Journal
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
Volume 116, Issue 22, Pages 10749-10756Publisher
NATL ACAD SCIENCES
DOI: 10.1073/pnas.1903875116
Keywords
metabolic engineering; Escherichia coli; Corynebacterium glutamicum; methyl anthranilate; two-phase fermentation
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Funding
- Technology Development Program to Solve Climate Changes on Systems Metabolic Engineering for Biorefineries from the Ministry of Science and ICT through the National Research Foundation (NRF) of Korea [NRF-2012M1A2A2026556, NRF-2012M1A2A2026557]
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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 plantderived metabolic pathway. Optimizing the key enzyme anthranilic acid (ANT) methyltransferasel (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.
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