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 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.

Exploiting nonionic surfactants to enhance fatty alcohol production in Rhodosporidium toruloides

“…A sustainable alternative to produce these products is through engineering microbial cell factories that can directly use renewable and cost‐effective feedstocks, such as lignocellulose, atmospheric CO 2 , and syngas (Peralta‐Yahya et al, 2012). A vast number of chemicals have been successfully produced in various microbes that feature diverse chemical structures and functional groups (Borodina et al, 2015; Jiang, Qiao, Bentley, Liu, & Zhang, 2017; Lee et al, 2019; Luo, Cho, & Lee, 2019; Wehrs et al, 2019). Recent advances in metabolic engineering and synthetic biology have provided a greatly expanded set of tools necessary to assemble and optimize metabolic pathways for improved titers, productivities and yields (D. Liu, Evans, & Zhang, 2015; Y. Liu & Nielsen, 2019; Salis, Mirsky, & Voigt, 2009).…”

Fermentative N-Methylanthranilate Production by Engineered Corynebacterium glutamicum

“…This is even more important for ANMT from R. graveolens because it is inhibited by SAH with a K I value of 37.2 µM [74]. As shown here and elsewhere [41], overexpression of one gene of the SAM regeneration system ( Figure 1A), S-adenosylhomocysteine (SAH) hydrolase gene sahH, partly overcame SAM limitation since conversion of anthranilate to NMA was improved 1.36-fold ( Figure 5). This may be due to reduced inhibition of ANMT from R. graveolens by SAH (see above) and/or better SAM regeneration.…”

“…Irrespective of sahH overexpression, not more than about 14 mol% of anthranilate was N-methylated to NMA ( Figure 5). As shown for OMA production [41], overexpression of SAM synthetase gene metK in addition to sahH improved SAM regeneration, whereas deletion of cystathionin-γ-synthase gene metB and of mcbR and cg3031 that code for transcriptional regulators involved in regulation of methionine biosynthesis were not beneficial. Addition of methionine even reduced the production [41].…”

“…A mobile phase of buffer A (0.1% trifluoroacetic acid dissolved in water) and buffer B (acetonitrile) was used with a flow rate of 1 mL•min −1 . The following gradient was applied: 0-1 min 10% B; 1-10 min a linear gradient of B from 10% to 70%; 10-12 min 70% B; 12-14 min a linear gradient of B from 70% to 10%; 14-18 min 10% B [41]. The injection volume was 20 µL, and detection was performed with DAD at 210, 280, and 330 nm.…”

Análise dos compostos voláteis presentes em bebidas isotônicas comercializadas em Belo Horizonte – MG

“…Identificou-se o α-terpineol como composto majoritário do sabor limão da marca E, enquanto para o sabor uva verde, o composto majoritário foi o antranilato de metila. Este composto é muito utilizado como aromatizante sintético de alimentos, pois confere aroma e sabor de uva (LUO et al, 2019). Também foi um composto comum nas amostras sabor uva, exceto na marca A. Observou-se que o α-terpineol foi o composto majoritário em todas as amostras de sabor limão com exceção da amostra da marca B. Os sabores iguais de mesma marca apresentaram os mesmos compostos majoritários e sabores iguais de marca distintas não apresentaram os mesmos compostos majoritários.…”