Cell free metabolic engineering for the production of hydrogen from cellulosic biomass
Abstract/Contents
- Abstract
- Establishing alternative methods to produce petrochemicals from renewable resources is a critical step toward a sustainable society. Hydrogen is a key commodity chemical with over 50 million metric tons produced annually with a market value of over $40 billion. The majority of this hydrogen is used for synthesis of ammonia and petroleum refining but also has the potential for use as a clean burning fuel. However, current methods rely primarily on the steam reformation of natural gas. We sought to develop a new platform for synthesis of hydrogen from cellulosic biomass using E.coli cell free extracts. By coupling the native E.coli metabolic pathways with a heterologous synthetic pathway, we established a method to produce hydrogen from glucose. In order to accomplish this, a synthetic pathway to produce hydrogen from a native redox source, NADPH, was developed using a ferredoxin NADP+ reductase, ferredoxin, and a [FeFe] hydrogenase. The pathway was constructed by screening natural proteins for the capacity to support maximal hydrogen production rates. Careful characterization of the interactions of the three protein synthetic pathway through UV/Vis spectroscopy and gas chromatography enabled further improvements. The synthetic pathway was then coupled to a cell free extract to demonstrate the feasibility of producing hydrogen from glucose with a cell extract. In order to maximize the yields of hydrogen from the cell extract, we knocked out three metabolic proteins, phosphofructokinase, 6-phosphogluconate dehydratase, and glyceraldehyde-3-phosphate dehydrogenase that eliminate undesirable side reactions in glycolysis and Entner Doudoroff pathways. With these cell lines, we established protocols for cell extract production and in vitro hydrogen production that demonstrate significant improvements in hydrogen yield. Finally, in order to further improve the cell extracts, we then developed a strategy for selective metabolic silencing (SeMs) of key enzymatic activities. Using this technique, specific enzymatic activities could be provided in vivo for efficient cell growth and enzyme production, but then these activities could be inactivated in the cell extract to avoid the loss of the reducing equivalents required for hydrogen production. To demonstrate this principle, we engineered a library of glyeraldehyde-3-phosphate dehydrogenase mutants with insertion of a recognition sequence for human rhinovirus 3C protease. Many of these mutants were active in vivo, but through protease treatment could be inactivated to improve hydrogen production. Together, these advances explore the feasibility of using cell free extracts for biochemical synthesis, as well as contribute to ongoing research on in vitro enzymatic biocatalysis.
Description
| Type of resource | text |
|---|---|
| Form | electronic; electronic resource; remote |
| Extent | 1 online resource. |
| Publication date | 2015 |
| Issuance | monographic |
| Language | English |
Creators/Contributors
| Associated with | Lu, Franklin |
|---|---|
| Associated with | Stanford University, Department of Chemical Engineering. |
| Primary advisor | Swartz, James R |
| Thesis advisor | Swartz, James R |
| Thesis advisor | Dunn, Alexander Robert |
| Thesis advisor | Smolke, Christina D |
| Advisor | Dunn, Alexander Robert |
| Advisor | Smolke, Christina D |
Subjects
| Genre | Theses |
|---|
Bibliographic information
| Statement of responsibility | Franklin Lu. |
|---|---|
| Note | Submitted to the Department of Chemical Engineering. |
| Thesis | Thesis (Ph.D.)--Stanford University, 2015. |
| Location | electronic resource |
Access conditions
- Copyright
- © 2015 by Franklin Lu
- License
- This work is licensed under a Creative Commons Attribution Non Commercial 3.0 Unported license (CC BY-NC).
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