Development And Application Of A High-throughput Screening Platform For Nitrilase Based On Niacin Biosensor

Nitrilase（EC 3.5.5.1）is a class of enzymes that can hydrolyze nitrile into carboxylic acid,and has been used in the production of various chemicals and pharmaceutical intermediates.However,the low activity of natural nitrilase is a bottleneck that limits its further application.Directed evolution based on mutant library construction and screening has been proven to be an effective strategy for enzyme molecular engineering,but the lack of high-throughput screening methods has slowed the evolution research of highly active nitrilases.Genetically encoded biosensors can detect the concentration of target molecules in cells in real-time and regulate the expression of reporter genes,and is an ideal connector between enzyme activity and cellular phenotype,allowing direct in vivo ultrahigh-throughput characterization of enzyme activity.Based on this,this study constructed a niacin biosensor using a transcription factor that specifically recognizes niacin,and a high-throughput screening platform for nitrilase was developed by adapting it to the needs of nitrilase activity screening.The platform was further used to carry out semi-rational directed evolution of the nitrilase Nit6803 from Synechocystis sp.PCC6803 substantially improves its enzymatic activity and obtains an engineered bacteria that can efficiently catalyze the production of nicotinic acid from 3-cyanopyridine.The main findings are as follows:（1）Using the Bacillus subtilis transcription factor Bs Nad R and its target promoter P_{nad B},and using the green fluorescent protein GFP as the reporter gene,a niacin biosensor was constructed heterologously in Escherichia coli.However,the extremely high sensitivity of Bs Nad R results in a very narrow detection range for niacin,making it difficult to use for enzymatic activity screening.Bs Nad R was engineered by error-prone PCR,and a series of mutants with significantly reduced sensitivity was obtained by screening.It was found that changes in ligand affinity and flexibility of the DNA-binding domain were the main factors for the different regulatory properties of mutants.Among them,the signal output of mutant AH6responded to a wide range of niacin concentrations(0-50 mmol·L^-1),exhibiting a gradient detection function.So it is suitable for the activity screening of nitrilase;（2）Develop an in vivo high-throughput screening platform for nitrilase based on the constructed high-performance niacin biosensor,and use this platform to evolve Nit6803 with high activity.First,a random combinatorial mutant library was constructed by grouping the key sites forming the catalytic pocket of Nit6803,and the constructed mutant library was screened by a high-throughput screening platform.Then,based on the iterative evolution of pivotal sites,a highly active combinatorial mutant C57-E10 was finally obtained,and its specific enzyme activity for catalyzing 3-cyanopyridine reached 32.3±0.11 U·mg^-1,which was 6.6 times that of the wild type.Through molecular docking and kinetic simulations,it was found that the catalytic pocket of C57-E10 can better bind 3-cyanopyridine,resulting in higher activity;（3）The characterization of whole-cell catalysis by wild-type Nit6803 and mutant C57-E10was done.It was found that wild-type Nit6803 could not completely catalyze 500 mmol·L^-1 of3-cyanopyridine within 600 min,while C57-E10 could catalyze 500 mmol·L^-1 of 3-cyanopyridine within 100 min,and 3-cyanopyridine of 1 mol·L^-1 can be completely converted into nicotinic acid in 285 min.The results show that the engineered bacteria obtained in this study can efficiently catalyze the production of niacin from 3-cyanopyridine.In addition,the specific enzyme activities of C57-E10 towards 2-cyanopyridine,benzonitrile,and acrylonitrile were also greatly improved compared with wild type,which was 34.3,1.7,and 1.8 times of the wild type,respectively.This indicated that C57-E10 has a larger application potential.