Part:BBa_K4390115
N-terminal L2NC-tagged 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
We designed the N-terminal L2NC-tagged Tri-PETase to make the construct functional for both PET degradation and silica immobilisation.
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 hydrolyzed 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 also 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).
L2NC is a truncated version of the L2 ribosomal protein from E. coli, designed for fusion to C-terminal of a protein using JUMP assembly. This tag contains just the N and C-terminal regions of L2 which were shown to have silica binding capacity in previous experiments, therefore allowing the use of a smaller tag without compromising on binding affinity. The attachment of L2NC silica tag on the C-terminus of the functional enzyme would result in the 14.19 kDa increasement in weight. From literature, the dissociation constant between L2NC silica tag and silica beads is 1.7nM. Therefore, this tag facilitates immobilisation to silica surfaces, enabling enzyme immobilisation or purification using silica-based spin columns (Kim et al., 2020).
We designed the N-terminal L2NC-tagged Tri-PETase aiming for produce Tri-PETase with both PET degradation and silica immobilisation function.
Design
N-terminal L2NC-tagged Tri-PETase was assembled by JUMP assembly with: T7 promoter (P part)-B0034 RBS (R part) - L2NC (N part)- [Tri-PETase] (O part)- L1U1H08 (CT part). All the codons were optimized for BioBrick and JUMP assembly.
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 110
- 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.
Kim S, Joo K, Jo B, Cha H. Stability-Controllable Self-Immobilization of Carbonic Anhydrase Fused with a Silica-Binding Tag onto Diatom Biosilica for Enzymatic CO2 Capture and Utilization. ACS Applied Materials & Interfaces. 2020;12(24):27055-27063.
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