Part:BBa_K4390114

N-terminal L2NC-linker-tagged 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.

Fusion protein with N-terminal L2NC-linker and FAST-PETase for PET degradation

Usage and Biology

We designed the N-terminal L2NC-linker-tagged FAST-PETase to make the construct functional for both PET degradation and silica immobilisation.

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 (Lu et al., 2022). For highly crystalline PET, a simple pre-treatment (e.g., melting) allows the PET to be feasibly degraded.

L2NC-linker is Part BBa_K3946002 but with the addition of a short linker (GSEGKSSGSGSESKST). 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-linker silica tag on the C-terminus of the functional enzyme would result in the 15.69 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).

Design

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

Characterization

All the Lv.0 parts for [N-terminal L2NC-linker-tagged FAST-PETase] were integrated into pJUMP29-1A(Laz), which is a JUMP Lv.1 backbone plasmid. The Blue-White screening was conducted to select the correct colony. The colony PCR was used to verify the band size of colony PCR product was the same as in silico simulation. The primers used were (PS1: AGGGCGGCGGATTTGTCC; PS2: GCGGCAACCGAGCGTTC), the general primers for all JUMP plasmids to amplify the insertion DNA. The size of [N-terminal L2NC-linker-tagged FAST-PETase] PCR product (Figure 1. K) was corresponding to 1728 bp in silico.

Figure 1. Agarose gel showed the PCR result of [N-terminal L2NC-linker-tagged FAST-PETase] fusion proteins (agarose concentration 1.2%). The lanes were labelled with letters, and the number behind each letter represented different colonies from Blue-White Screening. K: N-terminal L2NC-linker-tagged FAST-PETase. The ladder used: 1 kb DNA Ladder from NEB (N3232S).

We assessed the [N-terminal L2NC-linker-tagged FAST-PETase] activity based on para-nitrophenol-butyrate (pNPB) assay, since pNPB can be hydrolysed by PETase into para-nitrophenol (pNP) with maximum absorbance at 415 nm (Pirillo, V, et al., 2021). This is a preliminary assay to determine the activity of PETase, although pNPB has structural differences to the polyethylene terephthalate which is the real substrate of PETase.

Figure 2. The protein sample activities assessment based on para-nitrophenol-butyrate pNPB assay. The reaction system was set up in 96 wells plate with final volume 10ul in each well, and the reaction continued for 30 min in 37°C (45 mM Na2HPO4-HCl (pH 7.0) 90 mM NaCl, and 10% (v/v) DMSO; 2mM pNPB-para-nitrophenol butyrate). All the constructs were constructed with T7 promoter, B0034 RBS and L1U1H08 terminator, so only the shorten forms of construct name were on the X-axis. [Shuffle without Lv.1 plasmid. 1] is the protein sample from the empty Shuffle strain of the same batch. “U” is the amount of activity which releases one micromole of pNP per minute under these assay conditions. The error bar is derived from biological triplicates.

From the first set of pNPB assay, we observed protein sample containing [N-terminal L2NC-linker-tagged FAST-PETase] showed 3.81E-04 (±1.63E-03) U activity per mg proteins towards pNPB under the reaction condition. The mean activity of biological triplicates was lower than the empty control containing no Lv.1 plasmid. However, the SD of this construct data was larger than the mean value.

We immobilized the PETase mutants on the silica beads (Celite 545) after activity assessment. The immobilization was done by incubating cell lysate with silica beads on a rotating shaker for 30 mins, 4°C. The protein concentration was measured by Bradford assay before and after incubation.

Figure 3. The immobilization efficiency of different PETase constructs after 30mins incubation in 4°C. Immobilization efficiency= ([initial protein] - [protein in the washing buffer]) / [initial protein]. The protein concentration in the beginning solution and in the washing buffer was measured by Bradford assay. We load 500ug protein sample to each 20mg Celite545 silica beads for all constructs. [Shuffle without Lv.1 plasmid.2] was the protein sample from the empty SHuffle strain of the same batch for Tri-PETase. [Shuffle without Lv.1 plasmid.3] was the protein sample from the empty Shuffle strain of the same batch for FAST-PETase.

The [N-terminal L2NC-linker-tagged FAST-PETase] showed the immobilization efficiency (11.39%), lower than the empty control’s in the same batch. One possible reason might be the positively charged L2NC would interact with negatively charged PETase surface and inhibit the immobilization activity.

Figure 4. The fold-change of immobilized protein samples activity over the activity in empty control from the same batch. The fold changes of activity from [Dou_PETase] to [Tri_PETase-L2NC] were calculated by [activity of experimental group]/[SHuffle without Lv.1 plasmid.2]. The fold changes of activity from [FAST_PETase] to [FAST_PETase-L2NC] were calculated by [activity of experimental group]/[SHuffle without Lv.1 plasmid.3] The error bars on column from [FAST_PETase] to [FAST_PETase-L2NC] were calculated by data from plate reader (Figure. 2) and spectrometer (Figure.4). We don’t have biological replicates for constructs. [Shuffle without Lv.1 plasmid.2] was the protein sample from the empty SHuffle strain of the same batch for Tri-PETase. [Shuffle without Lv.1 plasmid.3] was the protein sample from the empty Shuffle strain of the same batch for FAST-PETase.

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 647
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal SapI site found at 876
  

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.

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.

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).

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.