Study On Fermentation Process For Production Of Shikimic Acid By Genetic Engineered E.Coli

Shikimic acid, a metabolic intermediate of shikimic acid pathway, is the key ingredient for synthesis of chiral pharmaceuticals. However, the supply of shikimic acid is limited, resulting in low yield and high costs. With the development of genetic engineering and metabolic engineering, microbial synthesis of shikimic acid has become an alternative method, which is short production time, low cost and environmentally friendly. In this paper, the engineered strain of Escherichia coli(E. coli) was used to production of shikimic acid, and fermentation process was optimized to improve the yield of shikimic acid. The conclusions are as follows:(1) Study on the effect of strains preservation stability and hereditary stability on yield of shikimic acid. The production strains were transferred to the slant medium after separation. After passaged for five generations, the production capacity was examined. The strain showed a good hereditary stability. In addition, the effect of preservation time in frozen tubes on production capacity was also investigated. The results indicated that the strains preserved within24months had good genetic stability.(2) The single factor experiment was applied to investigate the effect of slant culture time, seed age, inoculums, liquid volume and initial pH on shikimic acid fermentation in shake flasks culture. The optimum conditions for fermentation were slant culture time17-18h, seed age6-8h, inoculums5%, loading volume20mL/250mL and initial pH7-7.5.(3) The fed-batch fermentation of shikimic acid in50L fermentator was studied by investigating process conditions including sterilization method of basic carbohydrate, oxygen level, pH control and residue glucose concentration. The optimized conditions were basic carbohydrate autoclaved separately, relative oxygen20%-30%, and the pH7.5with ammonia, residual glucose0.5%-1%with feeding60%glucose solution. Under these conditions, the yield of shikimic acid could reach30000μg/L. (4) The amount of amino acids in fermentation medium of shikimic acid was studied in50L fermentation process. The optimum medium for production of shikimic acid were glucose20g/L, yeast extract lOg/L, tryptone lOg/L, Na2HPO45g/L, K2HPO43g/L, Mg2SO40.5g/L, NH4C11g/L, NaCl0.5g/L, L-phenylalanine0.5g/L, L-Tyrosine0.75g/L, L-Tryptophan0.75g/L and trace element1mL/L, in which the yield of shikimic acid was42000μg/L.