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C-terminal Car9-tagged Tri-PETase

Usage and Biology

We designed the C-terminal Car9-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).

Car9 is a short silica-binding tag to add to the C-terminal of a protein using JUMP assembly, including a short glycine-rich linker (GSGGGS). The tag facilitates immobilisation to silica surfaces with a dissociation constant (1 µM), enabling enzyme immobilisation or purification using silica-based spin columns. The advantage of using Car9 silica tag is its small size (1.87 kDa) would introduce smaller effect to the functional enzyme activity in theory.

Design

C-terminal Car9-tagged Tri-PETase was assembled by JUMP assembly with: T7 promoter (P part)-B0034 RBS (RN part) -[Tri-PETase] (O part)-[linker-Car9] (C part)-L1U1H08 (T part). All the codons were optimized for BioBrick and JUMP assembly.

Characterization

All the Lv.0 parts for [C-terminal Car9-tagged Tri-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 C-terminal Car9-tagged Tri-PETase PCR product (Figure 1. H1 and H2) was corresponding to 1344 bp in silico.

Figure 1. Agarose gel showed the PCR result of [C-terminal Car9-tagged Tri-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. H: C-terminal Car9-tagged Tri-PETase. The ladder used: 1 kb DNA Ladder from NEB (N3232S).

After being transformed with the Lv.1 plasmid [C-terminal Car9-tagged Tri-PETas] into E.Coli Shuffle strain, the cells grew and were sonicated for solubility test. The weight of C-terminal Car9-tagged Tri-PETase should be around 33.33 kDa, which is corresponding to the red line in Sample2 lane (Figure 2).

Figure 2. SDS-PAGE gel showed the solubility test result of [C-terminal Car9-tagged Tri-PETase] fusion proteins. The lanes were labelled with letters, and the number behind each letter represented different fractions of the cell samples. H: C-terminal Car9-tagged Tri-PETase. Sample 1: the cell sample before sonication. Sample 2: the supernatant of cell lysate after centrifuge. Sample 3: the resuspended insoluble portions of cell lysate after centrifuge. The ladder used: P7718S protein ladder from NEB.

After making sure [C-terminal Car9-tagged Tri-PETase] was expressing, we assessed its 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. Data for Tri-PETase and FAST-PETase were measured on different days (Figure 3). We observed an inconsistency in the empty control activity towards pNPB across different days. Therefore, we calculated the fold-change of individual protein sample activity towards the empty control in the same batch to reduce the inconsistency when comparing the data.

Figure 3. The protein sample activities result based on para-nitrophenol-butyrate pNPB assay. The figure presented the fold-change of protein samples activity over the activity in empty control from the same batch. The fold changes of activity from [Tri_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 reaction system was set up with final volume 1ml in each Eppendorf tube, 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). The absorbance was measured from the Spectrometer at 415nm. [Shuffle without Lv.1 plasmid.2] was the protein sample from the same batch of the empty SHuffle strain as Tri-PETase constructs. [Shuffle without Lv.1 plasmid.3] was the protein sample from the same batch of the empty SHuffle strain as FAST-PETase constructs. “U”is the amount of activity which releases one micromole of pNP per minute under these assay conditions. The activity of [Shuffle without Lv.1 plasmid.2] is 1.24E-03 U/mg protein sample. The activity of [Shuffle without Lv.1 plasmid. 3] is 4.44E-03 U/mg protein sample.

The [C-terminal Car9-tagged Tri-PETase] showed 4.04-fold higher activity towards pNPB over the empty control in the same batch (Figure 3).

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 4. 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 C-terminal Car9-tagged Tri-PETase showed the immobilization efficiency (44.29%), higher than the empty controls.

Figure 5. 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.

The C-terminal Car9-tagged Tri-PETase showed lower activity comparing to empty control in the same batch. We assumed that the protein loading on each silica bead should be low. If not, the enzymes may crowd together and inhibit each other’s activity. The data indicated that the protein loading per silica bead should be well defined to maintain the enzyme activity after immobilization, especially for Car9 silica tag which showed very strong binding activity to silica beads.

Sequence and Features

Reference

Coyle BL, Baneyx F. A cleavable silica-binding affinity tag for rapid and inexpensive protein purification. Biotechnol Bioeng [Internet]. 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.