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

Abstract

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.

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.…”

“…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.…”

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

“…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.…”

Impact of CRISPR interference on strain development in biotechnology

“…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].…”