Metabolic Engineering Of Corynebacterium Glutamicum For L-threonine Production

L-threonine is an essential amino acid for human and animal nutrition,widely used in various fields such as food,medicine,and feed.The global market size exceeds700,000 tons per year.L-threonine is the second limiting amino acid in pig feed and the third limiting amino acid in poultry feed,primarily used as a feed additive.Currently,industrial production of L-threonine is achieved through fermentation using Escherichia coli.Corynebacterium glutamicum,a bacterium commonly used as an industrial production chassis for amino acids such as glutamic acid and L-lysine,possesses advantages such as biosafety and strong environmental adaptability.It is considered a potential strain for the efficient production of L-threonine.However,current production of L-threonine by C.glutamicum often leads to the accumulation of significant amounts of other amino acid by-products,limiting the level of L-threonine production.Previous studies that aimed to block or weaken the pathways for by-product synthesis resulted in strains with nutritional deficiencies or only partial reduction in by-product accumulation.In this study,starting from the wild-type C.glutamicum,we first achieved the synthesis of L-threonine and its by-products by relieving the feedback inhibition of key enzymes in the L-threonine synthesis pathway and enhancing the expression of key genes.Secondly,we significantly reduced the accumulation of by-products by enhancing the feedback inhibition of two by-product synthesis pathways.Finally,by overexpressing L-threonine exporter and homoserine kinase,we further increased L-threonine production and reduced by-product accumulation.The main research content and results are as follows:（1）Modification of the L-threonine synthesis pathway:In wild-type C.glutamicum,the lys C gene（encoding aspartokinase）and the hom gene（encoding homoserine dehydrogenase）were targeted.The reported T311I and G378E point mutations were introduced to alleviate feedback inhibition.The production of L-Threonine reached 0.27g/L.By employing strong promoters P_pyc-20 and P_{gpm A-16} to enhance the expression of lys CT311I-asd and hom G378E-thr B operons,the production of L-threonine was increased to 2.33 g/L.Meanwhile,the by-products L-lysine,L-isoleucine,glycine,and L-homoserine also reached production levels of 0.47-0.96 g/L.（2）Modification of the by-product synthesis pathway in L-threonine fermentation:Heterologous replacement of key enzyme genes with strong feedback inhibition from external sources was employed in C.glutamicum,enabling control over by-product synthesis without causing nutritional deficiencies in the strain.By replacing the endogenous gene with the gene encoding dihydrodipicolinate synthase（Spdap A）from Streptococcus pneumoniae,feedback inhibition in the L-lysine synthesis pathway was enhanced.Plate fermentation showed no accumulation of L-lysine.Similarly,by replacing the gene encoding threonine dehydratase（Ecilv A）with the gene from E.coli,feedback inhibition in the L-isoleucine synthesis pathway was enhanced,reducing L-isoleucine production from 0.42 g/L to 0.03 g/L.After the modification,plate fermentation of the strain did not require the addition of any additional nutrients,and there was a slight increase in L-threonine or precursor L-homoserine production.（3）Overexpression of L-threonine exporter and homoserine kinase:Different sources of L-threonine exporters were overexpressed,including the endogenous Thr E and Ser E from C.glutamicum and Rht C from E.coli.The results showed that overexpression of Rht C had the most significant effect,increasing L-threonine production from 2.98 g/L to 9.07 g/L,an improvement of 2.04-fold.However,it also led to an increase in precursor L-homoserine production from 0.71 g/L to 2.10 g/L.To reduce the accumulation of L-homoserine,further overexpression of the homoserine kinase gene thr B was performed.Plate fermentation showed a decrease in L-Homoserine production to 0.15 g/L.The engineered strain Zcgl T11,constructed from scratch,was evaluated in a 5 L fermenter for production.The L-threonine production reached 67.63g/L,with a glucose conversion rate of 0.21 g/g and a production intensity of 1.21 g/L/h.There was no accumulation of L-lysine during fermentation,and the accumulated levels of L-isoleucine and glycine at the end of fermentation were approximately 1 g/L.The production of L-homoserine was 4.58 g/L.By reconstructing the feedback inhibition of L-threonine and by-product amino acid synthesis pathways and regulating their expression,this study has successfully engineered a non-nutritional-deficient strain of C.glutamicum for L-threonine production.The accumulation levels of various by-product amino acids have been significantly reduced,and both L-threonine production and production intensity surpass the highest reported levels in C.glutamicum.The developed strategy for by-product control in this study provides a new approach to address the issue of by-product accumulation in L-threonine production by C.glutamicum and can also serve as a reference for constructing other amino acid-producing strains.