Fermentative N-Methylanthranilate Production by Engineered Corynebacterium glutamicum

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.

Coenzyme Q10 Biosynthesis Established in the Non-Ubiquinone Containing Corynebacterium glutamicum by Metabolic Engineering

“…Cells were disrupted five times in a Precellys homogenizer (VWR, Darmstadt, Germany) at 6.5 m/s for 30 s. 100 µl of lysed cell extract was mixed with 100 µl of the organic solvent 2,2,2-trifluoroethanol and 5 µl 200 mM of the reducing agent dithiothreitol (DTT) and incubated for 60 min at 60 • C. Alkylation of cysteines was performed by adding 20 µl of 200 mM 2iodoacetamide and incubation for 90 min in the dark at room temperature. Alkylation was stopped by adding 5 ml of 200 mM DTT and incubation for 60 min at room pK19mobsacB with a construct for deletion of vdh (cg2953) and insertion of aroG D146N from E. coli K-12 under control of C. glutamicum promoter P ilvC , which has been amplified from APS529 (Purwanto et al, 2018) with the primers vdh-fw and vdh-rv This work pK19mobsacB-qsuABCD::P tuf -qsuC pK19mobsacB with a construct for deletion of qsuABCD (cg0501-cg0504) and insertion of qsuC (cg0503) under control of C. glutamicum promoter P tuf Walter et al, 2020 temperature. For tryptic digestion, samples were diluted 1:10 with 50 mM ammonium bicarbonate.…”

Physiological Response of Corynebacterium glutamicum to Indole

“…In contrast, naphthalene is converted via gentisate to fumarate and pyruvate [ 19 , 31 ]. Transport systems [ 32 , 33 , 34 , 35 ], transcriptional regulators [ 36 , 37 , 38 , 39 , 40 , 41 ], and production of aromatic compounds [ 42 , 43 , 44 , 45 , 46 , 47 , 48 ] have been described for C. glutamicum . In order to test if indole is metabolized by C. glutamicum or exerts a regulatory role as a putative signaling molecule, we determined the physiological and transcriptomic response of C. glutamicum to indole.…”

Sustainable Production of N-methylphenylalanine by Reductive Methylamination of Phenylpyruvate Using Engineered Corynebacterium glutamicum

“…The metabolic engineering strategy followed here to provide the precursor phenylpyruvate largely relies on optimizing entry into and conversion in the shikimate pathway. This approach enabled production of aromatic compounds to about g L −1 titers: 1.4 g L −1 shikimate, 3.0 g L −1 anthranilate, 0.5 g L −1 N-methyl-anthranilate [36] and 0.73 ± 0.05 g L −1 NMePhe in this study. Notably, the yield of NMePhe on glucose of 0.052 g g −1 was one order of magnitude higher than that for N-methylantranilate (4.8 mg g −1 glucose) [36].…”

“…Efficient provision of phenylpyruvate was expected to result from a high flux into the shikimate pathway coupled with conversion of chorismate solely to phenylpyruvate, but not to L-tryptophan, L-tyrosine or L-phenylalanine. As a base strain, C. glutamicum ARO9 was used, which was constructed for overproduction of anthranilate, an intermediate of the shikimate pathway, and N-methylated anthranilate [36]. To abolish synthesis of L-tryptophan, the anthranilate synthase genes trpEG were deleted.…”

“…Expression of dpkA in Corynebacterium glutamicum engineered to overproduce the respective 2-oxoacid precursor [24][25][26] allowed fermentative production of sarcosine [27], N-methyl-L-alanine [28] and N-ethylglycine [29] upon addition of either monomethylamine or monoethylamine to the growth medium. C. glutamicum is a logical choice for production of non-proteinogenic amino acids such as pipecolic acid [30,31], and trans-hydroxyproline [32,33], aromatic amino acids like 7-chloroor 7-bromo-tryptophan [34,35] or the N-alkylated amino acid N-methylanthranilate [36] since it is used for more than 50 years for safe production of the food and feed amino acids L-glutamate [37] and L-lysine [38] at the million-ton scale [39].…”