Engineering yeast platforms for enhancing biosynthesis and enzyme discovery
Abstract/Contents
- Abstract
- Recent advances in synthetic biology and metabolic engineering have enabled yeast to serve as a favourable platform for expression of multiple heterologous enzymes sourced from plants, fungi and bacteria; thereby, enabling the synthesis of valuable natural and semi-synthetic compounds. 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 RNA-based small molecule switches to develop a generalizable selection method for directed evolution of biosynthetic enzymes. Our design utilizes an RNA switch for detection of intracellular compound production, which then regulates the expression of a selection gene. Our data shows that the auxotrophy selection gene SpHIS5 exhibits the highest selective capability in combination with a theophylline-responsive RNA switch. Using the theophylline-responsive and a (S)-reticuline-responsive RNA switch, we demonstrated the enrichment of a high-producing variant of caffeine demethylase and a variant of norcoclaurine synthase, each in a population size of 10^3. Our work demonstrates the capability of RNA switches for enhancing biosynthetic enzyme activities via selection. In addition to a platform for biosynthesis, we also explored the potential of yeast for enzyme and compound discovery. Here, we characterized the biosynthetic potential of a putative tomato gene cluster computationally predicted from plant genome databases in yeast, and identified two previously unknown compounds from yeast culture, one being a hydroxycinnamic acid amide compound, dihydro-coumaroyl anthranilate amide. Further studies of the enzymes in the gene cluster reveal a previously uncharacterized amide synthesis activity for tomato chalcone synthase, which uses the same active site for synthesis of the amide compound and for its canonical synthesis of naringenin chalcone. Our work demonstrates the potential of yeast as a characterization tool for computationally aided discovery of compound structures and enzymatic activities from plant genomes.
Description
| Type of resource | text |
|---|---|
| Form | electronic resource; remote; computer; online resource |
| Extent | 1 online resource. |
| Place | California |
| Place | [Stanford, California] |
| Publisher | [Stanford University] |
| Copyright date | 2021; ©2021 |
| Publication date | 2021; 2021 |
| Issuance | monographic |
| Language | English |
Creators/Contributors
| Author | Kong, Deze |
|---|---|
| Degree supervisor | Endy, Andrew D |
| Degree supervisor | Smolke, Christina D |
| Thesis advisor | Endy, Andrew D |
| Thesis advisor | Smolke, Christina D |
| Thesis advisor | Covert, Markus |
| Degree committee member | Covert, Markus |
| Associated with | Stanford University, Department of Bioengineering |
Subjects
| Genre | Theses |
|---|---|
| Genre | Text |
Bibliographic information
| Statement of responsibility | Deze Kong. |
|---|---|
| Note | Submitted to the Department of Bioengineering. |
| Thesis | Thesis Ph.D. Stanford University 2021. |
| Location | https://purl.stanford.edu/fh513cm8153 |
Access conditions
- Copyright
- © 2021 by Deze Kong
- License
- This work is licensed under a Creative Commons Attribution Non Commercial 3.0 Unported license (CC BY-NC).
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