Construction Of High-yielding E.coli Strain For Shikimic Acid Production And Research In Fermentation

Shikimic acid is a metabolic intermediate of shikimic acid pathway, andalso key material for the synthesis of chiral drugs. At present, the productionof shikimic acid depends on extracting from Illicium verum. This methodhas not only limitations on plant material, but also high cost and low yieldon time-space. As the rise of genetic engineering and metabolic engineering,synthesizing shikimic acid through microbial metabolism has become afeasible method. Besides short production cycle and low cost, microbialmethod also has less pollution on the environment. This paper focus onconstructing shikimic acid high-yielding strains with genetic engineeringmethods, the finished research mainly included as following:1. Relying on Red recombination system, Successfully knocked out thegene aroL of wild E.c oli W3110,constructed E.coli W3110：aroL-. Tookshake flask fermentation under the same conditions, E.coli W3110: aroL-ha-d much higher shikimic acid production than E. coli W3110, but had less th-an E.coli GSH: aroL-, aroK-.2. Strengthened3-deoxy-d-arabino-heptulosonate-7-phosphate synthaseand transketolase A by constructing plasmid pFGT which contains genes:aroF, aroG, tktA. In addition, aroF and aroG are connected in series for thefirst time to strengthen the expression of3-deoxy-d-arabino-heptulosonate-7-pho sphate synthase at the greatest extent. 3. Strengthened shikimate dehydrogenase,3-dehydroquinate synthaseand PEP synthase by constructing plasmid pEBP which contains genes: aroE,aroB, ppS.4. Constructed shikimic acid fermentation strains: E.coli GSH：pFGT,pEBP；E.coli GSH：pFGT,pAEB；E.coli GSH：pKFG,pEBP；E.coli GSH：pKFG,pAEB. And chosen E.coli GSH: pFGT, pEBP as shikimic acid produ-ction strain,which had higher shikimic acid yield in flask fermentation.5. Took use of flask fermentation and the response surfacemethodology to optimize concentration of yeast extract, peptone, and aminoacids in the medium for E.coliGSH: pFGT, pEBP. The optimal value is:yeast extract3.82g/L；peptone3.61g/L；Tyrosine, phenylalanine,tryptophan6.85mg/L separately. Under these conditions, the production ofshikimic acid is up to705.32mg/L.6. Took fermentation in a1.2L fermenter with E.coliGSH: pFGT, pEBP.The production of shikimic acid could reach19.13g/L.