Plastics, widely used in almost every industry, have excellent physical properties, such as durability, flexibility, waterproof and lightweight. However, due to its low natural degradation rate and recovery rate, waste plastics have become a serious environmental problem, and plastics can be weathered and decomposed into micro plastics, which are easy to be ingested by marine organisms, so as to enter the human body and endanger human health.
Polyethylene terephthalate (PET), polymerized from ethylene glycol and terephthalic acid, is one of the most widely used synthetic plastics. By 2020, the global capacity of PET production is 106 million, while our national capacity of PET production is about 60 million.
Main method to solve PET pollution is recycling. In 2019, the amount of national plastic waste recycled is 18.9 million, among which PET takes around 33%. Researchers have developed several chemical or physical methods to solve the problems.
[Physical recycling] is to clean and crush a single polymer waste, granulate it, and then turn it into plastic products. It has high requirements for waste, low energy consumption for recycling, and lower cost than that of brand-new polymers. However, because the process is relatively simple, the recycled raw materials are mostly used for products that do not directly contact the human body, such as manhole covers.
[Chemical recycling] is to chemically degrade waste to obtain monomers and polymerize them to become products. The recycling process has high energy consumption and is often more costly than brand new polymers. Besides, this process may produce microplastics (PET polymer particles) that scatter in the environment, and are difficult to recycle, thus causing microplastics pollution.
[Biological recycling] In recent years, the use of keratinase and lipase to develop biodegradable plastics has become a research hotspot. However, these enzymes are limited by their low polyester degradation ability and need a temperature of about 70℃ to exert their activity. In 2016, Japanese scientists isolated a strain of bacteria Ideonella sakaiensis, which was proved to be able to effectively use PET as carbon source for growth, and identified two pet degrading enzymes PETase and MHETase, which can degrade PET into terephthalic acid (TPA) and ethylene glycol (EG).
Free Enzyme Method
In addition to the high substrate specificity of pet and maintaining enzyme activity at low temperatures such as 30 ℃, the most valuable feature of PET enzyme is that it shows enzyme activity for PET with high crystallinity. These unique properties make PETase a promising practical candidate in the field of polyester recycling. Recently, pioneering studies have been carried out to determine the crystal structure and construct enzyme mutants to clarify the molecular mechanism of polyester degradation and improve its enzyme activity. However, the enzyme activity of high crystalline polyester is still very low, which limits its application in large-scale recovery of polyester. Obviously, more research is needed to further improve the performance of PETase and more research on MHETase to accelerate its industrial application.
Surface Display System
Cell surface display technology can expose peptides or proteins to the cell surface. It is a powerful strategic tool for many fields such as industry and medicine. A large number of studies have shown that compared with free enzymes, the enzyme activity and stability anchored on the cell surface are better, and multi enzyme whole cell collaborative catalysis can be carried out, the step of enzyme purification is omitted, and the enzyme activity does not decrease significantly after repeated utilization, which meets the industrial demand. Recently, some researchers showed PETase on the surface of Pichia pastoris cells, and its activity was 36 times that of free enzyme, which proved that PETase was suitable for functional display on yeast cells.
Project Design
Overview
Our goal is to develop a whole-cell biocatalyst by displaying PETase and MHETase on the surface of yeast cell (Candida Tropicalis) to degrade PET waste. Firstly, we chose a robust Candida Tropicalis as the host and deleted the URA3 gene of genome to block the uracil synthesis by CRISPR-Cas9. In order to display the protein more efficiently, we must obtain the appropriate anchor protein. Therefore, we built a model to predict the anchor protein candidates. We used an enhanced green fluorescent protein (yeGFP) as the reporter and inserted the codon-optimized yeGFP gene to the genome of Candida Tropicalis by homologous recombination. Finally, we selected the best one, fused it with PETase, and then incubated the yeast with PET waste.
Step 1: Choose the Host: Candida Tropicalis
Candida Tropicalis (Candida) is a common diploid sample. It can grow by using organisms such as botanical gardens or unconventional organisms as a carbon source. It has strong tolerance to acid and water substances.
Step 2: Predicting Anchor Protein
We built a MODEL to predict the anchor protein from the geome of Candida Tropicalis based on the sequence analysis of signal peptide, intermembrane structure, and glycosyl phosphatidy linositol (GPI) sites.
Flowchart of GPI-anchored Protein Screening
Structure of GPI anchor protein
Step 3: Screening for a Suitable Anchor Protein by GFP Test Design
We constructed the plasmid of fusing yeGFP and anchor protein, and screened the suitable anchor protein by determining the fluorescence intensity.
Positive Control：yeGFP, gene of green fluorescent protein; V5-tag, simian virus 5;
Test: SS，Signal Peptide; yeGFP, gene of green fluorescent protein; V5-tag, simian virus 5;
References
Smith MR, Khera E, Wen F. “ Engineering Novel and Improved Biocatalysts by Cell Surface Display.” Ind Eng Chem Res, volume 53, issue 16, 29 April 2015, pp. 4021-4032.
Tanaka T, Yamada R, Ogino C, Kondo A. “Recent Developments in Yeast Cell Surface Display toward Extended Applications in Biotechnology.” Appl Microbiol Biotechnol, volume 75, issue 3, August 2012, pp. 577-591.
Chen Z, Wang Y, Cheng Y, Wang X, Tong S, Yang H, Wang Z. “Efficient Biodegradation of Highly Crystallized Polyethylene Terephthalate through Cell Surface Display of Bacterial PETase.” Sci Total Environ, 20 Mar 2020, 709:136138.
Andreu C, Del Olmo ML. “Yeast Arming Systems: pros and cons of different protein anchors and other elements required for display.” Appl Microbiol Biotechnol, volume 102, issue 6, Mar 2018, pp. 2543-2561.
Kawai F, Kawabata T, Oda M. “Current Knowledge on Enzymatic PET Degradation and Its Possible Application to Waste Stream Management and Other Fields.” Appl Microbiol Biotechnol, volume 103. issue 11, Jun 2019, pp. 4253-4268.
Cui Y, Chen Y, Liu X, Dong S, Tian YE, Qiao Y, Mitra R, Han J, Li C, Han X, Liu W. “Computational Redesign of A PETase for Plastic Biodegradation Under Ambient Condition by The GRAPE Strategy.” ACS Catalysis, Volume 11, issue 3, 13 Jan 2021, pp.1340-1350.
