Fine-Tuning of Transcription in Pichia pastoris Using dCas9 and RNA Scaffolds,ACS Synthetic Biology

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Fine-Tuning of Transcription in Pichia pastoris Using dCas9 and RNA Scaffolds
ACS Synthetic Biology ( IF 3.7 ) Pub Date : 2020-11-12 , DOI: 10.1021/acssynbio.0c00214
Michael Baumschabl _{1,

2} , Roland Prielhofer _{1,

2} , Diethard Mattanovich _{1,

2} , Matthias G Steiger _{1,

2,

3}

Affiliation

For metabolic engineering approaches, fast and reliable tools are required to precisely manipulate the expression of target genes. dCas9 can be fused via RNA scaffolds to trans-activator domains and thus regulate the gene expression when targeted to the promoter region of a gene. In this work we show that this strategy can be successfully implemented for the methylotrophic yeast Pichia pastoris. It is shown that the thiamine repressible promoter of THI11 can be activated under repression conditions using a scgRNA/dCas9 construct. Furthermore, the RIB1 gene required for riboflavin production was activated, leading to increased riboflavin production exceeding the riboflavin titers of a conventional RIB1 overexpression with a pGAP promoter.

中文翻译：

使用 dCas9 和 RNA 支架微调毕赤酵母中的转录

对于代谢工程方法，需要快速可靠的工具来精确操纵靶基因的表达。 dCas9 可以通过RNA 支架融合到反式激活子结构域，从而在靶向基因的启动子区域时调节基因表达。在这项工作中，我们表明这一策略可以在甲基营养酵母毕赤酵母中成功实施。结果表明，使用 scgRNA/dCas9 构建体可以在抑制条件下激活THI11的硫胺素抑制型启动子。此外，核黄素产生所需的RIB1基因被激活，导致核黄素产量增加，超过了带有pGAP启动子的常规RIB1过表达的核黄素效价。

更新日期：2020-12-18

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