Metabolic and Biomolecular Engineering National Research Laboratory, Department of Chemical and Biomolecular Engineering (BK21 Program), KAIST, Daejeon, Korea.
FEMS Microbiol Lett. 2010 Aug 1;309(2):193-200. doi: 10.1111/j.1574-6968.2010.02041.x. Epub 2010 Jun 16.
Ralstonia eutropha H16 is a Gram-negative lithoautotrophic bacterium and is one of the best biopolymer-producing bacteria. It can grow to high cell densities either under lithoautotrophic or under heterotrophic conditions, which makes it suitable for a number of biotechnological applications. Also, R. eutropha H16 can degrade various aromatic compounds for environmental applications. The mobile group II intron can be used for the rapid and specific disruption of various bacterial genes by insertion into any desired target genes. Here, we applied the mobile group II intron to R. eutropha H16 and developed a markerless gene knockout system for R. eutropha: RalsTron. As a demonstration of the system, the phaC1 gene encoding polyhydroxyalkanoate synthase was successfully knocked out in R. eutropha H16. Furthermore, this knockout system would be useful for knocking out genes in other bacteria as well because it is based on a broad-host-range vector and the mobile group II intron that minimally depends on the bacterial hosts.
产碱杆菌 H16 是革兰氏阴性的自养型细菌，也是生产生物聚合物的最佳细菌之一。它可以在自养或异养条件下生长到很高的细胞密度，这使其适用于许多生物技术应用。此外，产碱杆菌 H16 可以降解各种芳香族化合物，用于环境应用。移动的 II 组内含子可用于通过插入任何所需的靶基因，快速且特异性地破坏各种细菌基因。在这里，我们将移动的 II 组内含子应用于产碱杆菌 H16，并为产碱杆菌 H16 开发了一种无标记基因敲除系统：RalsTron。作为该系统的演示，成功地敲除了编码聚羟基烷酸合酶的 phaC1 基因。此外，由于该敲除系统基于广宿主范围载体和最小程度依赖于细菌宿主的移动的 II 组内含子，因此该系统也可用于敲除其他细菌中的基因。
