Overexpression Of Key Enzymes In Methionine Branch And Weakening Of Threonine Branch In Corynebacterium Glutamicum

Methionine,as a synthetic precursor of many substances in organisms,can effectively regulate the metabolic equilibrium of the body.It is widely used in food,feed,medical,and other industries.The production of amino acids by microbial fermentation has gradually replaced that of the traditional chemical separation method for its advantages of high utilization rate of raw materials,simple operation,and low environmental pollution.However,the industrial methionine production by biological fermentation has not been realized so far due to the long metabolic pathway as well as the complex regulation and inhibition of several enzymes in the synthesis process.This study was using Corynebacterium glutamicum with clear metabolic structure as the starting strain to amplify two enzymes obtained from previous studies:the Corynebacterium pekinense aspartate kinase(coding gene lys C~m)which has high enzyme activity and partial feedback-inhibition resistance;homoserine dehydrogenase(coding gene hom~m),also amplify homoserine acetyltransferase(coding gene:met X)from Corynebacterium glutamicum genome.By means of genetic engineering,the target fragment was connected with the shuttle expression vector PECXK99E in order to construct the amino acid-producing bacteria under the regulation of different overexpression genes.Analysize the carbon flow direction of each strain by fluorescence quantitative and liquid phase to find methionine metabolism repression sites.From this study,we found that the main reason of hindering the methionine production in its biosynthesis pathway was the significant increase of threonine after overexpressing key enzymes.Therefore,modification in vitro and weakening enzyme activity of the homoserine kinase which is the starting enzyme of the threonine branch-,were performed to reduce the consumption of the carbon flow in the threonine branch.The research would provide a reference for constructing methionine engineering strains.The conclusions of this study are as follows:1.Use the PCR amplification technology to obtain lys C~m and hom~m genes with high enzyme activity from Corynebacterium pekinense and met X genes of Corynebacterium glutamicum;then construct single-gene overexpression recombinant vectors PEC-lys C~mand PEC-met X;thus we could obtain the bi-gene overexpressed recombinant vectors PEC-lys C~m-hom~m,PEC-lys C~m-met X,and the three-gene overexpressed recombinant vector PEC-lys C~m-hom~m-met X by using the homologous recombination method to connect multiple target fragments.Then transfer the recombinant vector into Corynebacterium glutamicum Cg/WT.Finally,we obtained successfully 5 strains of amino acid-producing bacteria:Cg/PEC-lys C~m、Cg/PEC-met X、Cg/PEC-lys C~m-hom~m、Cg/PEC-hom~m-met X and Cg/PEC-lys C~m-hom~m-met X.2.Analysis of the 72-hour fermentation products of Cg/WT and 5 strains of amino acid-producing by fluorescence quantification,the results showed that all genes on the recombinant vector were successfully expressed in Corynebacterium glutamicum,which effectively increased the transcription level of all genes in the strain.HPLC(High performance liquid phase)results showed that the production of glutamic acid in Cg/PEC-lys C~m decreased significantly from 1.45g/L to 0.52g/L as compared with Cg/WT.But the production of the asp-derived amino acids is increased,and the overexpression gene lys C~m can make more carbon flow into the aspartic acid branch after the cycle of tricarboxylic acid,which is used for the synthesis of downstream amino acids;compared with Cg/WT,the production of threonine in Cg/PEC-met X decreased from 2.93g/L to 1.24g/L;methionine production increased from 1.83g/l to 2.4g/l,but there was no significant change in glutamate and lysine production.In a word,overexpression of gene met X could cause more carbon flow to methionine.3.Comparing the liquid phase of 5 strains of amino acid-producing bacteria The results from showed that the production of threonine in the metabolites of bi-gene over expressing strain Cg/PEC-lys C~m-hom~m increased significantly,reached 4.2g/l,and the threonine branch became the main consumption branch of carbon flow in Corynebacteriumglutamate.ComparedCg/PEC-lys C~m-hom~mto Cg/PEC-lys C~m-Hom~m-met X,we found that overexpression of met X could change the carbon flow direction from threonine branch to methionine branch,meanwhile increased the methionine production to 4.14g/L.However,the production of threonine is still significantly higher than the one of Cg/WT,the consumption of carbon flow of threonine branches might be the main reason for the impact of methionine production.Therefore,in order to increase the methionine production,it’s necessary to reconstruct the threonine branch to reduce the consumption of carbon flow of threonine branches.4.Using PCR technology to amplify the thr B gene of Corynebacterium glutamate,then construct the expression vector PET28a-thr B.Site-directed mutations,random mutations,and high-throughput screening were performed respectively on homoserine binding site A20 and ATP binding sites A27 and R240 from thr B gene substrates on the recombinant vector in order to obtain 2 high-enzyme active mutants(thr B-A20D and thr B-A20Q)as well as 10 low enzyme activity mutants(thr B-A20R,thr B-A20L,thr B-A20Y,thr B-A20E,thr B-A20V,thr B-A20H,thr B-D27W,thr B-D27C,thr B-R240L,and thr B-R240A).All mutants were transferred into E.coli for induced expression culture,and then we obtained pure protein after nickel column separation,SDS-PAGE,and Western blot verification.After enzymatic kinetics determination and enzyme property analysis of wild-type WT-thr B and its mutants,we analyzed the molecular structure of the variant thr B-A20Y which has a significant decrease in enzyme activity.When the alanine amino acid is changed into tyrosine,the number of hydrogen donors are increased to,meanwhile the side chain of tyrosine is large.This resulted in homoserine not entering the substrate binding pocket perfectly,and affected the binding efficiency of enzyme and substrate,and thus reduced the enzyme activity significantly.It provides a reference for the weakening of threonine branch in the production of methionine by Corynebacterium glutamicum.