Production Of L-Serine In Recombinant Escherichia Coli

L-serine is an important amino acid which is widely used in food, chemical and pharmaceutical field. Nowdays, L-serine is mainly synthetized by three methods comprising direct extraction, chemical synthesis, and biological synthesis. Due to toxic chemical agents involved in the process, direct extraction and chemical synthesis exhibit serious harm to the environment. In recent years, with the development of metabolic engineering, transcription genomics and synthetic biology, through the analysis of metabolic pathways, overexpression of an endogenous gene or the introduction of exogenous genes, and elimination of competition branch, several engineered strains for industrial production of the particular target compound have been constructed. However, the production and productivity of microbial fermentation is still low, and therefore a method with high substrate conversion rate and production is imperative for L-serine production.E. coli, because of its easy cultivation, clear genetic background and simple genetic manipulation, has been used in large-scale synthesis of a variety of valuable compounds. In E. coli, L-serine is synthesized from3-phosphoglycerate, a intermediate product of glycolysis, by use of3-phosphoglycerate dehydrogenase, phosphoserine aminotransferase and phosphoserine phosphatase, respectively.As an important intermediate metabolite in E. coli, L-serine is the precursor of many other metabolites, such as cysteine and glycine, which could be completely degraded by converting into pyruvate. As a result, only a small percentage of L-serine could be accumulated in the culture though a large amount are produced from glucose in vivo. L-tryptophan production in E. coli has been studied for a long time, but it had not been reached the industrial requirement until now.Plasmid, an important tools for gene overexpression, has been wildly used in metabolic engineering and synthetic biology. With differences of promoter strength, copy numbers and other aspects, plasmids could achieved different gene expression level. Although overexpression of genes with high-copy plasmid could indeed achieve high gene expression level, a great deal of energy and substance are needed to maintain the duplication and expression of plasmid which would be more obvious for high-copy plasmid. In addition, high-copy plasmid would be lost more easily during strain cultivation. In this study, we used E. coli DH5a as a parent strain. By deleting sdaA and overexpressing serABC in plasmid pBBR1MCS-2and pTrc99a, we consturcted L-serine producing strain Sp and SpTrc, respectively. After72hours batch cultivation, Sp produced4.46g/L L-serine, while the production of SpTrc reached6.36g/L. It indicated utilization of pTrc99a with stronger trc promoter improved the L-serine production. Otherwise, the accumulation of acetate was also increased in strain SpTrc.Glyoxylate cycle is a replenishment pathway of TCA cycle, which is inactive in E.coli K12due to the suppression by transcriptional repressor protein Ic1R and ArcA. In order to redistribute the metabolic flux and produce more L-serine in E. coli, we carried out gene deletion of Ic1R and ArcA to activate glyoxylate cycle. aceB, the first gene of aceBAK operon, encodes an important enzyme of glyoxylate cycle, malic acid synthase. It was reported that the absence of aceB would cause the accumulation of glyoxylate, and change the distribution of metabolism in turn. Therefore, we deleted aceB to elevated the glyoxylate level, and named consturcted strain3SpTrc. In batch fermentation, it produced8.34g/L L-serine after72h and exhibited an significantly improvement in glucose consumption and biomass accumulation.Due to the disadvantage of genetic instability of plasmids, a method for rapidly and convenietly integrating genes into genome is needed for microbial overproduction. In this study, we integrating serAsBC operon into genome by use of FLP/FRT recombination and constructed a strain with three copies operon on its genome. It could produced2.3g/L L-serine in batch fermentation. Then we randomLy intergrated the gene serAs and serC into strain3SR with five FRT sites in the genome, achieving the highest L-serine production in recombine strain which have10,6,4copies of serAs, serC, serB, respectively.