Metabolic Engineering For The Over-producing Shikimic Acid In Escherichia Coli

Shikimate (shikimic acid, SA)，as an important natural organic acid and medicialintermediate, plays a crtical role in the synthesis of aromatic amino acids, alkaloids and manyof chiral compound. In recent years, with the spread of the epidemic of avian flu in the global,shikimic acid, as the key raw materials of Tamiflu which is the only effective clinicalprevention and treatment of avian flu, have attracted more concerntration. Compared totraditional extract from Illicium plant fruit and chemical synthesis, microbial fermentationproduction of shikimic acid shows some merits such as short fermentation period, low costand less pollution of the environment. With the unparalleled advantages and potential, themicrobial fermentation has became a hotspot of solving the international demand forshikimate in recent years. Because of the low yield of SA and many other byproductsgenerated by fermentative production with the wild Escherichia coli, the use of geneticengineering, by altering the metabolism of the aromatic amino acid flux and metabolicpathways to produce shikimic acid, has broad application prospects.In this study, E. coli was metabolically engineered by rational design and geneticmanipulation for fermentative production of SA. Firstly, blocking the aromatic amino acidpathway after the production of SA was carried out by deletion of aroL and aroK genesencoding SA kinase. Secondly, the ptsG gene encoding protein EIICBglcwere removed in thearoL/aroK mutant strain to make the phosphotransferase system (PTS) system default. In theresulting strain, the phosphoenolpyruvate-dependent PTS pathway, a main pathway forglucose transport, were replaced by ATP-dependent GalP (galactose permease). Thus, morePEP flux was used to produce SA as a critical precursor of SA. Furthermore, ydiB gene(encoding quinic acid/SA dehydrogenase) was deleted to prevent SA precursors of3-dehyroquinic acid into the byproduct of quinic acid. Finally, the ackA-pta genes encodingacetate kinase-phosphate acetyltransferase were deleted to block the production of acetic acidby anaerobic pathway. Cell growth, glucose metabolism and accumulation of shikimic acid inthe mutant of deleting multiple genes were studied. Thus, the engineered strain B0013SA5with five genes deletion was constructed and150mg/L SA was produced in the shake flaskfermentation.In order to strengthen the SA synthesis pathway and guide more carbon flow to SA, theexpression of PEP synthase, transketolase A and DAHP synthase, by ppsA, tktA andaroG*(the site-directed mutagenesis aroG gene), were enhanced. DAHP synthase by aroGgene encoded is subjected to feedback inhibition by L-phenylalanine synthesized by theshikimate pathway producted. In this study, site-directed mutagenesis aroG gene (aroG*) isable to remove this inhibition. Overexpression of phosphoenolpyruvate synthase andtransketolase can increase the concentration of the shikimate synthesis of precursor PEP andE4P, which were catalyzed into the shikimate pathway by DAHP synthase. Compared tomutant SA5, the recombination strain SA5(pTH-aroG*-ppsA-tktA) could produce194mg/LSA in the shake flask fermentation which was1.29times compare to the former.By optimizing the composition and concentration of the medium on the basis of theinitial fermentation medium, we identified a fermentation medium suitable for the growth of the recombinant strain constructed in this experiment and producing shikimic acid. With thisfermentation medium, shikimic acid production of recombinant strainSA5(pTH-aroG*-ppsA-tktA) was1207mg/L in the shake flask fermentation and14.36g/L onthe tank fermentation, the latter is11.9times that of the former.