OVERVIEW

Plastic pollution has long been an old yet tricky problem that remains poorly tackled. However, traditional plastic materials, exemplified by polyethylene (PE), are still widely applied in different aspects of human activities in large quantities, especially in the booming express delivery industry. This inevitably caused severe resource loss and environmental contamination, as well as posted a great threat to species diversity. Therefore, it is of vital urgency to search for green and efficient methods to better degrade this kind of plastics.

PE belongs to other waste. Its major disposal methods under current situation are incineration and landfill, both of which are not the optimal way of disposing PE, for the reason that not only hazardous gas or harmful organic pollutants are produced, but also the occupancy of enormous land resources is observed.

Under the encouragement of government policy, recent years have seen a rise of PE recycling and reusing industry. However, its relatively small scale and high technological barrier have temporary hindered its further promotion, and constrained its ability to cleanse PE waste to a larger extent.

Therefore, in order to cope with the environment issue posted by the majority of unrecyclable PE, by integrating facts and data in literatures, as well as the on-site observations and feedback from our Human Practices members, we decided to take advantage of the power of nature, seeking specific agents that possess the unique ability of degrading PE.

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PRIMARY GOAL: TO SELECT A CENTRAL PE DEGRADATION AGENT

AGENT SELECTION

During our preliminary stage of literature research, strains of microorganism as well as enzymes that both had the potential of PE degradation were obtained by us. An either-or decision must be made upon the selection of the PE-degrading agent. Without much hesitation, we selected enzymes instead of strains due to a more definite origin and characteristics provided by online databases. After screening through potential candidates, the very manganese peroxidase (MnP) was selected as our central functional element.

It is a highly glycosylated lignin peroxidase with heme^[1,2] that can oxidize Mn²⁺ to Mn³⁺, the latter can be chelated by ligands like oxalic acid, forming the Mn³⁺-ligand chelate compound that can diffuse outside the enzyme for further degradation of lignin or other refractory chemicals^[3].

Fig. 1 The catalytic cycle of MnP.

It has been reported that MnP has a significant degradation efficiency on PE film. As reported before, the weight-average molecular weight (Mw) of PE was halved by MnP treatment for two days, showing its remarkable degradation efficacy^[4]. Thus, MnP was chosen by us as the key element for PE degradation.

USING AAO AS A BETTER APPROACH TO PROVIDE SUBSTRATE FOR MnP

It is shown on the catalytic cycle of MnP above that H₂O₂ is required as its essential substrate for activating the enzymatic reaction. Yet an abnormally high concentration of H₂O₂ could also inhibit, even deactivate the enzyme, which might happen when H₂O₂ was added into the system manually and periodically.

Therefore, a more in-depth investigation was carried out to seek a solution. As a result, we discovered a specific type of enzyme, namely aryl alcohol oxidase (AAO). It is an enzyme containing flavin-adenine-dinucleotide (FAD) that catalyzes the oxidation of aromatic and aliphatic allylic primary alcohols (which are far less oxidative when compared to Mn³⁺ and H₂O₂) to the corresponding aldehydes while reducing molecular oxygen to H₂O₂.

Fig. 2 The mechanism of AAO reducing molecular oxygen to H₂O₂ by oxidizing 4-methoxybenzyl alcohol.

We learned from the literature that AAO is able to produce H₂O₂ in a low but steady rate. Therefore, the inhibition of MnP due to an excess of H₂O₂ concentration can be effectively prevented when applying AAO as the source of H₂O₂. This would allow MnP to catalyze the PE-degrading reaction over a longer period of time, realizing a more complete degradation of PE. In addition, since the two enzymes work in tandem, the cascade reaction mediated by the two can only be initiated when substrates of AAO is introduced to the system. Therefore, we can achieve precise control to the onset and termination of the reactions via adding specific amount of substrates to the system in a given time, preventing uncontrollable situations from happening. As a result, we decided to select AAO as the assistant of MnP.

SECONDARY GOAL: TO ENHANCE THE PE-DEGRADING EFFICIENCY OF MnP.

OPTIMIZE THE DEGRADATION COMPETENCE OF MnP BY DIRECTED EVOLUTION

As our key PE-degrading enzyme, manganese peroxidase (MnP) undertakes a fundamental role of inflicting oxidation to PE by continuously producing Mn³⁺ ions. Therefore, enhancing the degradation efficiency of MnP is beneficial to reach a more complete destruction of PE films.

In theory, there are two approaches of reinforce the degradation efficacy of MnP, whether by increasing the activity of MnP to realize a stronger oxidative capacity, or by improving the stability of MnP to prolong its duration of effect. However, since the substrate and catalysate of MnP are both highly-oxidative, simply increasing its activity without restrictions is bound to cause irreversible harm not only to the MnP itself, but also to other affiliated elements in our design, AAO for instance. Therefore, we decided to improve the stability of MnP by proposing a semi-rational directed evolution strategy towards it, with the hope to increase its tolerance of high temperature, acidic pH, as well as different types of organic solvents, all of which are common inhibitory physiochemical properties that may severely impact the activity of MnP.

FACILITATE THE SURFACE ADHERENCE OF MnP BY INTRODUCING HFB1

Back to the stage where we were searching for agents with PE degradation efficacy, we noticed that certain bacterial or fungal strains capable of degrading PE could produce biosurfactant to assist their adherence and colonization on the hydrophobic surface of plastics, so that they could degrade PE in a faster pace. This inspired us to introduce biosurfactant into our design, aiming to increase the hydrophilicity of the surface of PE.

As a result, our focus was concentrated on hydrophobin-1 (HFB1), a kind of class Ⅱ HFBs derived from Trichoderma reesei. It is rich in hydrophobic amino acids, endowing its surface activity. By self-assembling at hydrophilic-hydrophobic interfaces autonomously, HFB1 can enhance the affinity between hydrophilic proteins and hydrophobic materials such as PE, thus facilitating its contact with aqueous environment, thereby facilitating MnP to degrade PE.

What's more, compared with other members of HFBs, HFB1 has better stability and higher surface activity, which means it can maintain its function of adherence on hydrophobic substances more firmly for a longer period of time.

Therefore, in our project, HFB1 is selected and used as a biosurfactant to produce consistent surface activity on PE, thereby promoting the adherence of MnP on PE surface, which helps to improve the degradation efficacy of this enzyme.

TERTIARY GOAL: CONVERGE THE ADVANTAGES OF THREE FUNCTIONAL PROTEINS

Now that the three functional proteins were selected, all of which possesses individual functions that could contribute to the degradation of PE, instead of directly applying all of them by simply adding them into the system separately, we began to consider the possibility of combining these discrete parts into a composite entity, enabling the production of a strong synergistic effect which may lead to an significant improvement on efficacy.

GETTING CLOSER TO THE SURFACE OF PE

The first idea that struck us was that we could minimize the spatial distance between MnP and PE by fusing HFB1 on the enzyme. Similar strategy could also be applied on AAO to generate fusion protein as well. In this way, our functional enzymes can simultaneously be anchored to the PE surface with the aid of fused HFB1, so that the diffusion distance of Mn³⁺-ligand chelate compound towards PE could be significantly lessened, enabling a more efficient degradation outcome. Meanwhile, the H₂O₂ generated by AAO can also become more accessible to MnP when the two enzymes are closely anchored to the surface of PE.

Therefore, we delved into literatures and previous iGEM projects to look for ideal solutions. It turned out that there existed a versatile protein ligation system, i.e. SpyCatcher/SpyTag system, that has been widely adopted by many laboratories and iGEM teams for construction of multi-domain protein. This system contains two essential elements:

• SpyCatcher: a modified immunoglobulin-like domain CnaB2 from a Streptococcus pyogenes surface protein
• SpyTag: a cognate 13-amino-acid peptide

Fig. 3 The isopepide-forming mechanism between the two Spy domains. Glu77 & Lys31 are the residues on SpyCatcher; Asp117 is the residue on SpyTag.

The two domains can autonomously form a covalent isopeptide bond between each other, thereby linking the two portions together. By linking the Spy domains on the N-terminal or C-terminal of the target protein with elastin-like protein (ELP) or serine/glycine link (Ser/Gly link), its structure and function are generally unaffected, while the formation of isopeptide bond between SpyCatcher and SpyTag remains effective and efficient. By adopting this system, MnP and AAO that was fused with HFB1 are able to stick to surface of PE, realizing a better spatial concentration on it.

GETTING CLOSER WITH EACH OTHER

Albeit introducing SpyCatcher/SpyTag connect system into our design could have a positive influence on accelerating PE degradation, several shortcomings are not yet solved. For example, the adherence of MnP-HFB1 and AAO-HFB1 fusion proteins on the surface of PE are likely to be unordered instead of evenly distributed. Protein clusters of the same type of fusion protein are likely to be formed on the PE surface, preventing thorough substrate exchange for those protein molecules in the middle of the cluster. Also, the maintenance of optimum functioning ratio between MnP and AAO cannot be guaranteed due to the arbitrary distribution on the PE surface. Both of the two uncontrollable conditions will exert an negative impact on the efficacy of PE degradation.

To enable the binding of MnP and AAO on the surface of PE in a more organized manner, we adopted a recently reported CRISPR/Cas-based DNA anchoring system to our design. This system utilizes an deactivated CRISPR-associated protein 9 linked to a SpyCatcher domain (dCas9-SpyCatcher), which can not only form covalent bond with proteins fused with SpyTag domain, but also recognize and bind to complementary DNA sequences after incorporating a single-guide RNA (sgRNA) without cleavage activity.
Therefore, by specially designing a double-stranded DNA with multiple sequence segments complementary to different sgRNAs, the dCas9-SpyCatcher incorporated with different types of sgRNAs and functional proteins can be anchored to the double-stranded DNA in a predetermined number and proportion, which

In our project,

THE OVERALL DIAGRAM

Eventually, by combining the three PE-degrading elements with the two assembly systems, we are able to construct a new type of protein-nucleic-acid complex that possesses an enhanced ability for PE degradation.

A demonstrative graph is shown below (Fig. 5).