Molecular breeding of Saccharomyces cerevisiae with high RNA content by harnessing essential ribosomal RNA transcription regulator.
Molecular breeding of Saccharomyces cerevisiae with high RNA content by harnessing essential ribosomal RNA transcription regulator.
复制标题
DOI：
10.1186/s13568-017-0330-4
复制
发表时间：
2017-12
期刊：
影响因子：
3.7
通讯作者：
Harashima S
中科院分区：
文献类型：
--
作者：
Sasano Y;Kariya T;Usugi S;Sugiyama M;Harashima S
由于酵母通常用于RNA生产，因此培育高RNA含量的菌株在工业上具有重要意义。上游激活因子（UAF）在核糖体RNA （rRNA）的转录中起着重要作用，rRNA是细胞内RNA种类的主要组成部分。在这里，我们针对酿酒酵母（Saccharomyces cerevisiae）的基本rRNA转录调节因子rn5 （UAF复合体的一个组成部分），并使用携带rn5基因的辅助质粒破坏基因组中的rn5基因。然后我们分离出9个rn5基因断裂抑制突变体（Sup突变体），导致rRNA转录缺陷。Sup突变体的RNA含量约为野生型的40%，rDNA重复数扩增至约400-700拷贝。将一个功能性的rn5基因重新引入Sup突变体，导致rDNA重复数减少到接近野生型水平，但没有改变RNA含量。然而，我们发现，将RRN5重新引入Sup16突变体（编码rDNA复制叉屏障位点结合蛋白的FOB1基因被破坏），与野生型菌株相比，RNA含量显著增加（17%），尽管rDNA重复拷贝数几乎与野生型菌株相同。在这种情况下，发生了非转录间隔子（NTS）的转录上调，特别是在NTS2区域；这可能是由RNA聚合酶II介导的，并解释了RNA含量的增加。因此，我们提出了一种新的育种策略，通过利用必需的rRNA转录调节因子来开发高RNA含量的酵母。本文的在线版本（doi:10.1186/s13568-017-0330-4）包含补充材料，可供授权用户使用。
As yeast is commonly used for RNA production, it is industrially important to breed strains with high RNA contents. The upstream activating factor (UAF) plays an important role in transcription of ribosomal RNA (rRNA), a major constituent of intracellular RNA species. Here, we targeted the essential rRNA transcription regulator Rrn5 of Saccharomyces cerevisiae, a component of the UAF complex, and disrupted the genomic RRN5 gene using a helper plasmid carrying an RRN5 gene. Then we isolated nine suppressor mutants (Sup mutants) of RRN5 gene disruption, causing deficiency in rRNA transcription. The Sup mutants had RNA contents of approximately 40% of the wild type level and expansion of rDNA repeats to ca. 400–700 copies. Reintroduction of a functional RRN5 gene into Sup mutants caused a reduction in the number of rDNA repeats to close to the wild type level but did not change RNA content. However, we found that reintroduction of RRN5 into the Sup16 mutant (in which the FOB1 gene encoding the rDNA replication fork barrier site binding protein was disrupted) resulted in a significant increase (17%) in RNA content compared with wild type, although the rDNA repeat copy number was almost identical to the wild type strain. In this case, upregulated transcription of non-transcribed spacers (NTS) occurred, especially in the NTS2 region; this was likely mediated by RNA polymerase II and accounted for the increased RNA content. Thus, we propose a novel breeding strategy for developing high RNA content yeast by harnessing the essential rRNA transcription regulator. The online version of this article (doi:10.1186/s13568-017-0330-4) contains supplementary material, which is available to authorized users.