Yang Jing, Tian Yujuan, Liu Huayi, Kan Yeyi, Zhou Yi, Wang Ying, Luo Yunzi
Frontier Science Center for Synthetic Biology and Key Laboratory of Systems Bioengineering (Ministry of Education), School of Chemical Engineering and Technology, Collaborative Innovation Center of Chemical Science and Engineering (Tianjin), Tianjin University, Tianjin, China.
Department of Gastroenterology, State Key Laboratory of Biotherapy, West China Hospital, Sichuan University, Chengdu, China.
Front Microbiol. 2021 Jun 18;12:679665. doi: 10.3389/fmicb.2021.679665. eCollection 2021.
pRS episomal plasmids are widely used in , owing to their easy genetic manipulations and high plasmid copy numbers (PCNs). Nevertheless, their broader application is hampered by the instability of the pRS plasmids. In this study, we designed an episomal plasmid based on the endogenous 2μ plasmid with both improved stability and increased PCN, naming it p2μM, a 2μ-modified plasmid. In the p2μM plasmid, an insertion site between the promoter and promoter was identified, where the replication (ori) of and a selection marker gene of were inserted. As a proof of concept, the tyrosol biosynthetic pathway was constructed in the p2μM plasmid and in a pRS plasmid (pRS423). As a result, the p2μM plasmid presented lower plasmid loss rate than that of pRS423. Furthermore, higher tyrosol titers were achieved in harboring p2μM plasmid carrying the tyrosol pathway-related genes. Our study provided an improved genetic manipulation tool in for metabolic engineering applications, which may be widely applied for valuable product biosynthesis in yeast.
pRS附加体质粒因其易于进行基因操作和具有高质粒拷贝数（PCNs）而在[具体应用领域未提及]中被广泛使用。然而，pRS质粒的不稳定性阻碍了它们更广泛的应用。在本研究中，我们基于内源性2μ质粒设计了一种附加体质粒，其稳定性得到提高且PCN增加，将其命名为p2μM，即一种2μ修饰质粒。在p2μM质粒中，鉴定出了位于[两个启动子名称未提及]启动子和[另一个启动子名称未提及]启动子之间的一个插入位点，在该位点插入了[某个质粒名称未提及]的复制起点（ori）和[另一个质粒名称未提及]的一个选择标记基因。作为概念验证，在p2μM质粒和一个pRS质粒（pRS423）中构建了酪醇生物合成途径。结果，p2μM质粒的质粒丢失率低于pRS423。此外，在携带酪醇途径相关基因的p2μM质粒的[具体宿主未提及]中获得了更高的酪醇滴度。我们的研究为[具体宿主未提及]中的代谢工程应用提供了一种改进的基因操作工具，其可能广泛应用于酵母中有价值产物的生物合成。
