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.
Highlights
N-methylated amino acids are non-proteinogenic amino acids found in plants, mammals, and microorganisms
Pre-cultures of C. glutamicum were cultivated in lysogeny broth (LB), and main cultures for growth and production experiments where grown in standard CGXII medium (Eggeling and Bott, 2005) with reduced nitrogen content (2 g L−1 ammonium sulfate and 0.5 g L−1 urea) and given concentrations of carbon source and/or monomethylamine (MMA) at 30◦C on a rotary shaker (120 rpm) in baffled shake flasks
We established fermentative production of sarcosine by methylamination of glyoxylate using DpkA and showed that xylose was superior as source of carbon and energy than glucose (Mindt et al, 2019)
Summary
N-methylated amino acids are non-proteinogenic amino acids found in plants, mammals, and microorganisms. In addition to the incorporated amino acid derivatives, monomeric N-alkylated amino acids occur in nature as intermediates of metabolic pathways such as sarcosine (N-methylglycine) in degradation of creatine and choline, or N-methylglutamate in the C1 assimilation from monomethylamine by methylotrophic bacteria like Methyloversatilis universalis (Latypova et al, 2010). Studies on the effect of the anesthetic lidocaine showed pain reducing properties (Eipe et al, 2016). Further studies on this mechanism showed that the lidocaine metabolite N-ethylglycine (NEtGly) acts as an inhibitor of glycine uptake and inhibits pain signaling. Is a promising candidate for chronical pain treatment (Werdehausen et al, 2015)
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