Part:BBa_K4989006

Butyryl-CoA:Acetyl-CoA transferase (butcoat)

It is an improved basic part of:

Alternative names of the enzyme

Other names that the butyryl-CoA:acetyl-CoA transferase enzyme could be found in the bibliography are:
Systematic name: Acetyl-CoA:butanoyl-CoA C-acetyltransferase
Else: Butyryl-coenzyme A transferase,
Butyryl-CoA transferase,
ACAT2 (short for Acetyl-CoA Acetyltransferase 2, which is a gene encoding this enzyme),
Mitochondrial acetoacetyl-CoA thiolase,
Mitochondrial 3-ketoacyl-CoA thiolase, 3-ketoacyl-CoA thiolase B,
Succinyl-CoA:3-ketoacid-coenzyme A transferase 2,
Beta-ketothiolase 2

Application in the field of biology

The butcoat gene encodes for a transferase that catalyzes the final step of the metabolic pathway production: Butyryl-CoA:Acetyl-CoA transferase.The primary function of Butyryl-CoA:Acetyl-CoA transferase is to facilitate the transfer of the acetyl group from butyryl-CoA to coenzyme A (CoA)[Figure A]. In this way the transferase can utilize the butyryl-CoA an the acetate as substrates in order to produce butyrate.
This process results in the formation of acetyl-CoA as a second product, which is a crucial molecule in the citric acid cycle (Krebs cycle) and oxidative phosphorylation, two central pathways of energy production in the cell. By converting butyryl-CoA into acetyl-CoA, this enzyme allows the cell to harness the energy stored in fatty acids more efficiently.

Figure A.The transfer of the acetyl group from butyryl-CoA to coenzyme A (CoA) for the production of butyrate

The expression of genes encoding Butyryl-CoA:Acetyl-CoA transferase in bacteria is regulated in response to the availability of specific carbon sources. The presence of medium-chain fatty acids or other substrates can trigger the upregulation of genes encoding this enzyme, allowing bacteria to adapt to their nutritional environment.
Butyryl-CoA:Acetyl-CoA transferase, often referred to as Butyryl-CoA transferase, is an enzyme found in both bacteria and eukaryotes. Its role in bacteria is primarily associated with metabolic pathways related to fatty acid degradation, energy production, and the catabolism of various carbon sources. This enzyme is particularly significant for individuals on diets rich in medium-chain triglycerides and for those with certain metabolic disorders, as it helps ensure the efficient utilization of these fatty acids for energy. In the human gastrointestinal tract, the enzyme plays a role in the microbial fermentation of dietary fibers and complex carbohydrates. Certain bacteria in the gut produce short-chain fatty acids, such as butyrate, which have important implications for gut health and host physiology.

Configuration of the new part

The previous part BBa_K2052018 was originated from Roseburia intestinalis L1-82.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 potentially a stop codon but due to the fact that iGEM 2016 METU_HS_Ankara team did some characterization experiments and omitted the stop codon, we decided to re-design the whole sequence of the transferase according to a research paper that provides all the sequence of the enzyme’s gene from the same strain Roseburia intestinalis L1-82 [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 2016 METU_HS_Ankara team, did perform a characterization showing that the transferase was produced (proceed with reviewing their experiments from the link provided in the top of the page). 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 any further, as something that is crucial to be conducted in the future.

References

[1]Duncan, Sylvia H., et al. “Acetate Utilization and Butyryl Coenzyme a (CoA):Acetate-CoA Transferase in Butyrate-Producing Bacteria from the Human Large Intestine.” Applied and Environmental Microbiology, vol. 68, no. 10, Oct. 2002, pp. 5186–90, https://doi.org/10.1128/AEM.68.10.5186-5190.2002. Accessed 20 Feb. 2021.
[2]Hippe, Berit, et al. “Quantification of Butyryl CoA:acetate CoA-Transferase Genes Reveals Different Butyrate Production Capacity in Individuals according to Diet and Age.” FEMS Microbiology Letters, vol. 316, no. 2, Jan. 2011, pp. 130–35, https://doi.org/10.1111/j.1574-6968.2010.02197.x. Accessed 6 June 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]“Roseburia Intestinalis L1-82 Chromosome 1, Complete Sequence.” NCBI Nucleotide, Mar. 2023, www.ncbi.nlm.nih.gov/nuccore/NZ_LR027880.1?report=genbank&from=430426&to=431769&strand=true.
[5]Louis, Petra, et al. “Diversity of Human Colonic Butyrate-Producing Bacteria Revealed by Analysis of the Butyryl-CoA:acetate CoA-Transferase Gene.” Environmental Microbiology, vol. 12, no. 2, Feb. 2010, pp. 304–14, https://doi.org/10.1111/j.1462-2920.2009.02066.x. Accessed 31 Oct. 2019.

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