Part:BBa_K4390113

N-terminal L2NC-tagged FAST-PETase

Usage and Biology

FAST-PETase (BBa_K4390073) 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). 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.

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 FAST-PETase aiming for produce FAST-PETase with both PET degradation and silica immobilisation function.

Design

N-terminal L2NC-tagged FAST-PETase was assembled by JUMP assembly with: T7 promoter (P part)-B0034 RBS (RN part)-[FAST-PETase] (O part)-L2NC (C part)-L1U1H08 (T part). All the codons were optimized for BioBrick and JUMP assembly.

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
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 110
  Illegal NgoMIV site found at 610
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal SapI site found at 839
  

Reference

Coyle BL, Baneyx F. A cleavable silica-binding affinity tag for rapid and inexpensive protein purification. Biotechnol Bioeng. 2014 Oct 1;111(10):2019–26.

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.