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

Mindt, Melanie; Risse, Joe Max; Gruß, Hendrik; Sewald, Norbert; Eikmanns, Bernhard J.; Wendisch, Volker F.

doi:10.1038/s41598-018-31309-5

Cited by 33 publications

(48 citation statements)

References 84 publications

(85 reference statements)

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“…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.…”

Section: Discussionmentioning

confidence: 56%

“…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.…”

Section: Discussionmentioning

confidence: 99%

“…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.…”

Section: Discussionmentioning

confidence: 99%

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Fermentative N-Methylanthranilate Production by Engineered Corynebacterium glutamicum

et al. 2020

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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.

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Section: Discussionmentioning

confidence: 56%

Section: Discussionmentioning

confidence: 99%

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Fermentative N-Methylanthranilate Production by Engineered Corynebacterium glutamicum

et al. 2020

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“…Recently, we have shown efficient fermentative production of another N -methylated amino acid, namely, N -methylalanine (Mindt et al, 2018 ). Although high titers (17.9 g L −1 NMeGlu as compared to 31.7 g L −1 NMeAla), yields (0.1 g NMeGlu per g glycerol as compared to 0.71 g NMeAla per g glucose), and volumetric productivities (0.13 g L −1 h −1 for NMeGlu as compared to 0.35 g L −1 h −1 for NMeAla) were reached for both methylated amino acids, the chosen pathways were different.…”

Section: Discussionmentioning

confidence: 99%

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

Mindt

Walter

Risse

et al. 2018

Front. Bioeng. Biotechnol.

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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.

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“…The model organism C. glutamicum is widely used in biotechnological industry, since it is a workhorse able to produce various compounds with high production titers [106–117], especially the amino acids l ‐glutamate [118–121] and l ‐lysine [122–127]. Globally, amino acids from C. glutamicum are produced in million ton scale [109].…”

Section: Metabolic Engineering Applications Of Crisprimentioning

confidence: 99%

Impact of CRISPR interference on strain development in biotechnology

Schultenkämper

Brito

Wendisch

2020

Biotech and App Biochem

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Genetic perturbation systems are of great interest to redirect metabolic fluxes for value‐added production, as well as genetic screening for the development of new drugs, or to identify new targets for biotechnological applications. Here, we review CRISPR interference (CRISPRi), a method for gene expression using a catalytically inactive version of the CRISPR‐associated protein 9 (dCas9) of the widely applied CRISPR‐Cas9 genome editing system. In combination with the appropriate sgRNA, dCas9 binds to specific DNA sequences without causing double‐stranded DNA breakage but interfering with transcription initiation or elongation. Besides manifold uses to interrogate the physiology of a bacterial cell, CRISPRi is used in applications for metabolic engineering and strain development in industrial biotechnology. Albeit in its infancy, CRISPRi has already delivered the first success stories; however, we also analyze limitations of the CRISPRi system and give future perspectives.

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

Cited by 33 publications

References 84 publications

Fermentative N-Methylanthranilate Production by Engineered Corynebacterium glutamicum

Fermentative N-Methylanthranilate Production by Engineered Corynebacterium glutamicum

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

Impact of CRISPR interference on strain development in biotechnology

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