An innovative protein expression system using RNA polymerase I for large‐scale screening of high‐nucleic‐acid content Saccharomyces cerevisiae strains

Abstract

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 0 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-nucleicacid content yeasts.

Enhanced Ribonucleic Acid Production by High-Throughput Screening Based on Fluorescence Activation and Transcriptomic-Guided Fermentation Optimization in Saccharomyces cerevisiae

“…Fluorescence determination of RNA has the characteristics of high sensitivity and rapidity, and the high-throughput screening method based on specific RNA staining will be more direct and effective than other screening methods that adopt indirect indicators to characterize cellular RNA content. 21 In addition, it could effectively eliminate the interference of DNA in yeast cells.…”

“…Thus, the FI could reliably represent the yeast cellular RNA content and shows that the RNASelect stain method is suitable for high-throughput FCS of yeast producing high levels of RNA. Fluorescence determination of RNA has the characteristics of high sensitivity and rapidity, and the high-throughput screening method based on specific RNA staining will be more direct and effective than other screening methods that adopt indirect indicators to characterize cellular RNA content . In addition, it could effectively eliminate the interference of DNA in yeast cells.…”

“…20 An S. cerevisiae strain with 58% improvement of cellular RNA content was obtained by FCS, which introduced a plasmid containing an RNA polymerase I-mediated GFP expression cassette, and the cellular RNA content reached 176.8 mg/g. 21 However, these previous studies were only carried out at the shake flask level, and determination of RNA was performed at the early stage of the logarithmic stage (samples for RNA determination were taken at OD 600 ≈ 1.0), which was not applicable to industrial scale production of RNA. Ying and Li achieved a volumetric productivity of 2.84 and 1.68 g/L RNA, respectively, by batch and continuous fermentation of Candida tropicalis.…”

Fed-Batch Fermentation of Saccharomyces pastorianus with High Ribonucleic Acid Yield

“…However, it still needed to be cultured on solid medium with a selectable property, which prolonged the selection period. A more efficient screening method or a higher RNA yield in S. pastorianus are the goals of scientific research, such as high-throughput screening methods [ 36 ]. In addition, the direction of mutation is uncertain in the mutation breeding technique.…”

Promoting Nucleic Acid Synthesis in Saccharomyces cerevisiae through Enhanced Expression of Rrn7p, Rrn11p, IMPDH, and Pho84p

“…We successfully applied this reporter system in S. cerevisiae for the large-scale screening of the HNAC laboratory strain (haploid, n), resulting in BXZ08m3 . In the current study, this expression system was further modified by adding a selectable marker for kanamycin resistance, which was then tested in a large-scale flow-cytometry-based screen for high RNA content in the industrial S.…”