Improving Saccharomyces cerevisiae ethanol production and tolerance via RNA polymerase II subunit Rpb7.
Improving Saccharomyces cerevisiae ethanol production and tolerance via RNA polymerase II subunit Rpb7.
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DOI：
10.1186/s13068-017-0806-0
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发表时间：
2017
影响因子：
6.3
通讯作者：
Jiang R
中科院分区：
文献类型：
--
作者：
Qiu Z;Jiang R
经典的菌株工程方法在改变多基因细胞表型方面往往有局限性。在这里，我们试图通过重新连接酿酒酵母的关键转录成分RNA聚合酶II(RNAP II)来重新编程其转录谱，从而提高酿酒酵母对乙醇的耐受性和生产率。这是首次使用定向进化方法来改造RNAP II来改变酿酒酵母菌株的表型。为了提高酵母对酒精的耐受性和产量，利用易错聚合酶链式反应技术对RNAP II的Rpb7亚基进行了改造。基于前人的研究和乙醇抗性提高会导致乙醇产量增加的假设，我们首先分离到对8%和10%乙醇的抗性有很大提高的变异体M1。在实验室超高密度(VHG)发酵条件下，M1的乙醇效价为122g/L(96.58%)，比对照提高40%。DNA芯片分析表明，VHG发酵12h后，M1中有369个基因有差异表达，涉及糖酵解、酒精发酵、氧化应激反应等。这是首次证明通过工程真核RNAP来改变全球转录谱和改善菌株表型的可能性。靶向RNAP II的Rpb7亚基能够在酿酒酵母的数百个基因中产生差异表达，最终导致酵母对乙醇的耐受性和产量的提高。本文的在线版本(doi：10.1186/s13068-0170806-0)包含补充材料，授权用户可以使用。
Classical strain engineering methods often have limitations in altering multigenetic cellular phenotypes. Here we try to improve Saccharomyces cerevisiae ethanol tolerance and productivity by reprogramming its transcription profile through rewiring its key transcription component RNA polymerase II (RNAP II), which plays a central role in synthesizing mRNAs. This is the first report on using directed evolution method to engineer RNAP II to alter S. cerevisiae strain phenotypes. Error-prone PCR was employed to engineer the subunit Rpb7 of RNAP II to improve yeast ethanol tolerance and production. Based on previous studies and the presumption that improved ethanol resistance would lead to enhanced ethanol production, we first isolated variant M1 with much improved resistance towards 8 and 10% ethanol. The ethanol titers of M1 was ~122 g/L (96.58% of the theoretical yield) under laboratory very high gravity (VHG) fermentation, 40% increase as compared to the control. DNA microarray assay showed that 369 genes had differential expression in M1 after 12 h VHG fermentation, which are involved in glycolysis, alcoholic fermentation, oxidative stress response, etc. This is the first study to demonstrate the possibility of engineering eukaryotic RNAP to alter global transcription profile and improve strain phenotypes. Targeting subunit Rpb7 of RNAP II was able to bring differential expression in hundreds of genes in S. cerevisiae, which finally led to improvement in yeast ethanol tolerance and production. The online version of this article (doi:10.1186/s13068-017-0806-0) contains supplementary material, which is available to authorized users.