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

Melanie Mindt,Volker F Wendisch,Joe Max Risse,Tatjana Walter

doi:10.3389/fbioe.2018.00159

Melanie Mindt, Volker F Wendisch + Show 2 more

Open Access

https://doi.org/10.3389/fbioe.2018.00159

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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.

Highlights

Alkylated amino acids became the focus of attention by the pharmaceutical and chemical industry over the last years
Inspection of the genome sequence of P. putida KT2440 revealed that no homologs of enzymes for biosynthesis of NMeGlu (GMAS and N-methylglutamate synthase (NMGS)) are encoded
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

Summary

Introduction

Alkylated amino acids became the focus of attention by the pharmaceutical and chemical industry over the last years. Peptidomimetics are imitating structures of natural bioactive peptides and have improved pharmaceutical properties (Gentilucci et al, 2010; Di Gioia et al, 2016). These advantages are present in naturally occurring alkylated amino acids and peptides such as the medically relevant compounds destruxin B or cyclosporine A. Cyclosporine A is one of the most important and medically relevant examples of an N-methylated peptide It shows potent immunosuppressant properties, low toxicity and metabolic stability as a medical drug for organ transplantation (Di Gioia et al, 2016). Besides the natural N-methylated molecules, N-methylation was applied to natural

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