Construction Of Shikimic Acid Producing Engineered Escherichia Coli Strains Based On Improved PTS System Mutants

Shikimic acid is an important intermediate in the synthesis of aromatic amino acids andhas emerged as a key chiral starting material for the synthesis of antiviral drug oseltamivirphosphatc (Tamiflu). Microbial fermentation production of shikimic acid has a variety ofadvantages, and Escherichia coli was commonly applied in both large scale fermentation andindustrial production. Metabolic engineering is one of the mainly technical methods toconstruct the industrialized high-yield shikimic acid strains. Phosphoenolpyruvate:carbohydrate phosphotransferase system (PTS) is the major active transport system involvedin the glucose internalization and phosphorylation in E. coli in which it effects the use ratio ofPEP in cell. Modification and transformation of the PTS system could regulate the flow ofintracellular metabolism and reduce the waste of PEP caused by the PTS system, in the sametime, a more ideal shikimic acid producing strain can be constructed combined with thespecific modifications of metabolic pathways.In this study, we successfully knocked-out the key genes in PTS system ptsI and ptsG byusing Red homologous recombination in E. coli and meanwhile we also knocked-in theglucose facilitator gene glf from Zymomonas mobilis in the E. coli chromosome. RecombinantE. coli strains were constructed and the effects of cell growth, glucose consumption andacetate accumulation were also evaluated in all recombinant strains. The deletion of gene ptsGand ptsI made some of PTS system functions inactive and the growth ability of the cell wasalso limited. Expressing the gene glf can help recombinant E. coli strains re-absorb theglucose through Glf-glk (glucose facilitator-glucokinase) pathway as it can use ATP tophosphorylate glucose and transport into cell. This pathway can improve the availability ofglucose and also reduce the accumulation of acetic acid; it can also broaden the carbon flux inthe metabolism pathway. The recombinant strain is supposed to be a high-productivity strainand can improve the yield of products.In order to enhance the yield of shikimic acid, the shikimic kinase gene aroL and aroKwere deleted. The growth and fermentation parameters were compared in the shake flask andthe recombined strain S5was constructed. Its production of shikimic acid was193.85mg L-1,far more than that in the wild type E. coli. In this study, the expression of the enzyme AroBand AroE were also strengthened as these two were the limit enzymes of shikimic pathway.The DAHP synthase AroFfbrwas also over-expressed to relieve the feedback inhibition ofsubstrate L-tyrosine. These three genes (aroB, aroE and aroFfbr) were ligated together afterthe tac promoter and constructed the plasmid pEtac-EBFfbr. This plasmid was introduced intothe S5strain and the fermentation parameters were detected. Compared to the mutant S5, therecombination strain S5(pEtac-EBFfbr) could produce345.36mg L-1shikimic acid in theshake flask fermentation,78%higher than the former.After optimizing the component and its concentration in the medium on the basis of theinitial fermentation medium, we confirmed a kind of suitable fermentation medium for bettergrowth and production of the recombinant strain S5(pEtac-EBFfbr). With this optimizedmedium, shikimic acid production of S5(pEtac-EBFfbr) was up to2766mg L-1, increased by 6.5times than before. On the tank fermentation its shikimic acid production was15.12g L-1.