Transformation Of Methionine Biosynthetic Pathway And Weakening Of Key Enzymes In Its Competitive Branch In Corynebacterium Glutamicum

Methionine has various physiological functions and high commercial value in the food,medicine and feed industries.Because of its complicated metabolic regulation,it has been unable to produce by fermentation,which has led to continuous investment of amino acid production enterprises and scientific researchers in the selection of methionine strains.The research ideas of strain selection basically include two aspects: on the one hand,the gene expression of the enzyme is regulated,and the gene expression is increased or decreased by methods such as promoter engineering and dynamic regulation;on the other hand,the catalytic properties of the enzyme are modified because Several key enzymes in the pathway are subject to strict feedback regulation,which is also very important for enzyme transformation.In this experiment,starting from Corynebacterium glutamicum,overexpression of aspartate kinase(AK)and homoserine O-acetyltransferase(HAT)increased the carbon flux of methionine synthesis.Among them,AK is a key enzyme derived from Corynebacterium pekinense,which was modified by this laboratory to release the synergistic feedback inhibition of lysine and threonine,and the enzyme activity was 20-50 times that of wild type.Through the determination of the amino acid yield of the constructed engineering bacteria,it was determined that lysine was the main by-product.In order to reduce the carbon flux of the lysine synthesis pathway,and at the same time avoid knocking out the pathway to cause cell death,select diaminopimelate dehydrogenase(Dap DH)which has an important influence on lysine synthesis for in vitro mutation Reducing enzyme activity,the data obtained will lay the foundation for weakening the lysine pathway in the future.The research results are as follows:1 Constructing 4 strains of engineering bacteria overexpressing AK and HAT increased the accumulation of methionine.Using wild-type Corynebacterium glutamicum ATCC13032 as the starting strain,over-expressing three different enzyme live AK mutants and AK-HAT co-expression mutants,constructing 4 strains of recombinant bacteria,namely Cg AK / p EC-NC,Cg AK / p EC-S59 F,Cg AK / p EC-T65 I and Cg AKM / p EC-T65I-met X and fermentation test.From the transcription results,the gene transcription levels of AK and HAT in the four strains increased,which proved that the genes were expressed after successful electroporation.In terms of fermentation products,the methionine yields of the four strains were 2.84 g / L,2.92 g / L,3.04 g / L and 3.29 g / L,which were 1.55 times,1.59 times,1.66 times and 1.79 times of the original bacteria.It was found that overexpression of HAT can significantly reduce the production of threonine,but it is not the main reason for reducing the carbon flow of the lysine pathway.In order to better reduce the carbon flow of the branch,further study the key enzymes of the lysine synthesis pathway.2 The Dap DH wild-type expression plasmid was constructed and 13 mutants were obtained by random mutation PCR and high-throughput screening,namely D120 N,D120R,D120 T,G151Q,T169 H,T169K,R195 G,R195H,H244 W,H244M,H244 S,N270T and N270 S.Induced expression and purification of WT Dap DH and mutants.SDS-PAGE and Western blot verified that Dap DH had been efficiently expressed in E.coli.3 Enzyme kinetic assays were performed on WT Dap DH and mutants.Enzyme activities of 6 mutants increased among 13 mutants,and enzyme activities of 7 mutants decreased.The enzyme activities of the mutants at D120,T169 and R195 were lower than Cg Dap DH,and the enzyme activities at G151,H244 and N270 were higher than Cg Dap DH.Among them,the enzyme activity was significantly reduced is T169 H,which is 11% of WT Dap DH,the Km value is 29.91,and the binding ability with the substrate becomes poor.In addition,the enzyme activity of the mutants at the D120 site all decreased,and D120 N decreased most significantly,which was 18% of WT Dap DH.Enzyme activity may be affected by the nature of key residue side chains and the ability to bind to the substrate.4 Molecular dynamics simulations were performed on WT Dap DH and mutants T169 H and D120 N to analyze the cause of the decrease in enzyme activity and provide a basis for future transformation.The results showed that the mutation of T169 H site disrupted the hydrogen bonding network of the substrate binding pocket,making the substrate unable to stabilize the binding of the enzyme,and the mutation caused the change of the structure of the substrate binding pocket.The hydrophobic microenvironment was destroyed,and the enzyme under the joint action of two aspects Live reduction.D120 is located at the entrance of the substrate binding pocket.Before the mutation,hydrogen bonds are formed with the residues on the nearby helix through the side chain to stabilize the protein conformation.At the same time,a narrow cavity is formed on the surface of the protein,which facilitates the substrate to enter the binding pocket in a specific form.After the mutation,the hydrogen bond at position 120 is broken,which increases the solvent accessible area in the substrate pocket,destroys the hydrophobic environment,and reduces the interaction between the enzyme and the substrate,thereby reducing the enzyme activity.