2021 Fiscal Year Annual Research Report
Electrochemical acceleration of ammonium biosynthesis from nitrogen gas
| Project/Area Number |
20F20105
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| Research Institution | National Institute for Materials Science |
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Principal Investigator |
岡本 章玄 国立研究開発法人物質・材料研究機構, 国際ナノアーキテクトニクス研究拠点, グループリーダー (70710325)
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| Co-Investigator(Kenkyū-buntansha) |
LONG XIZI 国立研究開発法人物質・材料研究機構, 国際ナノアーキテクトニクス研究拠点, 外国人特別研究員
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| Project Period (FY) |
2020-11-13 – 2023-03-31
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| Keywords | Nitrogen fixing / voltammetry / Multi-heme Cytochrome C / Exciton coupling / Circular dichroism |
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| Outline of Annual Research Achievements |
One progress on the influence of membrane to the MtrC, in charge of cross membrane electron transfer, was explored in strain Shewanella onedensis MR-1 (MR-1). The MR-1 strain was pre-cultured at 4 °C and 30 °C to initiate differentiation of membrane composition. The whole-cell difference electrochemical assay of wild-type and mutant strains lacking MtrC under 25 °C suggested that the rate of EET via MtrC increased approximately 18 times. Circular dichroism spectroscopy of whole cell firstly unveiled that the molar exciton coupling coefficient for inter-heme interaction in MtrC increased in MR-1 pre-cultured at 4°C than in those pre-cultured at 30 °C. Our work suggest that membrane modification may be a novel strategy for improving the uptake of the electrons of cells and columbic efficiency that helped enhancing the nitrogen fixing by rapid injection.
Moreover, the high throughput electrochemical system we proposed to screen the nitrogen fixing bacteria is also working well. Initiated with mutant’s library of MR-1, we verified our device and investigated the effect of unannotated proteins on extracellular electron transport. The random transposon mutant’s library was constructed by two methods of cooperation and self-construction. Combined with the customized 96-channel electrochemical system, six genes were discovered to be impactful to the electron transfer process, the high-throughput screening part is still in progress. This study provides an experimental means for the construction of a mutant library in this project to identify the nitrogen fixing pathway.
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| Current Status of Research Progress |
Current Status of Research Progress
2: Research has progressed on the whole more than it was originally planned.
Reason
Since we already know the influence of bacterial outer-membrane multi-heme cytochrome (OMC) mediate extracellular electron transport to the electrode from aforementioned part. We want to control inter-heme coupling in single OMC remains challenging, especially in intact cells. Therefore, a new method was explored by modifying the the heme coupling via mechanical interactions among OMCs by controlling their concentrations. Employment of whole-cell circular dichroism spectra of genetically engineered Escherichia coli reveals that upregulation of OMC decreased electron flux through MtrC, a cell-surface component of OMC at bacteria/electrode interface, approximately four-fold, associated with the molar circular dichroism and redox property alteration. The horizontal physical interaction among MtrCs was confirmed by the conductive current across biofilm on an interdigitated electrode via lateral inter-protein electron hopping. The importance of mechanobiological interaction presented herein for MtrC activity would open a novel strategy to increase microbial current production by mechanically enhancing the inter-heme coupling and upregulating a multi-center redox enzyme without its activity decrease.
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| Strategy for Future Research Activity |
Moreover, the high throughput electrochemical system we proposed to screen the nitrogen fixing bacteria is also working well. Initiated with mutant’s library of MR-1, we verified our device and investigated the effect of unannotated proteins on extracellular electron transport. The random transposon mutant’s library was constructed by two methods of cooperation and self-construction. Combined with the customized 96-channel electrochemical system, six genes were discovered to be impactful to the electron transfer process, the high-throughput screening part is still in progress. This study provides an experimental means for the construction of a mutant library in this project to identify the nitrogen fixing pathway.
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