菌株RG1，仍然具有野生型的约10%的KSTD酶活性，并且其在类固醇底物4-雄烯-3,17-二酮(AD)和9α-羟基-4-雄烯-3,17-二酮(9OHAD)上生长的能力不受影响。随后获得的生化证据表明红平红平菌 SQ1 中存在第二种 KSTD 酶 (KSTD2)。分离出不能在 AD 上生长的菌株 RG1 的 UV 突变体。其中一种突变体，即菌株 RG1-UV29，已丧失所有 KSTD 酶活性，并且也无法在 9OHAD 上生长。其按化学计量将 AD 转化为浓度高达 20 g l(-1) 的 9OHAD。这两种 KSTD 酶显然都在 AD 和 9OHAD 分解代谢中发挥作用。这些同工酶分别在菌株 RG1（KSTD1 阴性）和菌株 RG1-UV29（KSTD1 和 KSTD2 阴性）中失活。 (C) 2001 年欧洲微生物学会联合会。由 Elsevier Science B.V. 出版。保留所有权利。
This paper reports the first method for the construction of unmarked gene deletion mutants in the genus Rhodococcus. Unmarked deletion of the kstD gene, encoding 3-ketosteroid Delta (I)-dehydrogenase (KSTD1) in Rhodococcus erythropolis SQ1, was achieved using the sac B counter-selection system. Conjugative mobilization of the mutagenic plasmid from Escherichia coli S17-1 to R. erythropolis strain SQ1 was used to avoid its random genomic integration. The kstD gene deletion mutant, designated strain RG1, still possessed about 10% of the KSTD enzyme activity of wild-type and was not affected in its ability to grow on the steroid substrates 4-androstene-3,17-dione (AD) and 9 alpha -hydroxy-4-androstene-3,17-dione (9OHAD). Biochemical evidence subsequently was obtained for the presence of a second KSTD enzyme (KSTD2) in R. erythropolis SQ1. UV mutants of strain RG1 unable to grow on AD were isolated. One of these mutants, strain RG1-UV29, had lost all KSTD enzyme activity and was also unable to grow on 9OHAD. It stoichiometrically converted AD into 9OHAD in concentrations as high as 20 g l(-1). The two KSTD enzymes apparently both function in AD and 9OHAD catabolism. These isoenzymes have been inactivated in strain RG1 (KSTD1 negative) and strain RG1-UV29 (KSTD1 and KSTD2 negative), respectively. (C) 2001 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved.