Increased L-serine Production By Evolutionary Engineering And Metabolic Engineering In Escherichia Coli

Since it is an important amino acid,L-serine has been widely applied in many fields,including the cosmetics,pharmaceutical and food industries.With the rapid development of technology,microbial fermentation has attracted many reseachers'attention.In this study,two strategies were used to improve the L-serine biosynthesis.In the first part,based on the use of evolutionary engineering,E4G1 was engineered to relieve dependence on glycine for L-serine production.In the second part of this study,we improve the L-serine production in Escherichia coli through the metabolic engineering of glycolysis pathway and acetic acid synthesis pathway from glucose.In the first part of this study,firstly,E4G1,as the staring strain,was engineered to relieve dependence on glycine through evolutionary engineering.A mutant E-3which could grow normally and has strongly ability to produce L-serine was obtained.In addition,in order to study the effect of different pla smids on the accumulation of L-serine,the plasmids were introduced into the strain and the results showed that the production of L-serine is the highest when plasmid pPK10 was used.Simultaneously,E-3 was fermented in six different media,and the results showed that the maximum accumulation of L-serine was 608mg/l in M9 medium containing 1.0g/l YE,which was about 4.4 times compared with the starting strain E4G1.Then we studied the difference effects of serA^?197 from Corynebacterium glutamicum and serAmut which His³⁴⁴,Asn³⁴⁶ and Asn^364'residues were converted to alanine in Escherichia coli on the yield of L-serine.The accumulation of L-serine in E-3?p Z10?was 573 mg/l.The results showed that the replacement of serA^?197 on the pPK10 with serA_mut had no significant effect on the accumulation of L-serine.In the second research,through the engineering of glycolysis pathway and acetic acid synthesis pathway,we improved the L-serine production in Escherichia coli from glucose.In order to make more 3-phosphoglycerate of the glycolysis convert to L-serine,the gpm A in E4G2 which has been relieved glycine dependence through rational metabolic engineering was knocked out for the first time.The corresponding strain G24 produced 1037mg/l L-serine which was almost 4 times more than E4G2.In order to retain the glucose consumption rate and further down-regulate the EMP pathway and make more 3-phosphoglycerate flux to the L-serine biosynthesis pathway,the gene pyk F was deleted.The strain G25 could produce 1102mg/l L-serine,which was slightly higher than G24.Then by inactitivating acetate formation genes ack A-pta,the corresponding strain G30 produced 0.6 g/l acetate which was about 4.5times lower than that of the G25.And the yield of L-serine of G30 was about 17%higher than that of G25.The L-serine and acetate productivity had a little decrease compared with G25,we hypothesize that a certain amount of acetate is beneficial to the growth of strain.The plasmid loss rate of pPK10 increased gradually throughout the cultivation process.In order to make the expression of the L-serine synthesis pathway more stable,a copy of pgk,serAmut serC and serB with substitution of strong constitutive promoter trc was inserted to the genome of G25 and G30,resulting in strain G34 and G35.The engineering strain G34 could accumulate 1228mg/l L-serine in flask,which is about 11.5%higher than the engineered strain G25.The engineering strain G35 could accumulate 1069mg/l L-serine in flask,which is about 8%higher than the engineered strain G25.