Part:BBa_K5236025

BhrPETase

The sequence of BhrPETase was identified by the Shingo group in a metagenomic study on uncultured thermophiles, and was deposited into the NCBI database by the group in 2018 and annotated as a PET hydrolase [1]. This basic part encoding the BhrPETase, which has been predicted and optimized by Wu et al. Si-face binding is the main binding pose of PET in the active site of BhrPETase. And was constructed and modified as WT BhrPETase in our project.[2] The superior activity and thermostability of BhrPETase rendered it one of the most promising PETases for plastic waste recycling and bioremediation applications in the future [3].

Fig.1 The 3D protein structure of WT BhrPETase

Usage and Biology

We trained a Transformer model on 1007 homologous PETase protein sequences obtained from the UniProt Database using the masked language model (MLM) training method. This approach allows the model to learn contextual information about amino acid sequences and predict masked residues accurately [4]. The Transfer model will give out 10 most possible mutated points based on the contextual information. Then, those mutated points will be further selected by Meta’s Evolutionary Scale Modeling (ESM) 1b model[5].The useless mutated points who do not cause mutation to enzyme will be weed out. The BhrPETase mutants that scored in the top four in the trained model were used in the construction and tested. WT BhrPETase is the blueprint of our other mutants, therefore it is the reference when comparing the efficiency of mutated PETase.

Fig.2 The overall pipeline of our model training method.

To synthesize WT BhrPETase in E. coli we constructed plasmids using the pET28a vector.

Fig.3 The contructed plasmid with WT BhrPETase

The function of each parts is as follows:

T7 promoter: A Strong promoter recognized by T7 RNA polymerase, used to regulate gene expression of recombinant proteins.

Lac operator: Operator that can be activated by IPTG, used to control gene expression by lactose or IPTG.

RBS: Ribosome binding site.

WT BhrPETase:The basic part encoding the BhrPETase who had been mutated.

pelB: The sequence encodes a signal peptide that enables secretory expression of PETase.

6xHis: A label for protein purification

T7 terminator: Terminates transcription.

By conducting colony PCR, we are able to test if our parts have been transformed into E.coli successfully. The following result of electrophoresis proves that we’ve inserted the WT BhrPETase sequence into E.coli; the sequence containing our mutated genes has a total of 798 base pairs and the results are in the right location.

Fig.4 The sequence of BhrPETase WT
Fig.5 The DNA gel electrophoresis result

After completing plasmid construction and transformation. We tested the WT BhrPETase activity using the p-nitrophenyl butryte assay from the iGEM19_Toronto team (for more details, please see protocols). The results show that WT BhrPETase has higher activity than WT IsPETase.The relative enzyme efficiency (A415/protein concentration) that we are looking at takes into consideration both the efficiency of the enzyme itself and the PETase synthesis rate of the chassis, since our end goal is to implement the engineered organism in a self-sufficient PET degrading system as a whole.

Fig.6 Mutated BhrPETase Dynamic Curve

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]

Reference

[1] Kato, Shingo, et al. “Long-Term Cultivation and Metagenomics Reveal Ecophysiology of Previously Uncultivated Thermophiles Involved in Biogeochemical Nitrogen Cycle.” Microbes and Environments, vol. 33, no. 1, Jan. 2018, pp. 107–10. https://doi.org/10.1264/jsme2.me17165. [2]Wang, N., Li, Y., Zheng, M., Dong, W., Zhang, Q., & Wang, W. (2024b). 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 [3]Xi, X., Ni, K., Hao, H., Shang, Y., Zhao, B., & Qian, Z. (2020). 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 [4] Lu, Hongyuan, et al. “Machine Learning-aided Engineering of Hydrolases for PET Depolymerization.” Nature, vol. 604, no. 7907, Apr. 2022, pp. 662–67. https://doi.org/10.1038/s41586-022-04599-z. [5] Rives, A., Meier, J., Sercu, T., Goyal, S., Lin, Z., Liu, J., ... & Fergus, R. (2021). Biological structure and function emerge from scaling unsupervised learning to 250 million protein sequences. Proceedings of the National Academy of Sciences, 118(15), e2016239118.