Presentation Information
[P04-454]Engineering yeast for precise gene expression control
○Nanami Kawai1, Masahiro Tominaga1,2, Akihiko Kondo1,2,3,4, Jun Ishii1,2,3 (1. Graduate School of Science, Technology and Innovation, Kobe Univ. (Japan), 2. Engineering Biology Research Center, Kobe Univ. (Japan), 3. Department of Chemical Science and Engineering, Faculty of Engineering, Kobe Univ. (Japan), 4. Center for Sustainable Resource Science, RIKEN (Japan))
Keywords:
Synthetic biology,Yeast,Gene expression,Inducible promoters
[Purpose]
In the field of synthetic biology, precise gene expression control is fundamental for reconstructing/engineering synthetic biological systems. Among various regulatory strategies, artificial transcription systems which composed of engineered transcriptional regulators and synthetic promoters have been widely used in yeast to realize multidimensional gene regulation independent of endogenous regulatory networks in the cells, thereby enabling predictable and modular control of target gene expression. However, target gene expressions (i.e., synthetic promoter activities) are often influenced by their genetic contexts; upstream sequences of the synthetic promoters can act as enhancers, where endogenous factors bind to cause unintended transcriptional activation. This cryptic enhancer activities hamper a reliable gene expression control, making it challenging to construct multi-layered synthetic gene circuits in yeast. Although inserting insulator sequences upstream of the synthetic promoters could mitigate such effects (Tominaga, M., et al. Nat. Commun. (2024) 15, 10653), repetitive use of insulator sequences requires large DNA construct and causes genetic instability.
[Method]
In this study, we investigated whether the enhancer activities can be minimized by deleting the endogenous transcription factors. To this end, we performed screening of a single-gene knockout library in yeast Saccharomyces cerevisiae for reduced enhancer activities using the previously identified upstream sequence with strong enhancer activity together with the downstream inducible synthetic promoter (Tominaga, M., et al. Nat. Commun. (2021) 12, 1846).
[Results and Consideration]
Through the screening, we identified RPI1 knockout strain exhibited reduced enhancer activity. Together with the fact that Rpi1p is a transcriptional regulator containing a C-terminal activation domain, our results suggest that Rpi1p binds upstream of the enhancer and contribute to the activation of the downstream synthetic promoter. However, residual enhancer activity was still observed even in the RPI1 knockout strain, indicating that the endogenous factors other than Rpi1p bind to the enhancer sequence to activate transcription.
[Conclusion]
Future work will focus on identifying these endogenous factors and their binding sequences. Taken together, this study provides a novel framework for reducing context dependency of synthetic promoter activities only by deleting endogenous factors.
In the field of synthetic biology, precise gene expression control is fundamental for reconstructing/engineering synthetic biological systems. Among various regulatory strategies, artificial transcription systems which composed of engineered transcriptional regulators and synthetic promoters have been widely used in yeast to realize multidimensional gene regulation independent of endogenous regulatory networks in the cells, thereby enabling predictable and modular control of target gene expression. However, target gene expressions (i.e., synthetic promoter activities) are often influenced by their genetic contexts; upstream sequences of the synthetic promoters can act as enhancers, where endogenous factors bind to cause unintended transcriptional activation. This cryptic enhancer activities hamper a reliable gene expression control, making it challenging to construct multi-layered synthetic gene circuits in yeast. Although inserting insulator sequences upstream of the synthetic promoters could mitigate such effects (Tominaga, M., et al. Nat. Commun. (2024) 15, 10653), repetitive use of insulator sequences requires large DNA construct and causes genetic instability.
[Method]
In this study, we investigated whether the enhancer activities can be minimized by deleting the endogenous transcription factors. To this end, we performed screening of a single-gene knockout library in yeast Saccharomyces cerevisiae for reduced enhancer activities using the previously identified upstream sequence with strong enhancer activity together with the downstream inducible synthetic promoter (Tominaga, M., et al. Nat. Commun. (2021) 12, 1846).
[Results and Consideration]
Through the screening, we identified RPI1 knockout strain exhibited reduced enhancer activity. Together with the fact that Rpi1p is a transcriptional regulator containing a C-terminal activation domain, our results suggest that Rpi1p binds upstream of the enhancer and contribute to the activation of the downstream synthetic promoter. However, residual enhancer activity was still observed even in the RPI1 knockout strain, indicating that the endogenous factors other than Rpi1p bind to the enhancer sequence to activate transcription.
[Conclusion]
Future work will focus on identifying these endogenous factors and their binding sequences. Taken together, this study provides a novel framework for reducing context dependency of synthetic promoter activities only by deleting endogenous factors.
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