Part:BBa_K4989004

Electron flavoprotein beta unit (etfb)

It is an improved basic part of:

Application in the field of biology

The gene etfb encodes for the electron transfer flavoprotein subunit beta, which creates the heterodimeric electron transfer flavoprotein (ETF), when it is joined by the electron transfer flavoprotein subunit alpha. Those subunits are the fifth and sixth enzymes that are involved in the butyrate-producing metabolic pathway. Both subunits have orthologs in mammalian cells, which exist in the mitochondria and are included in various reactions[1]. The α subunit is a precursor protein in the mitochondria, while the β subunit is expressed in the cytosol and is transferred into the mitochondria[2].

The ETF protein is involved in electron burification which is a mechanism to couple endergonic to exergonic redox reactions widely distributed in anaerobic bacteria. ETF creates a complex with butyryl-CoA dehydrogenase (BCD), a so-called ETF-BCD complex which was the first to be studied. This complex couples the exergonic reaction of crotonyl-CoA to butyryl-CoA by NADH with the endergonic reaction of ferredoxin[3]. Current understanding indicates that the electron pair of NADH is split at the two-electron acceptor and one-electron donor FAD; one electron decreases the dehydrogenase FAD of Bcd and the other goes to ferredoxin (Fd), which has a low redox potential (E0′ = 405 mV). When this process is repeated, hydrogen transfer from FADH of Bcd to crotonyl-CoA results in a second decreased ferredoxin and butyryl-CoA. Two subunits make up the Etf complex from Acidaminococcus fermentans, according to structural and biochemical analyses. The α subunit coordinates one flavin adenine dinucleotide (FAD) (α-FAD), while the β subunit coordinates a second FAD (-FAD). Because NADH binds near this cofactor, the β-FAD was determined to be the location of hydride acceptance from NADH and electron bifurcation[4].

Below the crystal structure of the electron transferring flavoprotein of Acidaminococcus fermentans[Figure A] and the electron transferring flavoprotein/butyryl-CoA dehydrogenase complex from Clostridium difficile [Figure B] is shown:


Figure A.Electron transferring flavoprotein of Acidaminococcus fermentans

Figure B.Electron transferring flavoprotein/butyryl-CoA dehydrogenase complex from Clostridium difficile

Configuration of the new part

The previous part BBa_K1618042 originated from Coprococcus sp.L2-50 DSM. There were some issues in the sequence and its origin that we addressed and solved as follows:
1. The sequence did not contain a start and a stop codon, so we decided to change the sequence of the enzyme provided by the iGEM 2015 NRP-UEA-Norwich team, based on a research paper that provides all the sequences of the enzyme’s genes for the butyrate-producing pathway from the same strain Coprococcus sp.L2-50 [5,6].
2. We optimized our sequence in order to be expressed in both Lactobacillus species and E.coli.
3. We excluded all the restriction sites of the endonucleases that we used for cloning.
We used the GenSmart Optimization Tool to optimize our sequence and exclude the formation of the restriction sites of the enzymes that we used for cloning.

The obtainment of the sequence and its difficulties

We obtained our sequence from iGEM's 2023 sponsor Twist Bioscience, via synthesis. Due to the large size of the part we faced a small difficulty in synthesizing this part along with others of the butyrate-producing pathway as a whole and proceeded with gene cloning. However, we were able, through gBlocks, to synthesize the part.

Biosafety

Our part is safe to be synthesized and utilized on an open bench. Also, the product of the gene does not include any biohazard and does not pose any threat even if by chance there is a leak.

Characterization

The previous team, iGEM 2015 NRP-UEA-Norwich, did not perform any characterization. Our team, chose for the gold medal to create a new improved part and characterize it as well as possible for the competition standards and the other IGEM teams. So we did not characterize this specific part, as something that is crucial to be conducted in the future.

References

[1] George N. Bennett, Frederick B. Rudolph, The central metabolic pathway from acetyl-CoA to butyryl-CoA in Clostridium acetobutylicum, FEMS Microbiology Reviews, Volume 17, Issue 3, October 1995, Pages 241–249, https://doi.org/10.1111/j.1574-6976.1995.tb00208.x
[2] Henriques, Bárbara J et al. “Electron transfer flavoprotein and its role in mitochondrial energy metabolism in health and disease.” Gene vol. 776 (2021): 145407. doi:10.1016/j.gene.2021.145407
[3] Chowdhury, Nilanjan Pal, et al. “Studies on the Mechanism of Electron Bifurcation Catalyzed by Electron Transferring Flavoprotein (Etf) and Butyryl-CoA Dehydrogenase (Bcd) OfAcidaminococcus Fermentans.” Journal of Biological Chemistry, vol. 289, no. 8, Dec. 2013, pp. 5145–57, https://doi.org/10.1074/jbc.m113.521013. Accessed 13 June 2019.
[4] Garcia Costas, Amaya M., et al. “Defining Electron Bifurcation in the Electron-Transferring Flavoprotein Family.” Journal of Bacteriology, edited by Anke Becker, vol. 199, no. 21, Aug. 2017, https://doi.org/10.1128/jb.00440-17. Accessed 17 Dec. 2019.
[5]Petra Louis, Sheila I. McCrae, Cédric Charrier, Harry J. Flint, Organization of butyrate synthetic genes in human colonic bacteria: phylogenetic conservation and horizontal gene transfer, FEMS Microbiology Letters, Volume 269, Issue 2, April 2007, Pages 240–247,https://doi.org/10.1111/j.1574-6968.2006.00629.x
[6]“Butyrate-Producing Bacterium L2-50 Putative Fe-S Oxidoreductase Gene, Partial Cds; Thiolase, Crotonase, Beta Hydroxybutyryl-CoA Dehydrogenase, Butyryl-CoA Dehydrogenase, Electron Transfer Flavoprotein Beta-Subunit, and Electron Transfer Flavoprotein Alpha-Subunit Genes, Complete Cds; and Putative Multidrug Efflux Pump Gene, Partial Cds.” NCBI Nucleotide, July 2016, https://www.ncbi.nlm.nih.gov/nuccore/DQ987697.1/

Toolbox's links

Genscript's GenSmart Optimization Tool: https://www.genscript.com/tools/gensmart-codon-optimization

Other tools for optimizing a sequence:
IDT's Optimization tool: https://eu.idtdna.com/CodonOpt
PDB visualization tool for the proteins crystal structure:https://www.rcsb.org/structure/5OL2 & https://www.rcsb.org/structure/4L2I