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Riboflavin synthesis gene cluster

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. This gene cluster is comprised of key enzymes involved in the riboflavin synthesis pathway in Shewanella oneidensis without the regulatory elements.

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].

The riboflavin gene synthesis cluster comprises genes that are integral in the riboflavin synthesis pathway in S. oneidensis MR-1 without the regulatory elements. These include the riboflavin biosynthesis protein (ribD) which converts 2,5-diamino-6-(ribosylamino)-4(3h)-pyrimidinone 5'-phosphate into 5-amino-6-(ribosylamino)-2,4(1h,3h)-pyrimidinedione 5'-phosphate, the riboflavin synthase alpha subunit (SO_3468) which catalyzes the formation of riboflavin and 5-amino-6-(D-ribitylamino)uracil from two molecules of 6,7-dimethyl-8-ribityllumazine, the GTP cyclohydrolase-2 (ribA), 3,4-dihydroxy-2-butanone-4-phosphate synthase (ribB) and riboflavin synthase beta subunit (RibE). The latter three genes catalyzes the conversion of GTP to 2,5-diamino-6-ribosylamino-4(3H)-pyrimidinone 5'-phosphate , formate and pyrophosphate, Catalyzes the conversion of D-ribulose 5-phosphate to formate and 3,4-dihydroxy-2-butanone 4-phosphate and Catalyzes the formation of 6,7-dimethyl-8-ribityllumazine by condensation of 5-amino-6-(D-ribitylamino)uracil with 3,4-dihydroxy-2- butanone 4-phosphate, respectively.

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 initially begin as riboflavin and are then converted to flavin adenine dinucleotide (FAD) in the cytoplasm. FAD exits the cytoplasm where it is then reconverted to riboflavin in the periplasm or in the extracellular space through the intermediate flavin, flavin mononucleotide (FMN). The overexpression of the genes in the riboflavin synthesis gene cluster in S. oneidensis MR-1 has previously been shown to increase the concentration of extracellular flavins[3]. This increase in flavins has been shown to increase the rate of extracellular electron transfer in microbial fuel cells for bioelectricity generation.[3]

It is though that the overexpression of these genes in S. oneidensis MR-1 would lead to an increase in extracellular flavins that can then reduce GO, ultimately leading to an increase in the rate of reduction.

Bielefeld iGEM 2013 team expressed this riboflavin gene synthesis cluster in Escherichia coli (BBa_K1172303). They discovered that expression of the gene cluster in E coli resulted in a significant increase in extracellular flavins. We utilized the sequences from this biobrick which were initially obtained from the genome of S. oneidensis MR-1 and inserted a ribosome binding site (RBS) - BBa_0030 - for expression of each of the genes in the cluster and transformed this gene cluster into S. oneidensis MR-1. The gene cluster was successfully transformed into S. oneidensis MR-1 and was used for microbial reduction of GO.

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.