An integrated biosynthesis platform for tropane alkaloid drugs using yeast cellular engineering
Abstract/Contents
- Abstract
- Despite their medicinal use by humans long before the development of agriculture, plants remain a major source of bioactive molecules and pharmaceutical leads for modern medicine. One such group of bioactive molecules, the tropane alkaloids (TAs), are acetylcholine receptor inhibitors produced by certain nightshade plants and are classified as essential medicines by the World Health Organization for treating diverse neuromuscular dysfunctions. The current crop-based supply chain for TA drugs is ecologically unsustainable, vulnerable to environmental disturbances, and lacks the manufacturing agility needed to respond to drug shortages and public health crises. To address these limitations, we have developed a proof-of-concept biomanufacturing platform by genetically engineering brewer's yeast to convert sugar and amino acids into the most widely used TA medicines, hyoscyamine and scopolamine. Reconstructing the complete TA biosynthetic pathway in yeast necessitated rewiring yeast nitrogen and polyamine metabolism for overproduction of key precursors and intermediates, discovery of the final missing enzyme in the pathway using a functional genomics approach, and cellular and protein engineering to enable expression of a recalcitrant plant acyltransferase in the yeast vacuole using a novel transmembrane enzyme design. We then developed a neural network-based strategy to identify new TA transporters from a plant RNA sequencing dataset and showed that incorporating these transporters into our engineered yeast platform alleviates intracellular transport restrictions that limit TA production. Finally, we demonstrated the utility of our technology for exploring new drug leads by leveraging computational tools for biosynthetic pathway expansion and enzyme activity prediction to produce two classes of N-substituted TA derivatives in yeast. Our final yeast platform, capable of producing hyoscyamine and scopolamine at titers of 100-500 micrograms per liter, incorporates over 30 additional enzymes expressed across six cellular locations and constitutes a starting point for the development of a new TA supply chain via microbial biomanufacturing.
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 | Srinivasan, Prashanth |
|---|---|
| Degree supervisor | Sattely, Elizabeth |
| Degree supervisor | Smolke, Christina D |
| Thesis advisor | Sattely, Elizabeth |
| Thesis advisor | Smolke, Christina D |
| Thesis advisor | Swartz, James R |
| Degree committee member | Swartz, James R |
| Associated with | Stanford University, Department of Bioengineering |
Subjects
| Genre | Theses |
|---|---|
| Genre | Text |
Bibliographic information
| Statement of responsibility | Prashanth Srinivasan. |
|---|---|
| Note | Submitted to the Department of Bioengineering. |
| Thesis | Thesis Ph.D. Stanford University 2021. |
| Location | https://purl.stanford.edu/dp005hd1487 |
Access conditions
- Copyright
- © 2021 by Prashanth Srinivasan
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