Design And Construction Of Synthetic Microbial Constrium For Degrading Poly(Ethylene Terephthalate)

Poly(ethylene terephthalate)(PET)is one of the main sources of white pollution.His biodegradation has attracted more and more attention.Poly(ethylene terephthalate)hydrolase(PETase)from Ideonella sakaiensis shows very strong ability to degrade PET at room temperature and is considered to be a potential participant in solving polyester plastic contamination.The present study intends to achieve green degradation of PET by building a mixed bacteria system with rational design of PETase to improve its ability to degrade PET.In this study,the interaction between PETase and substrate 2PET was first explored based on the complex model of PETase and substrate 2PET binding.For this purpose,the modification was performed by six key residues around the substrate binding groove.The activity of the three variants(R61A,L88 F and I179F)was found to be increased by 1.4-fold,2.1-fold and 2.5-fold compared to the wild-type,respectively.Bacillus subtilis was used as the chassis to secret PETase.Subsequently,in order to efficiently secrete PETase,ten different signal peptides in Bacillus subtilis were screened that extracellular esterase had the best effect on the secretion of PETase.When esterase was used to secret PETase and MHETase to degrade PET,degradation product TPA had obvious inhibitory effect on PETase.In order to solve the inhibitory effect of TPA and the toxic effects of TPA and EG on the environment,a microbial consortium composed of Bacillus subtilis 168,Pseudomonas putida KT2440,and Rhodococcus jostii was constructed in this study.In this consortium,two Bacillus subtilis secret PETase and MHETase respectively to degrade PET,and degradation products TPA and EG are respectively used by Rhodococcus jostii and Pseudomonas putida to achieve green degradation of PET.In each process,the optimization of the culture medium,culture conditions and inoculation ratio were performed.It was found that P.putida strongly inhibited the growth of other bacteria in the presence of organic nitrogen,and the inoculum density R.jostii had a significant effect on the mixed bacteria degradation system.Finally,the optimized mixed system can effectively degrade PET,microbial consortium caused a weight loss of PET films of more than 26.3% after 7 days.The effect of mixed bacteria degradation increased by 6 times compared with the single B.subtilis degradation system.This study explored a method for the biodegradation of polyethylene terephthalate,which provided the basis for the subsequent biodegradation of the polymer.