Identification

Design

Currently, there are reports in the literature that certain peptides possess chemical sites capable of specifically binding to plastics, known as plastic-anchoring peptides. We aimed to leverage this concept to develop a biosensor that can enable portable and rapid detection.

The core of our detection method involves three categories of anchoring peptides (LCI, TA2 and Cg-Def). By modifying gold nanoparticles with these anchoring peptides, we grant them the ability to aggregate when they encounter the corresponding microplastics. The aggregation results in a visible color change. By measuring the RGB values and UV spectrum of the color reaction, we can quantitatively analyze the plastic concentration.

To further enhance the specificity of the anchoring peptides, we applied directed evolution technology to the TA2 anchoring peptide. We also utilized Raman spectroscopy in combination with machine learning to classify and identify plastics, as a supplement to the colorimetric method when dealing with mixed plastic samples, where specificity may be insufficient. For detailed information on these two aspects, please refer to the Modeling.

Vector Construction

We obtained the gene sequences of the anchor peptides LCI, TA2¹ , and Cg-Def² from the literature and constructed three plasmids: piGEM24_01, piGEM24_02, and piGEM24_03, all of which co-express the sfGFP-anchor peptide fusion protein. The main reason for co-expressing sfGFP with the anchor peptide is that the anchor peptides have a small molecular weight, making it difficult to verify their expression using conventional methods such as protein concentration assays. Therefore, the expression was monitored via fluorescence detection.

The only difference between the three plasmids is the sequence of the anchor peptide. Taking piGEM24_01 as an example, it contains the gene sequence encoding the LCI anchoring peptide, a TEV cleavage site, a His-tag, a CG-linker, and sfGFP. We chose pET-30a as the vector because it contains multiple T7 promoters and terminators, as well as kanamycin resistance. In this design, we introduced a GC-linker to ensure that the two components (sfGFP and the anchor peptide) can function as independent modules, while also enhancing their expression levels. Furthermore, because the substantial molecular weight of sfGFP could potentially impede subsequent gold nanoparticle modifications, we inserted a TEV protease cleavage site and a His-tag between the two to facilitate subsequent purification of the anchor peptide using TEV protease cleavage followed by nickel column purification.

PET Biodegradation

Multi-enzyme Surface Display System Design

Biodegradation of poly(ethylene terephthalate) (PET) can be achieved through the innovative technique of cell surface display, which anchors target proteins on the cell's outer membrane. This method not only preserves the functionality and activity of enzymes but also enhances their stability and reusability on the cell surface. Compared to intracellular expression and secretion, cell surface display technology offers unique advantages such as a streamlined preparation process, improved stability, enhanced catalytic efficiency, and the potential for reuse and regeneration in repeated batch processes. Additionally, it eliminates the need for laborious and time-consuming protein purification steps, further simplifying the production of enzymes for PET degradation³.

The Lpp-OmpA chimera is a fusion protein composed of the signal sequence and the first nine N-terminal amino acids from the major Escherichia coli lipoprotein (Lpp), fused to a transmembrane domain (amino acids 46-159) from outer membrane protein A (OmpA)⁴. This chimera serves as a vehicle for bacterial surface display. The Lpp-OmpA-based cell display system pioneered the successful display of full-length heterologous proteins on the surface of E. coli, providing a robust platform for biotechnological applications⁵.

Vector Construction

In our study, we addressed the challenge of PET plastic degradation by harnessing the potential of bacterial surface display technology. Specifically, we utilized the membrane protein Lpp-OmpA to anchor key PET-degrading enzymes, Z1-PETase and MHETase, on the surface of E. coli, named piGEM24_05. This innovative approach facilitates the efficient catalytic breakdown of PET directly on the bacterial cell surface, thereby enhancing enzyme stability and reusability. Our method not only improves the degradation efficiency of PET but also contributes to cost reduction and environmental sustainability.

To validate the efficiency and practicality of Lpp-OmpA for surface display, we engineered an Lpp-OmpA-sfGFP system (piGEM24_04). This system fuses Lpp-OmpA with the superfolder green fluorescent protein (sfGFP), enabling the visualization of the display level on the bacterial surface. The incorporation of sfGFP as a fluorescent tag allows for the real-time monitoring of protein expression and localization within living cells, which is essential for assessing the success of surface display and its impact on enhancing PET degradation. The stable fluorescence properties of sfGFP render it an effective tool for evaluating the surface display of Lpp-OmpA and its role in the enzymatic breakdown of PET plastics.

The Z1-PETase gene sequence, pivotal for PET degradation, was obtained from the research by Kyung-Jin Kim and colleagues⁴. This chimera serves as a vehicle for bacterial surface display. The Lpp-OmpA-based cell display system pioneered the successful display of full-length heterologous proteins on the surface of Escherichia coli, providing a robust platform for biotechnological applications⁶. We then independently designed the working plasmid piGEM24_05. Following the synthesis of the plasmid, we conducted polymerase chain reaction (PCR) and Sanger sequencing to verify the correct assembly of the plasmid, ensuring the accuracy of our genetic constructs.

Upcycling

Pathway Design

The upcycling pathway from terephthalic acid (TPA) to vanillin was realized by whole-cell catalysis and multi-enzyme cascade technology.

Multi-enzyme systems are widely available, and we transformed plastic pollutants based on multi-enzyme systems, and reused waste through the "one-pot conversion". Since all the reactions were carried out in one system, the separation and purification steps for intermediate products were omitted, result in a significant enhancement of reaction efficiency.

The upcycling pathway were divided into two parts. First, terephthalate 1, 2-dioxygenase (TPADO) and 1,2-dihydroxy-3, 5-cyclohexadiene-1, 4-dicarboxylic acid dehydrogenase (DCDDH) were expressed in E.coli. TPADO converted TPA to 1,2-dihydroxy-3, 5-cyclohexadiene-1, 4-dicarboxylate (DCD). The intermediate product protocatechuic acid (PCA) was formed by DCD under the action of DCDDH. PCA then generated vanillin with high added value by the combined action of carboxylate acid reductase (NiCAR) and catechol O-methyltransferase (S-COMT). In addition, we selected the gene encoding phosphopantetheinyl transferase (Sfp) from Bacillus subtilis, a cofactor required for post-translational modification of NiCAR ⁴. This chimera serves as a vehicle for bacterial surface display. The Lpp-OmpA-based cell display system pioneered the successful display of full-length heterologous proteins on the surface of Escherichia coli, providing a robust platform for biotechnological applications⁷.

Vector Construction

Three plasmids piGEM24_06, piGEM24_07, and piGEM24_08 were constructed to express the enzymes involved in the upengineering process. Among them, piGEM24_06 facilitated the conversion of TPA to the intermediate PCA, piGEM24_07 facilitated the conversion of PCA to the end product vanillin, and piGEM24_08 facilitated the expression of cofactor Sfp.

piGEM24_06 contains the sequence of the genes encoding the three subunits of TPADO, TphA1, TphA2, and TphA3, and the sequence of the gene encoding DCDDH, TphB. We selected pETDuet-1 as the vector of choice due to its inclusion of multiple T7 promoters and terminators, coupled with ampicillin resistance. We inserted TphA1 and TphA2 together into the cleavage sites of NcoⅠ and NruⅠ, and TphA3 and TphB together into the cleavage sites of SacⅡ and AsiSI, so that the four genes could be better expressed.

piGEM24_07 contains the gene sequences of S-COMT and NiCAR. We selected pCDFDuet-1 as the vector for its array of T7 promoters and terminators, as well as its streptomycin resistance, which streamlines the downstream screening procedures. The NiCAR sequence was inserted between NcoⅠ and NotⅠ restriction sites, and S-COMT was inserted between NdeⅠ and XhoⅠ restriction sites, so that the two genes could be expressed independently.

piGEM24_08 contains the gene Sfp encoding phosphopantetheinyl transferase, which is responsible for the expression of the cofactor Sfp. We selected pACYCDuet-1 as the vector because of its chloramphenicol resistance to facilitate differentiation from the first two plasmids. The Sfp gene sequence was inserted between the BamHI and NotI restriction sites.

Gene editing

Selection of Target Genes

Vanillin, also known as vanal, is metabolized in E. coli through pathways involving aldehyde-ketone reductases and ethanol dehydrogenases, with the YahK gene is one of the genes encoding aldehyde-ketone reductases. Its expression in E. coli reduces vanillin to vanillyl alcohol, thereby decreasing the retention and yield of vanillin. Therefore, knocking out the YahK gene in E. coli BL21(DE3) can effectively reduce the metabolism of vanillin through the aldehyde-ketone reductase pathway, thereby increasing the biosynthetic yield of vanillin and improving the overall yield.

Design of sgRNA Plasmid (PHCY-sgRNA-yahK/piGEM24_09)⁸

1. Design of sgRNA for YahK Gene in Escherichia coli BL21 Using Benchling
   On Benchling's CRISPR-sgRNA interface, select E. coli strain MG1655 and input the target gene YahK. Generate sgRNAs for the entire gene sequence and select the one with the highest efficiency and lowest off-target score.
2. Design of Homologous Arms for the Target Gene
   Isolate 500 base pairs upstream of the YahK gene to serve as the homology arm HA1, and 500 base pairs downstream as HA2. The position of these homology arms dictates the length of the sequence to be deleted during gene editing. Utilizing homologous recombination, the sequence between the two homology arms, including YahK, will be excised.
3. Synthesis of PHCY-sgRNA-yahK(piGEM24_09) Plasmid
   The sgRNA is linked to the HA1/2 fragments at designated sites in the PHCY26D plasmid using PCR, overlap PCR, and Gibson assembly techniques, resulting in the formation of the PHCY-sgRNA-yahK plasmid, also known as piGEM24_09 (Figure 9).