Improving functions of redox proteins for hydrogen production
Abstract/Contents
- Abstract
- The usage of hydrogen as an alternative energy carrier has become increasingly important to support environmental sustainability. A portion of this hydrogen could be produced from renewable biomass sources, particularly from the sugar components of cellulosic feedstocks. The electrons extracted from the sugars would be delivered to one of the best hydrogen producing catalysts available in nature, an [FeFe] hydrogenase from Clostridium pasteurianum, through a synthetic pathway involving NADPH and two other proteins: Ferredoxin-NADP+ Reductase (FNR) and the Clostridium pasteurianum ferredoxin (CpFd) as an electron carrier protein. This electron transfer pathway enables in vitro hydrogen production and, when coupled with unpurified cell extracts containing native E. coli metabolic pathway enzymes, will provide an economical advantage. We sought to improve the electron transfer efficiency between the proteins for more efficient hydrogen generation from biomass sugars. We focused on improving the electron transfer rate of the downstream NADPH to hydrogen pathway, because it has been shown to limit the overall productivity. We initially hypothesized that the rate-limiting step in the pathway lies in the FNR-catalyzed electron transfer, but also evaluated the CpFd-mediated electron transfer from FNR. Several mutagenesis strategies were employed to change the inherent properties of the two redox proteins, FNR and CpFd, to increase their electron transfer rates. First, two different FNR mutant libraries from Anabaena PCC 7119 and rice root were mutated and evaluated. Subsequently, a mutant library of CpFd was explored. Mutagenesis of both redox proteins has enabled a final ~2.3-fold improvement in hydrogen productivity compared to the wild-type proteins, with an FNR turnover rate of 0.9 s-1. Beneficial mutations consist of S154H in rice root FNR and S17V in CpFd, and significantly lower the KM of the rice root FNR mutant and the CpFd mutant. Electron transfer from NADPH to the FAD cofactor of FNR occurs by a hydride transfer, but the CpFd can only accept two electrons. The improved ability of the new histidine (in S154H mutant) to accept the un-transferred proton may be improving FNR functionality in our pathway. Since the interaction between rice root FNR and CpFd has been improved, future work will employ similar mutagenesis strategies to search for an [FeFe] hydrogenase variant that can improve the hydrogen production rate even further. Ultimately, the electron transfer between proteins is a complex process that needs to be optimized for hydrogen production. This study provides further understanding of how redox protein interactions influence electron transfer.
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 | Liong, Sylvie |
|---|---|
| Associated with | Stanford University, Department of Bioengineering. |
| Primary advisor | Swartz, James R |
| Thesis advisor | Swartz, James R |
| Thesis advisor | Lin, Michael |
| Thesis advisor | Smolke, Christina D |
| Advisor | Lin, Michael |
| Advisor | Smolke, Christina D |
Subjects
| Genre | Theses |
|---|
Bibliographic information
| Statement of responsibility | Sylvie Liong. |
|---|---|
| Note | Submitted to the Department of Bioengineering. |
| Thesis | Thesis (Ph.D.)--Stanford University, 2017. |
| Location | electronic resource |
Access conditions
- Copyright
- © 2017 by Sylvie Liong
- License
- This work is licensed under a Creative Commons Attribution Non Commercial 3.0 Unported license (CC BY-NC).
Also listed in
Loading usage metrics...