Z. Luo, Jae Sung Cho, S. Lee
May 13, 2019
Citations
4
Influential Citations
64
Citations
Journal
Proceedings of the National Academy of Sciences
Abstract
Significance Methyl anthranilate (MANT) is widely used in the flavoring and cosmetics industry to give grape scent and flavor. In an effort to replace the conventional petroleum-based synthesis of MANT, we report the direct fermentative production of MANT from glucose in metabolically engineered Escherichia coli and Corynebacterium glutamicum strains. A synthetic plant-derived metabolic pathway was introduced and extensive metabolic engineering was performed including fine-tuning key enzyme levels and increasing the availability of precursor and cofactor metabolites. A two-phase extractive cultivation was developed using an extractant solvent to recover MANT in situ, which led to high levels of MANT production. This work demonstrates a promising sustainable alternative to MANT production and presents strategies applicable toward production of other valuable natural compounds. 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 Escherichia coli and Corynebacterium glutamicum 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 S-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 E. coli and C. glutamicum 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.