Kinetic Characterization Of Partial Key Enzymes In The Central Metabolic Pathway Of Corynebacterium Glutamicum

Due to its wide substrate spectrum and easy to cultivate,Corynebacterium glutamicum is widely used in industrial production of L-glutamate,L-lysine and other amino acids.The purpose of this dissertation is to systematically characterize the kinetics of some key enzymes in the central metabolism of C.glutamicum type strain ATCC 13032 and a mutant strain with high L-lysine production,to determine the inhibition types and inhibition constants of some metabolites toward these key enzymes,and to provide data support for construction of C.glutamicum genome scale metabolic network model with enzyme restriction and designing of L-lysine high-yield strains by reverse metabolic engineering.The specific research objects of this dissertation are 6-phosphogluconate dehydrogenase(6-PGDH,the key enzyme of pentose phosphate pathway),L-malate dehydrogenase(MDH,the key enzyme of tricarboxylic acid cycle),and aspartate kinase(AK,the key enzyme of L-lysine synthesis pathway).The interconversion between L-malate and oxaloacetate catalyzed by MDH is one of the key reactions in TCA cycle,which connects multiple metabolic pathways in C.glutamicum.Oxaloacetate,the substrate of MDH,is the direct precursor of biosynthesis of aspartic acid family of amino acids like L-lysine.In the second chapter of this dissertation,the exogenous expression and purification of MDH in C.glutamicum ATCC 13032 and MDHm(272 Thr residue mutated to Ile residue)in a mutant strain with high L-lysine production were realized.It was confirmed that MDH and MDHm catalyze the reversible transformation between L-malate and oxaloacetate,but tend to use NADH as the cofactor to catalyze the reduction of oxaloacetate to L-malate.The kinetic parameters of MDH and MDHm with oxaloacetate and NADH as substrates were determined.It was found that the mutation of Thr residue at 272 position to Ile residue increased the affinity of MDHm for oxaloacetate while decreased its affinity for NADH.The inhibition type and inhibition constant of 2-ketoglutarate,ATP,ADP and AMP on reactions catalyzed by MDH and MDHm were also determined.Compared with MDH,the response mode of MDHm to intracellular energy level was changed.MDH was inhibited by ATP,while MDHm was inhibited by AMP.6-PGDH is one of the key enzymes in the pentose phosphate pathway of C.glutamicum.It catalyzes the production of ribulose-5-phosphate and CO2 from 6-phosphogluconate,accompanied by the production of NADPH.Some studies have shown that the mutation of 6-PGDH can significantly improve the production of L-lysine in C.glutamicum.In the third chapter of this dissertation,the exogenous expression and purification of 6-PGDH in C.glutamicum ATCC 13032 and 6-PGDHm(353 Ser residue mutated to Phe residue)in a mutant strain with high L-lysine production were realized.The kinetic parameters of 6-PGDH and 6-PGDHm with 6-phosphogluconateand NADP+as substrates were determined.It was found that the mutation of Ser residue at 353 position to Phe residue increased the affinity of 6-PGDHm for 6-phosphogluconic acid,but also significantly decreased its catalytic efficiency.The inhibition types and constants of fructose-1,6-diphosphate,ATP,NADPH,glyceraldehyde-3-phosphate and phosphoenolpyruvate on the reaction catalyzed by 6-PGDH and 6-PGDHm were also determined.It was found that 6-PGDH and 6-PGDHm were significantly inhibited by NADPH,but the Ki of NADPH did not change before and after mutation.AK is the first rate limiting enzyme in biosynthesis of aspartic acid family of amino acids.Its activity is feedback inhibited by L-lysine and L-threonine.Some studies have shown that AK mutation has a positive effect on the accumulation of L-lysine.In the fourth chapter of this dissertation,the exogenous expression of AK in C.glutamicum ATCC 13032 and AKm(311 Thr residue mutated to Ile residue)in a L-lysine high-yielding strain were carried out.It was found that only a subunit of AK and AKm could be expressed and purified when His tag was placed at the N-terminal.When His tag was change from the N-terminal to the C-terminal,both a subunit and ? subunit of AK and AKm could be expressed and purified,laying a foundation for the kinetic characterization of AK and AKm.