Construction And Investigation Of Genetic Circuit For Screening Quorum Quenching Enzyme,AiiA With High Activity

Many biological functions of bacteria are regulated and controlled by the quorum sensing system.Although antibiotics have obvious antibacterial effects,they can easily lead to the emergence of drug resistance.Studies have shown that some substances can shut down the virulence expression of pathogens by interfering with the quorum sensing system,instead of simply restricting cell growth,so that pathogens will not show the phenotype of overcoming drug toxicity,complex super-infection and antibiotic resistance.Among Gram-negative bacteria,AHL is a key signal molecule that regulates the quorum sensing system.AiiA enzyme is a quorum sensing quencher capable of degrading AHL.The development of highly effective enzyme for quenching of quorum sensing is a potentially effective solution to prevent bacterial resistance.In this study,a genetic circuit capable of screening high-activity quorum-sensing quenching enzyme AiiA was constructed with synthetic biology methods,realizing the directed evolution of quenching enzyme AiiA.5 highly active AiiA mutant strains selected by the genetic circuit were recombined in vitro to obtain excellent heterozygous traits by the staggered extension PCR technology.The effect of the mutation sites on the AiiA enzyme activity at the molecular level was investigated through enzyme activity determination and molecular docking.And through replacing the component in the genetic circuit the optimization of the circuit was achieved.The characterization in two strains of Escherichia coli showed that the changes of the component can enhance the stability of the circuit expression in the engineered bacteria and thus improve the efficiency of screening.This thesis mainly includes the following 4 main parts:First,synthetic biology methods were used to construct genetic circuit to screen high-throughput enzymes(AiiA enzymes)that can efficiently degrade the quorum sensing signal molecule homoserine lactone(AHL).The research results showed that this genetic circuit in the presence of high-concentrations signal molecules would initiate the death of host cell,and only when the receptor cell expressed the highly enough active AiiA enzyme could it survive.The concentration gradient experiment of the signal molecule showed that the sensitive concentration of the signal molecule(AHL,3-O-C6-(L)-HSL)in this circuit was 103 nM.Under this concentration,several mutants have been screened,including 115,10,14,22,and 191 site.Second,five AiiA mutants were recombined in vitro using StEP technology to obtain two recombinants(AA4:double-site mutation of Asn22Asp and Pro46His;AA63:three-site mutation of Cys14Ser,Leu97Val and Ala115Arg)and their enzyme activities were measured.We found 80 cycles was the optimal number of staggered extension cycles and 60℃ the optimal extension temperature.Among single point mutatants,Pro10Ala had the highest increase change fold of enzyme activity with a relative specific activity of 4.84,followed by Aln22Asp with a relative specific activity of 4.25.In the single point mutation,the activity of Ala115Arg and Cys14Ser increased slightly,which were 2.98 and 2.89 respectively.The double amino acid mutation AA4 is with the highest enzyme activity.AA4,a double-site mutant of Asn22Asp and Pro46His with specific activity 5.96,was only 1.71 fold higher compared with that of the single-point Aln22Asp.The specific activity of AA63 a three-site mutation of Cys14Ser,Leu97Val and Ala115Arg was 3.51,which is higher than the enzyme activity of two single-point mutations.Third,in order to further investigate the effect of mutation on the catalytic efficiency,molecular docking simulation was used to analyze binding of the active site of the mutant and the target substrate 3-oxo-C6-HSL.The shortened distance between Oμ atom and Zn2 in the Pro10Ala mutant may promote the formation of tetrahedrons to a certain extent and stabilize the enzyme and substrate complex.Asp at position 22 of Aln22Asp helps improve enzyme stability.The internal activity mechanism of Asp191Gly is temporarily unknown.The shortened Cα-Zn1 of Ala115Arg promotes nucleophilic attack.The hydrogen bonding between Tyr194-Oαin Cys14Ser is enhanced.Position 46 in AA4 may contribute to the non-polar hydrophobic region.The strong hydrogen-bond interaction in the Cys14Ser and the strengthening interaction between Cα and Zn1 of Ala115Arg are well reflected in AA63.Fourth,using the gene luxr that encodes the LuxR protein that specifically binds to AHL,the promoter PluxI-lacO that is both induced by the LuxR-AHL complex and inhibited by the LacI protein,the gene lysis that encodes the bacterial lytic protein Lysis,and the wild-type gene aiiA,a new genetic circuit that can screen high-activity quorum-sensing quenching enzyme was designed:Pconst(17H)-RBS-luxR-TT-PluxI-lacO-RBS-lysis-TT-Pconst(17H)-RBS-aiiA-TT,so that there would be no leakage leading to relatively high levels background expression of lysis,and it could stay sensitive to AHL,and also have a inducible response to low AHL concentrations.It alleviates the problem of instability of the circuit caused by high background expression of Lysis.