Team:Tianjin/Experiment/Protein Engineering

Experiment of Protein Engineering

Motivation

The degradation of PET is completed in two steps by PETase and MHETase. PETase hydrolyze PET to mono(2-hydroxyethyl) terephthalic acid (MHET), which will be further decomposed by MHETase into two monomers, terephthalic acid (TPA) and ethylene glycol (EG . In this two-step reaction system, MHET, the product of PETase-mediated hydrolysis of PET, was found to be a very minor component, which reveals rapid MHET, indicating the rate determining step in this reaction is the first step, hydrolysis of PET. So in order to accelerate the whole PET degradation speed, increasing the activity of PETase is rather crucial. To enhance PETase hydrolysis activity, we first tried to understand the mechanism of the hydrolysis reaction by generally confirming active sites of PETase and 3 dementional structure simulation.

Fig.1 Simulated 3D structure for PETase

Serine-based Catalytic Triad Mechanism & 3D Model Simulation

Since there is no x-ray structure for PETase , the mechanism of PETase hydrolysis activity can not be exactly identified. But the binding site and catalytic site can be generally inferred according to α/β hydrolase mechanism. Based on the efforts have been made to identify and characterize bacterial cutinases[2], α/β hydrolase fold family contain a highly conserved characteristic GXSXG motif. With sequence analysis, PETase was also found to contain an accordant GWSMG motif. Meanwhile, we simulated a best fit model for PETase by SWISSMODEL, an automated comparative protein modeling server[3]. The template was Thc_Cut2, which shares 52％ sequence identity with PETase [4]. As expected, the homology model of PETase displays a canonical α/β hydrolase fold with a Ser161-His237-Asp237 catalytic triad and a preformed oxyanion hole (Fig.1), suggesting a classic serine hydrolase mechanism.
Ribbon diagram of a predicted PETase model. The catalytic triad residues are shown as ball-and-sticks in green, formed by Ser161, His237 and Asp237, and the oxyanion hole binding site residues are in blue, formed by the main chain amides of Met161 and Tyr87.

Mutation Design Rationales

1.Hydrophobicity & space factor

Given that the hydrolysis of PET is a heterogeneous reaction, a prerequisite for its efficient reaction is an adequate interaction of the enzyme with the substrate at the solid-liquid interface. Since the substrate PET is hydrophobic polymer, both the hydrophobicity of the enzyme surface and the space around active site to accommodate PET may be the main factors for more efficient protein attachment and reaction activity[6]. In fact, tailoring enzymes for a more hydrophobic and larger substrate active site has been shown to facilitate PET hydrolysis by a fungal polyester hydrolase[5]. Also Silva et al. obtained a double mutant (Q132A/T101A) exhibiting a 2-fold increased hydrolysis activity against PET fibers by the substitution of spacious residues with alanine in its substrate binding site[6]. So we aimed to take the same strategy to modify hydrophobicity of the surface of PETase as well as broaden space near the active site to enhance the interaction between PETase and substrate.

2.Electrostatics factor

The electrostatic surface property in the vicinity to the active site was also found to be responsible for differences in hydrolysis efficiency. Exchange of the positively charged amino acids to the non-charged ones strongly increase the hydrolysis activity. In contrast, exchange of the uncharged amino acids by the negatively charged ones can lead to a complete loss of hydrolysis activity on PET films[7]. Changing the surface property is another strategy we took.

3.Conserved Residues

In another study, Wei and coworkers exchanged selected amino acid residues of TfCut2 involved in substrate binding with those in LC-cutinase(LCC) to relieve product inhibition and obtained enzyme variants with increased PET activity at 65 [8]. In the inspiration of their work as well as that the position of catalytic triad hold constant in different mature PET hydrolases with signal peptide excluded, we exchanged amino acid residues on the surface with highly conserved residues of the same position in other PET hydrolases, which can be a way to either increase the enzyme efficiency or confirm the essential sites which made PETase more efficient than other PET hydrolase.

Fig.2 Hydrophobicity of PETase surface
The deeper the bule is, the more hydrophobic the protein surface is; On contrast, the deeper the brown is, the more hydrophilic the protein surface is.

Based on the above rationales, our general strategies of mutation design are as follows:

1).According to the 3D structure of PETase we simulated, which shows the hydrophobicity of protein surface (Fig.2), we changed the hydrophilic amino acid residues on the surface with hydrophobic ones to enhance the surface hydrophobicity;

2). Modify the electrostatics of its surface by changing charged amino acid residues to the uncharged ones;

3). Based on 3D structure predicted and docking simulation with substrate, we found several spacious residues adjacent to active site and binding site, which may inhibit the interaction of protein and substrate due to its large body. We substituted them with smaller amino acids to expose the catalytic site and binding site;

4). We generated a multiple sequence alignment by ClustalX with seven serine hydrolases from NCBI conserved with GXSXG motif and catalytic triad and reported to be able to degrade PET (Table.1). Based on the multiple sequence alignment result(Fig.3), we exchanged some essential residues adjacent to active site and binding site with the conserved residues of the same position in the multiple sequences.

Table.1 Enzymes in multiple sequence alignmentr

Fig.3 Multiple Sequence Alignment
The catalytic triad is shaded in blue box and the binding site is shaded in green box. The position of catalytic triad hold constant throughout the multiple sequences in mature protein with signal peptide excluded.

Mutants

Based on the Rationales above, we designed 22 potential mutations (Table.2) in purposes to enhance the efficiency of PETase hydrolysis activity and confirm potential determining site of PETase hydrolysis.

Site-directed mutagenesis

Site-directed mutagenesis was performed using recombinant PCR technique[9]. Our modified approach is based on the PCR amplification of target PETase fragment by mutagenic primers divergently oriented but overlapping at their 5’ ends. The mutagenic nucleotides are located in both forward and reverse primers. The approach contained two rounds of PCR. In the PCR Round I, we use PETase fragment as template, respectively use original forward primer of PETase & mutagenic reverse primer, and original reverse primer & mutagenic forward primer as primers. The product of PCR Round I are portions of PETase gene with an overlapping region. In the PCR Round II, we use products of PCR Round I as two templates and original primers as primers to generate the final mutant fragment (Fig.4). And each specific mutation was verified by sequencing.

Fig.4 Site-directed Mutagenesis

References

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[2] Chen S, Tong X, Woodard R W, et al. Identification and characterization of bacterial cutinase[J]. Journal of Biological Chemistry, 2008, 283(38): 25854-25862.

[3] Schwede T, Kopp J, Guex N, Peitsch MC. SWISS-MODEL: an automated protein homologymodeling server. Nucleic Acids Research, 2003, 31 (13): 3381-3385

[4] Yoshida S, Hiraga K, Takehana T, et al. Supplementary Material For A bacterium that degrades and assimilates poly (ethylene terephthalate)

[5] Araújo R, Silva C, O’Neill A, et al. Tailoring cutinase activity towards polyethylene terephthalate and polyamide 6, 6 fibers[J]. Journal of Biotechnology, 2007, 128(4): 849-857.

[6] Silva C, Da S, Silva N, et al. Engineered Thermobifida fusca cutinase with increased activity on polyester substrates[J]. Biotechnology journal, 2011, 6(10): 1230-1239.

[7] Herrero Acero E, Ribitsch D, Dellacher A, et al. Surface engineering of a cutinase from Thermobifida cellulosilytica for improved polyester hydrolysis[J]. Biotechnology and bioengineering, 2013, 110(10): 2581-2590.

[8] Wei R, Oeser T, Schmidt J, et al. Engineered bacterial polyester hydrolases efficiently degrade polyethylene terephthalate due to relieved product inhibition[J]. Biotechnology and bioengineering, 2016.

[9] Ansaldi M, Lepelletier M, Méjean V. Site-specific mutagenesis by using an accurate recombinant polymerase chain reaction method [J]. Analytical biochemistry, 1996, 234(1): 110-111.

[10] Roth C, Wei R, Oeser T, et al. Structural and functional studies on a thermostable polyethylene terephthalate degrading hydrolase from Thermobifida fusca[J]. Applied microbiology and biotechnology, 2014, 98(18): 7815-7823.