5´-UTR introns enhance protein expression in the yeast Saccharomyces cerevisiae

Abstract

Saccharomyces cerevisiae is one of the most suitable microorganisms for recombinant protein production. To enhance protein production, various expression systems have been intensively studied. However, the effect of introns on protein expression has not been examined deeply in S. cerevisiae. In this study, we analyzed the effect of some introns on protein expression. RPS25A, RPS26A, and RPS26B contain single introns within the 5´-untranslated regions (5´-UTRs), and RPS24A has an intron just downstream of the initiation codon. Expression activity of the promoter regions containing introns (intron promoters) were analyzed by luciferase reporter assays. These intron promoters showed higher expression than the TDH3 promoter (TDH3p), which is one of the strongest promoters in S. cerevisiae. Deletion of the introns from these promoters decreased luciferase expression, indicating that introns have a role in enhancing protein expression. To develop artificial strong intron promoters, several chimeric promoters were constructed using the TDH3p and the RPS25A intron promoter. A construct containing the entire TDH3p followed by the RPS25A intron showed about 50-fold higher expression than the TDH3p alone. Inducible expressions driven by the GAL10 promoter and the CUP1 promoter were also enhanced by the RPS25A intron. However, enhancement of mRNA accumulation by the TDH3p and the GAL10 promoter with the RPS25A intron was lower than the effect on luciferase activity, suggesting that the intron affects post-transcriptionally. The chimeric promoter, TDH3p-RPS25A-intron, enhanced expressions of some, but not all proteins examined, indicating that 5'-UTR introns increase production of a certain type of recombinant proteins in S. cerevisiae.

Analysis of fungal genomes reveals commonalities of intron loss/gain and functions in intron-poor species

“…In addition, introns are more abundant in genes that have higher mRNA expression and translation efficiency, irrespective of their cellular functions ( Figure 9 ). This extends previous analyses of global gene expression of S. cerevisiae (Juneau et al 2006;Hoshida et al 2017) . In metazoa and plants, introns may enhance transcription or translation, in part, through EJCs (Wiegand et al 2003;Diem et al 2007;Chazal et al 2013;Le Hir et al 2016) .…”

“…However, S. cerevisiae has no EJCs, unlike complex eukaryotes or even the fission yeast S. pombe . It remains unclear how intron enhances transcription and translation in Saccharomycetes (Moabbi et al 2012;Hoshida et al 2017) .…”

5′ untranslated regions: the next regulatory sequence in yeast synthetic biology

“…Not only was expression confirmed by the intronic HXT1 promoter, but also a maximum twofold increase in reporter activity was observed in comparison to the intronless promoter. More recently, native 5′ UTR introns of RPS25A , RPS26A and RPS26B were used to increase gene expression (Hoshida et al ., ). When the 5′ UTR introns were placed downstream of the strong TDH3 promoter, a 17‐ to 50‐fold increase in luciferase activity was observed.…”

Systems and Synthetic Biology Approaches to Engineer Fungi for Fine Chemical Production

“…In this case, the authors demonstrated that the presence of 5′ untranslated regions (UTRs) of intronic sequences in the promoter of genes encoding 40S ribosomal proteins (RPs) increased the strength of the promoter. The luciferase activity was 17-fold higher when an intronic promoter was used to drive expression when compared with the strong wild type TDH3 promoter in S. cerevisiae (Hoshida et al, 2017 ). Furthermore, when the RPS25A intronic promoter and TDH3 promoter were merged, the luciferase activity increased, approximately, by 50-fold when compared with the control of the TDH3 promoter alone (Hoshida et al, 2017 ).…”