Disruption of a sugar transporter gene cluster in a hyperthermophilic archaeon using a host-marker system based on antibiotic resistance

Rie Matsumi, Kenji Manabe, Toshiaki Fukui, Haruyuki Atomi, Tadayuki Imanaka

研究成果: Contribution to journalArticle査読

86 被引用数 (Scopus)

抄録

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 (hmgTk) with the glutamate dehydrogenase promoter. With this system, we disrupted the T. kodakaraensis amylopullulanase gene (apuTk) or a gene cluster which includes apuTk 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 μM simvastatin were isolated. The transformants exhibited growth in the presence of 20 μM 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 hmgTk locus when the endogenous hmgTk gene was used. This could be avoided by using the corresponding gene from Pyrococcus furiosus (hmgpf) 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 ΔapuTk strain could not grow on pullulan and displayed only low levels of growth on amylose, suggesting that ApuTk is a major polysaccharide-degrading enzyme in T. kodakaraensis.

本文言語英語
ページ(範囲)2683-2691
ページ数9
ジャーナルJournal of bacteriology
189
7
DOI
出版ステータス出版済み - 4 2007
外部発表はい

All Science Journal Classification (ASJC) codes

  • 微生物学
  • 分子生物学

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