Microbial production of methyl anthranilate, a grape flavor compound.

Abstract

Methyl anthranilate (MANT) is a widely used compound to give grape scent and flavor, but is currently produced by petroleum-based processes. Here, we report the direct fermentative production of MANT from glucose by metabolically engineered <i>Escherichia coli</i> and <i>Corynebacterium glutamicum</i> strains harboring a synthetic plant-derived metabolic pathway. Optimizing the key enzyme anthranilic acid (ANT) methyltransferase1 (AAMT1) expression, increasing the direct precursor ANT supply, and enhancing the intracellular availability and salvage of the cofactor <i>S</i>-adenosyl-l-methionine required by AAMT1, results in improved MANT production in both engineered microorganisms. Furthermore, in situ two-phase extractive fermentation using tributyrin as an extractant is developed to overcome MANT toxicity. Fed-batch cultures of the final engineered <i>E. coli</i> and <i>C. glutamicum</i> strains in two-phase cultivation mode led to the production of 4.47 and 5.74 g/L MANT, respectively, in minimal media containing glucose. The metabolic engineering strategies developed here will be useful for the production of volatile aromatic esters including MANT.