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Building an RNA switch-based selection system for enzyme evolution in yeast

Deze Kong, Christina Smolke
doi: https://doi.org/10.1101/2022.06.14.496130
Deze Kong
1Department of Bioengineering, Stanford University, Stanford, CA, USA
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Christina Smolke
1Department of Bioengineering, Stanford University, Stanford, CA, USA
2Chan Zuckerberg Biohub, San Francisco, CA, USA
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  • For correspondence: csmolke@stanford.edu
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Abstract

Recent advances in synthetic biology and metabolic engineering have enabled yeast as a favorable platform for synthesis of valuable natural and semi-synthetic compounds through expression of multiple heterologous enzymes sourced from plants, fungi and bacteria. However, these heterologous enzymes can suffer from low activity, specificity, stability and solubility in yeast, resulting in arduous iterations of design-build-test-learn cycles to optimize their production often performed on a single enzyme basis. Laboratory directed evolution has proven to be a powerful and high-throughput method for protein engineering, albeit its limited application for biosynthetic enzymes. Here, we harness small molecule-sensing, RNA-based switches to develop a generalizable selection system facilitating enzyme evolution. Our design utilizes an RNA-based switch for detection of intracellular compound production, which then regulates the expression of a selection gene. Our initial data shows that the auxotrophy selection gene SpHIS5 exhibits the highest selective capability in combination with a theophylline-responsive RNA-based switch. Using the theophylline-responsive RNA-based switch, we demonstrated the enrichment of a high-producing variant of caffeine demethylase, in a population size of 103. We target to demonstrate the use of this RNA-based selection system as a general approach for enzyme evolution.

Competing Interest Statement

The authors have declared no competing interest.

Copyright 
The copyright holder for this preprint is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY-NC-ND 4.0 International license.
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Posted June 15, 2022.
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Building an RNA switch-based selection system for enzyme evolution in yeast
Deze Kong, Christina Smolke
bioRxiv 2022.06.14.496130; doi: https://doi.org/10.1101/2022.06.14.496130
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Building an RNA switch-based selection system for enzyme evolution in yeast
Deze Kong, Christina Smolke
bioRxiv 2022.06.14.496130; doi: https://doi.org/10.1101/2022.06.14.496130

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