Breeding L-methionine Over-producer By Corynebacterium Glutamicum Based On Metabolic Engineering

The sulfur-containing amino acid L-methionine is an essential amino acid that is irreplaceable in various of important metabolic activities.It has been used in many fields.As a feed additive,a large demand for L-methionine is met by chemical process or protein hydrolyzation.The production of L-methionine by submerged fermentation in large scale hasn't been achieved up to now.In order to solve problems mentioned above,a highly efficient L-methionine producing strain based on Corynebacterium glutamicum ATCC13032 and metabolic engineering principles was breeded.The effects of different genetic manipulations on L-methionine biosynthesis were analyzed in order to find the key targets of L-methionine biosynthesis pathway.The main research contents and results are as follows:Previously,in order to obtain an excellent platform for the production of L-methionine,the effect of deleting different L-methionine absorption system on the absorption activity and yield of L-methionine was studied.Met D protein complex was the main uptake system of L-methionine,and the fermentation results showed that the L-methionine yield of C.glutamicum ?met D was the highest.Based on C.glutamicum ?met D,the feedback inhibitions of metabolic end-products were removed by mutagenesis.A mutant named C.glutamicum 5-16 with analog-resistant for L-methionine producing was screened.The yield of L-methionine was 2.54 g/L which increased about 9 times to C.glutamicum ATCC13032,and the yield of product to substrate was 0.028 mol/mol.The transcription levels of the 7 enzymes in the L-methionine biosynthetic pathway were significantly increased in C.glutamicum 5-16.The genetic stability of C.glutamicum 5-16 was proved to be an excellent platform for the production of L-methionine.The competitive ways of L-methionine were blocked or weakened,then the feedback inhibitions of intermediate metabolites were removed by gene knockout or site-direct mutation,in order to enhance the synthetic metabolic pathway of L-methionine.The thr B gene was knocked out to block the competitive way of L-threonine.Then,we weakened the L-lysine branching pathway by changing the start codon of the dap A gene.Finally,the lys C gene and pyc gene were site-direct mutation to fixed the AK and PCx feedback inhibition.The AK node had a significant impact on the L-methionine biosynthesis.L-methionine yield was significantly increased,eventually.The fermentation results showed that the yield of L-methionine was increased to 5.89 g/L,and the yield of the product to substrate was 0.072 mol/mol.In addition,the knockout of the mcb R gene was not beneficial to the production of L-methionine on the basis of C.glutamicum LY-4.A new type of E.coli-C.glutamicum constitutive shuttle expression vector p LY-4 was constructed.p LY-4 has the following characteristics: the multiple cloning sites MCS with 11 restriction enzyme sites;two sets of screening system including chloramphenicol resistance system which was applicable to laboratory scale operation,and non-screening marker system based on alr gene which could meet fermentation without antibiotic;carring tac M promoter,which was a constitutive promoter removing induced expression of the lac Iq gene.Through verification of the performance of the expression vector p LY-4,it was proved that p LY-4 was a new vector for the study of the metabolism of C.glutamicum.On the basis of C.glutamicum LY-4,the effect of different methods of increasing NADPH supply on the yield of L-methionine was studied.The results showed that NADPH from glycolytic pathway is more conducive to the production of L-methionine than NADPH from pentose phosphate pathway.At last,the gap C gene was overexpressed using vector p LY-4 in C.glutamicum LY-4 to obtain recombinant strain LY-6.The fermentation results showed that L-methionine yield increased to 7.23 g/L and the yield of prouct to substrate was 0.110 mol/mol.The fermentation medium components and parameters of recombinant C.glutamicum LY-6 were optimized by response surface method.With the optimized fermentation medium composition and fermentation parameters,the final L-methionine production reached 9.88 g/L,26.82% higher than the original strain,moreover,the yield of product to substrate was increased to 0.133 mol/mol.