Metabolic Engineering Of Escherichia. Coli For L-serine Production

L-serine is an important intermediate metabolite in vivo, widely usd in medicine,cosmetics, food and other industries. Focusing on the L-serine biosynthetic pathways,Escherichia. coli was engineered as the starting strain, aiming at direct fermentationof L-serine from glucose by metabolic engineering. The sdaA、sdaB and tdcG geneswere disrupted, the pgk, serA△197, serC and serB of synthesis pathway wereoverexpressed, and the following gcvP deletion or glyA fine-tuning was alsoconducted to decrease the degradation of L-serine or increase the precursor supply,and finally enhance the biosynthis of L-serine.Applying the modified λ Red recombination system, the sdaA、sdaB and tdcGgenes were knocked out in E. coli MG1655successively to interdict the conversion ofL-serine to pyruvate. The resulting strain EJ3grew normally in the minimal medium,while the specific growth rate of which exhibited a slight decline; no L-serine wasdetected in the supernatant.Subsequently, the plasmid-based overexpression vector pJH04was transformedinto the strain EJ3, and the four genes of de nove synthesis pathway wereoverexpressed. The key gene serA was introduced from Cornebacterium glutamicumATCC13032exogenously which encoded the phosphoglycerate dehydrogenaseinsensitive to L-serine. The resulting strain EJ3(pJH04) could accumulate73.2mg/lL-serine in minimal medium, however, the accumulated L-serine was broken down inthe following fermentation. In addition, the biomass of EJ3(pJH04) went an apparentdecline mainly because of the introduction of plasmid.To attenuate the degradation of L-serine to glycine, two strategies were applied:(I)The fragment constructed to disrupt the gcvP gene was tranformd into the strainEJ3(pJH04) to interdict the glycine cleavagesystem and further indirectly inhance theL-serine accumulation by increasing the concentration of glycine. The gcvP deletionstrain EJ4(pJH04) produced200.5mg/l L-serine in14h, which was2.7-fold than thatof strain EJ3(pJH04).(II) The fragment constructed to replace the RBS sequence ofglyA gene was tranformd into the strain EJ3(pJH04) to improve the L-serineaccumulation by weakening the glyA expression in translational level. Of the obtained13RBS mutants, the strain S20(pJH04) accumulated the maxmium L-serine, reaching 778.4mg/l with a yield of102.4mg L-serine/g glucose. What’s more, the gainedL-serine was degraded in a trace level compared to the strains EJ3(pJH04) andEJ4(pJH04). In fermentor culture, S20(pJH04) could achieve a3.74g/l L-serineaccumulation with a increased yield of23.4%. The above results indicated theenhancement of L-serine production by E. coli could be acheved by metabolicengineering, and further provided guidances for other amino acids production.