Identification and Characterization of the Mitochondrial Replication Origin for Stable and Episomal Expression in Yarrowia lipolytica,ACS Synthetic Biology

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Identification and Characterization of the Mitochondrial Replication Origin for Stable and Episomal Expression in Yarrowia lipolytica
ACS Synthetic Biology ( IF 3.7 ) Pub Date : 2021-03-19 , DOI: 10.1021/acssynbio.0c00619
Zhiyong Cui ₁ , Huihui Zheng ₁ , Zhennan Jiang ₁ , Zhaoxuan Wang ₁ , Jin Hou ₁ , Qian Wang _{1,

2} , Quanfeng Liang ₁ , Qingsheng Qi _{1,

3}

Affiliation

Episomal plasmids are crucial expression tools for recombinant protein production and genome editing. In Saccharomyces cerevisiae, 2-μm artificial plasmids with a high copy number have been developed and used in metabolic engineering and synthetic biology. However, in unconventional yeasts such as Yarrowia lipolytica, episomal expression relies on a chromosome replication system; this system has the disadvantages of genetic instability and low copy numbers. In this study, we identified and characterized replication origins from the mitochondrial DNA (mtDNA) of Y. lipolytica. A 516-bp mtDNA sequence, mtORI, was confirmed to mediate the autonomous replication of circular plasmids with high protein expression levels and hereditary stability. However, the nonhomologous end-joining pathway could interfere with mtORI plasmid replication and engender genetic heterogeneity. In the Po 1f ΔKu70 strain, the homogeneity of the mtORI plasmid was significantly improved, and the highest copy number reached 5.0 per cell. Overall, mitochondrial-origin sequences can be used to establish highly stable and autonomously replicating plasmids, which can be a powerful supplement to the current synthetic biology tool library and promote the development of Y. lipolytica as a microbial cell factory.

中文翻译：

解脂耶氏酵母中稳定和游离表达的线粒体复制起源的鉴定和表征

附加型质粒是重组蛋白生产和基因组编辑的关键表达工具。在酿酒酵母中，已开发出具有高拷贝数的 2-μm 人工质粒并用于代谢工程和合成生物学。然而，在非常规酵母如解脂耶氏酵母中，附加型表达依赖于染色体复制系统；该系统具有遗传不稳定性和低拷贝数的缺点。在这项研究中，我们鉴定并表征了解脂耶氏酵母线粒体 DNA (mtDNA) 的复制起点. 516 bp 的 mtDNA 序列 mtORI 被证实介导具有高蛋白质表达水平和遗传稳定性的环状质粒的自主复制。然而，非同源末端连接途径可能会干扰 mtORI 质粒复制并产生遗传异质性。在Po 1f ΔKu70菌株中，mtORI质粒的均一性显着提高，最高拷贝数达到5.0/细胞。总体而言，线粒体起源序列可用于建立高度稳定且自主复制的质粒，可有力补充现有合成生物学工具库，促进解脂耶氏酵母菌作为微生物细胞工厂的发展。

更新日期：2021-04-16

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