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, inevitably causing severe environmental contamination, as well as posting a great threat to species diversity. Therefore, it is of vital urgency to search for green and efficient methods to better degrade these kinds of plastics.

As we were in search of ideal management method of disposed polyethylene waste, three functional proteins, including two enzymes, were selected to reach our goal:

• Manganese Peroxidase (MnP) : the key PE-degrading element.

  It is derived from fungi and utilizes hydrogen peroxide to produce high-redox-potential trivalent manganese ions that can oxidize a considerable variety of substances, including PE.

• Aryl Alcohol Oxidase (AAO): assists the function of MnP.

  It is a type of hydrogen-peroxide-producing enzyme for activating fungal peroxidases in the natural lignin decomposition process.

• Hydrophobin-1 (HFB1): enhances substrate adherence.

  A type of surface-activating protein. It is applied aiming to decrease the hydrophobicity of PE surface, thereby increasing the degradation efficacy of our enzymes.

To further converge the advantages provided by the three PE-degrading elements for improved performance, we began to consider the possibility of applying an integrated assembly system, consisting of the following two subsystems:

• SpyCatcher/SpyTag system

  It enables random proteins fused with reciprocal Spy domains to be linked together through the formation of a covalent bond.

• CRISPR/dCas9 system

  It promotes a programmable, specific binding of single strand RNA-guided deactivated CRISPR associated protein 9 (sgRNA:dCas9) towards a designed double-stranded DNA with variable interval, proportion and order.

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. 1).

Fig.1 Graphical abstract of our PE-degrading complex.

Further explanations and detailed information regarding elements, systems and chassis applied in our design can be found in the following contents.

PE DEGRADING ELEMENTS

As is briefly described earlier, each PE-degrading element plays a different but irreplaceable role in the whole integrated system. Their detailed characteristics and division of labor are displayed below.

MANGANESE PEROXIDASE (MnP)

Manganese Peroxidase (MnP) is a highly glycosylated lignin peroxidase with heme. It can oxidize Mn²⁺ to Mn³⁺, which can be chelated by ligands like oxalic acid, forming the Mn³⁺-ligand chelate compound that can diffuse outside the enzyme for further degrading of lignin or other refractory chemicals.


Fig. 2 The catalytic cycle of MnP.

Moreover, 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 after being treated for two days. Thus, MnP was chosen by us as the key element for PE degradation.

In our complex, MnP is assisted by two other elements. This enables MnP to gain H₂O₂ in a stable and consistent rate, as well as to get closer to PE. Under such coordination, MnP could achieve a better function and accelerate PE degradation.

ARYL ALCOHOL OXIDASE (AAO)

Aryl alcohol oxidase, a member of the glucose-methanol-choline oxidase/dehydrogenase (GMC) superfamily, is an enzyme containing flavin-adenine-dinucleotide (FAD) that catalyzes the oxidation of aromatic and aliphatic allylic primary alcohols to the corresponding aldehydes while reducing molecular oxygen to H₂O₂ (the corresponding mechanism is shown on Fig. 3).


Fig. 3 The mechanism of AAO reducing molecular oxygen to H₂O₂ by oxidizing 4-methoxybenzyl alcohol.

In our project, we plan to use AAO as a H₂O₂-producing enzyme to assist MnP to play its role.

HYDROPHOBIN-1 (HFB1)

Hydrophobin (HFB) is a type of biosurfactant rich in hydrophobic amino acids, possessing surface activity. By self-assembling at hydrophilic-hydrophobic interfaces autonomously, HFBs can enhance the affinity between hydrophilic proteins and hydrophobic materials, such as PE, thus facilitating its contact with aqueous environment. Hydrophobin-1 (HFB1) is a kind of class Ⅱ HFBs derived from Trichoderma reesei. 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 used as a biosurfactant to produce consistent surface activity on PE, therefore adhering the whole molecular machine on the PE surface, which will eventually improve the degradation efficacy of our protein-nucleic acid complex.

ASSEMBLY SYSTEM

In order to maximize the advantage of the three elements, two kinds of assembly systems were selected and cooperatively introduced into our system to integrate and align the enzymes and biosurfactant on one double-stranded DNA. In this way, the whole complex can be successfully constructed.

SPYCATCHER/SPYTAG CONNECT SYSTEM

SpyCatcher/SpyTag system is a convenient technique used for protein ligation. It contains two elements:

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

The two domains can autonomously form a covalent isopeptide bond between each other, thereby linking the two portions together. Moreover, scientists commonly apply elastin-like protein (ELP) or serine/glycine link (Ser/Gly link) as bridges between SpyCatcher/SpyTag and other functional proteins. By linking the Spy domains on the N-terminal or C-terminal of the target protein, its structure and function are generally unaffected, while the formation of isopeptide bond between SpyCatcher and SpyTag remains effective and efficient. In this way, both the enzyme and the SpyCatcher/SpyTag system can function orthogonally.
$$A+linker+SpyTag\quad\&\quad B+linker+SpyCather\\ \Downarrow \\ intracellular\quad expression\quad\&\quad connection\\ \Downarrow\\ a\quad multi-enzyme\quad complex$$
This unique covalent-bond-formation capacity between the two domains is capable of promoting the binding of two random proteins into one multi-enzyme complex both in vitro and in vivo. Therefore, this interaction has been utilized in several laboratories for bioligation, and the system has been reported in various applications such as vaccine optimization, hydrogel synthesis, and catalytic biofilm construction.

Therefore, in our project, we use SpyCatcher/SpyTag system with ELP and Ser/Gly links to construct various fusion proteins for assembly of our protein-nucleic-acid complex.

CRISPR/dCas9 ANCHOR SYSTEM

CRISPR/Cas9 technology is a genome engineering tool based on the adaptive immunity in prokaryotes:

• CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)

  It is a cluster of short palindromic repeats with regular intervals in the prokaryotic genome.

• CRISPR associated protein 9 (Cas9)

  It can cleave the target double-stranded DNA (dsDNA) complementary to CRISPR derived RNA (crRNA), under the guidance of tracrRNA/crRNA complex, which is formed by crRNA and trans-activating crRNA (tracrRNA) ^[1]; or the single-guide RNA (sgRNA).

• The deactivated CRISPR associated protein 9 (dCas9)

  It is a nuclease-deactivated variant of Cas9 created from S.pyogens. Though losing the DNA-cleavage activity, it can still specifically target and bind to DNA under the mediation of sgRNA^[3].

Previous studies have reported that^[4], random enzymes can be organized into a programmable assembly with the cooperation between dCas9 and the SpyCatcher/SpyTag system. In this complex, enzymes containing SpyTag are conjugated to the dCas9 containing SpyCatcher, and then anchored to a particular location on the DNA template by the guidance of appropriate sgRNA.

In our project, we intend to use this CRISPR/Cas-based strategy, in tandem with SpyCatcher/SpyTag system, to establish a multi-enzyme complex containing MnP, AAO, and HFB1. Together, the functions of the three elements could be aggregated, yielding a maximum PE degradation efficacy.

HOW DO OUR COMPLEX REALIZES ITS OPTIMAL FUNCTION?

In overview, a brief introduction of various elements and systems included in our project were displayed. Yet the reasons and considerations of putting forward such a design were not specified. Thus, it is of vital significance to state our explanation regarding how we managed to select and modify these PE-degrading elements, and to choose the right assembly system to merge their individual function into a fine-tuned symphony.

Initially, as our crucial PE-degrading enzyme, manganese peroxidase (MnP) undertakes a fundamental role of inflicting oxidation to PE by continuously producing Mn³⁺ ions. Therefore, increasing or maintaining its activity, as well as prolonging its stability should be the top priority for us in order to enlarge and reinforce the degradation efficacy of MnP towards PE. However, since the substrate and catalysate of MnP are both highly-oxidative, simply increasing its activity without restrictions is bound to cause irreversible harm to not only the MnP itself, but also other affiliated elements of our complex. Therefore, we decided to propose a semi-rational directed evolution strategy towards MnP, 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 (For detailed information about the results of directed evolution, see improvement page).

Secondary, in our design, for assisting MnP to perform its function, we selected aryl alcohol oxidase (AAO), a H₂O₂-producing enzyme that requires mainly aromatic alcohols as substrates for oxidation. This is because, due to the low but steady production rate of H₂O₂ by AAO, the inhibition of MnP due to an excess of H₂O₂ concentration, which could be the case when H₂O₂ is added manually, is effectively prevented. This will allow MnP to catalyze the PE-degrading reaction over a longer period of time, realizing a more complete degradation of PE. In addition, the cascade reaction mediated by the two enzymes 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.

Thirdly, as we were researching for means of enhancing PE degradation efficacy, we noticed that certain bacteria strains capable of degrading PE could produce biosurfactant to assist their adherence and growth on the hydrophobic surface of plastics. This inspired us to introduce hydrophobin-1, an amphipathic protein that could increase the hydropilicity of PE surface, thereby facilitating MnP to degrade PE.

Last but not least, instead of directly apply the three PE-degrading elements on PE, we selected the SpyCatcher/SpyTag connect system, as well as the CRISPR/dCas9 anchor system, to assemble the three elements into one compact complex. There are several advantages that can be provided by this assemblage. Firstly, the spatial distance between MnP and AAO is remarkably reduced by anchoring them in close proximity, which makes the H₂O₂ produced by AAO be more readily consumed by MnP, enhancing its catalytic activity; Secondary, the spatial distance between the complex and PE surface is also remarkably reduced, thanks to the surface activity of HFB1. This can lessen the the diffusion distance of Mn³⁺-chelate compound towards PE, in other words, lessen the occurrence of side reactions between Mn³⁺-chelate compound and other interfering substances before it reaches the surface of PE. In addition, the reduced Mn²⁺ after PE oxidation can be easily reabsorbed and oxidized to Mn³⁺ by activated MnP due to the close proximity of enzyme and substrate. In this way, the rate of PE degradation can be remarkably accelerated.

ELEMENT DESIGN

FUSION PROTEIN DESIGN

According to the sequence of SpyTag and SpyCatcher, combining with PE degradation elements described above, the following four fusion proteins are designed for the assembly of PE degradation complex.


Fig. 7 Fusion protein plasmid maps. A: pPIC9K-SpyTag-MnP; B: pPIC9K-SpyTag-AAO; C: pET-28a-SpyTag-HFB1; D: pET-28a-dCas9-SpyCatcher)

The genes that are fused with SpyTag in the N-terminus through the ELP sequence:

• mnp1 (Genbank accession number:AAA33744.1) from Phanerochaete chrysosporium strain ATCC20696
• peaao2 (Genbank accession number:MT711371.1) from Pleurotus eryngii strain P34
• hfb1 (Gene ID:18488188) from Trichoderma reesei 6MQa strain ATCC13631

Meanwhile, the TEV site and 6×His-tag are introduced to the N-terminus of SpyTag for His-tag removal and protein purification, respectively.

We inserted the gene sequence of SpyTag-MnP and SpyTag-AAO into the pPIC9K plasmid with the EcoRI at the N-terminus and NotI at the C-terminus. Subsequently, the recombinant plasmids will be transferred into Pichia pastoris for heterogenous expression.

We inserted the gene sequence of SpyTag-HFB1 into the pET-28a plasmid with the EcoRI at the N-terminus and NotI at the C-terminus. Subsequently, the recombinant plasmid will be transferred into Escherichia coli Rosetta(DE3) for heterogenous expression.

The gene that is fused with SpyCatcher in the C-terminus through a Ser/Gly link:

• dCas9

  The gene of cas9 (Gene ID: 57852564) is derived from Streptococcus pyogenes strain: NGAS638. Introducing single point mutations into each domain (D10A and H840A, correspondingly) to obtain the deactivated cas9 gene.

Meanwhile, the TEV site and 6×His-tag are introduced to the N-terminus of dCas9.

We inserted the gene sequence of dCas9-SpyCatcher into the pET-28a plasmid to form pET-28a-dCas9-SpyCatcher. Subsequently, the recombinant plasmid will be transferred into Escherichia coli BL21(DE3) for heterogenous expression.

sgRNA AND dsDNA TEMPLATE SYNTHESIS

The design of the sgRNA and dsDNA sequences was referred to Samuel Lim et al (2020)[1]. All three target sequences in dsDNA have a PAM sequence of CGG at their downstream. The sequences of the dsDNA and the gRNA scaffold used to synthesize sgRNAs were contained in one plasmid.

sgRNAs for each binding site were then transcribed from their corresponding templates using in vitro transcription. The dsDNA scaffold was similarly PCR amplified from a DNA plasmid (pUC-19) containing the target sequence using a forward primer and a reverse primer. The products were separated by agarose gel electrophoresis and the target bands were recovered by gel DNA extraction kit.