Gene Screening And Construction Of Genetically Engineered Bacteria For EE2 And BPA Degradation

The secondary effluent of urban sewage treatment plant generally contains residual environmental estrogen,which poses long-term ecological risk.Depth biological treatment of urban sewage is an important treatment method to ensure the safety of natural water quality.Most of the existing studies have focused on the isolation of environmental estrogen-degrading microorganisms,analysis of metabolites and pathways,and the bioaugmentation of activated sludge by wild-type bacteria,while the research exploring new enhanced application technologies and methods is relatively insufficient.In this study,EE2 and BPA,which are difficult to degrade environmental estrogens,were used as substrates,and eight degrading bacteria that can efficiently degrade E2were used as potential gene donors.In this study,we carried out a study on the deep biodegradation of EE2 and BPA through genetic engineering technology,aiming to establish a gene-enhanced activated sludge system to improve the removal efficiency of environmental estrogens from activated sludge and achieve deep biological treatment of municipal wastewater.Through the analysis and screening of EE2 and BPA degradation gene donors,we identified key enzyme coding genes for environmental estrogen degradation,constructed key gene vector plasmids,cultivated environmental estrogen genetically engineered degradation bacteria,and finally improved the efficiency of activated sludge estrogen degradation and removal.By analyzing the functional gene expression level and species abundance of activated sludge system,we explored the ecological stability and functional stability of gene-enhanced activated sludge microorganisms and provided the theoretical basis for urban wastewater biological treatment engineering practice.The main results obtained are as follows:1.Strains R-001 and P1 can efficiently degrade EE2 and BPA,respectively,and reduce the biological activity of EE2 and BPA through different pathways.In this study,two refractory environmental estrogens EE2 and BPA were used as substrates,and eight E2 high-efficiency degrading bacteria were selected to degrade EE2 and BPA at 30mg/L,respectively.Strain R-001 achieved effective degradation of EE2 through 2pathways,and the degradation rate could reach 92.01%within 120 hours.In pathway I,E1 is the main representative intermediate and estrogen biological activity is significantly reduced.In pathway II,hydroxylated EE2 is the main representative intermediate.Strains P1 can effectively degrade BPA through three pathways,and the degradation rate can reach 93.94%within 120 hours.In pathway I,1,2-bis（4-hydroxyphenyl）-2-propanol（P02）is formed.In pathway II,strain P1 could degrade BPA to 4-isopropylphenol,which was less toxic.In pathway III,representative intermediates are bis（4-hydroxyphenyl）methane or 4-benzylresorcinol.2.5 key genes for EE2 degradation and 9 key genes for BPA degradation were screened in the genomes of strains R-001 and P1,respectively.Dehydrogenase,oxygenase,and isomerase accounted for a higher proportion of the differentially expressed genes（DEGs）induced by EE2 and BPA,ranging from 68%to 87%.Based on the expression levels of DEGs and bioinformatics analysis,5 key genes for EE2degradation were identified in the genome of strain R-001,and their Log₂^FC ranged from 0.92 to 14.90,and the amino acid homology with known functional genes ranged from 21.62%to 81.57%.On the other hand,9 key genes for BPA degradation were identified in the genome of strain P1,and their Log₂^FC ranged from 2.70 to 6.88,and the amino acid homology with known functional genes ranged from 22.45%to 45.19%.The results showed that the 14 selected key genes were involved in the biodegradation of EE2 and BPA.3.fabG gene and sdr gene play a key role in the degradation of EE2 and BPA.The selected estrogen degradation key genes are respectively used for constructing recombinant bacteria for heterologous expression,and the degradation rate of the recombinant bacteria on various environmental estrogens is between 77%and 98%,and the recombinant bacteria have wide substrate spectrum characteristics.Among them,Strain-fabG showed the highest degradation rates for EE2 and E2,reaching 97.88%and 88.63%within 48 hours,respectively,and could convert both estrogens into E1,thereby significantly reducing estrogen activity.Strain-sdr showed the highest degradation rate of BPA,reaching 92.34%within 48 hours,and the main intermediates formed were 4-isopropenylphenol and bis（4-hydroxyphenyl）methane or 4-benzylresorcinol,which were also much less toxic than BPA.The results showed that the fabG gene from R-001 strain was the key gene for the degradation of EE2 by Rhodococcus equi,while the sdr gene from P1 strain was the key gene for the degradation of BPA by Pseudomonas asiatica.4.These two estrogen-degrading genetically engineered bacteria have the potential to bioenhance activated sludge.The genetic engineering bacteria B.-p MA5-fabG and B.-p MA5-sdr not only can stably grow and reproduce in the activated sludge,but also have the capability of efficiently degrading EE2 or BPA,can degrade 89%to 96%of environmental estrogen in the unique carbon source culture,and can increase the biodegradation capability of environmental estrogen in the activated sludge by 18.84%to 19.81%,while can make the SBR reactor remove environmental estrogens more completely and rapidly.In addition,the addition of genetically engineered bacteria did not adversely affect the activated sludge and even favored the water purification performance of the reactor to a certain extent.In summary,based on the 8 E2 high-efficiency degrading bacteria,R-001 was preferred to be the donor strain for EE2 degrading gene,and P1 was preferred to be the donor strain for BPA degrading gene.5 EE2 and 9 BPA degradation-related genes were identified using RNA-seq and molecular biology techniques.The degradation properties of EE2 and BPA were analyzed using gene recombination technology,and fabG and sdr were identified as the degradation key genes of EE2 and BPA,respectively.The results showed for the first time that short-chain dehydrogenase played an important role in the biodegradation of EE2 and BPA,and it could significantly reduce the estrogen activity of environmental estrogen,thus filling the research blank of short-chain dehydrogenase in the degradation of environmental estrogen.Based on genetic engineering and bioaugmentation theory,this study proposed to construct a gene-enhanced activated sludge system using genetic engineering technology,and then proposed a new method to enhance the environmental estrogen removal performance of activated sludge,which is an innovative study on the advanced biological treatment method of urban domestic sewage.It laid a theoretical foundation for the application of genetic engineering technology to improve the advanced biological treatment efficiency of specific pollutants,reduce the estrogen biological activity of secondary effluent,and carry out advanced biological treatment of urban sewage.