Abstract
The N-functionalized amino acid N-methylanthranilate is an important precursor for bioactive compounds such as anticancer acridone alkaloids, the antinociceptive alkaloid O-isopropyl N-methylanthranilate, the flavor compound O-methyl-N-methylanthranilate, and as a building block for peptide-based drugs. Current chemical and biocatalytic synthetic routes to N-alkylated amino acids are often unprofitable and restricted to low yields or high costs through cofactor regeneration systems. Amino acid fermentation processes using the Gram-positive bacterium Corynebacterium glutamicum are operated industrially at the million tons per annum scale. Fermentative processes using C. glutamicum for N-alkylated amino acids based on an imine reductase have been developed, while N-alkylation of the aromatic amino acid anthranilate with S-adenosyl methionine as methyl-donor has not been described for this bacterium. After metabolic engineering for enhanced supply of anthranilate by channeling carbon flux into the shikimate pathway, preventing by-product formation and enhancing sugar uptake, heterologous expression of the gene anmt encoding anthranilate N-methyltransferase from Ruta graveolens resulted in production of N-methylanthranilate (NMA), which accumulated in the culture medium. Increased SAM regeneration by coexpression of the homologous adenosylhomocysteinase gene sahH improved N-methylanthranilate production. In a test bioreactor culture, the metabolically engineered C. glutamicum C1* strain produced NMA to a final titer of 0.5 g·L−1 with a volumetric productivity of 0.01 g·L−1·h−1 and a yield of 4.8 mg·g−1 glucose.
Highlights
N-Functionalization of natural products as well as fine and bulk chemicals includes N-hydroxylation, N-acetylation, N-phosphorylation, or N-alkylation. These amine and amino acid modifications are found in all domains of life, and they fulfill various physiological roles such as resistance of bacteria to the antibiotic rifampicin by its N-hydroxylation [1], biosynthesis of the hormone melatonin via N-acetylated serotonin in plants and mammals [2], or assimilation of methylamine as carbon and energy source in methylotrophic bacteria [3]
Pre-cultures of E. coli and C. glutamicum were performed in lysogeny broth (LB) and brain heart infusion (BHI) medium at 37 or 30 ◦C in baffled shake flasks on a rotary shaker (160 rpm or 120 rpm)
Feeding with 400 g·L−1 glucose and 150 g·L−1 (NH4)2SO4 was activated when the relative dissolved oxygen saturation signal rose above 60% and stopped when rDOS fell below 60%
Summary
N-Functionalization of natural products as well as fine and bulk chemicals includes N-hydroxylation, N-acetylation, N-phosphorylation, or N-alkylation. Chemical synthesis of free N-alkylated amino acids is well studied, and various routes are known, such as by nucleophilic substitution of α-bromo acids, N-methylation of sulfonamides, carbamates or amides, reduction of Schiff bases generated with an amino acid and formaldehyde or other aldehydes, by direct alkylation of protected amino acids or by ring-opening of 5-oxazolidinones [6,7,8,9] These processes are often limited by low product yields, over-methylation, toxic reagents, or their incomplete enantiopurity [10,11]. The SAM-dependent transfer of a methyl group to anthranilate initiates the biosynthesis of NMA-dependent biosynthesis of N-methylated acridone alkaloids and avenacin in plants [16,17]. PEP, phosphoenolpyruvate; TCA, tricarboxylic acid; PPP, pentose phosphate pathway; E4P, erythrose-4-phosphate; DAHP, 3-deoxy-d-arabinoheptulosonate-7-phosphate; 3DHQ, 3-dehydroquinate; 3DHS, 3-dehydroshikimic acid; PCA, protocatechuic acid; iolR, transcriptional regulator; sugR, transcriptional regulator; ppc, phosphoenolpyruvate carboxylase; ldhA, lactate dehydrogenase; tkt, transketolase; aroF, DAHP synthase; aroGFBR, feedback-resistant DAHP synthase from Escherichia coli; aroB, 3-dehydroquinate synthase; qsuC, 3-dehydroquinate dehydratase; qsuB, 3-dehydroshikimate dehydratase; qsuD, shikimate dehydrogenase; aroE, shikimate dehydrogenase; qsuA, putative shikimate importer; aroK, shikimate kinase; aroA, 5-enolpyruvylshikimate-3-phosphate synthase; aroC, chorismate synthase; csm, chorismate mutase; trpEFBR, feedback-resistant anthranilate synthase from E. coli
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