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Revision as of 11:02, 12 October 2023

ASR1(node 59)-V265T

Usage and Biology

Profile Name: ASR1(node 59)-V265T

Base Pairs: 918 bp

Properties: Mutate V265 of ASR1 (node 59) to T265 by site-directed mutagenesis.

Inspiration In 2016, the discovery of I. sakaiensis-derived PET hydrolase (PETase) provided a new perspective for the biocatalytic degradation of plastic waste. IsPETase can degrade PET to bis (hydroxyethyl) terephthalate (BHET), mono (2-hydroxyethyl) terephthalic acid (MHET), and terephthalic acid (TPA). However, the degradation efficiency of natural IsPETase for PET is limited by poor thermostability and low substrate binding efficiency. Therefore, we employed the ancestral sequence reconstruction strategy to understand the diversity of plastic hydrolases during the evolutionary trajectory. In this process, machine learning was used to design promising ASR-PETase variants with great efficiency at a high temperature. To complete a closed-loop PET recycling, we developed a simple two-enzyme system to produce the homogeneous TPA.

characterization

Solution

1. The Ancestral Sequence Reconstruction of PETase

Recently, some researchers have suggested that profile-HMM algorithm could be used instead of BLAST local alignment algorithm for protein sequences in silico in order to circumvent low overall sequence similarity between them. Using the Pfam entry PF01738 (dienelactone hydrolase family) and the HMMER Web server to search Swiss-prot and NCBI public databases, we obtained 158 protein sequences from all species. Expected values (E-value) were between 2.4 × 10–76and 0.47. Lower E thresholds are more stringent, leading statistically to fewer chance-matches being reported. The 50 first bacterial protein sequences (2.4 × 10–76≤ E ≤ 2.9 × 10–6) were selected and we deleted three duplicate records. Among these protein candidates, 39 proteins have dienelactone hydrolase (DLH) domains. 21 homologous sequences were then manually added for ancestral sequence reconstruction (ASR). These sequences were aligned, and the corresponding phylogenetic tree was built using a maximum likelihood algorithm by Fireprot server(Fig. 1a).Next, we sought to evaluate the PET-hydrolytic activity of reconstructed ancestral sequence (ASR1-7). As shown in Fig. 1b, ASR1-PETase and ASR7-PETase exhibited high activity in degrading PET, releasing monomer quantities of 3.44 mM and 3.82 mM, respectively. Compared with the wild type PETase, the identity of ASR1-PETase (21.37%) significantly lower than that of ASR7-PETase (67.25%)(Fig. 1c).

Fig. 1 Evolutionary analysis of PETase on the basis of reported PET hydrolases. (a) A phylogenetic tree constructed on the basis of Swiss-prot and NCBI databases employing FireProt. Number 1-7 represents ASR1 (node 59), ASR2 (node 98), ASR3 (node 100), ASR4 (node 109), ASR5 (node 111), ASR6 (node 112), and ASR7 (node 115). (b) PET depolymerization by ASR1-7. Reaction conditions: The PET films (ø=6 mm) were soaked in 2000 μL of Na2HPO4-NaH2PO4(pH 8.0, 50 mM) buffer at 40 °C with 100 μL of 0.5 mg/mL enzyme solution for five days. Error bars correspond to the standard deviation (s.d.) of three measurements (n = 3). (c) Sequence identities between wide type PETase and ASR1-7. (d) 3D structures of PETase and ASR1-PETase. Red sticks represent the catalytic triad (S131-D177-H208 of PETase and C161-D210-H242 of ASR1-PETase).

2. A Machine Learning-guided Strategy to Engineer ASR1-PETase

As shown in (Fig. 2c), the heat map of the replacement of sequence sites will be obtained after employing ESM-1v model, and the potential variants could be obtained according to the heat map. We select top 20 variants (including W120R) with the highest scores, and PET-hydrolytic activity was detected through site-directed mutagenesis (Fig. 2b).

Fig. 2 Machine learning （ESM-1v）guided predictions improve enzyme performance using ASR1 scaffolds. (a) Comparison of algorithms for ESM-1v, Evmutation, and Deepsequence. (b) Potential positions of ASR1-PETase by machine learning. The red sticks represent the catalytic triad, and the blue sticks represent the variant position. (c) The schematic of ESM-1v algorithm.

3. The High Thermostability Variants by Site-Directed Mutagenesis In our study, we designed 20 variants to investigate the thermostability and catalytic efficiency. As shown in Fig. 3a-b, the Bis (2-Hydroxyethyl) terephthalate (BHET) conversion of both ASR1-PETase and its variants was generally higher than that of the wild type PETase. One of them, a variant ASR1-V265T exhibited increased BHET conversion at both 30 °C and 50 °C. Meanwhile, the corresponding terminal product Terephthalic acid (TPA) of four variants, was also maintained at high yields (2.58 to 2.88 mM). Furthermore, hydrolysis advantages of ASR1-V265T was also presented on PET film. As depicted in Fig. 3c, the ASR1-PETase variants maintained hydrolysis activity at 60 °C, whereas most of the wild type PETase activity was lost over 30 °C. In details, these four variants exhibited improved catalytic efficiencies on PET film at 40 °C, up to 1.58-fold (ASR1-V265T). Additionally, it was observed that the variants ASR1-V265T maintained PET degradation products at levels of 2.40 mM and 2.69 mM, respectively, at a temperature of 60 °C, exhibiting an average increase of 2.32-fold compared to ASR1-PETase. Considering the necessity of high thermostability for industrial enzymes, we adopted a scoring strategy to identify variants with greater thermostability. We assigned different weights to the data obtained at 30 °C, 40 °C, 50 °C, and 60 °C, which were 10%, 20%, 30%, and 40%, respectively, to determine the composite scores. Based on the final scores, ASR1-V265T (Score 4.258) was selected to analyze the product composition (Fig. 3d-f). We found that the terminal product TPA accounted for 63% to 75% of the total PET released monomers, while the remaining portion mainly comprised intermediates BHET and monohydroxyethyl terephthalate (MHET). These results indicated that enzymatic degradation of PET by a single enzyme will generate inhomogeneous products, resulting in contamination of the terminal product TPA. Considering that mixed products are unfavorable for subsequent reuse, we shifted our focus to the production of homogeneous TPA.

Fig. 3 Experimental verification of the variants upon BHET and PET film. 20 variants were validated through BHET experiments, including (a) conversion of BHET and (b) concentration of TPA. Reaction conditions: The BHET (5 mM) was dissolved in 2000 μL Na2HPO4-NaH2PO4buffer (pH 8.0, 50 mM) at 30 °C and 50 °C with 100 μL crude enzyme solution for 6 h. Error bars correspond to the standard deviation (s.d.) of three measurements (n = 3). (c) Product released from PET depolymerization by ASR1-V265T, ASR1-N81H, ASR1-W120R and ASR1-L122K. Score = C30 °C*10% + C40 °C*20% + C50 °C*30% + C60 °C*40%. Product composition of PET depolymerization by (d) ASR1, (e) ASR1-V265T, and (f) ASR1-W120R. Reaction conditions: The PET films (ø = 6 mm) were soaked in 2000 μL of Na2HPO4-NaH2PO4buffer (pH 8.0, 50 mM) at 30 °C, 40 °C, 50 °C and 60 °C with 100 μL of 0.5 mg/mL enzyme solution for five days. Error bars correspond to the standard deviation (s.d.) of three measurements (n = 3)

4. Mechanism Analysis of Increased Enzyme Activity by MD Simulations

The solvation of the active site of an enzyme was an important factor affecting its activity. Different enzymes had different characteristics at the active site, and the binding of solvent molecules to the active site were also varied. As showed in Fig. 4f, under the conditions of 30 °C and 60 °C, the number of water molecules around the substrate binding site of ASR1 variants was more than twice as much as PETase, and the number of water molecules around the substrate binding site of ASR1-V265T was 2 to 4 more than that in ASR1-WT. This suggested that the substrate binding site of the ASR1 variants could attract more water molecules. Meanwhile, as evident from (Fig. 4g), the active sites of the four enzymes kept getting closer to PET dimer in water as simulation time increased, and finally tended to be stable. When a stable state was reached, the distance between the active site in ASR1-WT and PET dimer was less than that between the active site in PETase and the PET dimer core. In addition, the distance between the active site and the PET dimer core of the two ASR1 variants was less than that of ASR1-WT. This suggested that compared with PETase, it was easier for the substrate to enter the active center of the ASR1 variants, therefore, promoting the transformation of the substrate. These results indicated that the activity of ASR1 variants designed by our team was better than PETase, and the activity of ASR1-V265T variants was superior to ASR1-WT.

Fig. 4 Overall structural change and solvation phenomena around the whole enzymes of PETase and ASR1 variants in water. (a) Time-average RMSD of the heavy atoms of four enzymes determined from the last 40 ns of simulation in water. (b) The radius of gyration (Rg) of four enzymes with respect to the initial structure as a function of time in water. (c) The time-averaged total SASA, the time-averaged hydrophobic SASA, and hydrophilic SASA of four enzymes at temperatures of 30 °C. RMSF of each residue of (d) PETase and (e) ASR1-W120R was determined from the last 40 ns simulation at temperatures of 30 °C and 60 °C with three independent MD runs. (f) The average number of water molecules around the substrate binding site during the MD simulations at temperatures of 30 °C and 60 °C. The number of water molecules averaged over the last 40 ns from three independent MD runs. (g) The distance between the active sites of the four enzymes and the centroid of the PET dimer in water. All error bars show the standard deviation from three independent MD runs.

5. Complete TPA Conversion of PET Achieved by A Simple Two-Enzyme System TPA produced by a single-enzyme degradation system often suffered from contamination by oligoethylene terephthalates, BHET, and MHET, which posed limitations on downstream applications. To overcome the obstacle, we have developed a two-enzyme degradation system that combines ASR1-V265T and ASR1- W120R, both performing better in preliminary validation and MD simulations, with BHETase (BsEst, identified in our previous study). It was proved that the system enables the production of sole homogeneous TPA, as illustrated in Fig. 5a. Coupling BsEst with two variants of ASR1 respectively in a two-enzyme system significantly increased TPA production compared to using variants alone in a single-enzyme system. As depicted in Fig. 5b, ASR1-V265T/BsEst increased TPA production by 1.7-fold over 120 h. Moreover, we observed that these two-enzyme systems produced 1.9-fold and 2.3-fold higher amounts of homogeneous TPA at 30 °C than at 60 °C. The discovery mentioned above was further supported by the analysis of the PET film surface using scanning electron microscopy (SEM), and the measurement of reduced the water contact angle (Fig. 5c). Notably, the two-enzyme system exhibited a remarkable efficiency degrading the PET film with up to a 15.6 °C water contact angle decrease in average at 60 °C, resulting in a visibly rougher surface compared to the single-enzyme system. To implement our team's sustainability philosophy and address the additional energy costs associated with high temperatures, we also investigated the PET degradation efficiency of the two-enzyme system at room temperature (30 °C) to achieve a more environmentally friendly process. We surprisingly observed better effects at room temperature compared to 60 °C. Specifically, the average reduction in water contact angle is 23.7° at 30 °C, 1.5 fold higher than at 60 °C.

Fig. 5 A two-enzyme degradation system at 30-60 °C. (a) An overview of the two-enzyme degradation system. Icon graphics of this figure was created by BioRender.com. (b) HPLC data of homogeneous TPA. Reaction conditions: The PET films (ø = 6 mm) were soaked in 2000 μL of 50 mM Na2HPO4-NaH2PO4(pH 8.0) buffer at 30 °C, 40 °C, 50 °C, 60 °C with 100 μL of 0.5 mg/mL enzyme solution for five days. Error bars correspond to the standard deviation (s.d.) of three measurements (n = 3). (c) HPLC data of homogeneous TPA in the reaction system after 120 h of degradation by the two-enzyme system. (d) The SEM images (up panel) and water contact angle analysis (down panel) of the PET film in two-enzyme degradation system with BsEst after 48 h at 60 °C.

Discussion

PET biodegradation represents a sustainable, low-energy solution for PET recycling, especially compared to current disposal routes such as landfill and incineration. Recent protein engineering and the more than 70 crystal structures of bacterial PET-degrading enzymes have improved our understanding of the enzyme degradation process, but there are remained challenge about the balance between high thermostability and catalytic efficiency. This project proposes to start from the ancestor sequence of PETase and deeply explore the evolution of PET, meanwhile, to provides the skeleton for the protein engineering design of PETase. Inspired by the achievements of artificial intelligence in solving the field of protein fitness to detect hidden evolutionary information, we adopted the machine learning ESM-1v model and redesigned PETase ASR1-V265T. Compared with wild-type ASR1-PETase, the four variants exhibited improved catalytic efficiencies on PET film at 40 °C, up to 1.58-fold (ASR1-V265T). Additionally, it was observed that the variants ASR1-V265T maintained PET degradation products at levels of 2.40 mM. After MD simulations, the mechanism of promoted enzyme performance has been proofed, emphasizing that the number of water molecules around the substrate binding site increased, and the distance between the substrate and the active center was closer. At the same time, the two-enzyme system constructed in this study provides homogeneous TPA for subsequent PET repolymerization, enabling the closed-loop PET recycle, and provides guidance for further investigation of other mass-produced polymer in this interesting research field.

Sequence and Features