Metabolic Engineering Of Corynebacterium Glutamicum For L-threonine Production

L-threonine is the third major amino acid(behind L-glutamate and L-lysine)produced by microbial fermentation throughout the world.L-glutamate and L-lysine are both exclusively produced with Corynebacterium glutamicum,whereas L-threonine is produced with Escherichia coli in industry.C.glutamicum is the generally-regarded-as-safe(GRAS)organism,and thus is acknowledged as ideal host for the production of amino acids,especially pharmaceutical-grade amino acids.However,the capacity of C.glutamicum for L-threonine production has achieved no breakthrough for the past two decades.Since L-threonine serves as a precursor for the synthesis of L-homoalanine and as such drug intermediates,investigating the high efficient biosynthesis of L-threonine in C.glutamicum would be of great significance.In this study,an L-isoleucine production strain C.glutamicum IWJ001 was metabolically engineered into an L-threonine high production strain by rationally rearranging its metabolic network.Furthermore,the difference between the mechanisms for L-isoleucine and L-threonine high production in C.glutamicum was indicated in this study.The main work was described as follows:1.The three key enzymes in L-threonine biosynthesis in C.glutamicum IWJ001 were characterized,namely,aspartate kinase(AK1),homoserine dehydrogenase(HD1)and homoserine kinase(HK1).Mutation sites were discovered in AK1 and HD1.Through heterologous expression,purification and enzyme tests,it was confirmed that: 1)the single mutation of A279 T in AK1 completely releases it from feed-back inhibition by L-threonine and/or L-lysine;at the absence of inhibitot,AK1 exhibited identical activity to the wild-type AK from C.glutamicum ATCC13869;2)the single mutation of G378 S in HD1 partially released it from feed-back inhibition by L-threonine,with the half maximal inhibitory concentration between 12 to 14 mM;at the absence of inhibitor,HD1 exhibited about 27% higher activity than the wild-type HD.2.A preliminary insight into the mechanism of high L-isoleucine production in C.glutamicum IWJ001 was achieved by studying the effects of deletion or overexpression of key genes on amino acids production: 1)deleting ilvA1 in IWJ001 failed to result in L-threonine accumulation,but caused carbon overflow at the pyruvate node,indicating threonine dehydratase TD1 encoded by ilvA1 plays the role of main driving force in L-isoleucine high production in IWJ001;2)TD1 as the main driving force.was confirmed by investigating the effects of overexpressing lysC1,hom1,thrB1,ilvA1 and ilvBN1 in different combinations on amino acids production in the wild-type ATCC13869.These results indicated that DXW001(IWJ001 derivative ddeficient in ilvA1)failed to accumulate L-threonine might be due to a lack of driving force in the L-threonine terminal pathway.3.DXW001 was metabolic engineered into an L-threonine production strain by 1)overexpressing the L-threnone exporter gene EcrhtC from E.coli and the native hom1 and thrB1 to pull carbon into L-threonine pathway;2)deleting the ilvBN1 C operon to push carbon into L-threonine pathway meanwhile reduce by-products accumulation.The resulting strain DXW002/p10-EcrhtC-hom1-thrB was able to produce 5.1 g/L L-threonine with a yield of 0.17 g per gram of glucose in shakeflask cultivation.4.The anaplerotic synthesis of oxaloacetate was strengthened and the TCA cycle flux was attenuated to enhance L-threonine production,by tuning biotin and D-panthothenate availability in the medium.Such a novel strategy was designed by utilizing: 1)the dependence of pyruvate carboxylase on biotin;2)the dependence of pyruvate dehydrogenase on D-pantothenate;3)the auxotrophic phenotype of DXW002/p10-EcrhtC-hom1-thrB for both biotin and D-pantothenate.RT-PCR revealed transcriptional regulation around the phosphoenolpyruvate-pyruvate-oxaloacetate node caused by tuning of co-factors availability.By addition of 0.15 mg/L biotin,D-pantothenate was shown dispensible for DXW002/p10-EcrhtC-hom1-thrB,and L-threonine titer was improved by 2.7 folds reaching 19.07 g/L,with a yield of 0.47 g per gram of glucose.5.Deleting pck and ddh in DXW002/p10-EcrhtC-hom1-thrB to minimize carbon loss and eliminating L-lysine by-production.The resulting strain DXW004/p10-EcrhtC-hom1-thrB was able to produce 20.1 g/L L-threonine with a yield of 0.53 g per gram of glucose in shakeflask cultivation.6.A solution to attenuating L-lysine accumulation in L-threonine and L-isoleucine production without sacrificing the cell growth was found.A base strain with eliminated L-lysine accumulation was constructed from C.glutamicum ATCC13869 by deleting ddh and lysE,and was further engineered for L-threonine or L-isoleucine production.L-lysine by-production was shown substantially attenuated in L-threonine and L-isoleucine production,with cell robustness maintained.Furthermore,deletion of ddh and lysE improved L-threonine production by 50%,;deletion of ddh boosted cell growth of the L-isoleucine production strain and improved its cell density by 21%.