An innovative protein expression system using RNA polymerase I for large-scale screening of high-nucleic-acid content Saccharomyces cerevisiae strains.,Microbial Biotechnology

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An innovative protein expression system using RNA polymerase I for large-scale screening of high-nucleic-acid content Saccharomyces cerevisiae strains.
Microbial Biotechnology ( IF 4.8 ) Pub Date : 2020-08-27 , DOI: 10.1111/1751-7915.13653
Duwen Zeng ₁ , Chenxi Qiu ₂ , Yu Shen ₂ , Jin Hou ₂ , Zailu Li ₁ , Jixiang Zhang ₃ , Shuai Liu ₃ , Jianli Shang ₃ , Wensheng Qin ₄ , Lili Xu _{1,

2,

3} , Xiaoming Bao _{1,

2}

Affiliation

Saccharomyces cerevisiae is the preferred source of RNA derivatives, which are widely used as supplements for foods and pharmaceuticals. As the most abundant RNAs, the ribosomal RNAs (rRNAs) transcribed by RNA polymerase I (Pol I) have no 5′ caps, thus cannot be translated to proteins. To screen high‐nucleic‐acid content yeasts more efficiently, a cap‐independent protein expression system mediated by Pol I has been designed and established to monitor the regulatory changes of rRNA synthesis by observing the variation in the reporter genes expression. The elements including Pol I‐recognized rDNA promoter, the internal ribosome entry site from cricket paralytic virus which can recruit ribosomes internally, reporter genes (URA3 and yEGFP3), oligo‐dT and an rDNA terminator were ligated to a yeast episomal plasmid. This system based on the URA3 gene worked well by observing the growth phenotype and did not require the disruption of cap‐dependent initiation factors. The fluorescence intensity of strains expressing the yEGFP3 gene increased and drifted after mutagenesis. Combined with flow cytometry, cells with higher GFP level were sorted out. A strain showed 58% improvement in RNA content and exhibited no sequence alteration in the whole expression cassette introduced. This study provides a novel strategy for breeding high‐nucleic‐acid content yeasts.

中文翻译：

使用 RNA 聚合酶 I 的创新蛋白质表达系统，用于大规模筛选高核酸含量的酿酒酵母菌株。

酿酒酵母是 RNA 衍生物的首选来源，广泛用作食品和药品的补充剂。作为最丰富的RNA，由RNA聚合酶I（Pol I）转录的核糖体RNA（rRNA）没有5'帽，因此不能翻译成蛋白质。为了更有效地筛选高核酸含量酵母，设计并建立了由Pol I介导的帽非依赖性蛋白表达系统，通过观察报告基因表达的变化来监测rRNA合成的调控变化。包括 Pol I 识别的rDNA启动子、蟋蟀麻痹病毒的内部核糖体进入位点（可以在内部招募核糖体）、报告基因（ URA3和yEGFP3 ）、 oligo-dT和rDNA终止子在内的元件被连接到酵母附加型质粒。通过观察生长表型，这个基于URA3基因的系统运行良好，并且不需要破坏帽子依赖性启动因子。表达yEGFP3基因的菌株在诱变后荧光强度增加并漂移。结合流式细胞术，筛选出GFP水平较高的细胞。菌株的 RNA 含量提高了 58%，并且引入的整个表达盒没有序列改变。该研究为培育高核酸酵母提供了一种新策略。

更新日期：2020-10-05

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