1. PET Biodegradation

1.1 Design of a Magnetic Nanoparticle-Based System for Sequential Multi-Enzyme Co-immobilization

To address the challenges of intermediate product accumulation and difficulty in recovery associated with free enzymes during PET degradation, we designed and constructed an immobilized dual-enzyme system based on functionalized magnetic nanoparticles, as illustrated in Figure 1. This strategy involves the sequential co-immobilization of the PET-degrading enzyme Tfh and the intermediate-degrading enzyme TfCa. This approach eliminates intermediate products while facilitating the reuse of the enzymes via simple magnetic recovery.

At the core of this system lies a functionalized magnetic carrier, K5C@Fe3O4, prepared via a one-step biomimetic mineralization method. The functionality of the carrier is derived from the K5C fusion protein, which consists of an elastin-like polypeptide (ELP) fused to a SpyCatcher protein. The ELP component leverages its thermo-responsive properties to enable a simple, non-chromatographic purification of the fusion protein via Inverse Transition Cycling (ITC), a method that uses simple temperature shifts to induce its reversible aggregation and precipitation; meanwhile, the SpyCatcher component on K5C@Fe3O4 serves as the primary anchoring site. To achieve the ordered co-immobilization of the dual PET-degrading enzymes (TfCa and Tfh) onto the carrier, we employed two specific covalent self-assembly systems: SpyTag/SpyCatcher and SnoopTag/SnoopCatcher. TfCa and Tfh were respectively engineered into the fusion proteins SpyTag-TfCa-SnoopTag and SnoopCatcher-Tfh. This design enables SpyTag-TfCa-SnoopTag to first be specifically and covalently immobilized onto K5C@Fe3O4. Subsequently, SnoopCatcher-Tfh specifically binds to the now-immobilized SpyTag-TfCa-SnoopTag, thereby achieving the sequential covalent co-immobilization of the dual enzymes onto K5C@Fe3O4. Furthermore, the specificity of this immobilization allows the process to be performed directly in crude enzyme lysates, thus bypassing tedious protein purification steps and further simplifying the PET degradation procedure.

1.2 Plasmid Construction

To implement our modular immobilized dual-enzyme system, we designed a set of three independent expression plasmids, each responsible for producing a key protein component, as depicted in Figure 2. The foundation of our system is the functionalized magnetic carrier, for which we constructed plasmid piGEM25_01. This plasmid directs the expression of the K5C fusion protein, which strategically combines an elastin-like polypeptide (ELP) with a SpyCatcher domain that serves as the covalent anchoring scaffold on the magnetic nanoparticles. For the enzymatic components, we engineered two separate plasmids to express the PET-degrading and MHET-degrading enzymes with specific tags for ordered assembly. Plasmid piGEM25_02 was constructed to express the first enzyme, TfCa, as a SpyTag-TfCa-SnoopTag fusion protein. Concurrently, plasmid piGEM25_03 was built to express the second enzyme, Tfh, fused with SnoopCatcher (SnoopCatcher-Tfh). All three constructed plasmids were cloned into the pET-30(a) expression vector. Following synthesis, the structural integrity and sequence accuracy of all three plasmids were rigorously verified by Polymerase Chain Reaction (PCR) and gene sequencing, confirming their successful construction.

2. Upcycling of EG to PEAs

2.1 Engineering E. coli to Utilize EG as Carbon Source

2.1.1 Metabolic Modeling-Guided Engineering Design of E. coli for EG Conversion

Ethylene glycol (EG) is one of the two primary products from PET depolymerization and serves as a vital chemical feedstock. The continuous accumulation of EG inhibits subsequent hydrolysis reactions. Therefore, utilizing microbial engineering to convert EG into high-value-added products represents a key strategy to overcome this bottleneck. This approach not only eliminates EG's inhibitory effects but also achieves the dual value of transforming non-degradable plastics into valuable resources. However, laboratory-common E. coli strains lack efficient EG assimilation pathways, limiting their application potential in PET upgrading.

This study aims to reconstruct metabolic networks through synthetic biology, enabling strains to grow using EG as the sole carbon source and further synthesize high-value-added products. Comprehensive literature evidence indicates that the glycolate/glyoxylate degradation pathway, comprising the four-gene combination “gcl–hyi–glxR–glxK,” has been validated as the core engine and minimal unit supporting E. coli growth with EG as the sole carbon source. In contrast, engineered strains containing only single modules (Module I (fucO-aldA), Module II (glcDEF), or Module IV (glcB)) failed to achieve effective growth in EG-only media^[1]. Thus, we adopted this quadruple gene construct as our reference benchmark. Furthermore, based on FDCA analysis results from the E. coli genome-wide metabolic model (GEM), we proposed a minimalization design: “Retain essential nodes gcl and glxK; replace glxR with isoenzyme garR; and remove hyi to optimize glyoxylate node flux and reduce genetic burden.” Based on this, we constructed a gcl–garR–glxK tri-gene combination and expanded it with computationally predicted fucO–aldA as needed. Through this integrated approach of metabolic modeling and experimental validation, we aim to uncover more streamlined and efficient E. coli metabolic pathways for EG utilization.

2.1.2 Plasmid Construction

For this purpose, we selected the pCDFDuet-1 (SmR) and pRSFDuet-1 (KanR) vector backbones to successfully construct the following three recombinant plasmids (Figure 3):

1. piGEM25_04: Carries the three-gene combination gcl–garR–glxK. This plasmid represents the optimal solution identified through our metabolically guided design and experimental validation, and it is engineered to achieve efficient EG utilization via a concise metabolic pathway;
2. piGEM25_05: Harbors the four-gene combination gcl–hyi–glxR–glxK. This construct was created to replicate and validate a strategy reported in the literature, serving as a comparative baseline;
3. piGEM25_06: Contains the fucO-aldA gene combination. This plasmid is designed to test the effect of enhancing the first step of EG oxidation (EG → glycolate) on the overall metabolic flux.

2.2 EG Detection

To conduct real-time monitoring of the concentration during the EG assimilation process, we have developed an EG detection system for this purpose，Gold nanoparticles (AuNPs) exhibit significant potential for applications in chemical and biological sensing, medical diagnostics, environmental monitoring, and photonic devices, owing to their unique optical properties, particularly the surface plasmon resonance (SPR) effect. When target molecules (such as ethylene glycol, EG) interact with the AuNP surface (e.g., via adsorption or coordination) or alter the local microenvironment, changes in the refractive index of the medium occur. This leads to shifts or intensity variations in the SPR absorption spectrum, enabling the quantitative detection of the target analyte.

Based on this principle, we designed a portable spectroscopic detection device utilizing AuNPs for the quantitative analysis of EG concentration. Using the AS7341 spectral sensor, we quantitatively analyzed the effect of EG concentration variation on the output value of the 525 nm channel. A distinct linear relationship was observed, and a fitted formula was ultimately derived for the reverse-calculation of EG concentration.

2.3 PEA Synthesis

2.3.1 PEA Synthesis Design

After establishing a minimal assimilation scheme for E. coli EG metabolism (gcl–garR–glxK) and verifying its ability to support EG-based one-carbon growth, we shifted our design focus from “feeding cells EG” to “efficiently directing EG carbon flux into intracellular polyesteramide (PEA) synthesis.” To achieve efficient conversion of EG into PEAs, we adopted a dual-plasmid, modularly loaded strategy. The core design logic of this system involves decomposing the complex PEA synthesis pathway into two functionally independent yet logically interconnected modules:

1. Monomer Synthesis Module: This module metabolizes endogenous amino acids into two key monomers required for PEAs polymerization: (R)-3-hydroxybutyrate-CoA (3HB-CoA), which forms ester bonds, and 3-aminopropionyl (3AP), which forms amide bonds. This is achieved through co-expression of the phaA, phaB, and panD genes;
2. Polymerization Module: This module activates and polymerizes the aforementioned monomers. We employed an engineered, substrate-broad PhaC synthase capable of simultaneously catalyzing the polymerization of both ester and amide monomers. This is complemented by the pct540 and act genes, which catalyze acyl transfer reactions to activate the monomers, enabling efficient PEAs synthesis.

2.3.2 Plasmid Construction

In this study, we designed and constructed two core plasmids:

1. piGEM25_07 (Monomer Synthesis Module): Constructed based on the pTrc99a vector (KanR) for expressing the phaA, phaB, and panD genes;
2. piGEM25_08 (Polymerization Module): Constructed based on the pGro7 vector (CmR) to express engineered phaC, pct540, and act genes. This module expresses the pct540 and act genes for monomer activation and the phaC gene for monomer polymerization.

3. Upcycling of TPA to PCA

3.1 Pathway Design

Building upon the aforementioned engineering for EG utilization, our UESTC-iGEM 2025 team's research also ensures project continuity. In 2024, the UESTC-iGEM team from our university achieved a low yield in the "TPA→vanillin" conversion pathway, which targets one of the two major products of PET hydrolysis. Based on process deconstruction and literature evidence, we identified insufficient precursor supply (i.e., limited PCA production) as the primary bottleneck. To address this, we re-engineered the upstream TPA→PCA module this year. We designed plasmid piGEM25_09 to co-express TphA1/TphA2/TphA3 (terephthalate 1,2-dioxygenase, TPADO) and TphB (1,2-dihydroxy-3,5-cyclohexadiene-1,4-dicarboxylate dehydrogenase, DCDDH) on a single vector. In this pathway, TPADO converts terephthalic acid (TPA) into 1,2-dihydroxy-3,5-cyclohexadiene-1,4-dicarboxylate (DCD). This intermediate is subsequently converted into protocatechuic acid (PCA) by TphB. We also optimized the expression conditions by introducing metals/cofactors, specifically supplementing with FeCl₃ and L-cysteine during the induction phase to promote the assembly of the Rieske [2Fe–2S] and mononuclear iron centers, and adding ZnSO₄ to ensure the proper metallation of TphB. Concurrently, the whole-cell catalyst loading (tested across an OD600 gradient) was optimized to enhance PCA production.

3.2 Plasmid Construction

Plasmid piGEM25_09 was constructed to express the enzymes involved in the catalytic pathway for the conversion of TPA to PCA. This plasmid incorporates the sequences encoding TPADO (TphA1, TphA2, and TphA3) and DCDDH (TphB). We selected the pETDuet-1 vector for this purpose, owing to its dual T7 promoters and ampicillin resistance marker. The TphA1/TphA2/TphA3 gene cluster was cloned into the NcoI and NruI restriction sites, while the TphB gene was inserted into the SacII and AsiSI sites. This arrangement facilitates the efficient expression of all four genes.

[1] Chi J, Wang P, Ma Y, Zhu X, Zhu L, Chen M, Bi C, Zhang X. Engineering Escherichia coli for utilization of PET degraded ethylene glycol as sole feedstock. Biotechnol Biofuels Bioprod. 2024 Sep 13;17(1):121. doi: 10.1186/s13068-024-02568-4. PMID: 39272202; PMCID: PMC11401383.