Study On The Fermentative Production Of Shikimic Acid By Brevibacterium Lactofermentum

Shikimic acid is a high valued compound used as a key starting material for the synthesis of the neuramidase inhibitor GS4104,which was developed under the name Tamiflus for treatment of antiviral infections.Isolation of shikimic acid from the fruits of the Illicium plant is cumbersome and costly and limited by the growth of the fruits of the Illicium plant,particularly,when we face an outbreak of Avian influenza and poor harvest of the fruits of the Illicium plant.In order to improve shikimic acid's availability,fermentative production processes from renewable resources like glucose present an excellent and even more sustainable alternative to meet the current market volume at a competitive price level.In this paper,the shikimate pathway and the production of shikimic acid by Brevibacterium lactofermentum was investigated.1.The effect of glyphosate on the yield of shikimic acid,phenylalanine,tryptophane and tyrosine in the shikimate pathway was investigated.With the addition of Glyphosate and urea,the yield of shikimic acid are substantially increase as aromatic amino acid are correspondingly decrease,which proved glyphosate can blocking the shikimate pathway of Brebacterium lactofermentum.The effects of the concentration of glyphosate,adding time and processing time on the fermentation of shikimic acid was studied.Adding glyphosate at the logarithms generate term,the yield of shikimic acid increases with increasing the concentration of glyphosate(in the low concentration range)and the processing time(in 24 hours)the that growth in the number of accession to the end of(in the low concentration range). Cultured 24 hours,added glyphosate to the broth(the concentration of glyphosate is 2000μmol·L^-1),the yield of shikimic acid is 377.26 mg/ L, which is 7 times as that of the glyphosate is absent.2.Designed a method of "compound mutagenesis-mixed fermented Breeding" to Screening strains of higher flux of the shikimate pathway. Choose Brebacterium lactofermentum(CICC10227)as the original strain for control of mutation breeding.Compound mutagenesis of UV and NTG under the designed conditions,and then all the strain fermented together,and optimum the fermentation conditions.Then we screened 8 strains.In this study, the better Mutation is 62.5%,greatly the method reduced the blindness and the workload of the traditional mutation breeding,and improved the efficiency of the mutagenesis.3.In this study,the conditions of fermentation is optimized.15 hours was be identified the best growth stage to inoculation.Through the uniform design to determine the optimal fermentation condition is:glucose16%,of ammonium sulphate2%,magnesium sulfate 0.04%,corn pulp0.5%,KH₂PO₄ 0.25%,K₂HPO₄ 0.25%,PH7.0～7.2.At this point the yield of shikimic acid is 648.12mg/L.The favorable fermentation condition was:inoculation amount 5%,media amount 60ml/250ml and pH7.0.