Part:BBa_K4989002

3-hydroxybutyryl-CoA dehydratase (cro)

It is an improved basic part of:

Alternative names of the enzyme

Other names that the 3-hydroxybutyryl-CoA dehydratase enzyme could be found in the bibliography are:
Systematic name: (3R)-3-hydroxybutanoyl-CoA hydro-lyase (crotonoyl-CoA-forming)
Else: 3-hydroxybutyryl-CoA dehydratase,
crotonase,
D-3-hydroxybutyryl coenzyme A dehydratase,
D-3-hydroxybutyryl-CoA dehydratase,
enoyl coenzyme A hydrase (D),
(3R)-3-hydroxybutanoyl-CoA hydro-lyase

Application in the field of biology

The cro gene encodes for an enzyme that belongs to the crotonase superfamily: 3-hydroxybutyryl-CoA dehydratase. Members of this superfamily catalyze different reactions such as dehalogenation, hydration/dehydration, decarboxylation, etc., while they are involved in variable metabolic pathways [1]. 3-hydroxybutyryl-CoA dehydratase belongs to the family of hydrolases which cleaves carbon-oxygen bonds. For the same reaction, there are different types of crotonases that perform the same reaction such as 4-hydroxybutyryl-CoA dehydratase from Clostridium aminobutyricum [2]. Crotonases may be active with different carbon chains of fatty acids. For example, C.acetobutylicum is active with short-chain fatty acyl-CoA compounds (C4-C6), while the equivalent crotonase from bovine liver is active with long-chain fatty acetyl-CoAs (C6-C16) [5].

The 3-hydroxybutyryl-CoA dehydratase is the third enzyme in the metabolic pathway of butyrate production and catalyzes the reaction of hydrolyzing 3-hydroxybutyryl-CoA to crotonyl-CoA[Figure A]. A corresponding enzyme catalyzes a similar reaction in eukaryotic cells for the beta-oxidation pathway.

The full reaction is shown below:

(3R)-3-hydroxybutanoyl-CoA ↔ crotonyl-CoA + H2O


Figure A. The reaction of hydrolyzing 3-hydroxybutyryl-CoA to crotonyl-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.

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 the origin of it 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 [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] Holden, Hazel M., et al. “The Crotonase Superfamily:  Divergently Related Enzymes That Catalyze Different Reactions Involving Acyl Coenzyme a Thioesters.” Accounts of Chemical Research, vol. 34, no. 2, Dec. 2000, pp. 145–57, https://doi.org/10.1021/ar000053l. Accessed 12 May 2022.
[2] Peter Willadsen, Wolfgang Buckel, Assay of 4-hydroxybutyryl-CoA dehydratase from Clostridium aminobutyricum, FEMS Microbiology Letters, Volume 70, Issue 2, July 1990, Pages 187–191, https://doi.org/10.1111/j.1574-6968.1990.tb13976.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