Creation of minimal genome yeast by chromosome splitting technology

染色体工程技术可以操纵大片段的染色体DNA，不仅对于研究真核生物基因组的组织，而且对于改良工业上重要的菌株都是有用的。为了开发能够有效操作大片段染色体的技术，我们在本研究中开发了一种单倍体酿酒酵母的一步染色体分裂技术。利用这项技术，我们可以成功地创造出一种具有多达21条染色体的新型酵母单倍体菌株。为了促进染色体上多个位置的染色体分裂，我们进一步关注了分布在酵母基因组中的200多个拷贝的δ序列。我们构建了一个含有δ序列和可重复使用的转化标记盒的染色体分裂载体，并成功地分离了多条染色体。我们还成功地开发了一种新的技术来改组两个酵母菌株所需的染色体区域。染色体改组技术为评估酵母中特定染色体区域替换的后果提供了一个强有力的工具。虽然这项研究的成果离最终目标，即创造最小基因组酵母和探索真核生物基因组结构与功能之间的关系还很遥远，但我们相信，通过这项研究，已经建立了实现这一目标的基因组工程的基础技术。

Chromosome engineering techniques that can manipulate a large segment of chromosomal DNA are useful not only for studying the organization of eukaryotic genomes but also for the improvement of industrially important strains. Toward the development of techniques that can efficiently manipulate a large segment of chromosome, we have developed in this study a one-step chromosome splitting technique in a haploid Saccharomyces cerevisiae. With this technique we could successfully to create a novel yeast haploid strain having up to 21 chromosomes. To facilitate chromosome splitting at multiple sites on chromosomes, we further focused on the δ sequences that are distributed in more than 200 copies throughout the yeast genome. We constructed a new chromosome-splitting vector harboring the δ sequence and the reusable marker cassette for transformation, and successfully split several chromosomes. We have also successfully developed a novel technique for shuffling a desired chromosomal region of two yeast strains. Chromosome shuffling technique provide a powerful tool to evaluate the consequence of replacement of a particular chromosomal region in yeast. Although achievement of this study is still far from ultimate aim, i.e., creation of minimal genome yeast and exploration of relationship between genome structure and function in eukaryote, we believe that fundamental technology for engineering genome toward the goal was established through this study.