We focus on enhancing the thermal stability of PETase which exhibits the highest degradation activity of highly crystallized PET among all enzymes reported to date. However, its low thermal stability limits its practical utilization. We are committed to develop a strategy to improve the thermal stability of PETase, and therefore enhance the degradation efficiency of PETase in practical applications, making industrial recycling of highly crystallized PET possible .
Fig.1 Overview of our project
Firstly, we analyzed the reported overall structure of PETase. Secondly, based on the structural information of PETase, we rationally developed five rational approaches to increase the thermal stability of PETase, including disulfide bond, hydrogen bond, salt bridge, proline and hydrophobic interaction. A total of 415 parts, each developed based on one of these strategies, were then obtained. Thirdly, these 415 parts were screened via several software and websites such as FoldX, Fireprot, CUPSAT, I-Mutant, Swiss model and so on. Fourthly, 587 parts were obtained by rational combination of these 415 mutants and were screened by FoldX. Finally, after evaluating by MD and docking, we developed our best PETase mutant, named PET-CRUSHER. The expected melting temperature (Tm) of the PET-CRUSHER is 80.08℃, increased greatly by 31.27 ℃ as never before.
Poly(ethylene terephthalate), commonly abbreviated PET, is produced by the chain polymerization of ethylene glycol and terephthalate. Due to its durability, low price, and convenient processability, PET becames one of the most widely used synthetic plastics worldwide, with an annual manufacturing capacity of over 30 million tons[2,3]. PET has various applications in our daily life and industries, such as in manufacturing bottles, fibers, films, containers and so on. According to its applications, PET has different crystallinities. For example, PET used for packaging has less crystallinity, e.g. approximately 8%. PET used for manufacturing bottles and textiles has high crystallinity of 30–40%, which our team aims to degrade.
The harm of PET, especially highly crystallized PET
Fig.2 The destruction of ecology of PET
The strong demand and wide applications of PET have brought a global problem that is the accumulation of discarded PET in the environment. Discarded PET never truly leave the environment even physically broken but are present as micro and nano plastics that are ingested by marine organisms and continue to accumulate in their bodies[6,7].
According to multiple studies, more than 690 species of marine organisms have been found to accumulate the PET debris and microplastics in their bodies, which means that PET are choking marine life and spreading in the food chain[8,9,10].
It should be mentioned that among all discarded PET, highly crystallized PET accumulate most due to its durability, therefore greatly harms soil, marine and human
PETase makes the degradation of highly crystallized PET possible
Fig.3 The discovery of PETase
Previous studies have shown that a number of PET degradation enzymes including esterases, lipases, and cutinases indeed can depolymerize the amorphous or low crystallinity PET to some extent, but they lack the ability to degrade the high crystallinity PET.
Surprisingly, Yoshida et al. isolated a novel bacterium, Ideonella sakaiensis 201-F6, which is able to use PET as its major energy and carbon source. The key to its PET biodegradation ability is the secreted PETase (PET-hydrolyzing enzyme).
Compared with other enzymes, PETase prefers PET to aliphatic esters and uniquely shows enzymatic activity towards high crystallinity PET, making the degradation of highly crystallized PET become possible.
Engineering PETase around pocket didn’t improve much
|Mutant||PETa used in reaction||Relative acticityb||Assay conditions||References|
|S238F/W159H||14.8 ± 0.2% (Crystallinities)||3.8||30℃, pH 7.2, 96h/weight loss||12|
|R280A||commercial PET film||1.2 (18 h), 1.3 (36 h)||30 °C, pH 9, 18 or 36 h/hydrolysis||1|
|Y87A||drinking bottle||3.1||30 °C, pH 9, 48 h/hydrolysis||13|
|R90A||biaxially oriented PET film||1.4||30 °C, pH 8.5, 48 h/weight loss||14|
a.The crystallinities of substrate PET are estimated as follows: >30% for drinking bottles; 35% for biaxially oriented PET film. b.Ratio of value of the PET degradation activity of the PETase mutant to that of the wild type.
Recent studies that increase the activity of PETase focus on modifying the PET-binding groove (active pocket) or close to this region.
Austin et al. mutated the two active site residues W159 and S238 into the conserved amino acids in cutinase to narrow the binding cleft, and the resulting mutant S238F/W159H improved the PET degradation performance.
Through molecular docking, Joo S et al. found that R280 is located around the substrate binding site, which seems to hinder the extension of the substrate and mutated it to Ala, leading to the degrading activity of the mutant R280A on PET increased by about 33%.
Liu et al. also succeeded in creating several mutants with higher hydrolytic activity toward PET bottles, including Y87A, W159A, W159H, A209I and S214H. Among them, mutation of Trp159 located in the substrate binding pocket to Ala or His can effectively increase the activity and substitution Ala for Tyr87 residue which forms the oxyanion pore showed 3.1 times higher activity.
Ma et al. focused on six key residues around the substrate-binding groove and successfully obtained three mutants R90A, L117F and I208F, whose enzymatic activities exhibited 1.4 fold, 2.1 fold, and 2.5 fold increases, respectively, in comparison with wild-type PETase. Particularly, the mutant I208F exhibits higher affinity to PET owing to enhanced hydrophobic interactions.
Despite these attempts to improved the degradation activity, PETase was still low in activity. It is quite clear that more studies need to be done to further improve the performance of PETase.
Highly crystallized PET is hard to degrade but heating up can turn this around
Fig. 5 The limitation and solution
The crystallinity of highly crystallized PET restricts enzymatic to attack against it. Ester bonds in the polymer backbone are usually more susceptible to biodegradation than the C−C bonds. However, PET contains a high ratio of aromatic terephthalate units that reduces the chain mobility, resulting in extremely low hydrolyzability of the backbone ester linkages[16,17]. Enzymes are thought to preferentially attack the flexible amorphous domain, so the biodegradation rate of plastics decreases with increasing crystallinity, making highly crystallized particularly hard to degrade.
It is well known that the polymer chain fluctuates at temperatures higher than the glass transition temperature (Tg). The Tg value of PET is 75 ℃. Therefore, when the temperature rises to 75 ℃, the flexibility of the PET chain increases, which increases their accessibility to PETase. However, it is impossible for natural PETase to have activity at 75 ℃. So we attempted to improve the Tm (melting temperature, which characterizes the thermostability) of PETase over 75 ℃ in order to have a better degradation of high crystallized PET.
A newly published article confirmed our idea.
Fig. 7 LCC: degrade low crystallized PET
When we were working on our project, we found a paper published in NATURE in April. The paper mentioned another PET depolymerase LCC that can decompose and recycle plastic. They successfully improved the degradation efficiency by improving the thermal stability of LCC. This, to some extent, proved the feasibility of our strategy to improved the degradation efficiency of PETase by improving the thermal stability as well. What distinguishes us is that they focused on low crystallinity PET but we focused on high crystallinity one which is harder to degrade. Therefore, we have great confidence in our project.
COVID-19 can’t stop us!
Due to COVID-19, we could not do any experiment this year. Nevertheless, we took some modeling measures and developed a series of strategy to evaluate the thermal stability of our mutants. COVID-19 can’t stop us! Though joint effort we still got our PET- eam that has done PETase-related projects in previous years, we have got the following
At present, there is much room for improvement of the degradation activity of PETase with high crystallinity. After reading numerous articles and referring to the iGEM team that has done PETase-related projects in previous years, we have got the following results.
Projects of previous iGEM teams related to PETase
The iGEM 2016 team TJUSLS_China created PETase mutants to improve degradation efficiency. And then they displayed them on prokaryotic (E. coli) and eukaryotic (Pichia pastoris) surfaces to perform whole-cell enzyme-catalyzed reactions.
The iGEM 2019 team TU Kaiserslautern revolutionized plastic degradation by introducing Chlamydomonas reinhardtii (Chlamydomonas reinhardtii) as a eukaryotic secretion platform.
These two projects mainly focused on enhancing the activity of PETase to degrade PET film, but have not yet solved the problem of degradation of highly crystallized PET e.g. PET bottles. Therefore, we decided to improve the degradation efficiency of highly crystallized PET by modifying the structure of PETase itself.
A large amount of articles enlightening us
We found that Seongjoon Joo et al.  designed mutations based on the structure information about active pocket, but the degradation efficiency of those mutants did not improve much. This led us to consider the reasons for the failure of the strategy of transforming pockets. For highly crystallined PET, it is difficult to combine well with PETase owing to the tight structure of PETase. As we mentioned above, when the temperature rises to 75 ℃, the flexibility of the PET chain increases, which increases their accessibility to PETase. This brings us a strategic idea: to improve the thermal stability of PETase.
At this time, we found a paper published in NATURE in April. This paper mentioned another PET depolymerase LCC that can decompose and recycle plastic. They successfully improved the degradation efficiency by improving the thermal stability of the protein. This coincides with our strategy, and it verifies the feasibility of our strategy! However, LCC only can break down the low crystallized PET and PETase is the key to degrade high crystallized PET. Therefore, we have great confidence in improving the degradation of the high crystallized PET.
Inspired by numerous literature[17,19-33], through structural observation, we can select potential mutation positions and design hydrophobic core, disulfide bond, hydrogen bond, salt bridge, etc. And then we use FoldX to evaluate and verify the effect of improving thermal stability of PTEase. Therefore, we searched for the possibility of a single mutation from these multiple types and successfully constructed a mutation library. Unfortunately, through analysis, we found that we were not as lucky as our predecessors to get good results. But even so, we were not discouraged. Then we referred to the research of others , proposed to design rational combinations of these single-type mutations and constructed multiple-type mutations. In this process, we used visual analysis of the structure, software evaluation of thermal stability and kinetic simulation activity. After multiple attempts, we finally selected our "KING" from hundreds of multi-point mutation combinations.
Fig. 8 Overview of methods
Rational strategy design single type mutation
Fig. 9 Rational design
(a. Hydrophobic Interaction. b. Salt Bridge. c. Disulfide Bond. d. Hydrogen Bond)
After several months of rational design, we have successively got 415 single-type mutations, and completed constructive work from visual screening to various software analysis. The specific results we have been recorded in the notebook. These single-type mutations are as below. 100 disulfide related parts, for example, got by homologous comparison with Tfcut2 and LCC calcium ion sites and software, forming disulfide bonds to stabilize the loop N233C/S282C [17,19-21]; 127 parts related to hydrophobic interaction, such as Q127L, which forms a new hydrophobic interaction with M128/L131/V156; and N275F, which forms pi-pi accumulation with F284 [22-25]. 131 parts related to hydrogen bond, such as K148R that stabilizes two loops and stabilizes one loop and α-helix at the same time; Y146R that stabilizes the connection between loop and β-sheet while increasing hydrophilicity [26-28]. 47 parts related to the salt bridge, such as L216K, which forms a new salt bridge with D220 [29,30]. 10 parts related to proline, for example, S58P and S141P [31-33] which are the proline replaces on β-turn .
Permutation and combination mutation
Fig. 10 New idea: WIN-WIN cooperation
After finding that the effect of single-type mutation on the improvement of thermal stability was close to the threshold, we soon turned to the rational design and combination of high-value single-type mutations, from 2 mutation sites to 3, 4..., 12... We also adopted molecular dynamics simulation (MD), molecular docking, modeling and other methods to further verify the structural rationality and thermal stability of the mutant. At this stage, we have got 587 groups of high-value combinations, all of which are embodied in the notebook. Through visual evaluation, software data supporting, and contribution comparison, we finally obtained the best combination— PET-CRUSHER with 12 mutation sites combined, whose Tm is 80.08 ℃, increased 31.27 ℃, and more than Tm of PET(75 ℃).
Aims & Meanings
We want to address the problem that there is currently no applicable high-efficiency and high-crystallinity PET degradation method. We hope that we can contribute to solving issues, especially the actual role of PETase in environmental governance. For this, we screened PETase mutants that are expected to be practically used to degrade PET with high crystallinity, which may solve the current global ecological problem of PET pollution. Besides, we evaluated and verified the activity of the mutants by combining structural screening and molecular simulation technology, and proved the rationality of our entire project and new contributions to synthetic biology. We have successfully found our PET-CRUSHER!
For further research, we will continue to carry out experiments to further validate our results.
We also hope that with the mature results, the improved PETase can establish a new circular chain and save the entire ecosystem. This will be a major progress in global environmental governance.
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