Difference between revisions of "Part:BBa K4989000"

Latest revision as of 22:10, 11 October 2023

Acetyl-CoA C-acetyltransferase (thl)

It is an improved basic part of:

BBa_K1618040 of iGEM 2015 NRP-UEA-Norwich.

Alternative names of the enzyme

Other names that the acetyl-CoA C-acetyl transferase enzyme could be found in the bibliography are:
Systematic name: acetyl-CoA:acetyl-CoA C-acetyltransferase
Else: 3-ketoacyl CoA thiolase,
3-ketoacyl coenzyme A thiolase,
3-ketoacyl thiolase, 3-ketoacyl-CoA thiolase, 3-ketothiolase,
3-oxoacyl-CoA thiolase,
3-oxoacyl-coenzyme A thiolase,
6-oxoacyl-CoA thiolase,
β-ketoacyl coenzyme A thiolase,
β-ketoacyl-CoA thiolase,
β-ketoadipyl coenzyme A thiolase,
β-ketoadipyl-CoA thiolase,
β-ketothiolase KAT,
acetoacetyl-CoA β-ketothiolase,
acetyl-CoA acyltransferase,
ketoacyl-CoA acyltransferase,
ketoacyl-coenzyme A thiolase,
long-chain 3-oxoacyl-CoA thiolase,
oxoacyl-coenzyme A thiolase,
pro-3-ketoacyl-CoA thiolase,
thiolase I, 2-methylacetoacetyl-CoA thiolase.

Application in the field of biology

The thl gene codes for an enzyme named acetyl-CoA C-acetyltransferase which belongs to the family of thiolases. This is the key enzyme for the condensation of two molecules of acetyl-CoA, which are produced from the glycolysis path, and the acetoacetyl-CoA production [Figure A]. This is a reaction that thermodynamically itself is not favored. It starts with the acylation of a nucleophilic cysteine at the active site by acetyl-CoA (or a different acyl group) with the release of the CoA group. Then the acyl group is transferred to an acetyl-CoA molecule.

The chemical reactions that take place are shown below:
1) acyl-CoA + acetyl-CoA = CoA + 3-oxoacyl-CoA
(1a) acetyl-CoA C-acyltransferase-S-acyl-L-cyteine + acetyl-CoA = 3-oxoacyl-CoA + acetyl-CoA C-acyltransferase-L-cyteine
(1b) acyl-CoA + acetyl-CoA C-acyltransferase-L-cyteine = acetyl-CoA C-acyltransferase-S-acyl-L-cyteine + CoA

Figure A. The chemical reaction of the acetoacetyl-CoA production

It's worth noting that different bacterial genera (such as Clostridium, Firmicutes, etc.) have various differences in the sequences and the molecular masses of the enzyme. Nonetheless, they catalyze the same reaction. Comparing the reaction of the butyrate and the acetate synthesis, which are both produced from the acetyl-CoA molecule, the latter produces a yield of ATP that is twice as much as that of the former[1].
During acidogenesis the acetyl-CoA C-acetyltransferase antagonizes with phosphotransacetylase for the remaining pool of acetyl-CoA. Also, the production of butyrate doesn't contribute to the pool of NADH while acetate production increases the levels of NADH [5].
Below there is a crystal structure of the enzyme acetyl-CoA C-acetyltransferase from the bacterium Clostridium acetobutylicum. It is worth noting that the enzyme's crystal structure is different from organism to organism [Figure B].

Figure B. Crystal structure of the enzyme acetyl-CoA C-acetyltransferase from the bacterium Clostridium acetobutylicum.

The enzyme is found both in the eukaryotes and the prokaryotes. Studies (primarily in a Clostridium strain) show that the change in the pH of the environment does not affect the regulation of the enzyme. It is inhibited by micromolar levels of CoA and ATP, as well as butanoyl-CoA. Also, the concentration of CoASH(the Coenzyme A synthetase) plays an important role in the net rate of the condensation of the two acetyl-CoA molecules [1,2].

Configuration of the new part

The previous part BBa_K1618040 was 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]Wiesenborn, Dennis P., et al. “Thiolase from Clostridium Acetobutylicum ATCC 824 and Its Role in the Synthesis of Acids and Solvents.” Applied and Environmental Microbiology, vol. 54, no. 11, 1988, pp. 2717–22,https://doi.org/10.1128/aem.54.11.2717-2722.1988. Accessed 21 Apr. 2021.
[2]Stim-Herndon, Kathleen P., et al. “Characterization of an Acetyl-CoA C Acetyltransferase (Thiolase) Gene from Clostridium Acetobutylicum ATCC 824.”Gene, vol. 154, no. 1, Feb. 1995, pp. 81–85,https://doi.org/10.1016/03781119(94)00838-j Accessed 20 Feb. 2021.
[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 visualization of crystal structures of the proteins:https://www.rcsb.org/structure/4wyr