Process Optimization Of Vanillin Fermentation By Recombinded Escherichia Coli

As an important flavour,vanillin is widely used in food and pharmaceutical industries.Chemical synthesis vanillin is cheap,but consumers prefer natural vanillin.Due to vanillin gained from vanilla can not meet the market demand for natural vanillin,vanillin producted by biotechnology may becom its alternative.Synthetic vanillin from ferulic acid is very popular nowadays.In this work,the vector is according the recorded gene of ech and fcs.We cloned the recombinant plasmid into Escherichia coli BW25113 and carried out the initial fermention.Vanillin production reached to 1240 mg/L after incubation for 48 h with 3 g/L ferulic acid in the optimized medium.Then we cloned gltA gene encoding citrate synthase to downstream of ech-fcs.The vanillin production reached to 1322 mg/L.PCR-mediated site directed mutation was used to make mutations of Cys-206 and Glu-207 of citrate synthase,as a result,vanillin production reached to 1668 mg/L.With the Red recombination system the icdA gene of BW25113 was knocked-out,which finally improved the production of vanillin to 1821 mg/L,which was 46.85%higher than before.Fermentation medium was also investigated through single factor experiment,orthogonal experimental design in shake-flask culture conditions.The optimal culture conditions were as follows:the fungus were induced by 1 mM IPTG,30?anaerobic fermentation for 10 h,then transfer to 37?until the fermentation is completed.The optimal constitution of culture medium was:add corn steep powder of 5 g/L,and sucrose of 3 g/L,ZnSO₄ of 0.5 mM as the final concentration to 2YT medium.Vanillin production reached to 2007 mg/L.