Characterization of essential elements for improved episomal expressions in Kluyveromyces marxianus
Characterization of essential elements for improved episomal expressions in Kluyveromyces marxianus
复制标题
DOI：
10.1002/biot.202100382
复制
发表时间：
2022-01
影响因子：
4.7
通讯作者：
Pingping Wu;Jungang Zhou;Yao Yu;Hong Lu
中科院分区：
文献类型：
--
作者：
Pingping Wu;Jungang Zhou;Yao Yu;Hong Lu
马克斯克鲁维酵母是一种很有前途的生产异源蛋白的细胞工厂。多拷贝质粒中适用于克雷伯氏菌的基本组分包括分配元件、拷贝数控制元件、启动子和终止子。马克思主义并没有广泛的特征。pKD 1是在K.基于pKD 1的质粒是唯一成功应用于K.马克思主义。环状质粒pKD 1含有三个主要的开放阅读框，即A、B和C。在这里，我们发现A基因负责维持K中基于pKD 1的质粒的高拷贝数。马克思主义。缺失B或C基因会损害基于pKD 1的质粒在克雷伯氏菌中的稳定增殖。marxianus，这种缺陷不能通过B和C基因的反式表达来挽救。在对一系列启动子和终止子的定量分析中，选择了来自毕赤酵母的AFT 1启动子、来自K. marxianus和一组合成终止符支持高级表达式。在基于pKD 1的质粒中，AFT 1或OM 45启动子与INU1终止子的组合实现了四种不同异源蛋白的高水平表达。我们的研究为K.马克思主义。
Kluyveromyces marxianus is a promising cell factory for producing heterologous proteins. Essential components including partition element, copy number control element, promoter, and terminator in multicopy plasmids applicable for K. marxianus are not extensively characterized. The pKD1 is an endogenous multicopy plasmid identified in K. lactis, and the pKD1‐based plasmid is the only multicopy plasmid successfully applied in K. marxianus. The circular plasmid pKD1 contains three major open reading frames, namely A, B, and C. Here, we showed that the A gene was responsible for maintaining the high‐copy number of pKD1‐based plasmid in K. marxianus. Deletion of the B or C gene impaired the stable propagation of pKD1‐based plasmid in K. marxianus, and this defect could not be rescued by the trans expression of B and C genes. In a quantitative analysis of a series of promoters and terminators, AFT1 promoter from Pichia pastoris, OM45 promoter and INU1 terminator from K. marxianus, and a set of synthetic terminators, supported high‐level expressions. A combination of AFT1 or OM45 promoter with INU1 terminator in a pKD1‐based plasmid achieved high‐level expressions of four different heterologous proteins. Our study provides valuable elements for high‐level episomal expressions in K. marxianus.