Part:BBa_K5117010
BhrPET
This part contains the gene of the uncultured bacterium HR29 encoding a polyethylene terephthalate hydrolase (PETase, EC 3.1.1.101). It is a homologue of the leaf-branch compost cutinase (LCC) and codon optimized for Bacillus subtilis (Xi et al. 2021). In addition to the coding sequence, the signal peptide of the aprE gene for secretion in Bacillus subtilis is included.
BhrPET only served for design purposes of the TU Dresden iGEM 2024 Team and was required for the construction of composite parts (see Contribution page).
Target organism: Bacillus subtilis
Main purpose of use: Gene expression and protein production using the host Bacillus subtilis
Design
For compatibility with the BioBrick RFC[10] standard, the restriction sites EcoRI, XbaI, SpeI, PstI and NotI were removed from the coding sequence. To make the part compatible with the Type IIS standard, BsaI and SapI sites were removed as well. This was achieved by codon exchange using the codon usage table of Bacillus subtilis (Codon Usage Database Kazusa).
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal AgeI site found at 579
Illegal AgeI site found at 624 - 1000COMPATIBLE WITH RFC[1000]
Enzyme characterization according to literature
In the study by Xi et al. (2021) titled "Secretory expression in Bacillus subtilis and biochemical characterization of a highly thermostable polyethylene terephthalate hydrolase from bacterium HR29", a polyethylene terephthalate (PET) hydrolase BhrPETase was heterologously overexpressed in B. subtilis and biochemically characterized (Xi et al. 2021).
The gene encoding BhrPETase was first discovered in a metagenomic study by Kato et al. (2018). Its sequence is homologous to that of the leaf-branch compost cutinase (LCC) variant (94 % sequence identity) (Xi et al. 2021).
In the work of Xi et al., a secretory expression of BhrPETase was performed in B. subtilis, which was improved by the development of the chaperone-overexpression mutant strain. After protein overexpression, supernatants were collected and subsequently purified with hydrophobic interaction chromatography (HIC) followed by ion exchange chromatography (IEC). The collected fractions from each purification step were analyzed with SDS-PAGE and a purified protein band corresponding to the expected molecular weight of 27.5 kDa was detected (Xi et al. 2021).
The optimal pH and temperature of BhrPETase were determined using 4-nitrophenyl octanoate (pNO) as substrate. The pH optimum of BhrPETase was found to be in the range between 6 and 8. A drastic loss of activity was observed at pH < 6 and pH > 9. Temperature profiling of BhrPETase showed an almost linear increase from 30 °C to 90 °C (Xi et al. 2021).
The thermostability study of BhrPETase was also conducted with pNO as substrate. It was shown that BhrPETase can retain 80 % of its activity when incubated at 80 °C for 2 h. A complete loss of activity was observed when BhrPETase was incubated at 90 °C. Notably, the thermostability of BhrPETase could be improved by the addition of CaCl2, which resulted in 80% residual activity after incubation at 90 °C (Xi et al. 2021).
More information related to this part can be found in the following publications and databases:
- Wang J. & Wang Y.H., HR-PETase from Bacterium HR29 (2021) https://doi.org/10.2210/pdb7EOA/pdb
- Wang N., Li Y., Zheng M., Dong W., Zhang Q., Wang, W. (2024): BhrPETase catalyzed polyethylene terephthalate depolymerization: A quantum mechanics/molecular mechanics approach. Journal of Hazardous Materials 477, 135414. https://doi.org/10.1016/j.jhazmat.2024.135414
- Gene sequence: Codon optimized sequence for Bacillus subtilis can be found in the supplementary data of Xi et al. (2021)
- Protein sequence: https://www.ncbi.nlm.nih.gov/protein/GBD22443
- UniProtKB: https://www.uniprot.org/uniprotkb/A0A2H5Z9R5/entry
References
Xi X., Ni K., Hao H., Shang Y., Zhao B., Qian Z. (2021): Secretory expression in Bacillus subtilis and biochemical characterization of a highly thermostable polyethylene terephthalate hydrolase from bacterium HR29. Enzyme and microbial technology 143, 109715. https://doi.org/10.1016/j.enzmictec.2020.109715
Kato S., Sakai S., Hirai M., Tasumi E., Nishizawa M., Suzuki K., Takai K. (2018): Long-term cultivation and metagenomics reveal ecophysiology of previously uncultivated thermophiles involved in biogeochemical nitrogen cycle. Microbes and environments 33(1), 107-110. https://doi.org/10.1264/jsme2.ME17165
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