Si-Qi Chen,Qing-Song Huang,Yang Li,Jing Wu,Sheng Chen,Zheng-Fei Yan
Polyethylene terephthalate (PET) is one of the most widely used plastics, but its fragmentation into microplastics poses significant environmental challenges. The recycling of PET microplastics is hindered by their low solubility and widespread dispersion in the environment, making microbial in-situ degradation a promising solution. However, existing PET-degrading strains exhibited the limited effectiveness, primarily due to the diffusion of secreted hydrolases away from the PET surface. In this study, Stenotrophomonas pavanii JWG-G1 was engineered to achieve the targeted aggregation of PET hydrolase PETase on the cell surface by fusing it with an endogenous anchor protein. This approach aims to maximise the local concentration of PETase around PET, thereby increasing the overall rate of PET degradation. The PETase surface-aggregated system, S. pavanii /PaL-PETase, demonstrated the highest degradation efficiency, achieving 63.3 % degradation of low-crystallinity PET (lcPET) and 27.3 % degradation of high-crystallinity PET bottles (hcPET) at 30 °C. This represents the highest degradation rate reported for a displayed whole-cell system at ambient temperature. Furthermore, this system exhibited broad-spectrum degradation activity against various polyesters. These findings suggest that this system offers a promising, eco-friendly solution to PET and other polyester pollution, with potential implications for environmental bioremediation strategies.
Kai Xue,Zhonghu Bai,Eric Fordour,Siqi Guo,Yuxuan Zhou,Yankun Yang,Xiuxia Liu,Ye Li,Chun-Li Liu
Biological degradation of PET plastic holds great potential for plastic recycling. However, the high costs associated with preparing free enzymes for degrading PET make it unfeasible for industrial applications. Hence, we developed various cell catalysts by surface-displaying PETase mutants and MHETase using autotransporters in E. coli and P. putida . The efficiency of surface display was enhanced through modifying the host, co-expressing molecular chaperones, and evoluting the autotransporter. In strain EC9F, PET degradation rate was boosted to 3.85 mM/d, 51-fold and 23-fold increase compared to free enzyme and initial strain ED1, respectively. The reusability of cell catalyst EC9F was demonstrated with over 38 % and 30 % of its initial activity retained after 22 cycles of BHET degradation and 3 cycles of PET degradation. The highest reported PET degradation rate of 4.95 mM/d was achieved by the dual-enzyme cascade catalytic system EC9F+EM2+R, a mixture of cell catalyst EC9F and EM2 with surfactant rhamnolipid.
The waste pollution problem caused by polyethylene terephthalate (PET) plastics poses a huge threat to the environment and human health. As plasticizers, Phthalate esters (PAEs) are widely used in PET production and become combined pollutants with PET. Synthetic biology make it possible to construct engineered cells for microbial degradation of combined pollutants of PET and PAEs. PET hydroxylase (PETase) and monohydroxyethyl terephthalate hydroxylase (MHETase) isolated from Ideonella sakaiensis 201-F6 exhibit the capability to depolymerize PET. However, PET cannot enter cells, thus enzymatic degradation or cell surface displaying technology of PET hydrolase are the potential strategies. In this study, Pseudomonas sp. JY-Q was selected as a chassis strain, which exhibits robust stress tolerance. First, a truncated endogenous outer membrane protein cOmpA and its variant Signal (OprF)-cOmpA were selected as anchor motifs for exogenous protein to display on the cell surface. These anchor motifs were fused at the N-terminal of PET hydrolase and MHETase and transformed into Pseudomonas sp. JY-Q, the mutant strains successfully display the enzymes on cell surface, after verification by green fluorescent protein labeling and indirect immunofluorescence assay. The resultant strains also showed the catalytic activity of co-displaying PETase and MHETase for PET biodegradation. Then, the cell surface displaying PET degradation module was introduced to a JY-Q strain which genome was integrated with PAEs degrading enzymes and exhibited PAEs degradation ability. The resultant strain JY-Q-R1-R4-SFM-TPH have the ability of degradation PET and PAEs simultaneously. This study provided a promising strain resource for PET and PAEs pollution control.
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