Fermentative Production of N-Alkylated Glycine Derivatives by Recombinant Corynebacterium glutamicum Using a Mutant of Imine Reductase DpkA From Pseudomonas putida

Abstract

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.

Fermentative N-Methylanthranilate Production by Engineered Corynebacterium glutamicum

“…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.…”

“…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.…”

“…Fermentation processes using simple mineral salts media have been developed for three different routes for de novo production of N -alkylated amino acids. Two metabolic engineering strategies for reductive alkylamination of 2-oxo acids with monomethylamine that either make use of a C1-assimilation pathway present in methylotrophic bacteria [ 13 ] or of the imine reductase DpkA [ 14 ] have been established. S -Adenosyl- l -methionine (SAM)-dependent methylation of aromatic amino acids by N -methyltransferases has also been described [ 15 ].…”

Recent Progress on Chemical Production From Non-food Renewable Feedstocks Using Corynebacterium glutamicum

“…Similarly, acid treatment of wheat bran generates 41 g/L of carbohydrate, containing 125 mM glucose, 62 mM xylose, and 64 mM arabinose, was also can be utilized by engineered C. glutamicum to produce L-glutamate (Gopinath et al, 2011). Inspired by this example, biobased production of N-ethylglycine from rice straw hydrolyzate was enabled in engineered C. glutamicum by introducing a mutant DpkA from P. putida (Mindt et al, 2019a). Moreover, small quantities of plant biomass hydrolyzates, such as those derived from sorghum, softwood lignin, and Miscanthus, also deserve attention in the fermentation field.…”

Overview on Multienzymatic Cascades for the Production of Non-canonical α-Amino Acids

“…In this sense, special consideration should be paid to natural secondary metabolic routes conducting to NcAAs (or using them) to expand established MECs for their synthesis. Metabolic engineering of microorganisms have also allowed production of NcAAs such as L -norvaline/ L -norleucine ( Anderhuber et al, 2016 ), L -citruline ( Eberhardt et al, 2014 ), hydroxyproline ( Falcioni et al, 2015 ), L -ornithine ( Wu et al, 2020 ) different D -α-AAs ( Mutaguchi et al, 2018 ) or different N -Alkylated AAs ( Mindt et al, 2019 , 2020 ; van der Hoek et al, 2019 ). Thus, isolated enzymes comprised in biosynthetic metabolic routes for the production of different compounds ( Hedges and Ryan, 2020 ) might find application on some of the existing MECs described in this paper.…”