| In the process of aspartate amino acids biosynthesis,aspartate kinase(AK)is the first key rate-limiting allosteric enzyme that determines carbon flow.It is regulated to the feedback inhibition by Lys and Thr,which affects the product accumulate excessively.This study analyzed the spatial structure of Corynebacterium pekinense aspartate kinase(Cp AK)and selected key residues around the catalytic center Asp and ATP for site-directed saturation mutations.In addition,we used high-throughput screening technology to obtain mutant strains with increased enzyme activity,constructed multiple mutants on the basis of the early research of our team,and studied enzymatic kinetics and enzymatic properties of wild-type(WT)and mutant AK.Then,we used genetic engineering technology to extract target fragments from mutant strains with good enzymatic properties of high enzyme activity and connected to expression vectors.Subsequently,Corynebacterium pekinense engineering strains were constructed through electrotransformation,and detected amino acid yields.The results were as follows:1.Determination of the catalytic active sites of mutants.Through the analysis of the Cp AK model and homologous sequence alignment,it was found that the catalytic active sites Glu92,Ser172,and Asp173 were the key residues of the substrate ASP,and Asp193 was the key residue around ATP.They were all highly conserved sites.Therefore,the four sites were selected for sitedirected saturation mutation to obtain mutant strains with improved enzyme activity.2.Construction of mutant strains.Through random mutation PCR,high-throughput screening,8 single mutants with increased enzyme activity were obtained,which were E92 V,E92R,S172 P,S172W,D173 G,D173R,D173 L,D193T,and quad-mutants T379N/A380C/T65I/D173 G,T379N/A380C/T65I/D173 R,T379N/A380C/T65I/S172 P,T379N/A380C/T65I/D193 T,T379N/A380C/T65I/E92 V were constructed.SDS-PAGE and Western blot verification showed that the mutant strain was successfully expressed and purified.3.Enzyme kinetics determination.The enzymatic kinetics determination of WT AK and mutant strains showed that compared with WT AK,the enzyme activities of D173 G,D173R,D173 L,S172P,S172 W,E92V,D193 T and E92 R,were increased by 13.99,11.56,9.11,8.49,7.85,7.23,5.81,1.54 times,respectively.The enzyme activities of the quad-mutants increased by 75.83,68.75,67.64,54.09 and 47.69 times,respectively.Except for S172 W,the Km value of other mutant strains all decreased,indicating that the affinity with the substrate increased.Except for D193 T,the Hill coefficient n value decreased,and the positive synergistic effect was weakened.4.Research on enzyme properties.The results showed that the optimum temperature of T379N/A380C/T65I/D173 G,T379N/A380C/T65I/D173 P,and T379N/A380C/T65I/D193 T was increased to 30℃ and the optimum temperature of D173 G and S172 W was increased to 28℃,compared with the wild type at 25℃.The heat resistance was enhanced.The optimal p H results showed that the optimal p H for D173 G,S172P,T379N/A380C/T65I/D173 G and T379N/A380C/T65I/E92 V was 8,which was the same as the wild type.The results of stability studies showed that the half-life of D173 R,E92V and T379N/A380C/T65I/S172 P were extended to 4.88 h,5.31 h,and 4.47 h,respectively,compared with the wild type of 4.24 h,which were beneficial to the fermentation production of the strain.The results of inhibitor studies suggested that the relative enzyme activity of the mutants increased in the presence of different concentrations of inhibitors.Especially in the presence of different concentrations of Lys+Thr,the mutants were all obviously activated and showed a dose-dependent relationship.5.Construction of engineering strains.Two strains of Corynebacterium pekinense engineering strains were successfully constructed by seamless cloning technology and electrotransformation methods,named Cp AK/p EC-T379N/A380C/T65I/D173 G and Cp AK/p EC-T379N/A380C/T65I/S172 P.After 48 hours of fermentation,the amino acid yield was analyzed.The results showed that the yield of engineering strain Cp AK/p EC-T379N/A380C/T65I/D173 G was higher.Compared with Cp AK,the yield of lysine increased by 83.05% and achieved to 1.08g/L,the yield of threonine was 1.41 g/L The increase rate was 29.36%;the output of methionine was increased by 30.77% and achieved to 0.17 g/L.The aspartate amino acid output of the engineered strain was higher than that of Cp AK,indicating that the spatial modification of AK has proved to be effective,and it provided a reference for the selection of high-yielding amino acid bacteriuria. |
