Access to N-alkylated amino acids by microbial fermentation

Conference Dates

July 14-18, 2019


N-methylated amino acids are found in many pharmaceutically active compounds and have been shown to improve pharmacokinetic properties as constituents of peptide drugs since N-methylation of amino acids may result in conformational changes, improved proteolytic stability and higher lipophilicity of the peptide drug.1 N-methylated amino acids are mainly produced chemically or by biocatalysis, however with low yields or high costs for co-factor regeneration. First, we established a fermentative route for production of N-mehtyl-L-glutamate by Pseudomonas putida from glucose and glycerol. Interception of the C1 assimilation pathway of Methylobacterium extorquence yielded N-methyl-L-glutamate titers of 17.9 g L-1 in fed-batch cultivation.2 Due to high substrate specificity of this C1 assimilation pathway genes, we continued with an independent pathway for extension of the product range. Therefore, we focus on pathway-design for N-methylated amino acids by the industrially relevant production host Corynebacterium glutamicum. Metabolic engineering of C. glutamicum led to an expanded product range of proteinogenic amino acids like L-valine2 but also ω-amino acids like γ-aminobutyrate and diamines like putrescine3. The rare imine reductase DpkA from P. putida KT2440 catalyzes the reductive methylamination of pyruvate as side activity. Implementation of DpkA into the central carbon metabolism of the pyruvate overproducing C. glutamicum strain ELB-P4 yielded N-methyl-L-alanine production. Optimization of carbon- and nitrogen ratios of the minimal medium allowed production of up to 10.5 g L-1 when cultivated in shake flasks. N-methyl-L-alanine titers of 31.7 g L-1 with a yield of 0.71 g per g glucose were achieved in fed-batch cultivation5. Due to the somewhat relaxed substrate scope of DpkA, the product portfolio of N-methylated amino acids produced by fermentation could be successfully extended. Changing the base strain to a glyoxylate producing C. glutamicum strain6 achieved production of 2.6 g L-1 sarcosine, the N-methylated glycine derivative, from glucose. Sarcosine production based on the second generation feedstocks xylose and arabinose led to higher product titers than glucose-based production and optimization of substrate composition led to a titer of 8.7 g L-1 sarcosine. This is the first example in which a C. glutamicum process using lignocellulosic pentoses is superior to glucose-based production. By mutation of the active site of DpkA, a mutant with higher specific activity towards glyoxylate (30.3 ± 2.7 U mg-1; wild type enzyme 25.7 ± 1.8 U mg-1) was identified. Therefore, the mutant DpkAF117L was incorporated into the production strain and enabled faster sarcosine production. Additionally, this mutation led to an increased activity towards reductive ethylamination of glyoxylate (31.2 ± 1.1 U mg-1; wild type enzyme 25.3 ± 3.2 U mg-1). As a result, the fermentative production of N-ethylglycine showed enhanced volumetric productivity compared to the strain harboring the wild type enzyme. Fermentative access to N-methylated amino acids was achieved by two independent pathway designs. First, we enabled N-methyl-L-glutamate production by pathway interception in P. putida. Additionally, introduction of the imine reductase gene dpkA from P. putida into various 2-oxoacid producing C. glutamicum strains extended the product range. Optimization of medium composition, preferred substrate specificity of the strain or the enzyme itself resulted in excellent production yields. 1 Chatterjee J, Rechenmacher F and Kesser H, Angew. Chem. Int. Ed., 2013, 52, 254-269. 2 Mindt M, Walter T, Risser JM and Wendisch VF, Front. Bioeng. Biotechnol., 2018, 6, 159. 3 Wendisch VF, Mindt M and Pérez-García F, Appl. Microbiol. Biotechnol., 2018, 102, 3583-3594. 4 Wieschalka S, Blombach B and Eikmanns BJ, Appl. Microbiol. Biotechnol., 2012, 94, 449-459. 5 Mindt M, Risse JM, Gruß H, Sewald N, Eikmanns BJ and Wendisch VF, Sci. Rep., 2018, 8, 12895. 6 Zahoor A, Otten A and Wendisch VF, J. Biotechnol., 2014, 192, 366-375

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