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

Abstract

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.

Keywords: GS/GOGAT; GlpR; Methylobacterium extorquens; N-methylglutamate; N-methylglutamate synthase; Pseudomonas putida; fed-batch fermentation; γ-glutamylmethylamide synthetase.

Figure 1

Schematic overview of the reaction catalyzed by γ-glutamylmethylamide synthetase (GMAS) and N-methylglutamate synthase (NMGS) and their incorporation in the metabolism of P. putida KT2440. N-methylglutamate formation (thick arrows) takes place by heterologous expression of gmaS and mgsABC from M. extorquens DM4 (green boxes) starting from l-glutamate. Deletion of the transcriptional repressor gene of the glycerol metabolism (glpR) is indicated by red cross. The second copy of endogenous glutamate dehydrogenase (gdhA) under control of Ptac was integrated in glpR locus (blue boxes). Reactions are indicated by an arrow, multiple reactions by dashed arrows.

Figure 2

Growth rates of P. putida KT2440 grown in the presence of varying concentrations of MMA. P. puida KT2440 wild type strain was grown in minimal medium supplemented with increasing MMA concentrations (0.05 m – 1.0 m) and growth rates were determined. Half maximal effect on growth rate was determined by linear fit ting (OriginLab, Northampton, MA).

Figure 3

Fed-batch cultivation of P. putida NMG3 using HSG medium supplemented with 100 mM MMA. Starting with an initial volume of 2 L, the feed solution was coupled to the rDOS value. The feed volume is indicated by the black line. After 24 and 48 h 100 mm MMA was added. The biomass formation (black open squares) and concentrations of glycerol (gray closed squares) and NMeGlu (green closed circles) are shown. All concentrations, the feed volume and the biomass formation were related to the initial volume.

Figure 4

Schematic overview of the two-step cascade of GMAS/NMGS (A) in comparison to the GS/GOGAT mechanism (B) and the proposed one-step reaction of NMGS (C) in comparison to the GDH (D) reaction. The γ-glutamylmethylamide synthetase (GMAS) amidates glutamate at C⁵ position followed by a transfer of the N-methylgroup of l-glutamine to 2-oxoglutarate catalyzed by N-methylglutamate synthase (NMGS) (A). Glutamine synthetase (GS) amidates l-glutamate to l-glutamate and the glutamate synthase catalyzes intermolecular transfer of the amino group to 2-oxoglutarate (C). NMGS methylamidates 2-oxoglutarate at C² position to NMeGlu (C). Glutamate dehydrogenase (GDH) reductively aminates 2-oxoglutarate for l-glutamate (D).