Difference between revisions of "Part:BBa K4995003"

Revision as of 02:22, 12 October 2023

Truncated ice nucleation prot+polyethylene terephthalate enzyme

The increasing commodification of modern society and the close association of plastic with various industries, according to research statistics, result in the global production of approximately 100 million tons of plastic products annually, with a year-on-year increase [1]. Especially in 2020, during the fight against the COVID-19 pandemic, products primarily made from plastic materials such as gloves, masks, and protective suits played a crucial role in controlling the spread of the virus [2]. Polyethylene terephthalate (PET), due to its excellent mechanical properties and practicality, has become one of the widely used plastic materials [3], with global PET production capacity surpassing 100 million tons in 2020 [4].

Currently, PET is applied in various fields such as beverage or mineral water bottles, films, and polyester clothing [5], resulting in a significant amount of PET waste. While many countries have started recycling PET waste, the quantity remains low, and most discarded PET is not effectively recycled. These materials undergo embrittlement due to atmospheric ultraviolet radiation, free radical oxidation, and seawater hydrolysis, resulting in microscopic plastic particles that are invisible to the naked eye. These microplastics can be absorbed by aquatic organisms, and as humans sit at the top of the food chain, the ingestion of these aquatic organisms may lead to the accumulation of a substantial amount of microplastics in the human body, posing unpredictable health risks [6].

As is well-known, microorganisms play a significant role in the removal of pollutants and material cycling in ecosystems. Since the 1990s, the application of microbial enzymes in the degradation of high-molecular-weight materials has begun to draw attention as a unique solution to address the environmental issues caused by plastics. From the discovery of the first PET-degrading enzyme in the thermophilic actinomycete Thermobifida fusca by Müller and others in 2005 [7], several research teams have conducted a series of studies on PET-degrading enzymes from various microbial sources. Recently, researchers in Japan found a bacterium (Ideonella sakaiensis) capable of degrading PET and isolated an enzyme from it called PETase. This enzyme has the ability to break down hcPET (high crystallinity PET), effectively dismantling its long-chain molecules, enabling degradation [8].

Cell surface display technology, also known as microbial surface display technology, is a biotechnological approach that enables the expression and localization of target proteins on the surface of microbial cells. This technology allows researchers to manipulate and alter proteins directly on the surface of living cells, making them more amenable to studies of biochemical and biophysical properties, enzyme catalysis, and cellular engineering. At the core of this technique is the utilization of naturally occurring signal peptides or carrier proteins capable of directing proteins to the cell surface. Typically, these proteins transport the target protein to the cell surface through specific pathways. In the field of genetic engineering, we can link the gene sequence of the target protein with the gene sequence of these signal peptides or carrier proteins through genetic engineering methods, creating fusion genes. These fusion genes are then introduced into microbes, allowing for the expression and localization of the fusion protein on the cell surface. Ice Nucleation Protein (INP) is a commonly used protein carrier in cell surface display technology, and it can display the target protein in the periplasmic space of bacteria [9].

Usage and Biology

Figure 1 Design of gene circuit of INP-PETase expression system.

To improve the efficiency of engineered E. coli as whole-cell biocatalysts for PET degradation, the ice nucleation protein encoding gene INP was fused upstream of the PETase gene. The recombinant plasmid was transformed into E. coli Rosetta.

Characterization

The recombinant E. coli were cultured overnight in LB medium (37°C, 180 rpm), and 1 mL of bacterial culture was taken, adjusted to OD600=1, and centrifuged (10,000 rpm, 1 min) to collect cell pellets, which were resuspended in 1 mL PBS (pH 7.0). In 1 mL of bacterial suspension, 1 mM pNPB was added, and the reaction was incubated at 30°C for 30 min. The supernatant was collected by centrifugation, and the absorbance at 405nm was measured using a microplate reader. A standard curve was prepared using 0-1 mM para-nitrophenol ester dissolved in PBS.

Figure 2 A: Comparison of whole-cell catalytic activity of wild-type Escherichia coli (WT), PETase overexpressing E. coli and INP-PETase overexpressing E. coli; B: Difference analysis of WT and engineered E. coli that express INP-PETase；C：Agarose gel electrophoresis image of INP-PETase.

The findings revealed that E. coli/INP-PETase is capable of degrading 1 mM p-nitrophenol butyrate (pNPB), resulting in the production of 0.3 mM p-nitrophenol ester. In contrast, both the wild-type Escherichia coli Rosetta and E. coli/PETase yield negligible amounts of para-nitrophenol esters. These experimental outcomes demonstrate that the catalytic efficiency of PETase can be substantially enhanced through the application of surface display technology (Figure 2).

To investigate the effective components of the whole-cell catalyst, E. coli Rosetta overexpressing INP-PETase was cultured overnight. From this culture, both extracellular and intracellular components were separated by centrifugation. The cell pellet underwent a washing, sonication, and further centrifugation process to separate intracellular components and cell membranes. Cell membrane proteins were then extracted using a Tris-HCl solution with Triton X-100, followed by centrifugation to remove insolubles. The protein concentrations of all three components (extracellular, intracellular, and cell membrane) were determined using a Bradford reagent kit. Enzyme activity was assessed using pNPB as a substrate.

Figure 3 Exploring the effective components of whole-cell catalyst.

From the results, we found that there was almost no PETase activity outside the cell, but in the cell contents, the activity of PETase was about 0.15U/mg, and the activity of PETase on the cell membrane was the highest, reaching 0.35U/mg. Thus, through the surface display, the main active components of PETase are located in the cell membrane and bacterial contents (Figure 3).

Potential application directions

One potential application direction stemming from these experiments is the optimization of surface display technology for enhanced enzymatic biodegradation of PET (polyethylene terephthalate) plastics by engineered E. coli. By fusing the ice nucleation protein encoding gene INP upstream of the PETase gene, we have successfully improved the catalytic efficiency of PETase, resulting in the degradation of 1 mM p-nitrophenol butyrate (pNPB) and the production of 0.3 mM p-nitrophenol ester. This approach offers promise for the development of more effective whole-cell biocatalysts for PET degradation, which is essential for addressing plastic pollution and recycling challenges. Additionally, our investigation into the localization of PETase activity within E. coli Rosetta overexpressing INP-PETase revealed that the majority of active components are situated in the cell membrane and bacterial contents. This knowledge can guide future research in optimizing the display and localization of key enzymes for enhanced biocatalytic applications in plastic degradation and other biotechnological processes.

References

[1] QUECHOLAC-PIÑA X, GARCÌA-RIVERA MA, ESPINOSA-VALDEMAR RM, VÁZQUEZ-MORILLAS A, BELTRÁN-VILLAVICENCIO M, CISNEROS�RAMOS AL. Biodegradation of compostable and oxodegradable plastic films by backyard composting and bioaugmentation[J]. Environmental Science and Pollution Research International, 2017, 24(33): 25725-25730. [2] WONG SL, NGADI N, ABDULLAH TAT, INUWA IM. Current state and future prospects of plastic waste as source of fuel: a review[J]. Renewable and Sustainable Energy Reviews, 2015, 50: 1167-1180. [3] ROCHMAN CM, BROWNE MA, HALPERN BS, HENTSCHEL BT, HOH E, KARAPANAGIOTI HK, RIOS-MENDOZA LM, TAKADA H, TEH S, THOMPSON RC. Policy: classify plastic waste as hazardous[J]. Nature, 2013, 494(7436): 169-171. [4] GEYER R, JAMBECK JR, LAW KL. Production, use, and fate of all plastics ever made[J]. Science Advances, 2017, 3(7): e1700782-e1700782. [5] MOHARIR RV, KUMAR S. Challenges associated with plastic waste disposal and allied microbial routes for its effective degradation: a comprehensive review[J]. Journal of Cleaner Production, 2019, 208: 65-76. [6] WEBB H, ARNOTT J, CRAWFORD R, IVANOVA E. Plastic degradation and its environmental implications with special reference to poly(ethylene terephthalate)[J]. Polymers, 2012, 5(1): 1-18. [7] WIERCKX N, PRIETO MA, POMPOSIELLO P, de LORENZO V, O’CONNOR K, BLANK LM. Plastic waste as a novel substrate for industrial biotechnology[J]. Microbial Biotechnology, 2015, 8(6): 900-903. [8] CHEN, ZHOUZI, et al. "Efficient biodegradation of highly crystallized polyethylene terephthalate through cell surface display of bacterial PETase." Science of The Total Environment 709 (2020): 136138. [9] DOU, JIAN-LIN, et al. "Surface display of domain III of Japanese encephalitis virus E protein on Salmonella typhimurium by using an ice nucleation protein." Virologica Sinica 26 (2011): 409-417.

Sequence and Features