Revisiting the unique structure of autonomously replicating sequences in Yarrowia lipolytica and its role in pathway engineering
Revisiting the unique structure of autonomously replicating sequences in Yarrowia lipolytica and its role in pathway engineering
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DOI：
10.1007/s00253-021-11399-4
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发表时间：
2021-08
影响因子：
5
通讯作者：
C. Lopez;Mingfeng Cao;Zhanyi Yao;Zengyi Shao
中科院分区：
文献类型：
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作者：
C. Lopez;Mingfeng Cao;Zhanyi Yao;Zengyi Shao
在微生物细胞工厂中生产工业相关化合物可以采用基因组或质粒作为表达平台。选择质粒作为途径载体有利于快速演示，但对稳定性提出了挑战。在过去的十年中，解脂耶氏酵母因生物合成与脂肪酸相关的化学物质的滴度对工业有吸引力而引起了极大的关注，并且已经开发了许多遗传工具来探索其产油潜力。我们最近对非常规酵母的自主复制序列（ARS）的研究表明，来自Y. lipolytica 展示了一种独特的结构，其中包括连接复制起点 (ORI) 和着丝粒 (CEN) 元件的先前未注释的序列（间隔区），并在调节质粒行为中发挥关键作用。与更普遍使用的最小化 ARS 相比，维持天然 645 bp 间隔区可使基因表达增加 2.2 倍，质粒稳定性提高 1.7 倍。测试 ARS 子元件的模块化表明质粒稳定性表现出明显的货物依赖性。不稳定性导致质粒丢失和分子内重排。总而言之，我们的工作为科学界阐明了各种 ARS 的适当应用，并揭示了以前未探索的 DNA 元素作为工程潜在目标的情况。要点•在解脂耶氏酵母中，ARS 包含复制起点、间隔区和着丝粒。•野生型 ARS 中存在的未注释间隔区可稳定附加型表达。•质粒稳定性和分子内重排表现出明显的货物依赖性。图形摘要
Production of industrially relevant compounds in microbial cell factories can employ either genomes or plasmids as an expression platform. Selection of plasmids as pathway carriers is advantageous for rapid demonstration but poses a challenge of stability.Yarrowia lipolyticahas attracted great attention in the past decade for the biosynthesis of chemicals related to fatty acids at titers attractive to industry, and many genetic tools have been developed to explore its oleaginous potential. Our recent studies on the autonomously replicating sequences (ARSs) of nonconventional yeasts revealed that the ARSs fromY. lipolyticashowcase a unique structure that includes a previously unannotated sequence (spacer) linking the origin of replication (ORI) and the centromeric (CEN) element and plays a critical role in modulating plasmid behavior. Maintaining a native 645-bp spacer yielded a 2.2-fold increase in gene expression and 1.7-fold higher plasmid stability compared to a more universally employed minimized ARS. Testing the modularity of the ARS sub-elements indicated that plasmid stability exhibits a pronounced cargo dependency. Instability caused both plasmid loss and intramolecular rearrangements. Altogether, our work clarifies the appropriate application of various ARSs for the scientific community and sheds light on a previously unexplored DNA element as a potential target for engineeringY. lipolytica.Key points•In Y. lipolytica, an ARS comprises an origin of replication, a spacer, and a centromere.•The unannotated spacer present in the wild-type ARS stabilizes episomal expression.•Plasmid stability and intramolecular rearrangements exhibit a clear cargo dependency.Graphical abstract