Study On The Production Of Citric Acid Using Escherichia Coli Metabolic Engineering

Citric acid,an organic acid with largest annual production,has been widely applied in the fields of food,medicine,detergent and cosmetics.At present,the industrial approach of citric acid was realized mainly by Aspergillus niger as the host with preferable production and yield.However,certain characteristics such as the slow growth of Aspergillus niger which in turn longer the fermentation cycle,oxygen demanded in the fermentation process have brought many inconveniences to industrial process.Therefore,improved methods and technology are in great demand to increase the production of citric acid.The charicteristcs of Escherichia coli such as rapid growth and simple cultivation could make up for the defects of Aspergillus niger.What's more,the metabolic engineering technology of E.coli is more mature,which facilitates the transformation of metabolic pathways.Therefore,E.coli was employed as start cell to reform through metabolic engineering approach and the expression strain wgc was acquired with highest citric acid yield of 1673 mg/L under optimizing the fermentation conditions.The main content and conclusions are as follows:(1)Citrate synthase gene glt A was cloned from E.coli using PCR technology and the promoter BBa?J23100,5'UTR and terminator BBa?B1001 at both ends were introduced to glt A gene to obtain the glt A expression cassette.Accurate glt A expression cassette was acquired through connecting glt A expression cassette to the p UCm-T vector for restriction digestion identification and sequencing.The expression vector pCOLA-glt A was successfully constructed through connecting glt A expression cassette and the gene fragment pCOLA,which derived from knocking out T7 promoter and terminator on the pCOLADuet-1 vector.The expression vector pCOLA-glt A was transformed into E.coli W to obtain the expression strain wg.(2)The cad expression cassette was acquired through introducing promoter tac,5'UTR and terminator BBa?B1001 at both ends of the chemically synthesized cad gene(the codon has been optimized)using the method of PCR.The expression vector pCOLA-glt A was successfully constructed through connecting cad expression cassette and gene fragment pCDF,which derived from knocking out T7 promoter and terminator on the pCDFDuet-1 vector by PCR.The expression vector pCDF-cad was transform into E.coli wg to obtain the expression strain wgc.(3)Shake flask fermentation of expression strains wg and wgc were carried out with highest citric acid yield of wg strain 149 mg/L and 244 mg/L of wgc strain,respectively.The expression strain was determined as wgc because the production of citric acid was 1.6 fold of wg.The fermentation conditions such as carbon source,initial sugar content,temperature,p H,inoculum amount,and exogenous addition of yeast extract,were optimized through single-factor experiments.The results showed that the highest citric acid yield was 1551 mg/L under the conditions of 8 g/L sucrose,temperature 35 ?,p H 7.0,inoculum size 4% and no exogenous yeast extract.The optimal fermentation conditions were determined as fermentation temperature 35 ?,fermentation time 95 h,inoculation amount 4% by response surface test on the basis of single factor experiment with the output of citric acid 1673 mg/L.In summary,a citric acid-producing E.coli engineered strain was acquired through metabolic engineering,gene optimization,and expression element replacement in this research.This is the first study of using E.coli to produce citric acid which possesses guiding significance for the exploration of using E.coli to produce citric acid and provides another promising method for industrial production of citric acid.