Polyethylene terephthalate (PET) is a kind of synthetic
polyester widely
used in packaging and textiles. It is composed of terephthalic acid (TPA) and ethylene glycol (EG) through ester
bond. Its low cost, portability, durability and gas barrier ability are widely used in beverage bottles, product
packaging and textile industries.
Despite its many advantages, the production and disposal of PET have significant
environmental impacts.
Microplastics will spread into the environment and accumulate along the food chain, affecting the health of organisms and the biogeochemical cycles of carbon, nitrogen, phosphorus and sulfur.
Landfill method buries plastic waste underground. Still used, it occupies space and wastes resources. Thermal degradation decomposes waste plastics into small molecules by heating, but it needs high temp, equipment and cost, reducing recycled PET's mechanical properties.
Chemical methods feature alcoholysis, demanding high temp, pressure, and acetate catalyst. DMT, the degradation product, necessitates additional hydrolysis, hiking costs.
Plastic garbage can also be made into handicrafts with certain economic and artistic value.
The biological enzyme degradation method breaks down plastics into small pieces using enzymes from microorganisms. Despite strict culture conditions and low product extraction rates, its efficient and eco-friendly degradation merits promotion.
Lipase
R.J.MÜLLER et al. found that lipase TfH from
Thermobifidafusca had degradation activity on PET film and could depolymerize PET effectively.
Esterase
S.YOSHIDA et al. discovered the strain
Ideonellasakaiensis201‐F6, and found that this strain can use the PET film with low crystallinity (1.9%) as the
main carbon source, and can degrade the PET film into oligomer or monomer with low molecular weight. In this
strain, they found IsPETase.
Keratinase
The researchers found a kind of foliar
compound cutinase (LCC) with high thermal stability, which originated from leaf compost. In the process of PET
degradation, the active site of LCC is not affected by thermal induction, and its degradation activity is high.
The disulfide bond contained in LCC is beneficial to its thermodynamic and kinetic stability.
PH=8, T=72℃, t=9.3h, degradation rate =90%.
In 2020, V.Tournier and others reported that ICCG, a mutant of LCC, had a good activity of degrading PET.
However, nowadays, the low output of PETase in the production process leads to high production cost, which is not conducive to the industrial application of PETase.
By integrating relevant literature resources and utilizing machine learning technology as an auxiliary design tool, we successfully optimized the promoter and signal peptide sequences and constructed a highly efficient expression strain. Based on optimized culture conditions, we established an efficient ICCG expression system.
Escherichia coli strain BL21 (DE3) is a widely used host strain for protein expression, particularly in the production of recombinant proteins. This strain features a DE3 site, which includes a gene for T7 RNA polymerase that, upon induction with an agent like isopropyl β-D-1-thiogalactopyranoside (IPTG), efficiently initiates the expression of genes under the control of a specific T7 promoter. BL21 (DE3) is chosen for its expression system due to its relatively simple genetic background and low protease activity, which aids in enhancing the expression and stability of the target protein.
【promoter】 DMtac is a series of mutant tac promoters designed using predictive models, with mutations located in the 16 bp sequence between the -35 and -10 regions. These mutants aim to optimize the expression of the PET-degrading enzyme ICCG in Escherichia coli. The DMtac series was designed based on the Mtac promoter library (BBa_K5292001 - BBa_K5292088) generated by random mutagenesis.
Compared to the T7 promoter, our random mutagenesis library of the Mtac promoter showed a 74% improvement, and Mtac was about 30% better than the wild-type tac promoter. Our DMtac series improved by up to 30% over the T7 promoter and had an 8.2% higher yield than the wild-type tac promoter. For the MnfaA signal peptides, we saw a 16% improvement compared to the wild-type nfaA peptide. Our project strongly demonstrating the potential of machine learning in genetic engineering. Our project provides an innovative solution for the large-scale application of PETase in plastic degradation, contributing new technological advancements to environmental conservation.
