Increased L-serine Production By Metabolic Engineering Of GlyA Knockout E.coli.

L-seriene is an important intermediate metabolite in vivo,and widely used in food,cosmitics and medicine.Focusing on cutting off the L-serine degradation and down-regulating the competing pathway,the genes which code L-serien deaminase deletion strain EJ3 was engineered as the starting strain to improve the L-serien production from glucose.Firstly,the glycine cleavage system and kbl-tdh were overexpressed to supply sufficient C1 unit,and the glyA which codes the L-serine hydroxymethyltransferase was knoked out to cut off the L-serien degradation.Then the serA of Escherichia coli was replaced by the serA?197 of Corynebacterium glutamicum.And the gpmA and pykF was deleted to make moer 3-phosphoglycerate flow to L-serine biosynthses pathway.To improve the glucose utilization efficiency,the ptsHIcrr of glucose transport system was deleted and galP was overexpressed.At last,one copy of L-serien biosynthses pathway genes was inserted to the chrosome of Escherricha coli to improve the stability of the L-serien biosynthses pathway and L-serien productivity of the strain.Firstly the gcvP and gcvT coding the glucine cleavage system of EJ3 were overexpressed to supply sufficient C1 unity by utilizing glycine.On the base of that,glyA was deleted,and the overexpression of kbl-tdh released the glycine dependent of the strain.The L-serine degradation pathway cutting down strain E4G2 produced 266.3mg/l L-serine,grew normally as the starting strain EJ3.In order to release the L-serine feedback-inhibitaion of phosphoglycerate dehydrogenase,the serA of Escherichia coli was replaced by ser A? 197 of Corynebacterium glutamicum,and the pgk,serA?197,serC and serB were conjuncted to the high copy plasmed.The activity of phosphoglycate dehydrogenase of the serA replacement strain E4G5 was decreased and the L-serine production decreased slightly.The flux from 3-phosphoglycerate to pyruvate was down-regulated to make more 3-phosphoglycerate flow to L-serine biosynthesis.Thus the gpmA and pykF was knocked out to get the strain G13.Compared with other strains,G13 produced 744.3mg/l L-serine but the glucose utilization ability was decreased.In view of the problem of glucose utilization efficiency,glucose transport system ptsHIcrr was knocked out,and galP was overexpressed to transport glucose at the same time.The corresponding strain G18 produced 1222.1mg/l L-serine,and the glucose utilization efficiency improved but the utilization ability was still weak.At last one copy of genes(pgk-serA? 197-serB-serC)in L-serien biosynthesis pathway was insterted to the chrosome of the engeering strain G18 to make up for the problem of plasmid untabilty.And L-serine biosynthesie ability was more stable and produced 1928.4mg/l L-serine.