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Revision as of 08:20, 8 October 2023
Contents
3-hydroxybutyryl-CoA dehydrogenase (bhbd)
It is an improved basic part of:
BBa_K1618041 of iGEM 2015 NRP-UEA-Norwich.
Alternative names of the enzyme
Other names that the 3-hydroxybutyryl-CoA dehydrogenase enzyme could be found in the bibliography are:
Systematic name: (S)-3-hydroxybutanoyl-CoA:NADP+ oxidoreductase
Else: beta-hydroxybutyryl coenzyme A dehydrogenase,
L(+)-3-hydroxybutyryl-CoA dehydrogenase,
BHBD or HBDH dehydrogenase,
L-3-hydroxybutyryl coenzyme A (nicotinamide adenine dinucleotide phosphate),
L-(+)-3-hydroxybutyryl-CoA dehydrogenase,
beta-hydroxybutyryl-CoA dehydrogenase.
Application in the field of biology
The bhbd gene encodes for the enzyme 3-hydroxybutyryl-CoA dehydrogenase, which belongs to the family of oxidoreductases and the superfamily of the dehydrogenase/reductase (SDR) and is the second enzyme involved in the butyrate production pathway. The enzyme catalyzes the asymmetric reduction of acetoacetate to 3-hydroxybutyryl-CoA in an NADPH-dependent manner[1]. It can be found in microorganisms such as bacteria as well as in the mitochondria of mammalian cells[2].
This enzyme can produce 3-hydroxybutyryl-CoA through two different mechanisms (in which two different enzymes are involved). Thus, 2 enantiomers are produced: (S)-3-hydroxybutyryl-CoA or (R)-3-hydroxybutyryl-CoA. For example in E.coli, there are two enzymes that catalyze the same reaction but have different products synthesized, which are enantiomers[3]. Generally, the above doesn't apply to all organisms, since some of them can produce only one from the two enantiomers. For example, C.acetobutylicum can produce only (S)-3-hydroxybutyryl-CoA as well as this part's coding sequence.
Important note:
Caution should be taken in regard to the details of the experimental and bibliographic details of the enzymatic reaction of 3-hydroxybutyryl-CoA.There are variabilities in the bibliography on whether NADPH or NADH is used as a co-substrate as well as the different genes and thus enzymes of 3-hydroxybutyryl-CoA that produce the two enantiomers in each organism.The gene bhbd which sequence is provided here according to BRENDA data base produces the (R)-3-hydroxybutyryl-CoA [6]. Therefore, further investigation should be conducted bibliographically in terms of the different potential chemical properties of the final product, butyrate.
The full reaction is shown below:
(S)-3-Hydroxybutyryl-CoA + NADP+ <=> Acetoacetyl-CoA + NADPH + H+
The crystal structure of the 3-hydroxybutyryl-CoA with acetoacetyl-CoA from Clostridium butyricum is shown below:
Configuration of the new part
The previous part BBa_K1618041 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 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 [4,5].
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] Machado, Teresa F. G., et al. “Dissecting the Mechanism of (R)-3-Hydroxybutyrate Dehydrogenase by Kinetic Isotope Effects, Protein Crystallography, and Computational Chemistry.” ACS Catalysis, vol. 10, no. 24, Dec. 2020, pp. 15019–32, https://doi.org/10.1021/acscatal.0c04736. Accessed 4 May 2022.
[2] Fottrell, P. F., and Ann O’Hora. “Multiple Forms of D(--)3-Hydroxybutyrate Dehydrogenase in Rhizobium.” Journal of General Microbiology, vol. 57, no. 3, Microbiology Society, Aug. 1969, pp. 287–92, https://doi.org/10.1099/00221287-57-3-287. Accessed 6 Oct. 2023.
[3] Tseng, Hsien-Chung, et al. “Metabolic Engineering of Escherichia Coli for Enhanced Production of ( R )- and ( S )-3-Hydroxybutyrate.” Applied and Environmental Microbiology, vol. 75, no. 10, May 2009, pp. 3137–45, https://doi.org/10.1128/aem.02667-08. Accessed 6 Oct. 2023.
[4]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
[5]“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/
[6] “Pathway Maps - BRENDA Enzyme Database.” Www.brenda-Enzymes.org,
https://www.brenda-enzymes.org/pathway_index.php?pathway=butanoate%20fermentation&ncbi_tax_id=2049024.Accessed 7 Oct. 2023.
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
Further bibliographic resources
[1]Lo, Jonathan, et al. “Acetogenic Production of 3-Hydroxybutyrate Using a Native 3-Hydroxybutyryl-CoA Dehydrogenase.” Frontiers in Microbiology, vol. 13, Frontiers Media, Aug. 2022, https://doi.org/10.3389/fmicb.2022.948369. Accessed 7 Oct. 2023.
[2]Colby, G. D., and J. S. Chen. “Purification and Properties of 3-Hydroxybutyryl-Coenzyme a Dehydrogenase from Clostridium Beijerinckii (‘Clostridium Butylicum’) NRRL B593.” Applied and Environmental Microbiology, vol. 58, no. 10, Oct. 1992, pp. 3297–302, https://doi.org/10.1128/aem.58.10.3297-3302.1992. Accessed 18 Oct. 2021.
[3]Kim, Eun-Jung, et al. “Crystal Structure of (S)-3-Hydroxybutyryl-CoA Dehydrogenase from Clostridium Butyricum and Its Mutations That Enhance Reaction Kinetics.” Journal of Microbiology and Biotechnology, vol. 24, no. 12, Dec. 2014, pp. 1636–43, https://doi.org/10.4014/jmb.1407.07027. Accessed 25 May 2022.
[4]Karel Olavarría, et al. “An NADH Preferring Acetoacetyl-CoA Reductase Is Engaged in Poly-3-Hydroxybutyrate Accumulation in Escherichia Coli.” Journal of Biotechnology, vol. 325, Elsevier BV, Jan. 2021, pp. 207–16, https://doi.org/10.1016/j.jbiotec.2020.10.022. Accessed 7 Oct. 2023.