Characterization Of Enzymatic Properties And Construction Of Engineering Bacteria Of Aspartokinase Mutants From Corynebacterium Pekinense

Aspartokinase?AK?is a key enzyme catalyzing the biosynthesis of the aspartate family of amino acids.Its activity is inhibited by synergistic feedback from Lys and Thr,which regulates the carbon flow of the whole metabolic pathway,thus limiting the accumulation of large amounts of products.Therefore,the elimination of allosteric inhibition is great challenges in designing and optimizing metabolic pathways.In this study,Corynebacterium pekinense?CpAK?was used as the research object,and seven kinds of AK mutants with significantly improved enzyme activity were successfully obtained by site-directed saturation mutation and high-throughput screening.The enzymatic kinetics and enzymatic properties of wild type?WT?and mutants AK were studied,respectively.The mechanism of the significant increase in enzyme catalytic activity were further discussed through molecular dynamics simulation.In addition,the mutant AK with high enzyme activity was transformed into Corynebacterium pekinense by electroporation,and two engineering strains were successfully constructed.The results were as follows:1.Through homology comparison and structural grid analysis,it was found that CpAK M372,T379 and E400 sites were the key residues of the inhibitor Lys binding pockets,which were highly conservative.Therefore,molecular modification of the above three sites was carried out to weaken the binding between binding pocket and Lys,so as to relieve the feedback inhibition of Lys on AK.2.Seven kinds of AK mutants with significantly improved enzymatic properties were successfully obtained by mutant PCR amplification,transformation and high-throughput screening,etc.These mutants were M372I,T379W,T379S,E400A,M372/E400A,M372I/T379S and M372I/T379W.By SDS-PAGE and Western blot,it was confirmed that the mutants AK were highly expressed in E.coli BL21.3.The enzymatic kinetics and enzymatic properties of WT and mutants AK were determined.The results showed that the maximum response rate V_max of mutants AK were higher than that of WT AK 2.78 U/mg·min^-1.The M372I/T379S and M372I/T379W increased15.60 and 17.68 fold,respectively.M372I/E400A and M372I/T379W both showed good heat resistance and increased from 28°C to 35°C.Except for M372I/T379W,the optimum pH of mutants AK increased slightly,but tolerance range was wider.The relative enzyme activity of M372I/E400A and M372I/T379W remained above 50%in the pH range of 7.5-9.5 and 6.5-9.0,respectively.Mutants AK were more stable,especially M372I/E400A,M372I/T379S and M372I/T379W,with better stability of 1.3,2.3 and 1.2 h longer than WT AK,respectively.4.The activity of WT AK was inhibited by the feedback of Lys and Thr.When the concentration was 5 and 10 mmol/L,the synergistic inhibition of Lys and Thr was the most significant.Compared with WT AK,mutants T379S,M372I/T379S,T379W and M372I/T379W AK all had weakened inhibitory effect of Lys in the range of 0.2-10 mmol/L concentration.Especially in the presence of Lys alone,the activation of AK increased with the increase of Lys concentration,showing a dose-dependent relationship.At the same time,Microscale thermophoresis analysis showed that M372I/T379W AK not only weakened the feedback inhibition of Lys,but also enhanced its binding activity with Asp.5.Molecular dynamics simulation results indicated that mutations M372I and T379W caused significant changes in the radius of gyration and solvent-accessible surface area of the whole protein as well as a structural rearrangement of the residues near the ATP/Asp binding pocket of aspartokinase.The angle between M372I/T379W AK residue clusters Leu288-Gly363and Lys364-Ala418 decreased from 107.9°to 93.1°,which induced the transition of AK from a closed state to an open state.The ratio of hydrogen bonds between M372I/T379W AK and ATP or Asp was higher,and the width of the Asp-binding pocket entrance gate Arg169-Ala60increased from 9.6?to 15.1?than that in WT AK,which made the steric hindrance disappear and promoted the binding of AK with substrate Asp,thereby improving the catalytic efficiency of M372I/T379W AK.6.The engineering strains M372I/T379S and M372I/T379W were successfully constructed by electroporation,and the fermentation products were analyzed.The total amino acid content in the fermentation broth of M372I/T379S and M372I/T379W was 53.64 g/L and 57.88 g/L,which was 4.92 g/L and 9.16 g/L higher than that of Corynebacterium pekinense,respectively.