Poly(ethylene terephthalate)hydrolase(PETase)from Ideonella sakaiensis exhibits a strong ability to degrade poly(ethylene terephthalate)(PET)at room temperature,and is thus regarded as a potential tool to solve the is...Poly(ethylene terephthalate)hydrolase(PETase)from Ideonella sakaiensis exhibits a strong ability to degrade poly(ethylene terephthalate)(PET)at room temperature,and is thus regarded as a potential tool to solve the issue of polyester plastic pollution.Therefore,we explored the interaction between PETase and the substrate(a dimer of the PET monomer ethylene terephthalate,2PET),using a model of PETase and its substrate.In this study,we focused on six key residues around the substrate-binding groove in order to create novel high-efficiency PETase mutants through protein engineering.These PETase mutants were designed and tested.The enzymatic activities of the R61A,L88F,and I179F mutants,which were obtained with a rapid cell-free screening system,exhibited 1.4 fold,2.1 fold,and 2.5 fold increases,respectively,in comparison with wild-type PETase.The I179F mutant showed the highest activity,with the degradation rate of a PET film reaching 22.5 mg perμmol·L^-1 PETase per day.Thus,this study has created enhanced artificial PETase enzymes through the rational protein engineering of key hydrophobic sites,and has further illustrated the potential of biodegradable plastics.展开更多
The management and recycling of plastic waste is a challenging global issue.Polyethylene terephthalate(PET),one of the most widely used synthetic plastics,can be hydrolyzed by a series of enzymes.However,upcycling the...The management and recycling of plastic waste is a challenging global issue.Polyethylene terephthalate(PET),one of the most widely used synthetic plastics,can be hydrolyzed by a series of enzymes.However,upcycling the resulting monomers is also a problem.In this study,we designed a co-cultivation system,in which PET degradation was coupled with polyhydroxybutyrate(PHB)production.First,PETase from Ideonalla sakaiensis was expressed in Yarrowia lipolytica Po1f with a signal peptide from lipase.The engineered PETase-producing Y.lipolytica was confirmed to hydrolyze bis(2-hydroxyethyl)terephthalate(BHET)and PET powder into the monomers terephthalate(TPA)and ethylene glycol(EG).Simultaneously,a TPA-degrading Pseudomonas stutzeri strain isolated from PET waste was transformed with a recombinant plasmid containing the phb CAB operon from Ralstonia eutropha,which encodes enzymes for the biosynthesis of PHB.The two co-cultivated engineered microbes could directly hydrolyze BHET to produce the bioplastic PHB in one fermentation step.During this process,5.16 g/L BHET was hydrolyzed in 12 h,and 3.66 wt%PHB(3.54 g/L cell dry weight)accumulated in 54 h.A total of 0.31g/L TPA was produced from the hydrolyzation of PET in 228 h.Although PHB could not be synthesized directly from PET because of the low hydrolyzing efficiency of PETase,this study provides a new strategy for the biodegradation and upcycling of PET waste by artificial microflora.展开更多
Polyethylene terephthalate(PET),one of the most widely used plastics in the world,causes serious environmental pollution.Recently,researchers have focused their efforts on enzymatic degradation of PET,which is an attr...Polyethylene terephthalate(PET),one of the most widely used plastics in the world,causes serious environmental pollution.Recently,researchers have focused their efforts on enzymatic degradation of PET,which is an attractive way of degrading and recycling PET.In this work,PET hydrolase Sb PETase from Schlegelella brevitalea sp.nov.was biochemically characterized,and rational design was performed based on its sequence similarity with the previ-ously reported Is PETase from Ideonella sakaiensis,resulting in a triple mutant with increased activity.Furthermore,using a sec-dependent signal peptide PeIB and colicin release protein Kil,we set up a high-efficiency secretion system of PETase in Escherichia coli BL21(DE3),enabling higher PETase secretion.Utilizing this secretion system,we established a high-throughput screening method named SecHTS(sec retion-based h igh-throughput s creening)and performed directed evolution of Is PETase and Sb PETase through DNA shuffling.Finally,we generated a mutant Is PETase S139T with increased activity from the mutant library.展开更多
基金National Program on Key Basic Research Project by the Ministry of Science and Technology of China(2014CB745100)the National Natural Science Foundation of China(21676190 and 21621004)the Innovative Talents and Platform Program of Tianjin(16PTGCCX00140 and 16PTSYJC00050).
文摘Poly(ethylene terephthalate)hydrolase(PETase)from Ideonella sakaiensis exhibits a strong ability to degrade poly(ethylene terephthalate)(PET)at room temperature,and is thus regarded as a potential tool to solve the issue of polyester plastic pollution.Therefore,we explored the interaction between PETase and the substrate(a dimer of the PET monomer ethylene terephthalate,2PET),using a model of PETase and its substrate.In this study,we focused on six key residues around the substrate-binding groove in order to create novel high-efficiency PETase mutants through protein engineering.These PETase mutants were designed and tested.The enzymatic activities of the R61A,L88F,and I179F mutants,which were obtained with a rapid cell-free screening system,exhibited 1.4 fold,2.1 fold,and 2.5 fold increases,respectively,in comparison with wild-type PETase.The I179F mutant showed the highest activity,with the degradation rate of a PET film reaching 22.5 mg perμmol·L^-1 PETase per day.Thus,this study has created enhanced artificial PETase enzymes through the rational protein engineering of key hydrophobic sites,and has further illustrated the potential of biodegradable plastics.
基金funding from the National Natural Science Foundation of China(grant numbers:Institute of Microbiology,Chi-nese Academy of Sciences:31961133016Beijing Institute of Technol-ogy:31961133015Shandong University:31961133014)and the Na-tional Key Research and Development Program of China(grant num-ber:2019YFA0706900)and was supported by European Union’s Hori-zon 2020 research and innovation programme under grant agreement No.870292(BIOICEP).
文摘The management and recycling of plastic waste is a challenging global issue.Polyethylene terephthalate(PET),one of the most widely used synthetic plastics,can be hydrolyzed by a series of enzymes.However,upcycling the resulting monomers is also a problem.In this study,we designed a co-cultivation system,in which PET degradation was coupled with polyhydroxybutyrate(PHB)production.First,PETase from Ideonalla sakaiensis was expressed in Yarrowia lipolytica Po1f with a signal peptide from lipase.The engineered PETase-producing Y.lipolytica was confirmed to hydrolyze bis(2-hydroxyethyl)terephthalate(BHET)and PET powder into the monomers terephthalate(TPA)and ethylene glycol(EG).Simultaneously,a TPA-degrading Pseudomonas stutzeri strain isolated from PET waste was transformed with a recombinant plasmid containing the phb CAB operon from Ralstonia eutropha,which encodes enzymes for the biosynthesis of PHB.The two co-cultivated engineered microbes could directly hydrolyze BHET to produce the bioplastic PHB in one fermentation step.During this process,5.16 g/L BHET was hydrolyzed in 12 h,and 3.66 wt%PHB(3.54 g/L cell dry weight)accumulated in 54 h.A total of 0.31g/L TPA was produced from the hydrolyzation of PET in 228 h.Although PHB could not be synthesized directly from PET because of the low hydrolyzing efficiency of PETase,this study provides a new strategy for the biodegradation and upcycling of PET waste by artificial microflora.
基金supported by the Qilu Youth Scholar Startup Funding of Shandong University(L.H.)National Natural Science Foundation of China(32170038)as well as the Sino-German mobility programme(M-0348).
文摘Polyethylene terephthalate(PET),one of the most widely used plastics in the world,causes serious environmental pollution.Recently,researchers have focused their efforts on enzymatic degradation of PET,which is an attractive way of degrading and recycling PET.In this work,PET hydrolase Sb PETase from Schlegelella brevitalea sp.nov.was biochemically characterized,and rational design was performed based on its sequence similarity with the previ-ously reported Is PETase from Ideonella sakaiensis,resulting in a triple mutant with increased activity.Furthermore,using a sec-dependent signal peptide PeIB and colicin release protein Kil,we set up a high-efficiency secretion system of PETase in Escherichia coli BL21(DE3),enabling higher PETase secretion.Utilizing this secretion system,we established a high-throughput screening method named SecHTS(sec retion-based h igh-throughput s creening)and performed directed evolution of Is PETase and Sb PETase through DNA shuffling.Finally,we generated a mutant Is PETase S139T with increased activity from the mutant library.
