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.
