Access to N-alkylated amino acids by microbial fermentation

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, 366-37

Fermentative production of N-methylglutamate from glycerol by recombinant Pseudomonas putida

Mindt M, Walter T, Risse JM, Wendisch VF. Fermentative production of N-methylglutamate from glycerol by recombinant Pseudomonas putida. Frontiers in Bioengineering and Biotechnology. 2018;6: 159.N-methylated amino acids are present in diverse biological molecules in bacteria, archaea and eukaryotes. There is an increasing interest in this molecular class of alkylated amino acids by the pharmaceutical and chemical industries. N-alkylated amino acids have desired functions such as higher proteolytic stability, enhanced membrane permeability and longer peptide half-lives, which are important for the peptide-based drugs, the so-called peptidomimetics. Chemical synthesis of N-methylated amino acids often is limited by incomplete stereoselectivity, over-alkylation or the use of hazardous chemicals. Here, we describe metabolic engineering of Pseudomonas putida KT2440 for the fermentative production of N-methylglutamate from simple carbon sources and monomethylamine. P. putida KT2440, which is generally recognized as safe and grows with glucose and the alternative feedstock glycerol as sole carbon and energy source, was engineered for the production of N-methylglutamate using heterologous enzymes from Methylobacterium extorquens. About 3.9 g L−1 N-methylglutamate accumulated within 48 h in shake flask cultures with minimal medium containing monomethylamine and glycerol. A fed-batch cultivation process yielded a N-methylglutamate titer of 17.9 g L−1

Fermentative Production of N-Methylglutamate From Glycerol by Recombinant Pseudomonas putida

N-methylated amino acids are present in diverse biological molecules in bacteria, archaea and eukaryotes. There is an increasing interest in this molecular class of alkylated amino acids by the pharmaceutical and chemical industries. N-alkylated amino acids have desired functions such as higher proteolytic stability, enhanced membrane permeability and longer peptide half-lives, which are important for the peptide-based drugs, the so-called peptidomimetics. Chemical synthesis of N-methylated amino acids often is limited by incomplete stereoselectivity, over-alkylation or the use of hazardous chemicals. Here, we describe metabolic engineering of Pseudomonas putida KT2440 for the fermentative production of N-methylglutamate from simple carbon sources and monomethylamine. P. putida KT2440, which is generally recognized as safe and grows with glucose and the alternative feedstock glycerol as sole carbon and energy source, was engineered for the production of N-methylglutamate using heterologous enzymes from Methylobacterium extorquens. About 3.9 g L−1N-methylglutamate accumulated within 48 h in shake flask cultures with minimal medium containing monomethylamine and glycerol. A fed-batch cultivation process yielded a N-methylglutamate titer of 17.9 g L−1

Fermentative production of N-alkylated glycine derivatives by recombinant Corynebacterium glutamicum using a mutant of imine reductase DpkA from Pseudomonas putida

Mindt M, Hannibal S, Heuser M, et al. Fermentative production of N-alkylated glycine derivatives by recombinant Corynebacterium glutamicum using a mutant of imine reductase DpkA from Pseudomonas putida. Frontiers in Bioengineering and Biotechnology. 2019;7: 232.Sarcosine, an N-methylated amino acid, shows potential as antipsychotic, and serves as building block for peptide-based drugs, and acts as detergent when acetylated. N-methylated amino acids are mainly produced chemically or by biocatalysis, with either low yields or high costs for co-factor regeneration. Corynebacterium glutamicum, which is used for the industrial production of amino acids for decades, has recently been engineered for production of N-methyl-L-alanine and sarcosine. Heterologous expression of dpkA in a C. glutamicum strain engineered for glyoxylate overproduction enabled fermentative production of sarcosine from sugars and monomethylamine. Here, mutation of an amino acyl residue in the substrate binding site of DpkA (DpkAF117L) led to an increased specific activity for reductive alkylamination of glyoxylate using monomethylamine and monoethylamine as substrates. Introduction of DpkAF117L into the production strain accelerated the production of sarcosine and a volumetric productivity of 0.16 g L−1 h−1 could be attained. Using monoethylamine as substrate, we demonstrated N-ethylglycine production with a volumetric productivity of 0.11 g L−1 h−1, which to the best of our knowledge is the first report of its fermentative production. Subsequently, the feasibility of using rice straw hydrolysate as alternative carbon source was tested and production of N-ethylglycine to a titer of 1.6 g L−1 after 60 h of fed-batch bioreactor cultivation could be attained

Efficient production of the dicarboxylic acid glutarate by Corynebacterium glutamicum via a novel synthetic pathway

Perez F, Jorge J, Dreyszas A, Risse JM, Wendisch VF. Efficient production of the dicarboxylic acid glutarate by Corynebacterium glutamicum via a novel synthetic pathway. Frontiers in Microbiology. 2018;9: 2589.The dicarboxylic acid glutarate is an important building-block gaining interest in the chemical and pharmaceutical industry. Here, a synthetic pathway for fermentative production of glutarate by the actinobacterium Corynebacterium glutamicum has been developed. The pathway does not require molecular oxygen and operates via lysine decarboyxylase followed by two transamination and two NAD-dependent oxidation reactions. Using a genome-streamlined L-lysine producing strain as basis, metabolic engineering was performed to enable conversion of L-lysine to glutarate in a five-step synthetic pathway comprising lysine decarboxylase, putrescine transaminase and γ-aminobutyraldehyde dehydrogenase from Escherichia coli and GABA/5AVA amino transferase and succinate/glutarate semialdehyde dehydrogenase either from C. glutamicum or from three Pseudomonas species. Loss of carbon via formation of the by-products cadaverine and N-acetylcadaverine was avoided by deletion of the respective acetylase and export genes. As the two transamination reactions in the synthetic glutarate biosynthesis pathway yield L-glutamate, biosynthesis of L-glutamate by glutamate dehydrogenase was expected to be obsolete and, indeed, deletion of its gene gdh increased glutarate titers by 10%. Glutarate production by the final strain was tested in bioreactors (n = 2) in order to investigate stability and reliability of the process. The most efficient glutarate production from glucose was achieved by fed-batch fermentation (n = 1) with a volumetric productivity of 0.32 g L-1 h-1, an overall yield of 0.17 g g-1 and a titer of 25 g L-1

Bromination of L-tryptophan in a fermentative process with Corynebacterium glutamicum

Veldmann K, Dachwitz S, Risse JM, Lee J-H, Sewald N, Wendisch VF. Bromination of L-tryptophan in a fermentative process with Corynebacterium glutamicum. Frontiers in Bioengineering and Biotechnology . 2019;7: 219.Brominated compounds such as 7-bromo-L-tryptophan (7-Br-Trp) occur in Nature. Many synthetic and natural brominated compounds have applications in the agriculture, food, and pharmaceutical industries, for example, the 20S-proteasome inhibitor TMC-95A that may be derived from 7-Br-Trp. Mild halogenation by cross-linked enzyme aggregates containing FAD-dependent halogenase, NADH-dependent flavin reductase, and alcohol dehydrogenase as well as by fermentation with recombinant Corynebacterium glutamicum expressing the genes for the FAD-dependent halogenase RebH and the NADH-dependent flavin reductase RebF from Lechevalieria aerocolonigenes have recently been developed as green alternatives to more hazardous chemical routes. In this study, the fermentative production of 7-Br-Trp was established. The fermentative process employs an L-tryptophan producing C. glutamicum strain expressing rebH and rebF from L. aerocolonigenes for halogenation and is based on glucose, ammonium and sodium bromide. C. glutamicum tolerated high sodium bromide concentrations, but its growth rate was reduced to half-maximal at 0.09 g L−1 7-bromo-L-tryptophan. This may be, at least in part, due to inhibition of anthranilate phosphoribosyltransferase by 7-Br-Trp since anthranilate phosphoribosyltransferase activity in crude extracts was half-maximal at about 0.03 g L−1 7-Br-Trp. Fermentative production of 7-Br-Trp by recombinant C. glutamicum was scaled up to a working volume of 2 L and operated in batch and fed-batch mode. The titers were increased from batch fermentation in CGXII minimal medium with 0.3 g L−1 7-Br-Trp to fed-batch fermentation in HSG complex medium, where up to 1.2 g L−1 7-Br-Trp were obtained. The product isolated from the culture broth was characterized by NMR and LC-MS and shown to be 7-Br-Trp

One-step process for production of N-methylated amino acids from sugars and methylamine using recombinant Corynebacterium glutamicum as biocatalyst

Mindt M, Risse JM, Gruß H, Sewald N, Eikmanns BJ, Wendisch VF. One-step process for production of N-methylated amino acids from sugars and methylamine using recombinant Corynebacterium glutamicum as biocatalyst. Scientific Reports. 2018;8(1): 12895.N-methylated amino acids are found in Nature in various biological compounds. N-methylation of amino acids has been shown to improve pharmacokinetic properties of peptide drugs due to conformational changes, improved proteolytic stability and/or higher lipophilicity. Due to these characteristics N-methylated amino acids received increasing interest by the pharmaceutical industry. Syntheses of N-methylated amino acids by chemical and biocatalytic approaches are known, but often show incomplete stereoselectivity, low yields or expensive co-factor regeneration. So far a one-step fermentative process from sugars has not yet been described. Here, a one-step conversion of sugars and methylamine to the N-methylated amino acid N-methyl-l-alanine was developed. A whole-cell biocatalyst was derived from a pyruvate overproducing C. glutamicum strain by heterologous expression of the N-methyl-l-amino acid dehydrogenase gene from Pseudomonas putida. As proof-of-concept, 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 cultivation. The C. glutamicum strain producing this imine reductase enzyme was engineered further to extend this green chemistry route to production of N-methyl-l-alanine from alternative feed stocks such as starch or the lignocellulosic sugars xylose and arabinose

Patchoulol production with metabolically engineered Corynebacterium glutamicum

Henke NA, Wichmann J, Baier T, et al. Patchoulol production with metabolically engineered Corynebacterium glutamicum. Genes. 2018;9(4): 219.Patchoulol is a sesquiterpene alcohol and an important natural product for the perfume industry. Corynebacterium glutamicum is the prominent host for the fermentative production of amino acids with an average annual production volume of ~6 million tons. Due to its robustness and well established large-scale fermentation, C. glutamicum has been engineered for the production of a number of value-added compounds including terpenoids. Both C40 and C50 carotenoids, including the industrially relevant astaxanthin, and short-chain terpenes such as the sesquiterpene valencene can be produced with this organism. In this study, systematic metabolic engineering enabled construction of a patchoulol producing C. glutamicum strain by applying the following strategies: (i) construction of a farnesyl pyrophosphate-producing platform strain by combining genomic deletions with heterologous expression of ispA from Escherichia coli; (ii) prevention of carotenoid-like byproduct formation; (iii) overproduction of limiting enzymes from the 2-c-methyl-d-erythritol 4-phosphate (MEP)-pathway to increase precursor supply; and (iv) heterologous expression of the plant patchoulol synthase gene PcPS from Pogostemon cablin. Additionally, a proof of principle liter-scale fermentation with a two-phase organic overlay-culture medium system for terpenoid capture was performed. To the best of our knowledge, the patchoulol titers demonstrated here are the highest reported to date with up to 60 mg L−1 and volumetric productivities of up to 18 mg L−1 d−1

Produktion und Analytik von N-Acetyl-phosphinothricin sowie N-Acetyl-phosphinothricyl-alanyl-alanin

Risse JM. Produktion und Analytik von N-Acetyl-phosphinothricin sowie N-Acetyl-phosphinothricyl-alanyl-alanin. Bielefeld; 2001

Proteinsekretion und -aufarbeitung

Risse JM, Friehs K. Sekretion und Affinitätschromatografie rekombinanter Proteine. BIOspektrum. 2010;5:550-552