Bio-depolymerization And Resource Utilization Of Polyethylene Terephthalate(PET)

Synthetic plastics bring great convenience to human life,but with the continuous growth of plastic production and consumption,the problem of plastic pollution is becoming more and more serious.Traditional disposal technologies have a variety of shortcomings,such as stringent reaction conditions,high equipment requirements,high energy consumption and easy to cause secondary pollution.Biological disposal method of plastic waste has the advantages of environmental friendliness and mild conditions,and the depolymerization products of plastics can be converted into high-value products by synthetic biology technology,turning waste into treasure.Polyethylene terephthalate(PET)is polymerized by repeating units of terephthalate(TPA)and ethylene glycol(EG)through ester bonds.It is currently the most consumed polyester plastic and is widely used in various fields.PET can be depolymerized by chemical methods such as pyrolysis,hydrolysis,methanolysis,glycolysis and ammonolysis to produce PET monomers or oligomers.In recent years,studies have found that PET can be hydrolyzed by various hydrolases such as cutinase and esterase.Among them,IsPETase,a PET hydrolase with high activity at ambient temperature from Ideonella sakaiensis 201-F6,and LCC,a thermophilic cutinase from the leaf branch compost,have received extensive attention.Their derived variants with excellent properties have made significant progress in promoting PET bio-depolymerization.TPA and EG,the depolymerization products of PET,can be metabolized by a variety of microorganisms and used for the synthesis of biological polyester polyhydroxyalkanoates(PHA),important unsaturated dicarboxylic acid muconate and other high-value chemicals.This thesis conducts research on the bio-depolymerization and resource utilization of PET by constructing artificial microorganisms,and provides feasible schemes for realizing the circular economy of PET plastics.In the natural environment,microorganisms depolymerize plastics by secreting extracellular enzymes,and convert high molecular weight polymers into small molecular compounds for metabolism.In the first part of our work,we studied the biological method for the depolymerization of PET and synthesis of bioplastic polyhydroxybutyrate(PHB)with artificial microorganisms by simulating the natural degradation process of plastics.Firstly,we engineered Yarrowia lipolytica Polf that naturally secretes lipases to realize the biodepolymerization of amorphous PET and bis(2-hydroxyethyl)terephthalate(BHET)during cultivation at 30℃ by expressing and secreting IsPETase.The production of extracellular IsPETase was enhanced through signal peptide optimization and random integration of IsPETase expression cassettes in the genome.Then,to realize the resource utilization of PET hydrolysates,we screened a PET monomers-degrading strain Pseudomonas stutzeri TPA3 from plastic waste samples,and the metabolic genes of TPA and EG were identified by genome sequencing analysis.By introducing the PHB synthesis gene cluster from Ralstonia eutropha to P.stutzeri TP A3,PHB was synthesized using acetyl-CoA produced by PET monomers metabolism as the precursor.Finally,we achieved the bio-depolymerization of BHET to synthesize PHB by co-culturing the two engineered strains.However,due to both the depolymerization rate of PET by engineered Y.lipolytica Polf and the synthesis rate of PHB using P.stutzeri TP A3 as a chassis were low,resulting in the failure of direct PHB synthesis from PET.Nevertheless,this was the first attempt at performing the bio-depolymerization of PET and the bioconversion of PET hydrolysates simultaneously.It proved the feasibility of using artificial microflora for the depolymerization and resource utilization of PET and provided a new strategy for realizing the PET circular economy.The high degree of polymerization of PET is one of the factors affecting the rate of enzymatic hydrolysis of PET.In the commercial application of PET glycolysis,not only BHET was produced for the synthesis of new PET materials,but also oligomers waste was produced.In the second part of the work,we used the model strains Escherichia coli and Pseudomonas putida as the hosts to express PET hydrolases and convert PET hydrolysates respectively,and realized depolymerizing PET oligomers waste from glycolysis to synthesize PHA by cocultivation.Firstly,two excellent PET hydrolase variants FAST-PETase(5 sites mutant of IsPETase)and LCCICCG(4 sites mutant of LCC)were expressed and secreted in E.coli BL21(DE3)to depolymerize PET oligomers.Considering the yield and activity of the protein,it was found that the E.coli BL21(DE3)expressing LCCICCG had the best performance in the hydrolysis of PET oligomers.In addition,the model strain P.putida KT2440 instead of the natural strain P.stutzeri TPA3 was used as the chassis for the bioconversion of PET monomers.The engineered P.putida KT2440 can use TPA and EG to produce PHA by introducing TPA conversion pathway from P.stutzeri TP A3,enhancing the native metabolism of EG,and improving PHA production.Finally,10 g/L PET oligomers were depolymerized to produce 1.10 g/L PHA within 72 h by co-culturing the two engineered microorganisms with glycerol as the supplemental carbon sources.In addition,we used fluorescent reporter proteins to characterize the growth dynamics of the strains,indicating that they functioned at different stages,laying a foundation for improving the bio-depolymerization and transformation efficiency of PET through dynamic regulation of the population ratio.High temperature condition can increase the flexibility of free molecular chains of PET and increase the accessibility of PET hydrolases,thus significantly improving the degradation efficiency of PET.The full biological upcycling strategies of PET have been developed coupling with thermophilic enzymatic hydrolysis of PET and the ambient bioconversion of monomers.However,in the existing schemes,the expression and purification of PET hydrolases and the bioconversion of PET monomers are carried out in different hosts and different batches of culture,resulting in a high cost.In the third part of the work,we used P.putida KT2440 as the chassis to construct a multi-functional strain which can not only express and secrete PET hydrolases,but also convert PET monomers to synthesize high-value chemical muconate.It can produce a constant yield of extracellular LCCICCG concurrently when fermented with PET hydrolysates to synthesize muconate.Crude LCCICCG in the culture supernatant can be separated by ultrafiltration and directly used in a new round of PET depolymerization without further purification,which has the advantages of simplifying operations and reducing costs.With the multi-functional engineered strain,PET can be continuously converted to the high-value chemical muconate through the process cycle of"biological fermentation-products separation-enzymatic hydrolysis".In each cycle,0.58 g TPA can be produced from 1 g PET and the converted to muconate at a 100%molar conversion.The muconate in the ultrafiltration permeate can be further purified by crystallization and desalting to obtain a high-purity product.In summary,we successively realized the bio-depolymerization and resource utilization of BHET,PET oligomers and PET polymer by coupling the constructed PET-depolymerization modules and PET monomer-conversion modules in this thesis.It proved that the biological scheme based on protein engineering,metabolic engineering and synthetic biology will play an important role to realize the upcycling of PET.