Use of expression-enhancing terminators in Saccharomyces cerevisiae to increase mRNA half-life and improve gene expression control for metabolic engineering applications.
Use of expression-enhancing terminators in Saccharomyces cerevisiae to increase mRNA half-life and improve gene expression control for metabolic engineering applications.
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DOI：
10.1016/j.ymben.2013.07.001
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发表时间：
2013-09
影响因子：
8.4
通讯作者：
Alper, Hal S.
中科院分区：
文献类型：
--
作者：
Curran, Kathleen A.;Karim, Ashty S.;Gupta, Akash;Alper, Hal S.
控制内源和异源基因的基因和蛋白质表达是代谢工程的关键组成部分。虽然为此目的已发表了大量表征启动子的工作，但阐明终止子在酵母中的作用的努力却较少。在这项研究中，我们对酿酒酵母代谢工程应用中使用的 30 多种终止子进行了表征，并确定 mRNA 半衰期变化是蛋白质和转录本表达水平变化的主要原因。我们证明，即使对于高强度启动子，转录水平的差异也可以超过 6.5 倍。当与低表达启动子偶联时，终止子选择的影响会被放大，高容量终止子和亲本质粒终止子之间的蛋白质表达最大差异为 11 倍，与无终止子基线相比，差异超过 35 倍。这是第一次在活性跨越多个数量级的多个启动子的背景下研究终止子。最后，我们通过使用突变木糖异构酶基因作为概念验证，证明了终止子选择在代谢工程中的实用性。通过将高容量终止子与低表达启动子配对，我们能够获得与强度高得多的启动子相同的表型结果。此外，我们可以通过将高强度启动子与高容量终止子配对来进一步增强高强度启动子的表型。这项工作强调了如何使用终止子元件来控制代谢途径，就像传统上在酵母中使用启动子一样。总之，这项工作表明终止子将成为未来酵母异源基因表达和代谢工程的重要组成部分。
Control of gene and protein expression of both endogenous and heterologous genes is a key component of metabolic engineering. While a large amount of work has been published characterizing promoters for this purpose, less effort has been exerted to elucidate the role of terminators in yeast. In this study, we characterize over 30 terminators for use in metabolic engineering applications in Saccharomyces cerevisiae and determine mRNA half-life changes to be the major cause of the varied protein and transcript expression level. We demonstrate that the difference in transcript level can be over 6.5-fold even for high strength promoters. The influence of terminator selection is magnified when coupled with a low-expression promoter, with a maximum difference in protein expression of 11-fold between a high-capacity terminator and the parent plasmid terminator and over 35-fold difference when compared with a no-terminator baseline. This is the first time that terminators have been investigated in the context of multiple promoters spanning orders of magnitude in activity. Finally, we demonstrate the utility of terminator selection for metabolic engineering by using a mutant xylose isomerase gene as a proof-of-concept. Through pairing a high-capacity terminator with a low-expression promoter, we were able to achieve the same phenotypic result as with a promoter considerably higher in strength. Moreover, we can further boost the phenotype of the high-strength promoter by pairing it with a high-capacity terminator. This work highlights how terminator elements can be used to control metabolic pathways in the same way that promoters are traditionally used in yeast. Together, this work demonstrates that terminators will be an important part of heterologous gene expression and metabolic engineering for yeast in the future.
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影响因子：
3.2
作者：
Karim AS;Curran KA;Alper HS
通讯作者：
Alper HS
DOI：
10.1016/j.bbagrm.2012.10.003
发表时间：
2013-01
影响因子：
4.7
作者：
Mischo, Hannah E.;Proudfoot, Nick J.
通讯作者：
Proudfoot, Nick J.
影响因子：
46.9
作者：
Pfleger, Brian F.;Pitera, Douglas J.;Keasling, Jay D.
通讯作者：
Keasling, Jay D.
影响因子：
4.4
作者：
Lee, Sun-Mi;Jellison, Taylor;Alper, Hal S.
通讯作者：
Alper, Hal S.
影响因子：
64.5
作者：
MONTGOMERY, DL;HALL, BD;SMITH, M
通讯作者：
SMITH, M