Team:CLSB-UK/Project/Riboflavin

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Riboflavin

Overview

Riboflavin is naturally found in the metal reducing bacteria Shewanella oneidensis and is used to transfer electrons primarily from one of the bacteria’s cytochromes to nearbye metals. This Mtr (metal reducing) respiratory pathway uses metals as the ultimate electron acceptors in the respiratory chain and MtrC cytochrome is the primary electron donor. S.oneidensis secretes riboflavin to increase the number of electrons transferred.

The 2013 Bielefeld iGEM team successfully cloned and created a Biobrick of the Riboflavin gene cluster that we planned to use in the cyanobacteria. In theory, if Synechocystis PCC6803 were able to produce and secrete riboflavin, there would be an increased rate of electron transfer from the cyanobacteria and the electrodes. We wanted to measure the potential of Synechocystis to excrete extracellular riboflavin and to reduce the molecule, and hence the anode, using its own cytochrome pathway, however it was unclear how effective Riboflavin would be without the S.oneidensis Mtr electron transport chain.

Theory

Riboflavin, otherwise known as Vitamin B2, is the precursor to the electron transferring molecules FMN and FAD, both essential to a variety of cellular processes including Oxidative Phosphorylation.

Riboflavin, otherwise known as Vitamin B2, is part of the flavin group of electron transferring molecules and is a precursor to other essential molecules like Flavin Mononucleotide and Flavin Adenine Dinucleotide. The group shares a characteristic isoalloxazine that allows the addition of several functional groups.

Figure 1.Oxidation and Reduction of Riboflavin

The molecule exists in three variably oxidised quinone forms: a fully reduced (2 added electrons) quinone, a semiquinone (1 added electron) and a hydroquinone with no added electrons. Reduction is made with the addition of hydrogen atoms to specific nitrogen atoms in the isoalloxazine ring system. The three molecules exist in a state of equilibrium between both the oxidised and reduced flavins and the radical semiquinone:

Fl_ox + Fl_redH₂ ⇌ FlH^•[1]

This equilibrium allows the riboflavin to rapidly gain and lose electrons in biochemical reactions and is the source of its utility in our project.

In S.oneidensis research suggests that decaheme cytochromes (MtrC and OmcA) are needed for the majority of electron transport^[2] but riboflavin may still be able to function in Synechocystic PCC6803. Electron transport rates may not reach the same levels as in S.oneidensis but may nevertheless be increased. We decided not to attempt to amplify cytochrome expression due to the difficulty of maturing cytochromes.

Riboflavin has been suggested to act as a metal chalator, binding to inorganic metals in the surroundings. Although in principle this may increase the rate of electron transport, a riboflavin coating may reduce the overall exposure of the electrodes to riboflavin molecules. Research indicates that carbon electrodes cause large and adverse amounts of riboflavin adsorption^[3].

Genetic Approach

Thanks to the Bielefeld team we have been able to use the BioBrick for the Riboflavin synthesis gene cluster (BBa_K1172303) ^[4]:

• ribD: is a 1145 base pair code which codes for the enzyme bifunctional diaminohydroxyphosphoribosylaminopyrimidine deaminase/5-amino-6-(5-phosphoribosylamino) uracil reductase RibD.
• SO_3468: is 656 base pairs and codes for the enzyme Riboflavin synthase alpha subunit RibC-like protein. ribBA: codes for both:
• • ribB: 3,4-dihydroxy-2-butanone-4-phosphate synthase; which catalyses the conversion of D-ribulose 5-phosphate to formate and 3,4-dehydroxy-2-butanone 4-phosphate.
  • ribA: GTP cyclohydrolase-2 enzyme. This enzyme catalyses the first step of the biosynthesis pathway, GTP to 2,5-diamino-6-ribosylamino-4(3H)-pyrimidinone 5’-phosphate, formate and pyrophosphate. The Enzyme is competitively inhibited by pyrophosphate and so its action is naturally capped. The use of multiple ribA genes is known to create overproduction of riboflavin in B.subtilis and so was used in this plasmid.
• ribE: 476 base pairs codes for the enzyme 6,7-dimethyl-8-ribityllumazine synthase.

The Promoter (J23119) was chosen for use with this gene cluster in Synechocystis 6803 for its high levels of expression in cyanobacteria. In order for the highest rate of electron transport to be achieved, there is need for a high concentration of riboflavin.

Figure 2.Riboflavin Operon

Results

Unfortunately, we were unable to find a ribosome binding site suitable for the part and for use in cyanobacteria. This meant that although we completed the assembly of the promoter and rib operon, it has no functional purpose.

References

• [1] Massey, Vincent, M. Stankovich; Peter Hemmerich (January 1978). "Light-Mediated Reduction of Flavoproteins with Flavins as Catalysts" Biochemistry. 17
• [2] Dan Coursolle , Daniel B. Baron, et al; (January 2010). “The Mtr Respiratory Pathway Is Essential for Reducing Flavins and Electrodes in Shewanella oneidensis” Journal of Bacteriology 192
• [3]Enrico Marsili, Dnaiel Baron, et al; (March 2008) "Shewanella secretes flavins that mediate extracellilar electron tranfer" Proceedings of the National Academy of Sciences of the United States of America, 105
• [4] https://2013.igem.org/Team:Bielefeld-Germany/Project/Riboflavine