Design

Background

PETase is an enzyme capable of degrading polyethylene terephthalate (PET). PETase biodegrades PET by hydrolyzing the ester bonds in the PET molecule and breaking it down into smaller molecules. MHETase is an enzyme capable of degrading mono(2-hydroxyethyl) terephthalic acid (MHET). It can further degrade the small molecule produced by PETase degradation of PET into p-phthalic acid (TPA) and ethylene glycol (EG), which is part of the PET-degrading enzyme system.

Escherichia coli (E. coli) is currently the most widely used chassis cell in the field of synthetic biomanufacturing due to its mature genetic manipulation tools. In addition, E. coli is an excellent host cell for a range of membrane protein targets, and its genetic ease of handling permits the screening of a variety of gene constructs for optimal expression conditions, resulting in relatively high yields of membrane proteins in a short period of time.For strain selection, we used strain BL21(DE3) for efficient expression of PETase and MHETase as well as TPA transporter proteins for PET degradation and TPA enrichment. We constructed a TPA-responsive plasmid by combining eGFP with a TPA-inducible promoter.

Abstract: Signaling pathway

The signaling pathway we designed is divided into three parts: extracellular, cell membrane and intracellular.Extracellular:We secreted PETase and MHETase into the extracellular area.Under the action of PETase,most of the PET is degraded to MHET,and a small part of it is directly degraded to TPA and EG^[1]. Cell membrane: Since wild-type E. coli can only transport a small amount of TPA or no TPA, in order to achieve the effect of enriching and detecting TPA, we would like to express membrane proteins that transport TPA on E. coli. Intracellular: We introduced an exogenous inducible promoter that can respond to TPA, and attached an Enhanced Green Fluorescent Protein (eGFP) to the downstream of the promoter to indirectly detect the intracellular TPA content by detecting the fluorescence intensity.

Part Ⅰ: Extracellular pathway

For extracellular part, we secreted PETase and MHETase into extracellular

For the selection of PETase, we used TurboPETase^[2] published this year. TurboPETase was modified to obtain strong thermal stability, which greatly improved the rate of PET decomposition under higher temperature environment.

However, since PET as a polymer has too large to enter the cell, we need to secrete PETase to extracellular to react with PET. We fused a signal peptide sequence at the N-terminal of the PETase, since the TurboPETase we used is a new type of PETase published this year and the optimal signal peptide may be different from one enzyme to another, we designed four signaling peptides to verify the effect of different signaling peptides on TurboPETase.

We did not include MHETase in the signaling pathway in our pre-test. We used BHET as the substrate and mixed with PETase for 1 hour, and the liquid chromatography analysis showed that the concentration of MHET in the reaction solution was very low, so we thought that the TurboPETase degraded PET very well and had part of the MHETase activity, but after repeated experiments, the liquid chromatography showed that there was still a large amount of MHET in the reaction solution, and the conclusion drawn in the pre-test was inaccurate.

In order to improve the efficiency of PET degradation to monomer, we still chose to take MHETase into consideration and selected the following two signal peptides as the signal peptides of MHETase.

Part Ⅱ: Cell membrane pathway

To transport TPA into E. coli, we used a tripartite aromatic acid transporter from Comamonas sp. strain E6^[5], which is attribute to tripartite tricarboxylic acid transporter (TTT) family^[6].

This transport system consists of two multiple transmembrane proteins (TpiA and TpiB) and a binding protein (TphC).TphC first binds to TPA to form a complex, and then is transported to the intracellular compartment through the complex formed at the membrane by TpiA and TpiB.

We expect the transporter proteins to fulfill two roles.
1. Enrichment of TPA.
2. Binding to TPA-responsive promoters to assist in the detection of intracellular TPA concentration.

We placed TpiAB and TphC on the same plasmid and used the T7 expression system to express the target proteins. Because of the difficulty in heterologous expression of membrane proteins, especially multiple transmembrane proteins^[7], we fused HA-tag and Myc-tag after TpiA and TpiB, respectively, to facilitate the detection of membrane proteins.

Part Ⅲ: Intracellular pathway

To detect intracellular TPA concentration, we used the promoter Ptphc from Comamonas sp. strain E6 that can respond to TPA^[8].

TPA that enters the cytosol binds to the regulatory protein TphR and promotes the transcription of PtphC. We added eGFP downstream of PtphC to detect indirectly cytosolic TPA content.

In the genome of Comamonas sp. strain E6, PtphC connects upstream to regulatory proteins and downstream to enzymes that degrade TPA. Therefore, we initially thought that PtphC is a bidirectional promoter, so when we constructed the plasmid, we still put TphR upstream of Ptphc, and added a terminator sequence after the CDS of TphR to reduce the length of transcription and shorten the transcription time.

Since the specific functions of each part of PtphC element are not yet clear, and it is impossible to confirm whether there is an E. coli Ribosome Binding Site (RBS) in PtphC, we decided to add a section of RBS in front of the CDS of both TphR and eGFP.

Reference

• 1. Yoshida S, Hiraga K, Takehana T, Taniguchi I, Yamaji H, Maeda Y, Toyohara K, Miyamoto K, Kimura Y, Oda K. A bacterium that degrades and assimilates poly(ethylene terephthalate). Science. 2016 Mar 11;351(6278):1196-9. doi: 10.1126/science.aad6359. PMID: 26965627.
• 2. Cui Y, Chen Y, Sun J, Zhu T, Pang H, Li C, Geng WC, Wu B. Computational redesign of a hydrolase for nearly complete PET depolymerization at industrially relevant high-solids loading. Nat Commun. 2024 Feb 15;15(1):1417. doi: 10.1038/s41467-024-45662-9. PMID: 38360963; PMCID: PMC10869840.
• 3. Cui L, Qiu Y, Liang Y, Du C, Dong W, Cheng C, He B. Excretory expression of IsPETase in E. coli by an enhancer of signal peptides and enhanced PET hydrolysis. Int J Biol Macromol. 2021 Oct 1;188:568-575. doi: 10.1016/j.ijbiomac.2021.08.012. Epub 2021 Aug 8. PMID: 34371048.Facey SJ, Kuhn A. Membrane integration of E. coli model membrane proteins. Biochim Biophys Acta. 2004 Nov 11;1694(1-3):55-66. doi: 10.1016/j.bbamcr.2004.03.012. PMID: 15546657.
• 4. Tan Y, Henehan GT, Kinsella GK, Ryan BJ. Extracellular secretion of a cutinase with polyester-degrading potential by E. coli using a novel signal peptide from Amycolatopsis mediterranei. World J Microbiol Biotechnol. 2022 Feb 23;38(4):60. doi: 10.1007/s11274-022-03246-z. PMID: 35195792; PMCID: PMC8866283.
• 5. Hosaka M, Kamimura N, Toribami S, Mori K, Kasai D, Fukuda M, Masai E. Novel tripartite aromatic acid transporter essential for terephthalate uptake in Comamonas sp. strain E6. Appl Environ Microbiol. 2013 Oct;79(19):6148-55. doi: 10.1128/AEM.01600-13. Epub 2013 Aug 2. PMID: 23913423; PMCID: PMC3811363.
• 6. Winnen B, Hvorup RN, Saier MH Jr. The tripartite tricarboxylate transporter (TTT) family. Res Microbiol. 2003 Sep;154(7):457-65. doi: 10.1016/S0923-2508(03)00126-8. PMID: 14499931.
• 7. Facey SJ, Kuhn A. Membrane integration of E. coli model membrane proteins. Biochim Biophys Acta. 2004 Nov 11;1694(1-3):55-66. doi: 10.1016/j.bbamcr.2004.03.012. PMID: 15546657.
• 8. Dierkes RF, Wypych A, Pérez-García P, Danso D, Chow J, Streit WR. An Ultra-Sensitive Comamonas thiooxidans Biosensor for the Rapid Detection of Enzymatic Polyethylene Terephthalate (PET) Degradation. Appl Environ Microbiol. 2023 Jan 31;89(1):e0160322. doi: 10.1128/aem.01603-22. Epub 2022 Dec 12. PMID: 36507653; PMCID: PMC9888244.

Background

Our genetically engineered E. coli BL21 strain has been modified to express and secrete turboPETase and MHETase enzymes, which are capable of degrading polyethylene terephthalate (PET). Additionally, it expresses the TPA transporter on its cell membrane to accumulate terephthalic acid (TPA) from the surrounding liquid. However, due to the microscale dimensions of E. coli, which are approximately 1 micrometer in diameter, their collection poses a significant challenge. In contrast, the Caenorhabditis elegans (C. elegans), a well-established model organism, presents a more feasible collection option. The adults of C. elegans can grow up to 1 millimeter in length^[1]. This size difference makes C. elegans significantly easier to be harvested by natural sedimentation or filter interception. Considering that C. elegans feeds on E. coli, we propose to employ C. elegans as a biorecycler and amplifier within our system. Once the engineered E. coli has accomplished the targeted degradation of PET and the accumulation of TPA, we introduce C. elegans into this system. By feeding on the modified bacteria, C. elegans not only prevents the escape of engineered bacteria into the environment but also facilitates the recycling and accumulating of TPA, thereby enhancing the overall efficiency of the process.

Step 1: To test if C. elegans could survive in the reaction system

Recent studies have reported that exposure to 250 μm PET microfilaments (MFs) can reduce the locomotion speed and increase intestinal reactive oxygen species (ROS) of C. elegans^[2], indicating micron-scale PET is toxic to C. elegans. However, the impact of nano-scale PET and its degradation product, TPA, on C. elegans remains uncertain. Given our goal to utilize C. elegans as a functional component in a co-culture system with bacteria and their degradation products, it is imperative to assess the toxic effect on C. elegans to determine if C. elegans suitable for this co-culture system.

We design experiments to evaluate the toxic effects of nano-size PET and TPA on C. elegans by measuring intestinal ROS production^[3]. We culture a transgenic C. elegans strain expressing hsp-6::GFP, a specific reporter sysytem for the mitochondrial unfolded protein response (UPR^mt), which can be induced by ROS, on solid NGM plates containing varying concentrations of these substances. Given that the co-culture system is liquid, we next test the intestinal ROS production in the liquid culture system with supplementation of PET or TPA^[4]. At last, when the co-culture reaction finishes, we plan to test the survival rate of C. elegans to confirm that C. elegans could survive in the reaction system.

Step 2: To determine the ability of colonization of E. coli BL21 strain inside the intestine of C. elegans

While it is well-documented that C. elegans commonly feeds on the E. coli strain OP50, it remains uncertain whether our genetically modified E. coli BL21 strain can be similarly consumed. Therefore, we first aim to verify whether E. coli BL21 strain can well colonize inside the intestine of C. elegans through our experiment.

We culture C. elegans strain N2 on nematode growth medium (NGM) plates, seeded with both modified E. coli BL21 and OP50 strains expressing the fluorescent protein tdTomato. tdTomato is a red fluorescent protein that is widely used in molecular and cellular biological studies to label and track the expression of cells, proteins or genes. Upon the C. elegans growing to the day 1 adult stage, the intestinal fluorescent intensity is detected and quantified to assess bacterial colonization.

Additionally, an intestine lysis spreading assay would be performed to estimate the number of living bacteria within the C. elegans intestine^[5], providing further insights into the potential for successful colonization of our modified E. coli.

According to our current intestinal colonization results, we have successfully demonstrated the hypothesis that the engineered E. coli BL21 strain, capable of transporting TPA into the bacterial cell, can be ingested by C. elegans, thereby enabling the accumulation of bacteria and TPA within the intestine of C. elegans.

Step 3: Estimation of the ability of C. elegans to enrich TPA both from the engineered bacteria and from the liquid co-culture system

After assessing the colonization of E. coli BL21 strain inside the intestine of C. elegans, we will proceed with our final experiment: using C. elegans to TPA - one of the final degradation products of PET.

Since E. coli is difficult to recycle in liquid, we leverage the feature that C. elegans feed on E. coli and let C. elegans N2 strain directly ingest the modified E. coli BL21 strain that has transported TPA inside itself in the liquid environment. Then liquid chromatography is performed to detect the difference in the concentration of TPA in the liquid with or without C. elegans, to assess the ability of C. elegans to enrich TPA.

To further increase the TPA enrichment efficiency, we would also like to perform synthetic biology modification of C. elegans to absorb TPA directly into the worm intestine cells. We attempt to construct a plasmid containing a complete TPA transport system and an intestine-specific promoter to achieve successful TPA transport into C. elegans intestinal cells.

The key components of the TPA transport system in C. elegans (TpiA, TpiB, and TphC) are designed similarly to those in E. coli, but due to the differences between the expression systems of C. elegans and E. coli, we employ different promoter and signal peptides.

First, we use the intestine-specific promoter vha-6, ensuring that the system is only expressed in the intestinal cells of C. elegans. Next, we employ the gbb-1 and glr-2 signal peptide to localize the TpiA and TpiB transporter protein to the cell membrane, respectively. The dsl-1 signal peptide is used for the secretion of the TphC binding protein. The selection of these signal peptides is based on the number of transmembrane domains predicted from the protein structures.

What’s more, to avoid misfolding of functional proteins, we insert Splice Leader 2 (SL2) elements between genes coding different proteins. SL2 is trans-splicing sequence which can help to express the neighboring genes into single proteins instead of fused one.

To monitor the system’s expression, we also include mCherry gene, a red fluorescent protein derived from coral DsRed, which has excitation and emission peaks around 587 nm and 610 nm, respectively. mCherry serves as a reporter gene, allowing us to visualize gene expression; by detecting red fluorescence, we can confirm whether the system is successfully expressed in the intestine of C. elegans.

Through this design, we aim to enable C. elegans to transport and accumulate TPA, positioning it as a secondary guardian of aquatic environments.

Reference

• 1. Stiernagle, T., Maintenance of C. elegans. WormBook, 2006: p. 1-11.
• 2. Liu, H., et al., Multigenerational effects of polyethylene terephthalate microfibers in Caenorhabditis elegans. Environ Res, 2021. 193: p. 110569.
• 3. Djapovic, M., et al., Synthesis and characterization of polyethylene terephthalate (PET) precursors and potential degradation products: Toxicity study and application in discovery of novel PETases. Chemosphere, 2021. 275: p. 130005.
• 4. Jiang, W., et al., The toxic differentiation of micro- and nanoplastics verified by gene-edited fluorescent Caenorhabditis elegans. Sci Total Environ, 2023. 856(Pt 1): p. 159058.
• 5. O'Donnell, M.P., et al., A neurotransmitter produced by gut bacteria modulates host sensory behaviour. Nature, 2020. 583(7816): p. 415-420.