Fermentative N-Methylanthranilate Production by Engineered Corynebacterium glutamicum

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.


Bacterial Strains and Culture Conditions
All bacterial strains used are listed in Table 1. Escherichia coli DH5α [43] was used for plasmid construction. C. glutamicum C1* was used as host organism for shikimate, anthranilate, and NMA production. 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). Cultures were inoculated freshly from LB agar plates. When necessary, spectinomycin (100 µg·mL −1 ) and kanamycin (25 µg·mL −1 ) were added to the medium. For induction of gene expression from vectors pEKEx3 and pGold, isopropyl-β-d-1-thiogalactopyranoside (IPTG) was added to the medium. For the performance of growth or production experiments of C. glutamicum, pre-cultures were inoculated as described above. After cell harvesting (3200× g, 7 min), cells were washed with TN-buffer pH 6.3 (50 mM Tris-HCL, 50 mM NaCl) and inoculated to an optical density at 600 nm (OD 600 ) of 1 in CGXII minimal medium [44] and 40 g glucose as sole carbon source. C. glutamicum grown in 500 mL baffled shake flasks was followed by measuring OD 600 using a V-1200 spectrophotometer (VWR, Radnor, PA, USA). An OD 600 of 1 was determined to be equivalent to a biomass concentration of 0.25 g cell dry weight per liter. Evaluation of the effects of anthranilate and NMA on C. glutamicum growth was performed in the microbioreactor system Biolector (m2p-labs; Aachen, Germany). Pre-cultures were grown in BHI-rich medium overnight and transferred to second pre-culture of CGXII minimal medium with 40 g·L −1 glucose until the early exponential phase before inoculating to the main medium of CGXII minimal medium and 40 g·L −1 glucose with addition of varying anthranilate (solved in water) and NMA (solved in methanol) concentrations. Each condition with NMA contained 1.65 M methanol. Growth experiments in the Biolector were carried out using 48-well flower plates (MTP-48-B; m2p-labs) with a filling volume of 1 mL, at 30 • C, and 1200 rpm shaking frequency. Humidity was kept constant at 85%, and online biomass measurements of scattered light were monitored with backscatter gain of 20.

Fed-Batch Cultivation
Fed-Batch fermentation of C. glutamicum NMA105 was performed in an initial volume of 2 L in a bioreactor (3.7 L KLF, Bioengineering AG, 8636 Wald, Switzerland) at 30 • C, 0.2 bar overpressure, and an aeration rate of 2 NL·min −1 . We did not perform off-gas analysis. To maintain relative dissolved oxygen saturation at 30%, stirrer speed was controlled during growth. The pH was maintained at pH 7.0 due to controlled addition of KOH (4 M) and phosphoric acid (10% (w/w)). To avoid foaming, the antifoam Sruktol ® J647 was added manually when necessary. Feeding with 400 g·L −1 glucose and 150 g·L −1 (NH 4 ) 2 SO 4 (total volume: 500 mL) was activated when the relative dissolved oxygen saturation (rDOS) signal rose above 60% and stopped when rDOS fell below 60%. Samples were taken automatically every 4 h during the whole cultivation and cooled down to 4 • C until further use. C. glutamicum NMA105 cells were transferred from a first pre-culture grown in LB in shake flasks to a second pre-culture in standard CGXII (pH 7.0) medium with 40 g·L −1 glucose (without IPTG) and the required antibiotics. For the bioreactor culture, standard CGXII medium without addition of 3-(N-morpholino)propanesulfonic acid (MOPS) and antibiotics was used. The fermenter was inoculated with the second pre-culture to an OD of 1.5 and immediately induced with 1 mM of IPTG.

Molecular Genetic Techniques and Strain Construction
Standard molecular genetic techniques were performed as described [46]. Competent E. coli DH5α [43] was performed with the RbCl method and transformed by heat shock [46]. Transformation of C. glutamicum was performed by electroporation [44]. The gene trpE FBR was amplified using specific primers ( Table 2) with ALLin TM HiFi DNA Polymerase (highQu GmbH, Kraichtal, Germany). The PCR products were assembled with BamHI restricted pEKEx3 via Gibson Assembly [44].
For heterologous expression of the N-methylanthranilate transferase gene, firstly, the pEC-XK99E vector was modified to be suitable for Golden Gate based modular assembly of multiple genes simultaneously. To this end, the three BsaI sites present in the vector located in the rrnB terminator, the vector backbone, and the repA ORF were removed. Next, a linker containing two BsaI sites (CAGATGAGACCGCATGCCTGCAAGGTCTCAGTAT) was added to the MCS between EcoRI and SacI restriction sites. The resulting vector was named pGold (GenBank: MT521917). The coding sequence (CDS) of the plant gene anmt (GenBank: DQ884932.1) encoding the N-methylanthranilate transferase of Ruta graveolens was codon-harmonized to the natural codon frequency of C. glutamicum ATCC13032 with the codon usage table of kazusa database [47] and synthesized with Golden Gate assembly compatible flanking regions including recognition site for the restriction enzyme type 2 BsaI and pGold complementary sequences and an optimized RBS [48,49] (Supplementary Data  Table S1). The gene anmt was amplified using specific primers ( Table 2) with ALLin TM HiFi DNA Polymerase according to the manufacturer (highQu GmbH, Kraichtal, Germany). The PCR products were assembled with digested pGold-anmt with BamHI via Gibson Assembly [44].
Ribosomal binding sites are in bold, and binding regions of Gibson oligonucleotides are underlined.
Chromosomal gene deletions and replacements in C1*-derived strains were performed by two-step homologous recombination [44] using the suicide vector pK19mobsacB [50]. The genomic regions flanking the respective gene for homologous recombination were amplified from C. glutamicum WT as described elsewhere [51] using the respective Primer pairs containing artificial RBS ( [48,49], Table 2). The purified PCR products were assembled and simultaneously cloned into restricted pK19mobsacB by Gibson Assembly resulting in the plasmids listed in Table 3. Transfer of the suicide vectors was carried out by trans-conjugation using E. coli S17 as donor strain [33]. For the first recombination event, integration of the vector in one of the targeted flanking regions was selected via kanamycin resistance. The resulting clones showed sucrose sensitivity due to the levansucrase gene sacB. Suicide vector excision was selected by sucrose resistance. Gene deletions or replacements were verified by PCR and sequencing with respective primers ( Table 2). Table 3. List of plasmids used in this study.

Quantification of Amino Acids and Organic Acids
Extracellular amino acids and carbohydrates were quantified by high-performance liquid chromatography (HPLC) (1200 series, Agilent Technologies Deutschland GmbH, Böblingen, Germany). The culture supernatants were collected at different time points and centrifuged (20,200× g) for HPLC analysis.

Corynebacterium glutamicum as Suitable Host for NMA Production
C. glutamicum is widely used in amino acid fermentation, which operates at a million tons per annum scale [56]; however, it has not been engineered so far for NMA production. As expected, inspection of the genome revealed that there was no gene(s) encoding for a native enzyme that may N-methylate anthranilate to yield NMA. To study the growth responses of C. glutamicum to anthranilate and NMA, the wild-type strain ATCC13032 (WT) was cultivated with addition of varying anthranilate and NMA concentrations to CGXII minimal medium and 40 g·L −1 glucose. Neither anthranilate nor NMA were utilized or converted by C. glutamicum WT, since their concentrations in supernatants analyzed at the beginning and the end of cultivation were comparable. Maximal biomass concentrations (expressed as ∆OD 600 ) were hardly affected by addition of anthranilate or NMA. By extrapolation, the concentrations of anthranilate (about 36 mM) and NMA (about 34 mM), which reduced the specific growth rate in glucose minimal medium to half-maximal, were determined ( Figure 2). Based on the observed tolerance, C. glutamicum is a suitable candidate for production of anthranilate and NMA.

Construction of a C. glutamicum Platform Strain for Production of Anthranilate
Since anthranilate, an intermediate of the tryptophan branch in the shikimate pathway, is a direct precursor of NMA, C. glutamicum C1* was engineered for increased supply of shikimate

Construction of a C. glutamicum Platform Strain for Production of Anthranilate
Since anthranilate, an intermediate of the tryptophan branch in the shikimate pathway, is a direct precursor of NMA, C. glutamicum C1* was engineered for increased supply of shikimate pathway intermediates by eliminating bottlenecks and minimizing formation of by-products (Figure 1). Hence, in sequential steps, aroG D146 encoding feedback resistant 3-deoxy-d-arabino-heptulosonate-7-phosphate (DAHP) synthase from E. coli [57] was inserted into the locus of vdh coding for vanillin dehydrogenase, which oxidizes vanillin and other aromatic aldehydes such as protocatechic aldehyde [58]. Next, an in-frame deletion of ldhA to reduce l-lactate formation (ARO02) and an sugR deletion to increase glycolytic gene expression and sugar uptake [59] were introduced to yield strain ARO03.
Upon transformation with pEKEx3 as an empty vector control and pEKEx3-trpE FBR for expression of feedback-resistant anthranilate synthase from E. coli [60], strains were evaluated regarding their growth behavior, anthranilate production, and formation of by-products. After 48 h of shake flask cultivation, ARO03(pEKEx3) exhibited decreased biomass formation and increased trehalose and α-ketoglutarate accumulation as compared to ARO01(pEKEx3). Expression of trpE FBR further decreased biomass formation (i.e., 16.4% less than in empty vector). Comparing strains C1* to ARO03 carrying pEKEx3-trpE FBR revealed a stepwise increase both in anthranilate and in shikimate production ( Figure 3). For example, ARO03 strain harboring pEKEx3-trpE FBR produced 17.6 ± 1.0 mM anthranilate and 6.8 ± 0.8 mM shikimate as compared to C1*(pEKEx3-trpE FBR ) that accumulated only 9.0 ± 0.2 mM anthranilate and 1.7 ± 0.1 mM shikimate.  To further increase the carbon flux towards shikimate, several further metabolic engineering steps were undertaken. In ARO04, the gene aroR, which codes for a translational regulatory leader peptide and is located upstream of DHAP synthase gene aroF [61], was replaced by an ilvC promoter followed by an optimized RBS in order to relieve negative translational control of aroF by phenylalanine and tyrosine. As described previously [36], the qsuABCD operon was replaced by qsuC transcribed from the constitutive strong tuf promoter in strain ARO05. This blocked conversion of 3dehydroshikimate (3-DHS) to the unwanted by-product protocatechuate (PCA) on the one hand and increased the flux from 3-dehydroquinate (3-DHQ) to 3-DHS on the other hand. The replacement of ppc encoding phosphoenolpyruvate (PEP) carboxylase by a second copy of endogenous aroB encoding 3-DHQ synthase in ARO06 probably increased supply of PEP as precursor for the shikimate pathway, and overexpression of aroB increased conversion of DHAP to 3-DHQ. To increase supply To further increase the carbon flux towards shikimate, several further metabolic engineering steps were undertaken. In ARO04, the gene aroR, which codes for a translational regulatory leader peptide and is located upstream of DHAP synthase gene aroF [61], was replaced by an ilvC promoter followed by an optimized RBS in order to relieve negative translational control of aroF by phenylalanine and tyrosine. As described previously [36], the qsuABCD operon was replaced by qsuC transcribed from the constitutive strong tuf promoter in strain ARO05. This blocked conversion of 3-dehydroshikimate (3-DHS) to the unwanted by-product protocatechuate (PCA) on the one hand and increased the flux from 3-dehydroquinate (3-DHQ) to 3-DHS on the other hand. The replacement of ppc encoding phosphoenolpyruvate (PEP) carboxylase by a second copy of endogenous aroB encoding 3-DHQ synthase in ARO06 probably increased supply of PEP as precursor for the shikimate pathway, and overexpression of aroB increased conversion of DHAP to 3-DHQ. To increase supply of erythrose-4-phosphate (E4P) as second precursor of the shikimate pathway [62], the native promoter upstream of transketolase gene tkt was exchanged by the constitutive strong promoter Ptuf with an artificial RBS. Since tkt is co-transcribed with other genes of the pentose phosphate pathway as operon tkt-tal-zwf-opcA-pgl, this promoter exchange is expected to increase flux into the pentose phosphate pathway towards E4P in strain ARO07.
In ARO08, shikimate dehydrogenase gene aroE was overexpressed from the strong constitutive promoter Ptuf and used to replace iolR. In the absence of IolR, the inositol catabolism operon (cg0197-cg0207), cg1268, and PEP carboxykinase gene pck are deregulated [63,64], and iolT1, which codes for a non-phosphoenolpyruvate dependent phosphotransferase transporter (non-PTS) inositol uptake system, is derepressed. Non-PTS uptake of glucose is known to improve availability of PEP. The final strain, ARO09, is a sugR-positive derivative of ARO08. ARO09(pEKEx3-trpE FBR ) grew faster than ARO7(pEKEx3-trpE FBR ) ( Figure 3) and accumulated less trehalose as unwanted by-product. The maximum anthranilate titer of 22.0 ± 1.4 mM (equivalent to about 3.1 g·L −1 anthranilate) was achieved with ARO09(pEKEx3-trpE FBR ) after 48 h of shake flask cultivation. This titer was 2.5 times more than that obtained with C1*(pEKEx3-trpE FBR ). Taken together, an anthranilate producing C. glutamicum strain converting 12.7% of carbon from glucose ( Figure 4) to about 3.1 g·L −1 of anthranilic acid, the direct precursor for NMA, was constructed.
In order to test for NMA production, strains NMA100 to NMA105 were cultivated in CGXII minimal medium supplemented with 40 g·L −1 glucose as carbon source. HPLC analysis of supernatants after cultivation for 48 h revealed that NMA100 and NMA103 did not produce NMA, which was expected since they lacked anmt from R. graveolens ( Figure 5). Expression of anmt alone or in combination with endogenous sahH resulted in production of about 0.5 mM NMA by strains NMA101 and NMA102, respectively. This indicated functional expression of anmt from R. graveolens in C. glutamicum. with ARO09(pEKEx3-trpE FBR ) after 48 h of shake flask cultivation. This titer was 2.5 times more than that obtained with C1*(pEKEx3-trp   Figure S1; Figure S2).

Establishing Fermentative Production of NMA by C. glutamicum
NMA is synthesized from anthranilate in a single SAM-dependent methylation reaction at its amino group (Figure 1). Therefore, the anthranilate producing C. glutamicum strain ARO09(pEKEx3-trpE FBR ) was used for heterologous expression of the anthranilate N-methyltransferase gene ANMT Figure 4. Fate of carbon from glucose in cultivations of C. glutamicum ARO strains carrying the empty vector (left) or pEKEx3-trpE FBR (right). Carbon (given in mol%) derived from glucose found after 48 h in secreted products anthranilate (blue), shikimate (maroon), α-ketoglutarate (light red), trehalose (green), lactate (black) as well as in the formed biomass (yellow) are shown for C. glutamicum strains C1*, ARO03, ARO07, and ARO09 harboring either pEKEx3 (left) or pEKEx3-trpE FBRfbr (right). Carbon that could not be accounted for is depicted in gray (other). Values were determined from duplicate cultures. Experimental error was less than 20%. Abbreviations used: α-KG, α-ketoglutarate. Carbon distribution of all ARO strains can be found in the Supplementary Data ( Figure S1; Figure S2).
Coexpression of trpE FBR to boost anthranilate production with anmt alone (strain NMA104) resulted in production of 1.7 ± 0.1 mM (0.25 ± 0.02 g·L −1 ) NMA. The finding that the anthranilate concentration was reduced from 20.8 ± 0.0 mM as obtained with NMA103 to 17.3 ± 0.9 mM (NMA104) indicated that conversion of anthranilate to NMA was incomplete (at about 10 mol%). Upon coexpression of trpE FBR with both anmt and sahH in strain NMA105, 15.8 ± 1.9 mM anthranilate remained as unconverted precursor (Figure 5), and a significantly increased NMA titer of 2.2 ± 0.2 mM was obtained. This maximal titer in shake flasks corresponds to 0.34 ± 0.02 g·L −1 . Thus, metabolic engineering of C. glutamicum for NMA production was achieved. resulted in production of 1.7 ± 0.1 mM (0.25 ± 0.02 g·L −1 ) NMA. The finding that the anthranilate concentration was reduced from 20.8 ± 0.0 mM as obtained with NMA103 to 17.3 ± 0.9 mM (NMA104) indicated that conversion of anthranilate to NMA was incomplete (at about 10 mol%). Upon coexpression of trpE FBR with both ANMT and sahH in strain NMA105, 15.8 ± 1.9 mM anthranilate remained as unconverted precursor (Figure 5), and a significantly increased NMA titer of 2.2 ± 0.2 mM was obtained. This maximal titer in shake flasks corres

Fed-Batch Production of NMA in Bioreactors
For industrial applications, a production in larger volumes is preferable, which runs under controlled conditions to obtain a constant production titer. The stability of the NMA production of Figure 5. Production of anthranilate (light blue) and NMA (dark blue) by C. glutamicum strains NMA100 to NMA105. Cultivation was performed in minimal medium supplemented with 40 g·L −1 glucose as carbon source. 1 mM IPTG was added for induction of gene expression. Means and standard deviations of triplicate cultures determined after 48 h cultivation are depicted. Significance has been determined for NMA concentrations based on a two-sided unpaired Student's t-test (*: p < 0.05; ns: not significant).

Fed-Batch Production of NMA in Bioreactors
For industrial applications, a production in larger volumes is preferable, which runs under controlled conditions to obtain a constant production titer. The stability of the NMA production of the metabolically engineered strain NMA105 was investigated in a fed-batch cultivation. Starting with a working volume of 2 L CGXII minimal medium containing 40 g·L −1 glucose as carbon source, 160 mL feed (400 g·L −1 and 150 g·L −1 (NH 4 ) 2 SO 4 ) was added in a controlled manner depending on the rDOS (see Section 2.2). In total, 104 g glucose was consumed during 48 h fed-batch cultivation with no residual substrate concentrations detectable in the cultivation broth. The strain showed slow growth to OD 600 5 in the first 24 h. In the following phase, growth was faster (growth rate of 0.12 h −1 , which was comparable to the growth rate observed in shaking flasks), and a maximal optical density of 53 was reached (Figure 6). High concentrations of by-products accumulated, i.e., 1.4 g·L −1 of the intermediate shikimate and 2.6 g·L −1 of the direct precursor anthranilate ( Figure 6). Compared to production in shaking flasks ( Figure 5), a reduced product yield on glucose (4.8 mg·g −1 as compared to 8.4 mg·g −1 in shaking flask) and a comparable volumetric productivity were observed, but NMA accumulated to an about 1.5-fold higher titer (0.5 g·L −1 as compared to 0.34 g·L −1 ). Taken together, the fed-batch fermentation with the newly constructed C. glutamicum strain NMA105 showed stable production of NMA in bioreactors at the 2 L scale ( Figure 6). 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 was achieved.
intermediate shikimate and 2.6 g·L −1 of the direct precursor anthranilate (Figure 6). Compared to production in shaking flasks ( Figure 5), a reduced product yield on glucose (4.8 mg·g −1 as compared to 8.4 mg·g −1 in shaking flask) and a comparable volumetric productivity were observed, but NMA accumulated to an about 1.5-fold higher titer (0.5 g·L −1 as compared to 0.34 g·L −1 ). Taken together, the fed-batch fermentation with the ne Figure 6. NMA production by C. glutamicum strains NMA105 in bioreactors operated in fed-batch mode. The cultivation (initial volume of 2 L) was performed in minimal medium supplemented with 40 g·L -1 glucose (dark grey line with squares). The feed (dark grey line) contained 400 g·L −1 glucose and 150 g·L −1 (NH4)2SO4. 1 mM IPTG was added for induction of gene expression during inoculation. OD600 (yellow) and concentrations of anthranilate (light blue), shikimate (maroon), and NMA (dark blue) in the culture broth are depicted. One of two representative fermentations is shown.

Discussion
N-methylanthranilate production was achieved by applying the plant enzyme Nmethylanthranilate transferase ANMT of R. graveolens in a newly metabolically engineered C. glutamicum anthranilate overproducer. N-methylanthranilate is known as precursor for several industrially and medically relevant compounds. ANMT of R. graveolens showed a narrow substrate specificity when various amino benzoic or benzoic acids or phenolic derivatives were tested as substrates [16]. However, feeding O-methylanthranilate (OMA) to E. coli expressing ANMT led to production of the flavoring compound O-methyl-N-methylanthranilate [15]. Hypothetically, ANMT could also be an interesting candidate to produce the pharmaceutically interesting compounds Opropyl-or O-isopropyl-N-methylanthranilate [22,23]. In the biosynthesis of acridone alkaloids, e.g.,
Biosynthesis of N-alkylated amino acids can be catalyzed by other enzymes besides N-methyltransferases. However, while reductive amination using free ammonia is known for many enzymes, only few enzyme classes accept alkyl amines for N-alkylation, e.g., opine dehydrogenases, N-methyl amino acid dehydrogenases, ketimine reductases, pyrroline-5-carboxylate reductases, or imine reductases [12]. These processes differ regarding the substrate spectra of the involved enzymes. For example, anthranilate N-methylation described here as well as N-methylglutamate production established in Pseudomonas putida using N-methylglutamate synthase and γ-glutamylmethylamide synthetase of the methylamine assimilation pathway of Methylobacterium extorquens [13] have narrow substrate spectra (e.g., GMAS from Methylovorus mays also forms γ-glutamylethylamide, also known as theanine [69]) compared with N-alkylation using the imine reductase DpkA of Pseudomonas putida [12]. Several methylated or ethylated amino acids could be produced by C. glutamicum using the wild-type or a mutant version of DpkA and either MMA or ethylamine as substrates [14,34,35]. With respect to aromatic amino acids, N-methyl-l-phenylalanine could be obtained from phenylpyruvate by enzyme catalysis using DpkA and MMA [12]; however, production of NMA via DpkA by N-alkylamination of a carbonyl precursor of NMA has not been described.
The NMA process described here showed lower titers (0.5 g·L −1 ) than the processes depending on reductive alkylamination using MMA (about 32 g·L −1 N-methylalanine [34] and about 9 g·L −1 sarcosine [14]). This may be due to (a) higher activity of DpkA compared with ANMT, (b) better provision of the precursors pyruvate and glyoxalate than of anthranilate, and/or (c) the requirement of SAM for ANMT as compared to MMA for DpkA. Indeed, purified DpkA has a much higher activity (about 40 U·mg −1 ) [70] than purified ANMT (about 0.04 U·mg −1 ) [16]. Moreover, while ARO09(pEKEx3-trpE FBR ) produced 3 g·L −1 anthranilate (Figure 3), the precursor strains used for production of N-methylalanine and sarcosine produced up to 45 g·L −1 pyruvate [71] and about 5 g·L −1 glycolate [72], respectively. Third, reductive methylamination using DpkA requires addition of MMA as methyl donor to the medium. This is beneficial since MMA has a low price, is readily available, is tolerated well by C. glutamicum [34], and because stoichiometric excess of MMA can be used to drive reductive N-methylation by mass action law.
Compared to NMA production by an engineered E. coli strain expressing the N-methyltransferase of R. graveloens [15], the NMA production by engineered C. glutamicum using the same enzyme described here resulted in about 12 times higher titers in shaking flask cultivation (370 mg·L −1 as compared to 29 mg·L −1 ). This may be due to the fact that, in this study, C. glutamicum was metabolically engineered for improved supply of the direct NMA precursor anthranilate as, e.g., strain ARO09(pEKEx3-trpE FBR ) produced about 3 g·L −1 anthranilate. Moreover, while the E. coli relied on native SAM regeneration [15], in C. glutamicum the endogenous gene for SAM regeneration sahH was overexpressed to increase SAM regeneration, and NMA production was improved 1.36-fold (compare 0.34 ± 0.02 g·L −1 for NMA105 with 0.25 ± 0.02 g·L −1 for NMA104 in Figure 5). Two bottlenecks observed with the C. glutamicum strain engineered here may be overcome by future metabolic engineering: incomplete conversion of shikimate to anthranilate and incomplete N-methylation of anthranilate by SAM-dependent ANMT. To improve conversion of shikimate to anthranilate from about half to full conversion (compare about 1.4 g·L −1 of shikimate and 2.6 g·L −1 anthranilate produced by NMA105 in bioreactor cultivation; Figure 6), expression of the operon aroCKB encoding chorismate synthase, shikimate kinase, and 3-dehydroquinate synthase may be boosted, e.g., by changing the endogenous promoter for the strong promoter Ptuf and using shikimate kinase from Methanocaldococcus jannaschii as shown previously [36]. In addition, various studies have shown that deletion of the chorismate mutase will increase the carbon flux towards tryptophan biosynthesis [36,40,73].
SAM-dependent N-methylation of anthranilate by ANMT from R. graveloens represents the second bottleneck. ANMT from R. graveolens shows high affinity for its substrates (K M of 7.1 µM for anthranilate and K M of 3.3 µM for SAM), and inhibition by its product NMA has not been described [16]. On the other hand, the inherently low activity of ANMT as compared, e.g., to DpkA (see above) may limit conversion of anthranilate to NMA. Importantly, regeneration of the methyl donor SAM ( Figure 1A) is critical in all SAM-dependent methylation reactions. 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]. These changes and abolishing pathways competing for SAM and its precursor by deletion of homoserine kinase gene thrB along with overexpression of metK and vgb, coding for methionine adenosyltransferase and Vitreoscilla hemoglobin, led to a C. glutamicum strain secreting about 0.2 g·L −1 SAM within 48 h [75]. In addition to improving SAM regeneration (as shown here by sahH overexpression), it may be beneficial for NMA production to increase SAM biosynthesis and, therefore, the intracellular concentration of SAM. Thus, possibly, NMA production may be improved by overexpression of SAM biosynthesis genes such as metK, or by de-repression of SAM biosynthesis, e.g., via deletion of mcbR, or by deletion of genes for enzymes competing with use of SAM or of SAM biosynthetic precursors such as thrB.
NMA may inhibit anthranilate biosynthesis since NMA was not produced in addition to anthranilate, while the combined titer of NMA and anthranilate remained similar when comparing strains NMA103, NMA104, and NMA105 ( Figure 5). Enzymes that are inhibited by NMA have not been described to date. However, product inhibition of anthranilate synthase by anthranilate is known, e.g., in Streptomyces [76], which belongs to the actinobacteria as C. glutamicum, and in Salmonella typhimurium with a K I of 0.06 mM anthranilate [77]. Here, we used the E. coli enzyme TrpE, which is known to be inhibited by tryptophan, which binds at a site distant from the active center (allosteric regulation) [78]. In the mutant TrpE S40F , Trp binding is lost as well as allosteric inhibition by Trp [78]. Product inhibition by anthranilate is expected to involve binding to the active center. Since NMA differs from anthranilate just by the N-methyl group, it is conceivable that NMA inhibits in a similar way as anthranilate. This may explain that upon NMA production the anthranilate titer decreased ( Figure 5).
NMA also affected growth of C. glutamicum (34 mM or 5 g·L −1 reduced the growth rate to half-maximal; Figure 2), but to a lesser extent than OMA, for which a complete growth inhibition was observed at 2 g·L −1 OMA [41]. Inhibition of growth by OMA was overcome by application of a tributyrin-based extraction method [41]. This approach likely cannot be transferred directly to the NMA process since OMA contains a methylated carboxy group, whereas the amino group is methylated in NMA. Adaptive laboratory evolution (ALE) is an efficient method to select more tolerant strains and has been applied to C. glutamicum to select strains with improved tolerance to methanol [79][80][81] or lignocellulose-derived inhibitors [82].
Taken together, this study characterized NMA production by metabolically engineered C. glutamicum, and a first bioreactor process leading to a final titer of 0.5 g·L −1 NMA with a volumetric productivity of 0.01 g·L −1 ·h −1 and a yield of 4.8 mg·g −1 glucose was achieved. This strain provides the basis to develop an industrially competitive NMA process and shows potential to enable access to a fermentative route to pharmaceutically relevant secondary metabolites such as the acridone alkaloids.