Building Mutant Library Of LysR-type Transcriptional Regulator DntR And High-throughput Screening The Biosensor Of Improved Catechol-Response

Aromatic pollutants(aromatic pollution,AP)has been a hot topic in the field of Environmental pollution & Control,and it is important role to establish a safe and effective metabo Lic regu Lation mechanism for the detection and removal the residues of aromatic pollutants in the environment.DntR is a bacterial transcription factor that has been isolated from Burkholderia species and regulated the oxidative degradation pathway of DNT,which can mediate the transcriptional activity and expressed the downstream of the promoter(PDNT)in the presence of salicylic acid.The aerobiotic degradation metabo Lism of aromatic pollutants requires bacteria,through the ring fission reaction,yielding the centra L metabolic intermediates catechol.However,catechol has a similarities structural to salicylate.a whole-cell catechol biosensor molecules was constructed by modifiding the DntR regulatory protein,which can be used to detect the standard emission of industrial sewage and residues of pollutants in the Environment,but also extended to the high-throughput screening of the AP key degradation gene modified by direct evolution.A whole-cell p RB1k-DntR-gfp sensor was contructed holding plasmids harboring the DntR gene and gfp reporter gene,where gfp gene was under the control of the PDNT promoter.This sensor induced by external inducer,which can transform unobservable genetic transmission into a detectable fluorescence signal.Based on the knowledge of the three-dimensional structure of the DntR-Salicylate and the inducer-binding pocket has been analysised.Choosing 151?153?206?230 of DntR as mutation site,which being used to have a four site saturated mutations at the same time,and constructed the resultant Libraries size of 106 by using the method is called Megaprimer PCR of whole plasmid.A gfp gene was used as a reporter for transcriptiona L activity mediated by DntR and cells with higher GFP expression after addition of catechol were sorted out using fluorescence-activated cell sorting(FACS).A DntR mutant C5-5,which displayed 1.6 times higher induction Levels than wild-type DntR in response to catechol was isolated.The detection Limit was ~10?M.Analysising the amino acid sequence of mutant C5-5,which was compared to the WT DntR,the results as expected that the mutation site at L151 I,L153P,H206 T,I230T.Select the highest similarity protein(PDB ID: 1utb)as a temp Late,replacing the amino acid residues to create a plausible three-dimensional structural model of mutant C5-5 by Pymol,and combination with catechol to have a structure-activity relationship using by Auto Dock.The results has been showed that the polarity of the mutant was enhanced and the free energy change value was increased among amino acids,urging the hydrogen bonds formation between the oxygen atoms of carboxyl group and hydrogen atom of water molecules,which increased the cavity diameter of inducer-DNA binding,making the inducer coductive easily.