Study On The Mechanism Of L-lysine Over-production In Corymebacteriun Glutamicum

Lysine is one of the essential amino acids for humans and animals and has been widely used in feed and pharmaceutical industries.The Corynebacterium glutamicum strain is a kind of important lysine-producer.A large number of lysine-producing strains have been obtained by mutagenesis and metabolic engineering in the past several decades.In the previous work,18 C.glutamicum strains with different lysine-producing levels have been sequenced and divided into different groups according to the genomic relationship,and the mechanism of high yield of lysine about the strains has been predicted.In this study,the activities of several key enzymes in the lysine synthesis pathway were tested.Reversion mutation tests were carried out to test the effects of the key enzyme mutations on the production of lysine.By analyzing the aspartokinase(LysC)encoded by the lysC gene and the homoserine dehydrogenase(Hom)encoded by the hom gene of ZL5 and A12,which were a lysine-producing strain of C.glutamicum with different lysine producing levels.It was found that LysC and Hom of ZL5 were mutated——LysC(G359D)and Hom(Q399*,399-position glutamine mutation to stop codons),respectively.LysC-ZL5(G359D)retained about 80%activity when lysine and threonine concentration was 10 mM,while the activity of LysC-A12 was loss of more than 90%under the same condition.The reversion mutation of lysC gene of ZL5 strain were carried out,which caused the lysine production of ZL5 strain decreased by 16%,it shown that LysC(G395D)mutation significantly relieved the coordinate inhibition by lysine plus threonine,thereby significantly increased lysine production.The reversion mutation of hom gene of ZL5 strain was also carried out,which leaded to the lysine production of ZL5 strain decreased by 88.57%.Hom as a key enzyme for threonine synthesis,it means that the weakening of the threonine pathway was also an important reason for the high yield of lysine.Reversion mutation the dapA gene in lysine-producing strain D was carried out,the result shown that the lysine yield decreased by 8.57%.Dihydrodipicolinate synthase(DapA)catalyzed one of the rate-limiting steps of lysine synthesis pathway,and it mean that DapA(K293E)mutation was beneficial to lysine synthesis.Compared with the lysine-hypoproducing strain A14 and the lysine-hyperproducing strain C,the aspartate-semialdehyde dehydrogenase(Asd)encoded by the asd gene of C strain had a point mutation Asd(V183A),and the meso-Diaminopimelate Dehydrogenase(Ddh)encoded by the ddh gene of C strain had a point mutation Ddh(A175V),respectively.The specific activity of the Asd mutant was retain 91%compared with that of the wild type,and the specific activity of the Ddh mutant was 1.25 times higher than that of the wild type.The reversion mutations of asd and ddh of C strain were carried out respectively,and it leaded to the lysine production of C strain decreased by 5.6%and 12%,respectively.Although the specific activity of two mutant enzymes that were important to lysine synthesis were changed differently,they all lead to a negative impact on the lysine production.It indicated that only the activities of enzymes were at the right level,and they could promote lysine production.In this study,two key enzyme mutants,LysC(G395D)and DapA(K293E),were confirmed to be important for the high yield of lysine.Meanwhile,it confirmed that the weakening of the key enzyme Hom could greatly promote the synthesis of lysine.The mutations of the asd and ddh genes of the C strain may be introduced during evolution but have negative impacts on the lysine production,which indicating that not all point mutations in the high yield strain are good.The study find some new targets for lysine strain engineering and provide theoretical basis for the further transformation of lysine industrial strains.