γ‐Glutamylation of Isopropylamine by Fermentation

Abstract

Glutamylation yields N‐functionalized amino acids in several natural pathways. γ‐Glutamylated amino acids may exhibit improved properties for their industrial application, e. g., as taste enhancers or in peptide drugs. γ‐Glutamyl−isopropylamide (GIPA) can be synthesized from isopropylamine (IPA) and l‐glutamate. In Pseudomonas sp. strain KIE171, GIPA is an intermediate in the biosynthesis of l‐alaninol (2‐amino‐1‐propanol), a precursor of the fluorochinolone antibiotic levofloxacin and of the chloroacetanilide herbicide metolachlor. In this study, fermentative production of GIPA with metabolically engineered Pseudomonas putida KT2440 using γ‐glutamylmethylamide synthetase (GMAS) from Methylorubrum extorquens was established. Upon addition of IPA during growth with glycerol as carbon source in shake flasks, the recombinant strain produced up to 21.8 mM GIPA. In fed‐batch bioreactor cultivations, GIPA accumulated to a titer of 11 g L−1 with a product yield of 0.11 g g−1 glycerol and a volumetric productivity of 0.24 g L−1 h−1. To the best of our knowledge, this is the first fermentative production of GIPA.

Establishment of synthetic microbial consortia with Corynebacterium glutamicum and Pseudomonas putida: Design, construction, and application to production of γ‐glutamylisopropylamide and l‐theanine

“…strain KIE171 (de Azevedo Wäsch et al., 2002 ; Pan et al., 2020 ). For the production of both amines, monocultures of P. putida Thea1 have been described (Benninghaus et al., 2021 , 2023 ) and the strain was developed further for use in consortia with C. glutamicum . The enzyme γ‐glutamyl‐methylamide synthetase (EC 6.3.4.12, GMAS), encoded by gmaS , from Methylorubrum extorquens , was used and monoethylamine (MEA) or isopropylamine (IPA) was added as amine donor for production of theanine or GIPA, respectively.…”