Graduate School of Science, Technology and Innovation, Kobe University, Kobe, Japan.
Biotechnology Research Laboratories, Kaneka Corporation, Takasago, Japan.
Appl Environ Microbiol. 2018 Jul 17;84(15). doi: 10.1128/AEM.02882-17. Print 2018 Aug 1.
The methylotrophic yeast is widely used to produce recombinant proteins, taking advantage of this species' high-density cell growth and strong ability to secrete proteins. Circular plasmids containing the -specific autonomously replicating sequence () permit transformation of with higher efficiency than obtained following chromosomal integration by linearized DNA. Unfortunately, however, existing autonomously replicating plasmids are known to be inherently unstable. In this study, we used transcriptome sequencing (RNA-seq) data and genome sequence information to independently identify, on each of the four chromosomes, centromeric DNA sequences consisting of long inverted repeat sequences. By examining the chromosome 2 centromeric DNA sequence () in detail, we demonstrate that an ∼111-bp region located at one end of the putative centromeric sequence had autonomous replication activity. In addition, the full-length sequence, which contains two long inverted repeat sequences and a nonrepetitive central core region, is needed for the accurate replication and distribution of plasmids in Thus, we constructed a new, stable, autonomously replicating plasmid vector that harbors the entire sequence; this episome facilitates genetic manipulation in , providing high transformation efficiency and plasmid stability. Secretory production of recombinant proteins is the most important application of the methylotrophic yeast , a species that permits mass production of heterologous proteins. To date, the genetic engineering of has relied largely on integrative vectors due to the lack of user-friendly tools. Autonomously replicating plasmids are expected to facilitate genetic manipulation; however, the existing systems, which use autonomously replicating sequences (ARSs) such as the -specific ARS (), are known to be inherently unstable for plasmid replication and distribution. Recently, the centromeric DNA sequences of were identified in back-to-back studies published by several groups; therefore, a new episomal plasmid vector with centromere DNA as a tool for genetic manipulation of is ready to be developed.
甲醇营养型酵母被广泛用于生产重组蛋白，利用该物种高密度细胞生长和强分泌蛋白的能力。含有 - 特异性自主复制序列 () 的环状质粒允许转化的效率比线性化 DNA 进行染色体整合获得的效率更高。然而，不幸的是，现有的自主复制质粒已知是固有不稳定的。在这项研究中，我们使用转录组测序 (RNA-seq) 数据和基因组序列信息，分别在四个染色体上独立识别包含长反向重复序列的着丝粒 DNA 序列。通过详细检查染色体 2 的着丝粒 DNA 序列 ()，我们证明位于假定着丝粒序列一端的约 111-bp 区域具有自主复制活性。此外，全长 序列，包含两个长反向重复序列和一个非重复的核心区，对于质粒在 中的准确复制和分配是必需的。因此，我们构建了一个新的、稳定的、自主复制的质粒载体，该载体包含整个 序列; 这个附加体促进了 在 中的遗传操作，提供了高转化效率和质粒稳定性。分泌生产重组蛋白是甲醇营养型酵母最重要的应用，这种酵母能够大规模生产异源蛋白。迄今为止，由于缺乏用户友好的工具， 的遗传工程主要依赖于整合载体。自主复制的 质粒有望促进遗传操作；然而，现有的系统，使用自主复制序列 (ARSs) 如 - 特异性 ARS ()，已知对质粒复制和分配是固有不稳定的。最近，几个小组发表的背靠背研究确定了 的着丝粒 DNA 序列; 因此，一种带有着丝粒 DNA 的新型附加体质粒载体作为 遗传操作的工具，已经准备好开发。
