Part:BBa_K4390073
FAST-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.
FAST-PETase is a mutant form of the PETase, which has faster activity. We immobilised this part to Silica beads as part of our cell free PET biodegradation device.
Usage and Biology
FAST-PETase is an engineered mutant of PETase (290 amino acids) with (S121E/D186H/R224Q/N233K/R280A).
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 (Lu et al., 2022). For highly crystalline PET, a simple pre-treatment (e.g., melting) allows the PET to be feasibly degraded .
Design
The FAST-PETase (BBa_K4390073) encode the peptide sequence of PETase with mutations on (S121E/D186H/R224Q/N233K/R280A) and 2 additional base pairs to fit in JUMP assembly O part design. The codon is optimized for BioBrick and JUMP assembly.
Characterization
The FAST-PETase (BBa_K4390073) was used to assemble the following Lv.1 JUMP assembly for activity assessment and immobilization.
| Functional Part | Part number |
|---|---|
| Untagged FAST-PETase | K4390090 |
| N-terminal L2NC-tagged FAST-PETase | K4390113 |
| N-terminal L2NC-linker-tagged FAST-PETase | K4390114 |
| N-terminal Car9-tagged FAST-PETase | K4390084 |
| C-terminal L2NC-tagged FAST-PETase | K4390076 |
| C-terminal L2NC-linker-tagged FAST-PETase | K4390077 |
| C-terminal Car9-tagged FAST-PETase | K4390075 |
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 139
- 1000INCOMPATIBLE WITH RFC[1000]Illegal SapI site found at 368
References
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.
Improvement on BBa_K3946023 by Edinburgh-UHAS_Ghana 2022
We improved upon the Dou-PETase part (Part:BBa_K3946023) with the FAST-PETase part (Part:BBa_K4390073).
PETase-silica tag fusion protein Activity Test
We assessed the various PETase activities using a para-nitrophenol-butyrate (pNPB) assay, since PETases can hydrolyse pNPB into para-nitrophenol (pNP), which strongly absorbs at 415 nm. pNPB is not the PETase’s true substrate, but this preliminary assay is still representative of PETase activity. Figure 1 shows that untagged FAST-PETase has higher activity than Dou-PETase in this assay. Hence, FAST-PETase is an improvement over Dou-PETase for degradation of PET. The data also shows that PETase activity is diminished, but still exists when immobilised on silica beads, as the activity is higher than SHuffle E. coli lysate.
Improvement on BBa_K5023003 by UNILA-Latam 2023
We improved upon the FAST-PETase part (Part:BBa_K4390073) with the FAST-PETase part (Part:BBa_K5023003) optimized for Chlamydomonas reinhardtii.
FAST-PETase is a mutant variant derived from the PETase enzyme, optimized with specific codon changes to enhance its expression efficiency in Chlamydomonas reinhardtii. This enzyme showcased superior PET-hydrolytic activity in comparison to both the wild-type and other engineered alternatives between 30 and 50°C across various pH levels. At 50°C, FAST-PETase exhibited the most significant degradation among all mutants tested, releasing 33.8 mM of PET monomers, which is the combined amount of terephthalic acid (TPA) and mono-(2-hydroxyethyl) terephthalate (MHET). Its activity against post-consumer PET (pc-PET) surpassed other enzymes like WT PETase, ThermoPETase, DuraPETase, LCC, and ICCM under identical conditions. Remarkably, FAST-PETase managed to degrade nearly all untreated post-consumer PET from 51 distinct thermoformed products within a week. Additionally, it proved its ability to depolymerize the untreated, amorphous sections of a commercial water bottle. A time-course analysis highlighted an almost linear PET degradation rate and a simultaneous rise in crystallinity over time. The introduction of FAST-PETase presents a promising approach to PET plastic degradation, especially considering its amplified activity and stability across diverse conditions. This enzyme holds significant potential in addressing the environmental issues caused by the accumulation of PET plastics.
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