Part:BBa_K4390074
Tri-PETase
This part is not compatible with BioBrick RFC10 assembly but is compatible with the iGEM Type IIS Part standard which is also accepted by iGEM.
Usage and Biology
Tri-PETase is an engineered mutant of PETase (290 amino acids) with (T140D/R224Q/N233K).
PETase was discovered in 2016 in Ideonella sakaiensis, which uses PET as a single carbon source (Yoshida, 2016). The PETase hydrolyses PET polymers and produces mono(2-hydroxyethyl)-TPA (MHET) majorly, and minorly two final products shown below: terephthalic acid (TPA), and ethylene glycol (EG) (Joo et al., 2018). However, since only a very small amount of MHET can be continued to be hydrolysed to TPA by PETase, we need to add MHETase to the device to increase TPA yield and purity in our cell-free device (Puspitasari, Tsai and Lee, 2021).
From literature search, we learnt Lu's team has enhanced the activity of PETase with CNN-based machine learning algorithms and developed FAST-PETase, the most efficient enzyme available today with five mutations comparing to wild-type PETase (S121E/D186H/ R224Q/N233K/R280A). Untreated post-consumer PET from 51 different thermoformed products is almost always completely degraded by FAST-PETase at 50 ºC for periods ranging from 24 h to 1 week. FAST-PETase can also depolymerize the untreated amorphous fraction of a commercial water bottle and an entire heat pre-treated water bottle at 50 ºC. For highly crystalline PET, a simple pre-treatment (e.g., melting) allows the PET to be feasibly degraded. We selected another Triple mutant PETase (T140D/R224Q/N233K) with similar activity as FAST-PETase under 40°C to compare their performance (Lu et al., 2022).
Design
The Tri-PETase (BBa_K4390074) encode the peptide sequence of PETase with mutations on (T140D/R224Q/N233K) and 2 additional base pairs to fit in JUMP assembly O part design. The codon is optimized for BioBrick and JUMP assembly.
Characterization
The Tri-PETase (BBa_K4390074) were used to assemble the following Lv.1 JUMP assembly for activity assessment and immobilization.
| Functional Part | Part number |
|---|---|
| Untagged Tri-PETase | K4390089 |
| N-terminal L2NC-tagged Tri-PETase | K4390115 |
| N-terminal L2NC-linker-tagged Tri-PETase | K4390116 |
| N-terminal Car9-tagged Tri-PETase | K4390086 |
| C-terminal L2NC-tagged Tri-PETase | K4390082 |
| C-terminal L2NC-linker-tagged Tri-PETase | K4390083 |
| C-terminal Car9-tagged Tri-PETase | K4390081 |
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
Reference
Yoshida S, Hiraga K, Takehana T, Taniguchi I, Yamaji H, Maeda Y et al. A bacterium that degrades and assimilates poly(ethylene terephthalate). Science. 2016;351(6278):1196-1199.
Joo S, Cho I, Seo H, Son H, Sagong H, Shin T et al. Structural insight into molecular mechanism of poly(ethylene terephthalate) degradation. Nature Communications. 2018;9(1).
Puspitasari N, Tsai S, Lee C. Class I hydrophobins pretreatment stimulates PETase for monomers recycling of waste PETs. International Journal of Biological Macromolecules. 2021;176:157-164.
Lu H, Diaz D, Czarnecki N, Zhu C, Kim W, Shroff R et al. Machine learning-aided engineering of hydrolases for PET depolymerization. Nature. 2022;604(7907):662-667.
| None |