Disruption of a sugar transporter gene cluster in a hyperthermophilic archaeon using a host-marker system based on antibiotic resistance
Disruption of a sugar transporter gene cluster in a hyperthermophilic archaeon using a host-marker system based on antibiotic resistance
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DOI：
10.1128/jb.01692-06
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发表时间：
2007-04-01
影响因子：
3.2
通讯作者：
Imanaka, Tadayuki
中科院分区：
文献类型：
--
作者：
Matsumi, Rie;Manabe, Kenji;Imanaka, Tadayuki
我们已经开发了一个基因破坏系统中的超嗜热古菌Thermococcus kodakaraensis使用抗生素辛伐他汀和一个融合基因设计过表达的3-羟基-3-甲基戊二酰辅酶A（HMG-CoA）还原酶基因（hmg（TK））与谷氨酸脱氢酶启动子。有了这个系统，我们打乱了T。kodakaraensis淀粉支链淀粉酶基因（apu（TK））或包括apu（TK）和编码推定的糖转运蛋白组分的基因的基因簇。将破坏质粒导入野生型T. kodakaraensisKOD 1细胞和对4 μ M辛伐他汀显示抗性的转化体。转化体在20 μ M辛伐他汀存在下生长，我们观察到细胞内HMG-CoA还原酶活性增加30倍。通过双交换重组的预期基因破坏发生在靶基因座，但当使用内源性hmg（TK）基因时，我们也观察到hmg（TK）基因座的重组事件。这可以通过使用来自激烈火球菌（Pyrococcus furiosus）的相应基因（hmg（PF））或通过在转化之前线性化质粒来避免。虽然两种基因破坏菌株在氨基酸或丙酮酸盐上显示正常生长，但没有糖转运蛋白基因的细胞不能在麦芽寡糖或多糖上生长，这表明基因簇编码参与这些化合物摄取的唯一糖转运蛋白。Delta apu（TK）菌株不能在支链淀粉上生长，而在直链淀粉上仅显示低水平的生长，这表明Apu（TK）是T.科达卡拉
We have developed a gene disruption system in the hyperthermophilic archaeon Thermococcus kodakaraensis using the antibiotic simvastatin and a fusion gene designed to overexpress the 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase gene (hmg(TK)) with the glutamate dehydrogenase promoter. With this system, we disrupted the T. kodakaraensis amylopullulanase gene (apu(TK)) or a gene cluster which includes apu(TK) and genes encoding components of a putative sugar transporter. Disruption plasmids were introduced into wild-type T. kodakaraensis KOD1 cells, and transformants exhibiting resistance to 4 mu M simvastatin were isolated. The transformants exhibited growth in the presence of 20 mu M simvastatin, and we observed a 30-fold increase in intracellular HMG-CoA reductase activity. The expected gene disruption via double-crossover recombination occurred at the target locus, but we also observed recombination events at the hmg(TK) locus when the endogenous hmg(TK) gene was used. This could be avoided by using the corresponding gene from Pyrococcus furiosus (hmg(PF))or by linearizing the plasmid prior to transformation. While both gene disruption strains displayed normal growth on amino acids or pyruvate, cells without the sugar transporter genes could not grow on maltooligosaccharides or polysaccharides, indicating that the gene cluster encodes the only sugar transporter involved in the uptake of these compounds. The Delta apu(TK) strain could not grow on pullulan and displayed only low levels of growth on amylose, suggesting that Apu(TK) is a major polysaccharide-degrading enzyme in T. kodakaraensis.