[P006] Poster

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

[P03-392] A Modular Expression Architecture for Tunable Single- and Multi-gene Expression in Yeast Using T7 RNA Polymerase

○Eunha Jeon^1,3, Sujin Hong¹, Seung-Gyun Woo², Dae-Hee Lee^1,3 (1. Synthetic Biology Research Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB), Daejeon 34141, Korea (Korea), 2. Department of Biological Science and Biotechnology, Hannam University, Daejeon 34430, Korea (Korea), 3. Graduate School of Engineering Biology, Korea Advanced Institute of Science and Technology (KAIST), Daejeon 34141, Korea (Korea))

Keywords:

Orthogonal gene expression,T7 RNA polymerase(T7 RNAP),Internal ribosome entry site(IRES),5' transcript architecture,Multi-gene expression

Orthogonal gene expression systems in yeast based on bacteriophage-derived T7 RNA polymerase (T7 RNAP) enable transcription independently of host regulatory networks and provide a compact promoter architecture well suited for modular and multigene expression. However, their broader application remains limited by low expression efficiency and the lack of scalable cassette design principles. In this study, we systematically optimized the expression cassette architecture of a yeast T7 RNAP-based system to identify key determinants of expression output. While plasmid copy number influenced overall expression levels, protein production was determined primarily by plasmid and transcript architecture rather than by polymerase activity alone. In particular, the sequence context proximal to the T7 promoter and the 5′ end structure of the transcript strongly affected expression efficiency. Modification of the 5′ transcript architecture markedly influenced protein output despite sustained transcript production, indicating that efficient translation depends on proper transcript configuration. To overcome this limitation, we introduced and screened synthetic internal ribosome entry site (IRES) elements and identified variants that enhanced protein expression from T7 RNAP-derived transcripts in yeast. This led to a modular expression architecture in which translational output can be tuned through IRES selection. Using this framework, we further demonstrated multigene expression with repeated T7 promoter-based expression units and showed that combinatorial IRES configurations allow independent control of individual gene expression levels. Together, this work establishes a practical design framework for tunable and scalable orthogonal multigene expression in yeast.