Background

The first synthetic plastic, Bakelite, was produced in 1907, marking the beginning of the global plastics industry. Due to the excellent physicochemical properties and economic viability, plastic quickly conquered several industrial sectors, including packaging, healthcare, fisheries, and agriculture, over the 20th and 21st centuries. According to industry data, plastic production in the USA, Europe, and Asia currently accounts for 85% of the global total. Grand View Research has estimated that the global plastics market will experience a compound annual growth rate of 3.2% from 2020 to 2027. Unexpectedly, the coronavirus pandemic outbreak in 2019, abbreviated as COVID-19, has been considered an unprecedented healthcare crisis, which has severely disrupted nearly every aspect of daily life. Healthcare-related plastic products, especially disposable personal protective equipment (PPE) like medical-grade masks, gloves, medicinal syringes, antigen test kits, medical swabs, and medical protective clothing are applied to avoid millions of people suffering from the infection of SARS-CoV-2. The global usage of face masks reached a staggering 129 billion per month during the early stages of the pandemic (Table 1). According to the report of the Oceans Asia in December 2020, about 52 billion masks were produced worldwide in 2020, and at least 1.56 billion masks were discarded.

These phenomena unmasked the fact that plastic pollution is ubiquitous, that it interacts and reacts with a larger number of living organisms frequently, and thereby threatens the public’s health and the ecosystem. All these observations might trigger the dreadful “Plastic Pandemic” (Fig. 1). According to estimates from relevant authorities, the production of every 1 ton of plastic consumes 3 tons of oil, placing a significant strain on our resources. When plastic bags are discarded and burned, they emit a substantial amount of toxic and harmful gases, polluting the air. If buried in the ground, these bags take approximately 200 years to decompose, negatively affecting soil pH levels. Once plastic packaging and masks enter the natural environment, they prove resistant to degradation, causing persistent and deep-seated ecological issues. Surveys have revealed that plastic can be found almost everywhere on Earth's surface, including the deepest ocean depths, and even in the Antarctic and Arctic regions.

The abandonment of plastic and masks not only inflicts irreversible harm on the ecological environment but also poses a threat to the survival of marine and terrestrial creatures. Over 800 marine and coastal species suffer from this pollution through ingestion, entanglement, and other hazards. There have been documented cases of entanglement for at least 344 species, including 233 marine species directly or indirectly affected by consuming prey containing plastic. Moreover, under natural conditions, these plastic waste items gradually break down into minuscule plastic particles due to the effects of wind, sunlight, and ocean waves. Clinical studies have provided evidence that these particles can enter the human body through ingestion, inhalation, and skin absorption, accumulating in organs, including the placenta, causing significant harm to human health.

Problems

Currently, plastic processing methods are primarily categorized into three distinct groups: physical mechanical processes, chemical processes, and biological processes. Besides, incineration is a conventional method for managing discarded plastic waste. In the short term, incineration serves as a means to partially recover the energy stored within plastic waste. However, this approach falls short in generating additional economic value from waste or addressing long-term resource depletion concerns. Furthermore, it releases carbon dioxide and various volatile toxins, contributing to the deterioration of air quality over time. From a chemical decomposition perspective, the requirements for this method are quite stringent, often necessitating substantial quantities of thermal energy and organic solvents to break down plastics. This, unfortunately, leads to secondary environmental pollution. In contrast, biodegradation involving microbes and enzymes has emerged as a more environmentally friendly and sustainable alternative. It boasts reduced energy consumption, lower CO₂ emissions, and eliminates the risk of groundwater contamination. Notably, significant advancements have been made in recent years in the field of biodegradation, making it a promising solution for addressing the plastic waste challenge.

Inspiration

In 2016, the discovery of I. sakaiensis-derived PET hydrolase (PETase) provided a new perspective for the biocatalytic degradation of plastic waste (Fig. 2). 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.

Solution

1. The ancestral sequence reconstruction of PETase

We used profile-HMM algorithm for protein sequences in silico and obtained the ancestral sequence of PETase (ASR-PETase) by FireProt (https://loschmidt.chemi.muni.cz/fireprotweb/). We proceeded to investigate the evolutionary trajectory of PETase's functionality, pinpointing pivotal amino acid residues and structural domains through both structural and functional analysis. Our findings unveiled the catalytic mechanism of PETase, elucidated the evolutionary path of substrate specificity, and illuminated the characteristics of its reactions. Experimentally, ancestral sequences of PETase were heterologously expressed in E. coli for PET degradation activity validation.

2. A machine learning-guided strategy to engineer ASR1-PETase

The last few years have witnessed impressive progress in the tailoring of natural enzymes by computational redesign strategies. Inspired by the achievements in artificial intelligence for addressing the protein fitness landscape to probe hidden evolutionary information, we employed machine learning algorithms (ESM-1v model) to predict potentially beneficial variants of ASR-PETase. Experimentally, we detected PET-hydrolytic activity of potential variants through site-directed mutagenesis.

3. Mechanism analysis of increased enzyme activity by MD simulations

To demonstrate the driving force behind the increased degradation capacity, we investigated the Molecular Dynamics (MD) simulations of PETase and ASR1 variants. The optimal conformation was selected as the preparation material for MD simulations, followed by trajectory analysis to observe the dynamic changes and binding patterns of enzyme molecules with plastic substrates during the catalytic process. Based on the experimental findings, software GROMACS was utilized to construct a system consisting of plastic-degrading enzymes, plastic molecules, and solvent molecules. After system equilibration, the MD simulation of 100 ns was conducted. Through the structural observables and solvation observables analysis, the stability and activity of PETase and ASR1 variants were studied. Thus, the mechanism of the increased enzyme activity of the variants were identified (Fig. 3).

4. The closed-loop PET recycling 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 ASR-PETase and BHETase (BsEst, identified in our previous study) to produce the homogeneous TPA. Finally, we designed a chemical-enzymic approach based on the homogeneous TPA to repolymerize PET, achieving the closed-loop PET recycling (Fig. 4).

Reference

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