Abstract

Vanillin is an important flavouring agent applied in food, spices, pharmaceutical industries and other fields. Microbial biosynthesis of vanillin is considered a sustainable and economically feasible alternative to traditional chemical synthesis. In this study, Escherichia coli K12 MG1655 was used for the de novo synthesis of VAN by screening highly active carboxylic acid reductases and catechol O-methyltransferases, optimising the protocatechuic acid pathway, and regulating competitive metabolic pathways. Additionally, major alcohol by-products were identified and decreased by deleting three endogenous aldo-keto reductases and three alcohol dehydrogenases. Finally, a highest VAN titer was achieved to 481.2 mg/L in a 5 L fermenter from glucose. This work provides a valuable example of pathway engineering and screens several enzyme variants for the first time in E. coli.