Developing new expression parts and biosynthetic pathways for yeast synthetic biology
Abstract/Contents
- Abstract
- Baker's yeast (Saccharomyces cerevisiae) has been used in bioproduction for millenia. Current efforts in synthetic biology seek to further extend yeast's capabilities, enabling applications such as producing useful and valuable plant natural products in a cheaper and less resource-intensive manner than current methods, which rely on cultivation of the native producer plants. This dissertation opens with a review (Chapter 1) of current synthetic biology strategies for producing plant natural products in heterologous hosts: model plants, bacteria, and yeast. As a case study of heterologous phytochemical production, we describe a functional reconstitution of the dhurrin pathway from sorghum (Sorghum bicolor) in yeast (Chapter 2). We produced this plant defense compound at titers of over 80 mg/L, and demonstrated a workflow using our dhurrin-producing strain to explore the activities of other S. bicolor genes. Further, we developed a method for model-driven generation of artificial yeast promoters (Chapter 3). Promoters - DNA sequences that appear 5' to genes and modulate their expression - play a central role in controlling gene regulation; however, a small set of native promoters is used for most genetic construct design in S. cerevisiae, limiting engineers' ability to control gene expression in this organism. The ability to generate and utilize models that accurately predict protein expression from promoter sequence may enable rapid generation of novel useful promoters, facilitating synthetic biology efforts in yeast. We measured the activity of over 675,000 unique sequences in a constitutive promoter library, and over 327,000 sequences in a library of inducible promoters. Training an ensemble of convolutional neural networks jointly on the two datasets enabled very high predictive accuracies on multiple prediction tasks. We developed model-guided design strategies which yielded large, sequence-diverse sets of novel promoters exhibiting activities similar to current best-in-class sequences. In addition to providing large sets of new promoters, our results show the value of model-guided design as an approach for generating DNA parts. The final chapter discusses the outlook for the field and possible extensions to the work presented here (Chapter 4). Taken together, our work shows the value of yeast as a heterologous host for producing plant natural products, and offers a means to develop novel genetic parts to further expand its usefulness.
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 | 2019; ©2019 |
| Publication date | 2019; 2019 |
| Issuance | monographic |
| Language | English |
Creators/Contributors
| Author | Kotopka, Benjamin John |
|---|---|
| Degree supervisor | Smolke, Christina D |
| Thesis advisor | Smolke, Christina D |
| Thesis advisor | Endy, Andrew D |
| Thesis advisor | Zou, James |
| Degree committee member | Endy, Andrew D |
| Degree committee member | Zou, James |
| Associated with | Stanford University, Department of Bioengineering. |
Subjects
| Genre | Theses |
|---|---|
| Genre | Text |
Bibliographic information
| Statement of responsibility | Benjamin J. Kotopka. |
|---|---|
| Note | Submitted to the Department of Bioengineering. |
| Thesis | Thesis Ph.D. Stanford University 2019. |
| Location | electronic resource |
Access conditions
- Copyright
- © 2019 by Benjamin John Kotopka
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