Difference between revisions of "Part:BBa K4989003"

Latest revision as of 23:21, 11 October 2023

Butyryl-CoA dehydrogenase (bcd)

It is an improved basic part of:

BBa_K1618043 of iGEM 2015 NRP-UEA-Norwich.

Alternative names of the enzyme

Other names that the butyryl-CoA dehydrogease enzyme could be found in the bibliography are:
Systematic name: short-chain acyl-CoA:electron-transfer flavoprotein 2,3-oxidoreductase
Else: short-chain acyl-CoA dehydrogenase,
butyryl-CoA dehydrogenase,
butanoyl-CoA dehydrogenase,
butyryl dehydrogenase,
unsaturated acyl-CoA reductase,
ethylene reductase,
enoyl-coenzyme A reductase, unsaturated acyl coenzyme A reductase,
butyryl coenzyme A dehydrogenase,
short-chain acyl CoA dehydrogenase, short-chain acyl-coenzyme A dehydrogenase,
3-hydroxyacyl CoA reductase,
butanoyl-CoA:(acceptor) 2,3-oxidoreductase, ACADS (gene name)

Application in the field of biology

The bcd gene encodes for a flavoprotein oxidoreductase that has great specificity for short-chain fatty acids: butyryl-CoA dehydrogenase. The enzyme catalyzes the a and b desaturation of acyl-CoA substrates[1]. This enzyme doesn’t utilize NAD(H) directly but requires electron transfer flavoproteins to form a complex and transfer electrons from butyryl-CoA dehydrogenase to NAD(H)[2].In mammalian cells, in mitochondria, there is a similar enzyme that catalyzes a similar reaction in β-oxidation of short-chain fatty acids[5].
The full reaction is shown below:

2 NADH+2Fdox + crotonyl−CoA↔2 NAD+2 Fdred+ butyryl−CoA

Important note:

Caution should be taken in regard to the implementation of this biochemical reaction. Since it is bidirectional, it can be affected by different variables. So in an in vivo situation the environment and the different conditions that this organism might live in can affect the balance of the reaction. Thus further bibliographic research is recommended.

The crystal structure of butyryl-CoA dehydrogenase from Megesphaera elsdenii is shown below:

Configuration of the new part

The previous part BBa_K1618043 originated from Coprococcus sp.L2-50 DSM. There were some issues in the sequence and the origin of it that we addressed and solved as follows:
1. The sequence did contain a start and a stop codon but due to the results of the iGEM 2015 NRP-UEA-Norwich team, we decided to change them according to 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 [3,4].
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] Boynton, Z. L., et al. “Cloning, Sequencing, and Expression of Clustered Genes Encoding Beta-Hydroxybutyryl-Coenzyme a (CoA) Dehydrogenase, Crotonase, and Butyryl-CoA Dehydrogenase from Clostridium Acetobutylicum ATCC 824.” Journal of Bacteriology, vol. 178, no. 11, June 1996, pp. 3015–24, https://doi.org/10.1128/jb.178.11.3015-3024.1996. Accessed 13 June 2019.
[2] 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
[3]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
[4]“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/
[5]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

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 tool for the visualization of the protein crystal structure:https://www.rcsb.org/structure/1BUC

@@ Line 27: / Line 27: @@
 <br>
 <br>
-The crystal structure of butyryl-CoA dehydrogenase from <Megesphaera elsdenii</i> is shown below:
+<b>The crystal structure of butyryl-CoA dehydrogenase from <i>Megesphaera elsdenii</i> is shown below:</b>
 <br>
 <br>
@@ Line 34: / Line 34: @@
 ==<strong>Configuration of the new part</strong>==
 The previous part BBa_K1618043 originated from <i><b>Coprococcus sp.L2-50 DSM</b></i>. There were some issues in the sequence and the origin of it that we addressed and solved as follows:
+<br>
 . The sequence did contain a start and a stop codon but due to the results of the iGEM 2015 NRP-UEA-Norwich team, we decided to change them according to a research paper that provides all the sequences of the enzyme’s genes for the butyrate-producing pathway from the same strain <i>Coprococcus sp.L2-50</i> [3,4].
+<br>
 . We optimized our sequence in order to be expressed in both <i>Lactobacillus</i> species and <i>E.coli</i>.
+<br>
 . We excluded all the restriction sites of the endonucleases that we used for cloning.
 <br>
 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.
 ==<strong>The obtainment of the sequence and its difficulties</strong>==
@@ Line 72: / Line 74: @@
 Other tools for optimizing a sequence:<br>
 IDT's Optimization tool: https://eu.idtdna.com/CodonOpt
+<br>
+PDB tool for the visualization of the protein crystal structure:https://www.rcsb.org/structure/1BUC