[P008] Poster

1:00 PM - 1:50 PM JST(4:00 AM - 4:50 AM UTC)

[P04-452] Engineering yeast to enhance gene expression mediated by internal ribosome entry site (IRES)

○Masahiro Tominaga^1,2, Kosuke Kaji², Tomohiko Hori², Akihiko Kondo^1,2,3,4, Jun Ishii^1,2,3 (1. Engineering Biology Research Center, Kobe University (Japan), 2. Kobe University, Graduate School of Science, Technology and Innovation (Japan), 3. Kobe University, Faculty of Engineering (Japan), 4. Center for Sustainable Resource Science, RIKEN (Japan))

Keywords:

Internal ribosome entry site,Ribosome,Gene expression,Yeast,Synthetic biology

Introduction
Internal ribosome entry site (IRES) is an RNA sequence that initiate translation in a cap-independent manner, enabling the polycistronic gene expression from a single transcribed RNA in eukaryotic cells. However, IRES-dependent translation is inefficient in yeast thereby limiting the use of IRES in yeast gene expression systems. In this study, we investigated the reason why the IRES-dependent translation is not efficient in yeast, using IRES from intergenic region of the Plautia stali intestine virus RNA (PSIV-IGR-IRES) as a model.

Results
To evaluate IRES-dependent gene expression in yeast, we constructed a yeast Saccharomyces cerevisiae strain expressing RNA polymerase from T7 bacteriophage (T7 RNAP) under the control of an inducible promoter to produce uncapped RNA in yeast. By placing the gene encoding green fluorescent protein (GFP) downstream of T7 promoter and PSIV-IGR-IRES, we could monitor the levels of IRES-dependent gene expression as GFP fluorescence of yeast cells dependent of the T7 RNAP expression. When using wild-type yeast, the yeast exhibited a detectable but faint fluorescence upon T7 RNAP induction.

We hypothesized that the interaction of IRES with yeast ribosome is weaker than that with the mammalian ribosome and tested this by constructing yeast expressing Rps25p derived from human (hRPS25), since Rps25 protein is known to directly binds to IRES and essential for IRES-dependent gene expression. As expected, this slightly increased IRES-dependent translation in yeast. Further protein engineering of hRPS25, including directed evolution strategy enabled an increase in IRES-dependent gene expression by more than 10-fold. Moreover, we performed genome-wide screening for gene knockdown target using a CRISPR interference library system (Momen-Roknabadi A., et al, Commun. Biol. (2020) 3, 723), revealing that CRISPRi system targeting to TIF34 gene enabled further increase in IRES-dependent gene expression. Based on these insights, we constructed a yeast strain that exhibited >100-fold stronger IRES-dependent gene expression than the wild-type yeast strain, which is approximately 40% of the gene expression level obtained using a well-characterized strong yeast promoter, PGK1 promoter, instead of the T7 promoter together with PSIV-IGR-IRES.

Conclusions
We have successfully engineered yeast S. cerevisiae to enhance IRES-dependent gene expression. The resulting yeast strain provides a robust platform for investigating sequence-function relationship of different IRESs.