Enhancing the copy number of episomal plasmids in Saccharomyces cerevisiae for improved protein production
Enhancing the copy number of episomal plasmids in Saccharomyces cerevisiae for improved protein production
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DOI：
10.1111/j.1567-1364.2012.00809.x
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发表时间：
2012-08-01
影响因子：
3.2
通讯作者：
Nielsen, Jens
中科院分区：
文献类型：
--
作者：
Chen, Yun;Partow, Siavash;Nielsen, Jens
基于2μm的附加型表达载体广泛用于酿酒酵母中的重组蛋白生产和合成途径优化。在这项研究中，我们报告了一种增加质粒拷贝数（PCN）的新方法，从而提高质粒编码蛋白的表达。这是通过将标记蛋白的不稳定与降低标记基因转录水平相结合来实现的。通过将泛素/N-降解决定子标签 (ubi-tag) 融合到 Ura3 标记蛋白的 N 末端来单独破坏标记蛋白的稳定性，可以增加同一载体表达的 LacZ 的 PCN 和活性。当泛素裂解后标记蛋白的 N 末端暴露出精氨酸时，PCN 和 LacZ 活性增加了 7080%。用 HXT1、KEX2 或 URA3-d 启动子替换天然 URA3 启动子导致 PCN 和 LacZ 活性增加约 30100%。结合标记基因的ubi标签和启动子修饰，PCN和LacZ活性增加了三倍。我们还证明，这种新的表达载体可用于通过将广藿香醇产量提高三倍来提高酶活性。
2 mu m-based episomal expression vectors are widely used in Saccharomyces cerevisiae for recombinant protein production and synthetic pathway optimization. In this study, we report a new approach to increase the plasmid copy number (PCN) and thus improve the expression of plasmid-encoded proteins. This was achieved by combining destabilization of the marker protein with decreasing the marker gene transcription level. Destabilization of the marker protein alone by fusing a ubiquitin/N-degron tag (ubi-tag) to the N-terminus of the Ura3 marker protein could increase the PCN and activity of LacZ expressed from the same vector. When arginine was exposed at the N-terminus of the marker protein after cleavage of ubiquitin, the PCN and LacZ activity were increased by 7080%. Replacement of the native URA3 promoter with the HXT1, KEX2 or URA3-d promoter resulted in an increase in the PCN and LacZ activity by about 30100%. Combining the ubi-tag and promoter modification of the marker gene, increased the PCN and LacZ activity by threefold. We also demonstrated that this new expression vectors can be used to increase enzyme activity by improving patchoulol production by threefold.