Department of Synthetic Chemistry and Biological Chemistry, Graduate School of Engineering, Kyoto University, Katsura, Nishikyo-ku, Kyoto, Japan.
Japan Collection of Microorganisms, RIKEN BioResource Research Center, Tsukuba, Ibaraki, Japan.
Extremophiles. 2020 May;24(3):339-353. doi: 10.1007/s00792-020-01159-z. Epub 2020 Feb 28.
In this study, a transformation system enabling large-scale gene recombination was developed for the hyperthermophilic archaeon Thermococcus kodakarensis. Using the uracil auxotroph T. kodakarensis KU216 (∆pyrF) as a parent strain, we constructed multiple host strains harboring two 1-kbp DNA regions from the genomes of either the hyperthermophilic archaeon Pyrococcus furiosus or Methanocaldococcus jannaschii. The two regions were selected so that the regions between them on the respective genomes would include pyrF genes, which can potentially be used for selection. Transformation using these host strains and genomic DNA from P. furiosus or M. jannaschii were carried out. Transformants with exogenous pyrF were obtained only using host strains with regions from P. furiosus, and only when the distances between the two regions were relatively short (2-5 kbp) on the P. furiosus genome. To insert longer DNA fragments, we examined the possibilities of using P. furiosus cells to provide intact genomic DNA. A cell pellet of P. furiosus was overlaid with that of T. kodakarensis so that cells were in direct contact. As a result, we were able to isolate T. kodakarensis strains harboring DNA fragments from P. furiosus with lengths of up to 75 kbp in a single transformation step.
在这项研究中，开发了一种可用于大规模基因重组的转化系统，用于嗜热古菌 Thermococcus kodakarensis。我们以尿嘧啶营养缺陷型 T. kodakarensis KU216（∆pyrF）为亲本菌株，构建了多个携带来自嗜热古菌 Pyrococcus furiosus 或 Methanocaldococcus jannaschii 基因组中两个 1-kbp DNA 区域的宿主菌株。选择这两个区域，是因为它们在各自基因组上的位置彼此相邻，包含潜在可用于选择的 pyrF 基因。使用这些宿主菌株和来自 P. furiosus 或 M. jannaschii 的基因组 DNA 进行转化。只有使用来自 P. furiosus 的宿主菌株，并且在 P. furiosus 基因组上两个区域之间的距离相对较短（2-5 kbp）时，才能获得具有外源 pyrF 的转化体。为了插入更长的 DNA 片段，我们研究了使用 P. furiosus 细胞提供完整基因组 DNA 的可能性。将 P. furiosus 的细胞沉淀覆盖在 T. kodakarensis 的细胞沉淀上，使细胞直接接触。结果，我们能够在单个转化步骤中分离出携带来自 P. furiosus 的长达 75 kbp 的 DNA 片段的 T. kodakarensis 菌株。
