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Riboflavin biosynthesis protein RibF

Two extracellular electron transfer pathways have been identified in the reduction of graphene oxide (GO) by Shewanella oneidensis. These are indirect electron transfer, mediated by secreted electron shuttles, and direct extracellular electron transfer (DET) which involves direct contact with the extracellular material. It has been proposed that flavins may act as electron shuttles in the reduction of extracellular material by Shewanella oneidensis. These flavins can exit the cell as Flavin adenine dinucleotide (FAD) where they are then reconverted to riboflavin. RibF converts riboflavin to FAD to be shuttled out. Increasing expression of RibF should increase the conversion of riboflavin to FAD and would therefore increase the electron donors available for reduction.

Usage and Biology

Shewanella oneidensis MR-1 are gram-negative bacteria at the center of studies of microbial reduction due to their ability to transfer electrons extracellularly to reduce materials such as graphene oxide (GO)[1]. Such characteristics have made S. oneidensis MR-1 an organism of interest in microbial fuel cells for bioelectricity generation and potential applications in bioremediation[2]. Two extracellular electron transfer pathways have been identified in the reduction of GO by Shewanella oneidensis MR-1. These are indirect electron transfer, mediated by secreted electron shuttles, and direct extracellular electron transfer (DET) which involves direct contact with the extracellular material[3]. It has been proposed that flavins may act as electron shuttles in the reduction of extracellular material by S. oneidensis MR-1.[4] These flavins can exit the cytoplasm as flavin adenine dinucleotide (FAD) where they are then converted to riboflavin in the periplasm or in the extracellular space. RibF codes for the riboflavin biosynthesis protein which catalyzes the phosphorylation of riboflavin to flavin mononucleotide (FMN) then adenylation of FMN to FAD.[5] This FAD can then be transported into the periplasm by inner membrane flavin transporters. From the periplasm, FAD is converted to FMN where it is shuttled out of the cell then converted to riboflavin. Riboflavin has been seen to be the dominant flavin involved in reduction of extracellular material where it acts as a reducing electron shuttle eventually transferring its electrons to the terminal electron acceptor. Riboflavin has also been shown to be a key component in the transfer of electrons from biofilm in contact with electrodes as the terminal electron acceptor.[6] It was thought that increasing expression of RibF would increase the conversion of riboflavin to FAD and would therefore increase the amount of FAD shuttled into the periplasm to be converted to riboflavin for a faster rate of reduction.

We therefore synthesized the ribF gene from the S. oneidensis MR-1 genome and inserted it into the kanamycin resistant vector pcD8 under the control of an IPTG-inducible promoter (Keitz lab, University of Texas Austin)[7].

Sequence and Features

References

[1] Wang, G.; Qian, F.; Saltikov, C. W.; Jiao, Y.; Li, Y. Microbial Reduction of Graphene Oxide by Shewanella. Nano Research 2011, 4, 563–570.
[2] Schwalb, C.; Chapman, S. K.; Reid, G. A. The Tetraheme Cytochrome Cyma Is Required for Anaerobic Respiration with Dimethyl Sulfoxide and Nitrite in Shewanella Oneidensis. Biochemistry 2003, 42, 9491–9497.
[3] Lin, T.; Ding, W.; Sun, L.; Wang, L.; Liu, C.-G.; Song, H. Engineered Shewanella Oneidensis-Reduced Graphene Oxide Biohybrid with Enhanced Biosynthesis and Transport of Flavins Enabled a Highest Bioelectricity Output in Microbial Fuel Cells.
[4] Kouzuma, A.; Kasai, T.; Hirose, A.; Watanabe, K. Catabolic and Regulatory Systems in Shewanella Oneidensis MR-1 Involved in Electricity Generation in Microbial Fuel Cells. Frontiers in Microbiology 2015, 6.
[5] UniProt Consortium European Bioinformatics Institute Protein Information Resource SIB Swiss Institute of Bioinformatics. Riboflavin biosynthesis protein.
[6] Marsili, E.; Baron, D. B.; Shikhare, I. D.; Coursolle, D.; Gralnick, J. A.; Bond, D. R. Shewanella Secretes Flavins That Mediate Extracellular Electron Transfer. Proceedings of the National Academy of Sciences 2008, 105, 3968–3973.
[7] Dundas, C. M.; Walker, D. J. F.; Keitz, B. K. Tuning Extracellular Electron Transfer by Shewanella Oneidensis Using Transcriptional Logic Gates. ACS Synthetic Biology 2020, 9, 2301–2315.