Metabolic Engineering Of Corynebacterium Glutamicum To Produce L-homoserine

Although L-homoserine does not participate in protein synthesis and belongs to non essential amino acids,it is the synthetic precursor of L-threonine,L-methionine and L-isoleucine.It has very important application value in the fields of medicine,agriculture and chemical industry.At present,the industrial production of L-homoserine mainly depends on chemical synthesis method.Chemical synthesis method has the disadvantages of high pollution,low product purity and long time-consuming.Compared with chemical method,microbial fermentation meets the needs of green development and is the trend of industrial production of L-homoserine in the future.Studies on the production of L-homoserine by microbial method mainly take Escherichia coli as the chassis strain,but Corynebacterium glutamicum,as a main industrial production strain,can be produced by fermentation with cheaper substrates in the fermentation process,with high safety,no pathogenicity to humans and animals,and is suitable for the synthesis of amino acids,It has been used to produce a variety of amino acids and various important compounds,which has very important research value.In this study,C.glutamicum ATCC13032 was used as the starting strain.Firstly,the thr B gene encoding L-homoserine kinase was knocked out by using the common gene editing tool of Corynebacterium glutamicum,so as to block the metabolic pathway of L-homoserine to L-threonine in Corynebacterium glutamicum.The thrb deletion strain C.glutamicum H1 was successfully constructed.No L-homoserine accumulation was found in the fermentation broth of wild bacteria and the cells of the strain,mall amount of L-homoserine can be accumulated in C.glutamicum H1 fermentation broth.Using different specifications of shake flasks,the dissolved oxygen environment in the culture medium was changed,and the concentration of L-homoserine and cell in the fermentation broth of C.glutamicum H1were detected under different dissolved oxygen environment.The results showed that the concentration of L-homoserine in ordinary shake flask was 44.6 mg/L,and the concentration of L-homoserine in baffle shake flask was 861.8 mg/L,which increased the yield of L-homoserine by nearly 20 multiple;In the ordinary shake flask,the cell concentration OD₆₀₀was 16.03,and in the baffle shake flask,the cell concentration OD₆₀₀was 46.4,which increased by 2 multiple.Under the environment of high dissolved oxygen,the yield and cell concentration of L-homoserine increased greatly,indicating that high dissolved oxygen is very important to improve the yield of L-homoserine.In C.glutamicum H1 strain,the mutant thr A^Fbr（Fbr,anti feedback inhibition）encoding aspartate kinase and homoserine dehydrogenase bifunctional enzyme gene thr A,which is released from threonine feedback inhibition,enhances the carbon metabolic flow of L-homoserine synthesis in C.glutamicum H1,so that more carbon sources can be transformed into L-homoserine in the metabolic process of the strain,To further improve the L-homoserine production of C.glutamicum H1.By verifying the concentration of L-homoserine in the fermentation broth under the induction conditions of different concentrations of inducers,it is concluded that the optimal IPTG induction concentration in the fermentation process of engineering bacteria is 1 mmol/L.at this concentration,the induced plasmid expression can effectively improve the yield of L-homoserine.Through shake flask fermentation,the concentration of L-homoserine in the fermentation broth is 8.09 g/L,which is nearly multiple of 13 higher.By comparing from the Rht B family of Corynebacterium glutamicum the coding genes of NCgl0143,NCgl2262,NCgl2566 and two-component transporter Brn EF and the coding gene of amino acid transporter Rht A from Escherichia coli,it was found that the overexpression of amino acid transporter Rht A in Escherichia coli increased the yield of L-homoserine most significantly,and the concentration of L-homoserine in fermentation broth increased by 11%after overexpression of Rht A.The yield of L-homoserine reached 15.28 g/L by controlling the fermentation conditions,which was nearly 70%higher than that of shake flask.