Improving heterologous expression in yeast through a selective assay
Abstract/Contents
- Abstract
- Heterologous expression of valuable proteins and biosynthetic pathways is a worthwhile endeavor of modern biotechnology, with pharmaceutical, industrial, and agricultural applications. But the process of expressing heterologous protein often requires significant optimization. Here, I have sought to express several proteins of bacterial, fungal, and human origin. In Chapter 2, I demonstrate the successful expression of two genes from the disorazole biosynthetic pathway from Sorangium cellulosum ce12: the first module (dszM1) and the trans-acting acyltransferase (dszD). I developed a growth-based reporter for protein expression by fusing the yeast URA3 gene to the C-terminus of dszM1 and explored the role of codon optimization in improving protein expression. In Chapter 3, I describe an assay for host improvement by screening the yeast homozygous deletion collection for strains that improve the heterologous expression of proteins fused to selectable markers. I performed this screen for three genes: dszM1; the core enzyme of the cytochalasin biosynthetic cluster (ccsA); and an isoform of human fibrinogen (FGA). I identified two (Δtif2 and Δecm25) strains that improve the heterologous expression of a fungal (ccsA) and a bacterial (dszM1) biosynthetic gene and proceeded to test these strains under alternate promoters. These results demonstrate the utility of unbiased screens coupled to selectable reporters and may be useful for researchers seeking to express large, complex pathways in yeast.
Description
| Type of resource | text |
|---|---|
| Form | electronic; electronic resource; remote |
| Extent | 1 online resource. |
| Publication date | 2017 |
| Issuance | monographic |
| Language | English |
Creators/Contributors
| Associated with | Kolesnikov, Dmitriy |
|---|---|
| Associated with | Stanford University, Department of Biology. |
| Primary advisor | Davis, Ronald W. (Ronald Wayne), 1941- |
| Thesis advisor | Davis, Ronald W. (Ronald Wayne), 1941- |
| Thesis advisor | Khosla, Chaitan, 1964- |
| Thesis advisor | Stearns, Tim |
| Advisor | Khosla, Chaitan, 1964- |
| Advisor | Stearns, Tim |
Subjects
| Genre | Theses |
|---|
Bibliographic information
| Statement of responsibility | Dmitriy Kolesnikov. |
|---|---|
| Note | Submitted to the Department of Biology. |
| Thesis | Thesis (Ph.D.)--Stanford University, 2017. |
| Location | electronic resource |
Access conditions
- Copyright
- © 2017 by Dmitriy Kolesnikov
- License
- This work is licensed under a Creative Commons Attribution Non Commercial 3.0 Unported license (CC BY-NC).
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